Hindawi Publishing CorporationMediators of InflammationVolume 2013 Article ID 159349 13 pageshttpdxdoiorg1011552013159349
Research ArticleTNF-120572 Regulates Natriuretic Peptides and Aquaporins inHuman Bronchial Epithelial Cells BEAS-2B
Letizia Mezzasoma1 Lucio Cagini2 Cinzia Antognelli1 Francesco Puma2
Eugenio Pacifico3 and Vincenzo Nicola Talesa1
1 Department of Experimental Medicine and Biochemical Sciences University of Perugia Polo Unico SantrsquoAndrea delle Fratte06156 Perugia Italy
2Thoracic Surgery Unit Ospedale S Maria della Misericordia University of Perugia S Andrea delle Fratte 06156 Perugia Italy3 Clinical Pathology and Hematology Unit Ospedale S Maria dellaMisericordia of Perugia S Andrea delle Fratte 06156 Perugia Italy
Correspondence should be addressed to Vincenzo Nicola Talesa vincenzotalesaunipgit
Received 15 May 2013 Revised 26 August 2013 Accepted 7 October 2013
Academic Editor Yong Jiang
Copyright copy 2013 Letizia Mezzasoma et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
Postoperative-fluid retention is a severe complication frequently reported in patients undergoing major surgical procedures Thecomplex network of molecules involved in such a severe surgery-induced condition remains poorly understood Inflammation hasbeen proposed among the various causes of fluid retention Since TNF-120572 is one of the main proinflammatory cytokine initiallyreleased after major surgery it is reasonable to assume its involvement in fluid overload Here we showed that TNF-120572 selectivelyregulates keymolecules involved in fluids balance such as natriuretic peptides (NPs) and aquaporins in human bronchial epithelialcells BEAS-2B In particular we found that TNF-120572 induced a decrease of arial natriuretic peptide natriuretic peptide receptor-1aquaporin-1 and aquaporin-5 and an increase of brain natriuretic peptide with a different involvement of nuclear factor-120581B andmitogen-activated protein kinases signaling pathway activation Moreover the observed changes in NPs expression demonstrateinflammation as an additional cause of brain natriuretic peptide elevation adding an important piece of information in the novelarea of study regarding NPs and inflammation Finally we suggest that inflammation is one of the mechanisms of Aquaporin-1 and aquaporin-5 expression regulation Therefore in this exploratory study we speculate that TNF-120572 might be involved inpostoperative-fluid retention related to major surgery
1 Introduction
Weight gain with edema formation is frequently reportedin patients undergoing major surgical procedures with anincidence as high as 40 [1] Postoperative weight gain andfluid overload have been associated with poor survival [2]and complications [3 4] The causes of fluid retention arevarious and not completely clear One of them could berelated to the systemic response induced by surgical stressand operative trauma regulated by a complex network ofendocrine neuronal and immunological mechanisms [5ndash7] Such surgery-induced reaction leads to an early hyperin-flammatory status that is essential for tissue repair and hostdefense [5] Cytokines are thought to play a pivotal role inthe pathogenesis of surgical trauma They have local effects
of mediating and maintaining the inflammatory response totissue injury and also initiate some of the systemic changeswhich occur [8ndash10] After major surgery TNF-120572 is one ofthe main proinflammatory cytokines initially released in thedamaged tissuewhere it stimulates the production and releaseof more cytokines responsible for inducing the systemicchanges known as the acute phase response [5 10]Thereforeit is reasonable to assume TNF-120572 involvement among thevarious causes of fluid retention and thus it would be veryimportant to understand the mechanisms underlying itsinvolvement in this area
In a recent study patients undergoing pulmonary lobec-tomy showed a significant weight gain correlated with fluidretention and an early rise in the plasma concentrations ofbrain natriuretic peptide (BNP) a member of the natriuretic
2 Mediators of Inflammation
peptides (NPs) family [11] In particular a significant weightgain was found to be correlated with large volumes of fluidsaccumulation on the postoperative day 2 despite a negativeintraoperative fluid balance and peroperative strict fluidrestriction [11] Moreover the patients none of whom devel-oped signs or symptoms of heart failure during the postop-erative period showed immediately after surgery (on day 1)a significant increase in BNP plasma concentration [11] NPsare hormoneparacrine factors that are released by the heartin response to myocardial stretch and overload modulatingbody fluid homeostasis [12 13] BNP secreted from the car-diac ventricles and atrial natriuretic peptide (ANP) secretedfrom the cardiac atria activate the same transmembraneguanylyl cyclase-Anatriuretic peptide receptor-A (NPR-A orNPR-1) [14ndash17] In addition to vasodilation cardiovascularhomeostasis sodium excretion and inhibition of aldosteronesecretion it is becoming increasingly recognized that NPspossess a much broader range of biological activities includ-ing effects on endothelial function and inflammation [1819] The genetic expression and secretion of ANP and BNPhave been studied mainly in the context of cardiac diseasesassociated with neuroendocrine and hemodynamic changes[12 13 20] however it has been pointed out that changes inBNP also occur in a context of an acute inflammatory process[19 21ndash24]
Another family of proteins aquaporins (AQPs) is deeplyinvolved in the physiological response to change of fluidvolume and osmolarity [25 26] AQPs are widely distributedin various tissues throughout the body and facilitate osmot-ically driven water transport across cell membranes [25ndash27]Recently AQPs involvement in edema development has beenpointed out In particular it has been shown that AQP4 isan essential mediator in the formation and resorption ofedema fluid from brain parenchyma [28] and that AQP1 andAQP5 might play an important role in lung edema [29] Inaddition AQP1 and AQP5 expression is decreased in lunginflammation [30 31]
The aim of our work was to investigate the potentialinvolvement of TNF-120572 in the regulation of ANP BNP andtheir receptor NPR-1 as well as AQP1 and AQP5 keymolecules involved in body fluid homeostasis In order toexclude any hemodynamic change able to modulate NPsexpression we carried out an in vitro study in humanbronchial epithelial cells BEAS-2B
2 Materials and Methods
21 Reagents Human TNF-120572 was obtained from Immuno-Tools GmbH (Friesoythe Germany) The Nuclear Factor-120581B (NF-120581B) inhibitors BAY 11ndash7082 and QNZ as wellas the p38 mitogen-activated protein kinases (p38 MAPK)inhibitor SB 203580 the c-Jun N-terminal kinases 12(JNK 12) inhibitor SP 600125 and the extracellular-signal-regulated kinases 12 (ERK 12) inhibitor U-0126 wereobtained from Santa Cruz Biotechnology Inc (HeidelbergGermany) andwere dissolved in dimethyl sulfoxide (DMSO)Human BNP was obtained from Phoenix Europe GmbH(Karlsruhe Germany) Dexamethasone (DEX) was fromSigma-Aldrich (Milan Italy) Rabbit polyclonal antibodies
(Abs) against ANP NPR-1 AQP1 and AQP5 as well as themouse monoclonal Ab against 120573-actin and the appropriateHRP-conjugated secondary Abs were purchased from SantaCruz Biotechnology Inc (Heidelberg Germany) Rabbitmonoclonal Abs against Phospho-I120581B-120572 and Phospho-p38MAPK were purchased from Cell Signaling Technology Inc(Danvers MA)
22 Cell Culture and Drug Treatments Human bronchialepithelial cell line BEAS-2B was purchased from AmericanType Culture Collection (ATCC) and was routinely main-tained at 37∘C in 5 CO
2in RPMI 1640 supplemented
with 10 heat inactivated (1 h at 56∘C) fetal calf serum1X Lglutamine 1mM sodium pyruvate 1X nonessentialamino acids 100 unitsmL of penicillin and 01mgmL ofstreptomycin (Invitrogen Milan Italy) Forty-eight hoursbefore study cells were seeded onto six-well culture dishes at300000 cellswell TNF-120572was dissolved in distilledwater andused at the concentrations of 10 20 and 40 ngmL for 24 hInhibitors BAY 11-7082 QNZ SB 203580 SP 600125 and U-0126 were dissolved in 05 DMSO The anti-inflammatoryglucocorticoid DEX was dissolved in 01 methanol Inindependent experiments BAY 11-7082 QNZ SB 203580 SP600125 and U-0126 and DEX were added to cells 60minbefore TNF-120572 administration at the concentration of 1120583Mfor BAY 11-7082 or DEX and 10 120583M for QNZ SB 203580 SP600125 or U-0126 DMSO or methanol final concentrationsin each assay were 0005 and 0001 respectively Controlcells with DMSO or methanol did not show any significantdifference respect to control cells in RPMI 1640 mediumtherefore all the relative treatments were compared to theselatter controls
23 Cell Viability The effects of TNF-120572 BAY 11-7082 QNZSB 203580 SP 600125 U-0126 DEX and BNP treatmentswere measured with a standard trypan blue uptake assayCell cultures were also examined morphologically via lightmicroscopy
24 RNA Isolation and cDNA Synthesis Total cellular RNAwas isolated using TRIzol Reagent (Invitrogen Milan Italy)according to the manufacturerrsquos instructions and 1 120583g wasreverse transcribed using the RevertAidHMinus First StrandcDNA Synthesis Kit (Fermentas Hanover MD) and randomprimers System (Invitrogen Milan Italy) according to themanufacturerrsquos instructions
25 Quantitative Real Time SYBR Green PCR Analysis Weemployed quantitative Real Time SYBR Green PCR analysis(qRT-PCR) on Mx3000P QPCR Systems (Agilent Tech-nologies Milan Italy) to evaluate the expression of ANP(NM 006172) BNP (NM 002521) NPR-1 (NM 000906)AQP1(NM 198098) and AQP5 (NM 001651) versus ACTB(NM 001101)The sequences of oligonucleotide primers usedfor qRT-PCR and the thermal cycling conditions are sum-marized in Table 1 All primers were designed using BeaconDesigner 4 software (Stratagene La Jolla CA) starting frompublished sequences data supplied by the NCBI database
Mediators of Inflammation 3
Table 1 Primer sequences primer concentrations and qRT-PCR cycling conditions
Gene Primer sequences Primer concentrations qRT-PCR cycling conditions
ANP F 51015840-tcagcccagcccagagag-31015840 200 nM 1 cycle at 95∘C for 10min 40 cycles at 95∘C for 1minR 51015840-gctccaatcctgtccatcctg-31015840 200 nM 60∘C for 30 sec and 72∘C for 30 sec
BNP F 51015840-gagggcaggtgggaagcaaac-31015840 200 nM 1 cycle at 95∘C for 10min 40 cycles at 95∘C for 1minR 51015840-gcaagaagagcaggagcaggag-31015840 200 nM 60∘C for 30 sec and 72∘C for 30 sec
NPR-1 F 51015840-ccctggaggtgctggctttgg-31015840 200 nM 1 cycle at 95∘C for 10min 40 cycles at 95∘C for 1minR 51015840-ctctcaaggctactgggctcaacg-31015840 200 nM 59∘C for 30 sec and 72∘C for 30 sec
AQP1 F 51015840-ttggacacctcctggctattgact-31015840 400 nM 1 cycle at 95∘C for 10min 40 cycles at 95∘C for 1minR 51015840-ccagtggttgctgaagttgtgtgt-31015840 400 nM 60∘C for 30 sec and 72∘C for 30 sec
AQP5 F 51015840-cgctcaacaacaacacaacgc-31015840 200 nM 1 cycle at 95∘C for 10min 40 cycles at 95∘C for 1minR 51015840-ccagtgaagtagattccgacaagg-31015840 200 nM 59∘C for 30 sec and 72∘C for 30 sec
ACTB F 51015840-cactcttccagccttccttcc-31015840 600 nM 1 cycle at 95∘C for 10min 40 cycles at 95∘C for 1minR 51015840-acagcactgtgttggcgtac-31015840 600 nM 59∘C or 60∘C for 30 sec and 72∘C for 30 sec
qRT-PCR quantitative real time polymerase chain reaction F forward R reverse
Reactionswere performed in a total volume of 25120583L contain-ing 250 ng or 500 ng of cDNA 1X Reagent Brilliant II SYBRGreen QPCRMaster Mix and the appropriate concentrationof the specific primers (Medical Milan Italy) Data for com-parative analysis of gene expression were obtained using theΔΔCt method as described in the ABI Prism 7000 sequencedetection system user guide [32] Agarose gel electrophoreticanalysis was used to check the predicted size amplicons forANP (160 bp) BNP (148 bp) NPR-1 (151 bp) AQP1 (98 bp)and AQP5 (180 bp)
26Western Blot Analysis Total protein extracts (40120583g)wereseparated by 12 sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) andblotted onto a nitrocellulosemembrane using iBlot Dry Blotting System (InvitrogenMilan Italy) Nonspecific binding sites were blocked in Roti-Block (Roth GmbH Karlsruhe Germany) for 2 h at roomtemperature The membranes were blotted overnight at 4∘Cwith a 1 100 dilution of the primary specific Abs and washedwith TBSTThe antigen-Ab complex was detected by incuba-tion of the membranes for 2 h at room temperature with theappropriated HRP-conjugated secondary Abs and revealedusing the enhanced chemiluminescence (ECL) system byAmersham Pharmacia Biotech (Uppsala Sweden)
As internal loading controls and for protein expressionnormalizing purpose all membranes were subsequentlystripped of the first antibody in a stripping buffer (100mM2-ME 2 SDS and 625mM Tris-HCl pH 68) for 30minat 50∘C followed by washings with TBST The membraneswere then reprobed with anti-120573-actin antibody followed byincubation with HRP-conjugated Ab
27 BNP Protein Concentration in Culture Medium BNPprotein concentrationwasmeasured in culturemediumusingthe chemiluminescent enzyme immunoassay kit TRIAGEBNP (Biosite Incorporated San Diego USA) The minimumquantity of human BNP detectable with this system is10 pgmL The intraassay and the interassay coefficients ofvariations were 31 and 45 respectively
28 Statistical Analysis Results were expressed as means plusmnSD of three independent experiments The statistical signifi-cance of differences between treated and untreated cells wasassessed by Studentrsquos t-test Differences between groups wereconsidered significant when 119875 lt 005
3 Results
31 TNF-120572 Selectively Modulates ANP BNP and Their Recep-tor NPR-1 Expression in BEAS-2B Cells We firstly demon-strated that BEAS-2B cell line expresses ANP BNP NPR-1AQP1 and AQP5 By qRT-PCR analysis fragments of thepredicted molecular size were generated (Figure 1) To deter-mine whether the expression of ANP BNP and NPR-1 wasaffected by TNF-120572 BEAS-2B cells were treated with 10 20or 40 ngmL TNF-120572 for 24 h ANP mRNA was significantlydecreased about 75 (119875 lt 00001) and about 40 (119875 lt005) of control after treatment with 10 and 20 ngmL TNF-120572 respectively Forty ngmL TNF-120572 induced an upregulation(about 20 of control) of ANP expression even though notstatistically significant (119875 gt 005) (Figure 2(a)) A similartrend was also observed at protein level (Figure 2(b)) BNPmRNA expression was significantly (119875 lt 001) increased(62 of control) since 10 ngmL TNF-120572 treatment maximumstimulation (80 of control) being obtained with 40 ngmL(119875 lt 00001) (Figure 2(c)) Treatment with TNF-120572 at theabove described concentrations also increased BNP proteinsecretion in culture medium (119875 lt 001) (Figure 2(d))NPR-1 gene expression level was dramatically decreased (70ndash80 of control) after TNF-120572 exposure at all the used doses(119875 lt 0001) (Figure 2(e)) paralleled by a decrease of proteinexpression (Figure 2(f))
32 NPs and NPR-1 Modulation by TNF-120572 May RequireActivation of NF-120581B Signaling Pathway in BEAS-2B CellsAs known TNF-120572 regulates numerous genes essential tothe inflammatory process through the activation of mul-tiple signal transduction pathways including NF-120581B [33]Therefore to investigate the possible involvement of NF-120581B signaling pathway in TNF-120572-mediated regulation of NPs
4 Mediators of Inflammation
2 3 4M
242190147
(bp) (bp)M
111110147190
67
5 6
Figure 1 Identification of ANP BNP NPR-1 AQP1 and AQP5transcripts in BEAS-2B cells qReal-Time PCR was performed usinggene specific primers for ANP (lane 2) BNP (lane 3) NPR-1 (lane 4)AQP1 (lane 5) sand AQP5 (lane 6) in BEAS-2B cells Single bands ofthe predicted molecular size for ANP (160 bp) BNP (148 bp) NPR-1(151 bp) AQP1 (98 bp) andAQP5 (180 bp) transcripts were detectedLane M DNA molecular marker
and NPR-1 we used the specific NF-kB inhibitor BAY 11-7082 that diminishes the activation of NF-120581B by preventingphosphorylation of its inhibitory I120581B-120572 protein As shownin Figure 3(a) TNF-120572-induced downregulation of ANP wascompletely reverted by BAY 11-7082 at both mRNA and pro-tein level (Figure 3(b)) suggesting that this response requiresNF-120581B activation pathway Regarding TNF-120572-induced BNPupregulation this was only partially reverted by BAY 11-7082at mRNA level (Figure 3(c)) while was completely revertedat protein level (Figure 3(d)) Conversely TNF-120572-induceddecrement in NPR-1 expression was not affected by BAY 11ndash7082 either at mRNA or protein level (Figures 3(e) and 3(f))
Western blot analysis for the Ser32-phosphorylated I120581B-120572protein proved the biochemistry evidence of the inhibitoryaction of BAY 11-7082 on NF-120581B activity (Figure 3(g)) Tofurther investigate the role of NF-120581B in the modulation ofBNP and NPR-1 in BEAS-2B cells after TNF-120572 treatmentwe employed theNF-120581B-DNA binding inhibitor QNZ whichprevents free NF-120581B from binding to DNA [34] The resultsobtained with such additional inhibitor confirmed the previ-ous findings obtained with BAY 11-7082 In particular TNF-120572-induced BNP up-regulation was not reverted by QNZ atmRNA level (Figure 4(a)) while it was completely reverted atprotein level (Figure 4(b)) Accordingly also TNF-120572-induceddecrement of NPR-1 expression was not affected by QNZeither at mRNA or protein level (Figures 4(c) and 4(d))Western blot analysis for the Ser32-phosphorylated I120581B-120572protein proved the biochemistry evidence of the inhibitoryaction of QNZ on NF-120581B activity (Figure 4(e))
33 BNP and NPR-1 Modulation by TNF-120572 May RequireActivation of MAPKs Signaling Pathway in BEAS-2B CellsBecause TNF-120572 is able to activate multiple signal transduc-tion pathways including MAPKs and because the activationof p38 MAPK and JNK participates in the regulation ofinflammatory processes in bronchial epithelial cells [35 36]we then investigatedMAPKs pathway involvement in TNF-120572modulation of BNP and NPR-1 expression TNF-120572-mediated
up-regulation of BNP was not affected by SB 203580 or SP600125 p38 MAPK and JNK 12 inhibitors respectivelywhile it was completely reverted by the ERK 12 inhibitorU-0126 (Figures 5(a) and 5(b)) suggesting that ERK 12activation pathway is required for BNP TNF-120572-mediatedmodulation Regarding NPR-1 the TNF-120572-mediated down-regulation was completely reverted by the use of all inhibitors(Figures 5(c) and 5(d)) Western blot analysis for theThr180Thr182-phosphorylated form of p-38 MAPK provedthe biochemistry evidence of the inhibitory action of SB203580 on p38 MAPK activity (Figure 5(e))
34 TNF-120572 Decreases AQP1 and AQP5 Expression in BEAS2B Cells We found that TNF-120572 induced a dose dependentdown-regulation of AQP1 expression (119875 lt 001) at mRNA(Figure 6(a)) and protein level (Figure 6(b)) and a dramaticdecrease in AQP5 gene expression level (about 80 ofcontrol) with all the used doses (119875 lt 0001) (Figure 6(c)) par-alleled by a comparable trend at protein level (Figure 6(d))
35 Decreased AQP1 andAQP5 Expression by TNF-120572DoesNotRequire Activation of NF-120581B Signaling Pathway in BEAS-2BCells To examine a possible involvement of NF-120581B in TNF-120572-mediated down-regulation of AQP1 and AQP5 BEAS-2Bcells were incubated with BAY 11-7082 Cotreatment of suchNF-120581B inhibitor with TNF-120572 resulted in a decrease of AQP1expression which was similar to that observed with TNF-120572alone (Figures 7(a) and 7(b)) suggesting that the observedresponse does not require NF-120581B activation Conversely co-treatment of BAY 11-7082 with TNF-120572 affected AQP5 only atprotein level (Figures 7(c) and 7(d))
36 BNP Effect on ANP NPR-1 and AQP1 or AQP5 mRNAExpression in BEAS-2BCells In order to determine a possibledirect involvement of BNP on the gene expression of ANPNPR-1 and AQP1 or AQP5 BEAS-2B cells were treated with001 nM BNP This concentration was previously found tobe accumulated in culture medium after 40 ngmL TNF-120572treatment the dose that induced the maximum effect on theexpression of the studied genes BNP administration induceda significant (119875 lt 005) increase (100 of control) on ANPmRNA level (Figure 8(a)) and did not affectNPR-1 expression(Figure 8(b)) while induced a marked decrease (45 ofcontrol) on AQP1 mRNA level (119875 lt 005) (Figure 8(c))A trend in decreasing was also observed for AQP5 geneexpression even though not statistically significant (119875 gt005) (Figure 8(d))
37 Dexamethasone (DEX) Effect on BNP mRNA and ProteinLevels in BEAS-2B Cells In order to study the capability ofthe anti-inflammatory drug DEX to affect TNF-120572-mediatedup-regulation of BNP mRNA and protein levels BEAS-2Bcells were treated with 1120583MDEX As shown in Figure 9 DEXinduced a down-regulation of BNP expression and proteinlevels alone or in combination with 40 ngmL TNF-120572 (119875 lt001) demonstrating the capability of this glucocorticoid torevert TNF-120572 effects
Mediators of Inflammation 5
0 10 20 40TNF-120572 (ngmL)
AN
P (
of c
ontro
l)
160
140
120
100
80
60
40
20
0
lowast
lowastlowastlowastlowast
(a)
0 10 20 40
ANP
Actin TNF-120572 (ngmL)
(b)
0
50
100
150
200
0 10 20 40
BNP
( o
f con
trol)
TNF-120572 (ngmL)
lowastlowastlowastlowastlowastlowast
lowast
(c)
0
50
100
150
200
0 10 20 40BN
P (
of c
ontro
l)
TNF-120572 (ngmL)
lowastlowast
lowastlowast
lowastlowast
(d)
0 10 20 40TNF-120572 (ngmL)
120
100
80
60
40
20
0
NPR
-1 (
of c
ontro
l)
lowastlowast
lowastlowastlowastlowastlowastlowastlowastlowast
(e)
0 10 20 40
NPR-1
Actin TNF-120572 (ngmL)
(f)
Figure 2 TNF-120572 selectively modulates ANP BNP and NPR-1 expression BEAS-2B cells were treated with 10 20 and 40 ngmL TNF-120572 for24 h ((a) (b)) ANP ((c) (d)) BNP and ((e) (f)) NPR-1 gene expression at mRNA ((a) (c) and (e)) and protein level ((b) (d) and (f))Western blots were obtained by using the specific rabbit polyclonal Abs The blots were stripped of the bound Ab and reprobed with mouseanti-120573-actin to confirm equal loading Western blots are representative of three separate experiments All histograms indicate meansplusmn SD ofthree different cultures each one tested in quadruplicate and expressed as percentage of control (lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 andlowastlowastlowastlowast
119875 lt 00001 versus untreated cells)
4 Discussion
Postoperative-fluid retention is a severe complication fre-quently reported in patients undergoing major surgical pro-cedures [1ndash4] The complex network of molecules involvedin such a severe surgery-induced condition remains poorlyunderstood After major surgery TNF-120572 is one of the mainproinflammatory cytokines initially released in the damagedtissue where it stimulates the production and release of morecytokines responsible for inducing the systemic changesknown as the acute phase response [5 10] Therefore it isreasonable to assume TNF-120572 involvement among the variouscauses of fluid retention and very important to understandthemechanisms underlying its involvement in such an ambit
In the present study we demonstrated for the first time toour knowledge that TNF-120572modulates the expression ofANPBNP NPR-1 AQP1 and AQP5 in human bronchial epithelialcells BEAS-2B via different involvement of NF-120581B andMAPKs signaling pathway activation Here we also providedthe first demonstration that human bronchial epithelial cellsexpress ANP BNP NPR-1 and AQP1 and that BNP is able tomodulate AQP1 expression
The observed expression of NPs and NPR-1 suggestsan autocrine andor paracrine function for these moleculesand indicates important roles for NPs in several biologicalfunctions including regulation of fluid movement across theairway epithelial cells bronchial relaxation [37] vasodilata-tion and pulmonary vascular permeability About the former
6 Mediators of Inflammation
020406080
100120140160
C BAY 10 20 40
AN
P (
of c
ontro
l)
TNF-120572 (ngmL)
TNF-120572TNF-120572 + BAY
lowast
lowastlowastlowastlowast
(a)
ANP
Actin 40
BAY 0 0 10 20 40 10 20minus + +minus minus minus + +
Figure 3 TNF-120572 modulation of NPs and NPR-1 may require activation of NF-120581B pathway BEAS-2B cells were treated with 10 20 and40 ngmL TNF-120572 alone or in combination with NF-120581B inhibitor BAY 11-7082 (1120583M) for 24 h ((a) (b)) ANP ((c) (d)) BNP ((e) (f)) NPR-1gene expression at mRNA ((a) (c) and (e)) and protein level ((b) (d) (f)) and (g) Phospho-I120581B-120572 protein level Western blots were obtainedby using the specific rabbit polyclonal or monoclonal AbsThe blots were stripped of the bound Ab and reprobed with mouse anti-120573-actin toconfirm equal loading Western blots are representative of three separate experiments All histograms indicate means plusmn SD of three differentcultures each of one tested in quadruplicate and expressed as percentage of control (lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowastlowast119875 lt 00001 versus Controls)(∘119875 lt 005 ∘∘119875 lt 001 and ∘∘∘∘119875 lt 00001 versus BAY) C Controls (untreated cells)
process the role of ANP and BNP could be analogous to thathypothesized forNPs in transepithelial ion flux in the choroidplexus [38] in the kidney [39] colon [40] and retina [41]
The observed TNF-120572 capability of selectively regulatingANP BNP and NPR-1 gene expression in BEAS-2B cellline is of particular interest because it demonstrates that
inflammation alters NPs expression levels In particular thesignificant up-regulation of BNP expression and proteinsecretion after TNF-120572 treatment shows that this modulationcan occur independently of hemodynamic influences andthat inflammation should be considered an extracardiaccause of BNP elevation Besides in vitro studies conducted in
Mediators of Inflammation 7
020406080
100120140160180200
C QNZ TNF
BNP
( o
f con
trol)
QNZ + TNF
lowastlowast
∘∘
(a)
0
20
40
60
80
100
120
140
160
180
BNP
( o
f con
trol)
C QNZ TNF QNZ + TNF
lowastlowast
(b)
0
20
40
60
80
100
120
C QNZ TNF QNZ + TNF
∘∘
lowastlowastlowastlowast
lowastlowastlowastlowast
NPR
-1(
of c
ontro
l)
(c)
NPR-1 Actin
0 0QNZ
10 10TNF-120572+minus +minus
(d)
Actin
QNZ TNF-120572 + +
minus ++
minusminus
minus
P-I120581B-120572
(e)
Figure 4 TNF-120572 modulation of protein levels of BNP and NPR-1 may require activation of NF-120581B pathway BEAS-2B cells were treated10 ngmL TNF-120572 alone or in combination with the inhibitor QNZ (10120583M) for 24 h ((a) (b)) BNP ((c) (d)) NPR-1 gene expression at mRNA((a) (c)) and protein ((b) (d)) level and (e) Phospho-I120581B-120572 protein level Western blots were obtained by using the specific rabbit polyclonalor monoclonal AbsThe blots were stripped of the bound Ab and reprobed with mouse anti-120573-actin to confirm equal loading Western blotsare representative of three separate experiments All histograms indicatemeansplusmnSDof three different cultures each one tested in quadruplicateand expressed as percentage of control (lowastlowast119875 lt 001 and lowastlowastlowastlowast119875 lt 00001 versus Controls) (∘∘119875 lt 001 versus QNZ) C Controls (untreatedcells)
rat cardiomyocytes have shown that not only hemodynamicfactors but also neurohumoral factors activated during heartfailure such as angiotensin II endothelin and cytokinescause BNP secretion [42ndash44] In addition in patients withcardiovascular diseases plasma BNP levels have been shownto be also affected by low-grade inflammation [24] and aselective increase inBNPplasma levels has been proposed as ageneral feature of inflammation [19]The lack on any apparentTNF-120572 dose-depending increase in BNP gene expression thatwe observed in our system could be due to an autocrinenegative feedback action mediated by BNP itself followingbinding to the NPR-1 receptor expressed in BEAS-2B cells
The biological role of BNPup-regulation after TNF-120572 treatment remains to be elucidated However since it
has been demonstrated that BNP regulates the productionof major inflammatory molecules such as IL-12 IL-10leukotriene B and prostaglandin E2 in human macrophages[45] and that BEAS-2B are able to release inflammatorymolecules [46] it could be reasonable to assume that theobserved BNP increase after TNF-120572 treatment may stimu-late the production and release of such cytokines also fromhuman epithelial bronchial cells In addition in in vitrostudies a potent BNP inhibitory action on the productionof the glucocorticoid anti-inflammatory hormone cortisolhas been described [47] Therefore BNP could be con-sidered a proinflammatory molecule by inducing cytokinesexpression in target cells and inhibiting the production ofanti-inflammatory hormones Therefore the observed BNP
8 Mediators of Inflammation
0
50
100
150
200
250BN
P (
of c
ontro
l)
C
TNF SB
SB+
TNF SP
SP+
TNF
U-0
126
U-0126+
TNF
lowastlowast
lowastlowast
∘∘
sect
(a)
020406080
100120140160180
BNP
( o
f con
trol)
C
TNF SB
SB+
TNF SP
SP+
TNF
U-0
126
U-0126+
TNF
lowastlowast
lowastlowast
∘∘∘
sectsect
(b)
0
20
40
60
80
100
120
140
160
C
TNF SB
SB+
TNF SP
SP+
TNF
U-0
126
U-0126+
TNF
lowastlowastlowast
lowastlowastlowast
lowast
lowast
sect
NPR
-1(
of c
ontro
l)
(c)
+
SB 203580
+
SP 600125 U-0126
+ActinTNF-120572
NPR-1
minus minus minus minus +
(d)
SB 203580
Actin
P-p38 MAPK
TNF-120572 + +
minus ++
minusminus
minus
(e)
Figure 5 TNF-120572 modulation of BNP and NPR-1 expression may require activation of MAPKs pathway BEAS-2B cells were treated with10 ngmL TNF-120572 alone or in combination with 10 120583M SB 203580 (p38 MAPK inhibitor) SP 600125 (JNK 12 inhibitor) or U-0126 (ERK12 inhibitor) for 24 h Panels show ((a) (b)) BNP ((c) (d)) NPR-1 gene expression at mRNA ((a) (c)) and protein ((b) (d)) level and (e)Phospho-p38 MAPK protein level Western blots were obtained by using the specific rabbit polyclonal or monoclonal Abs The blots werestripped of the bound Ab and reprobed with mouse anti-120573-actin to confirm equal loading Western blots are representative of three separateexperiments All histograms indicate means plusmn SD of three different cultures each one tested in quadruplicate and expressed as percentage ofcontrol (lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 versus Controls) (∙∙119875 lt 001 versus SB) (sect119875 lt 005 sectsect
119875 lt 001 versus SP) (∘∘119875 lt 001∘∘∘
119875 lt 0001 versus U-0126) C Controls (untreated cells)
up-regulation after TNF-120572 treatment could concur in poten-tiating an inflammatory statusThe increased production andrelease of specific cytokines causing systemic changes knownas the acute phase response [5 8 10] induced by TNF-120572 maythus also occur via BNP up-regulation
Recently a BNP rise has been observed in patientsundergoing pulmonary lobectomy for lung cancer [11] Inparticular the patients none of whom developed signs orsymptoms of heart failure during the postoperative period
showed an early significant increase on day 1 immediatelyafter surgery of the plasma concentrations of BNP whichwas followed on the postoperative day 2 by a significantweight gain correlated with large volumes of fluids accumu-lation despite a negative intraoperative fluid balance andperoperative strict fluid restriction [11] We suggest thatsuch an increase could be at least in part explained bythe ability of proinflammatory cytokines such as TNF-120572 toupregulate BNP gene expression and secretion and that this
Mediators of Inflammation 9
0
20
40
60
80
100
120
0 10 20 40TNF-120572 (ngmL)
AQP1
( o
f con
trol)
lowast
lowastlowastlowast
lowastlowastlowast
(a)
Actin 0 10 20 40
AQP1
TNF-120572 (ngmL)
(b)
0
20
40
60
80
100
120
AQP5
( o
f con
trol)
0 10 20 40
TNF-120572 (ngmL)
lowastlowast
lowastlowastlowastlowast
(c)
AQP5
Actin 0 10 20 40TNF-120572 (ngmL)
(d)
Figure 6 TNF-120572 decreases AQP1 and AQP5 expression in BEAS 2B cells BEAS-2B cells were treated with 10 20 and 40 ngmL TNF-120572 for24 h Gene expression of AQP1 ((a) (b)) and AQP5 ((c) (d)) at mRNA ((a) (c)) and protein ((b) (d)) level Western blots were obtained byusing the specific rabbit polyclonal Abs The blots were stripped of the bound Ab and reprobed with mouse anti-120573-actin to confirm equalloading Western blots are representative of three separate experiments All histograms indicate means plusmn SD of three different cultures eachone tested in quadruplicate and expressed as percentage of control (lowast119875 lt 001 lowastlowast119875 lt 0001 and lowastlowastlowast119875 lt 00001 versus untreated cells)
up-regulation could enhance the major surgery-related earlyhyperinflammatory response essential for tissue repair andhost defense [5]
The observed dexamethasone capability to completelyrevert TNF-120572-induced BNP up-regulation suggests that thisanti-inflammatory glucocorticoid may prevent BNP-relatedeffects
Regarding ANP the observed significant down-regula-tion of mRNA and protein levels after 10 20 ngmL TNF-120572treatment suggests that also this condition could affect localinflammation status In fact ANP administration determinesanti-inflammatory effects in airway epithelial cells [48] play-ing important roles in modulating inflammatory responseIn consideration of such ANP anti-inflammatory action wespeculate that the observed TNF-120572-induced ANP down-regulation could concur in enhancing an inflammatory sta-tus Differently from the above reported doses 40 ngmLTNF-120572 administration induced ANP up-regulation eventhough not statistically significant We suggest that this resultmight be due to a direct BNP involvement In fact a markedANP up-regulation was observed after BNP treatment at the
dose accumulated in culture medium after 40 ngmL TNF-120572treatment
The observed selectivemodulation inANP and BNP geneexpression after TNF-120572 treatment in line with other studiesin different cell models [19 49 50] could be related to adifferent regulation mechanism at transcriptional level assuggested by our observation on the different involvementof NF-120581B and MAPKs signaling pathways In particular wedemonstrated that ANP down-regulation TNF-120572-mediatedoccurs via NF-120581B activation pathway whereas BNP modula-tion requires ERK 12 activation pathway NF-120581B involvementin TNF-120572-induced ANP down-regulation is not opposed tothe proinflammatory role of this transcription factor beingANP a molecule that exerts an anti-inflammatory effect inairway epithelial cells [48] Regarding themechanism linkingNF-120581B to the downregulation of ANP gene expression wesuggest that it could be similar to that recently proposedfor Fibroblast Growth Factor 10 (FGF-10) where NF-120581Bactivationmay lead to reduced gene expression by recruitinginhibitory factors to specific gene promoters [51]
10 Mediators of Inflammation
0
20
40
60
80
100
120
C BAY 10 20 40
AQP1
( o
f con
trol)
TNF-120572 (ngmL)
TNF-120572TNF-120572 + BAY
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowast
(a)
BAY
AQP1Actin
0 0 10 20 40 10 20 40TNF-120572 (ngmL)minus + +minus minus minus + +
(b)
0
20
40
60
80
100
120
AQP5
( o
f con
trol)
C BAY 10 20 40
TNF-120572 (ngmL)
TNF-120572TNF-120572 + BAY
∘∘∘∘∘∘
∘∘∘
lowastlowastlowastlowast
lowastlowast
(c)
AQP5Actin
0 0 10 20 40 10 20 40TNF-120572 (ngmL)BAY minus + +minus minus minus + +
(d)
Figure 7 TNF-120572 downregulation of AQP1 and AQP5 expression may require activation of NF-120581B pathwayBEAS-2B cells were treated with10 20 and 40 ngmL TNF-120572 alone or in combination with NF-120581B inhibitor BAY 11-7082 (1 120583M) for 24 h Gene expression of AQP1 ((a) (b))and AQP5 ((c) (d)) at mRNA ((a) (c)) and protein ((b) (d)) level Western blots were obtained by using the specific rabbit polyclonal AbsThe blots were stripped of the bound Ab and reprobed with mouse anti-120573-actin to confirm equal loading Western blots are representative ofthree separate experiments All histograms indicate means plusmn SD of three different cultures each one tested in quadruplicate and expressed aspercentage of control (lowast119875 lt 001 lowastlowast119875 lt 0001 lowastlowastlowast119875 lt 00001 versus Controls and ∘∘∘119875 lt 00001 versus BAY) C Controls (untreated cells)
ANP and BNP biological functions occur through theirbinding to the same NPR-1 [13] Here we demonstratedfor the first time to our knowledge TNF-120572 capability todownregulateNPR-1mRNAandprotein expression in BEAS-2B cells and that this regulation occurs via MAPKs signalingpathway activation The consequence of the complex TNF-120572-induced NPs and NPR-1 observed modulation associatedwith their binding to the same receptor on BEAS-2B cellsmakes it difficult to predict what would be the net resultingeffect both in vitro and in vivo in relation also to the presenceof a possible autocrine regulation by NPs andor to the effectsof different cytokines activated by both BNP and TNF-120572We hypothesize that the TNF-120572-induced downregulation of
NPR-1 paralleled by an up-regulation of BNP could alsolead to a paracrine action of this peptide possibly on themicrovascular endothelium of the lung where NPR-1 isdensely expressed [18] In this context we speculate that theinduction of local modification on BNP or ANP by TNF-120572 inhuman bronchial epithelial cells could alter the barrier per-meability of the pulmonary microcirculation Hence TNF-120572 acting on epithelial cells could also indirectly contributeto endothelial cells barrier dysfunction and permeability byenhancing the local production of proinflammatory BNPand by down-regulating the anti-inflammatory expressionof ANP Besides exogenous synthetic ANP has been shownto protect from endothelial barrier dysfunction in in vivo
Mediators of Inflammation 11
0
50
100
150
200
250
300
0 001
AN
P (
of c
ontro
l)
BNP (nM)
lowast
(a)
0
50
100
150
0 001BNP (nM)
NPR
-1(
of c
ontro
l)
(b)
0
20
40
60
80
100
120
0 001
BNP (nM)
lowast
AQP1
( o
f con
trol)
(c)
0
20
40
60
80
100
120
0 001
BNP (nM)
AQP5
( o
f con
trol)
(d)
Figure 8 BNP effect on ANP NPR-1 AQP1 and AQP5 expressionBEAS-2B cells were treated with 001 nM BNP for 24 h Histograms show(a) ANP (b) NPR-1 (c) AQP1 and (d) AQP5 gene expression at mRNA level All histograms indicate means plusmn SD of three different cultureseach one tested in quadruplicate and expressed as percentage of control (lowast119875 lt 005 versus untreated cells)
020406080
100120140160180200
DEX
BNP
( o
f con
trol)
C TNF-120572 TNF-120572 + DEX
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowast
lowast
lowast
Figure 9 Dexamethasone effect on BNP expression BEAS-2B cellswere treatedwith 1 120583Mdexamethasone (DEX) and 40 ngmLTNF-120572alone or in combination with DEX for 24 h Histograms show BNPgene expression at mRNA (gray bars) and protein (black bars) levelAll histograms indicate means plusmn SD of three different cultures eachone tested in quadruplicate and expressed as percentage of control(lowast119875 lt 001 lowastlowast119875 lt 0001 and lowastlowastlowast119875 lt 00001 versus Controls) CControls (untreated cells)
and in vitro models of lung injury [18] and the therapeu-tical relevance of these experimental observations has beenshown in intensive care patients without heart disease whereintravenous ANP infusion diminished pulmonary vascularpermeability and pulmonary edema [18]
Regarding AQPs we demonstrated that BEAS-2B cellsexpress AQP1 and AQP5 and that TNF-120572 directly down-regulates their expression at mRNA and protein levelsRegarding AQP1 also an additional BNP-mediated TNF-120572involvement can be proposed In fact AQP1 down-regulationwas observed with BNP treatment at the dose accumulatedin culture medium after 40 ngmL TNF-120572 treatment Themechanisms that underlie AQPs regulation are of consider-able interest Several AQPs has recently been demonstratedto undergo complex regulation [27] Our results show thatAQP1 and APQ5 are subject to inflammation regulationadding an important piece of information in this contextOur results are in agreement with other studies describinga marked reduction in the expression levels of AQP1 andAQP5 inmouse lung [30] andmouse lung epithelial cells [31]The biological significance of the observed TNF-120572-inducedAQP1 and AQP5 down-regulation is at present unclear Ithas been recently suggested that the decrease in both AQPsmay play an important role in the development of lung edema[52] which is known to be related with fluid retention Wetherefore speculate that TNF-120572-induced AQP1 and AQP5down-regulation might play a role also in the excess of fluidaccumulation related to major surgical procedures
5 Conclusions
In conclusion our results provided evidence that in humanbronchial epithelial cells BEAS-2B TNF-120572 selectively mod-ulates mRNA and protein expression levels of different
12 Mediators of Inflammation
molecules involved in body fluid homeostasis ANP BNPand their receptor NPR-1 AQP1 and AQP5 Moreoverour data pointed out that the changes specifically affectingNPs expression occur independently from hemodynamicinfluences and that inflammation should be considered anextracardiac cause of BNP elevation adding an importantpiece of information in the novel area of study regarding NPsand inflammation In addition we demonstrated that such amodulation occurs via different involvement of NK-120581B andMAPKs signaling pathways Finally we suggested inflamma-tion among the mechanisms involved in the regulation ofAQP1 and AQP5 Therefore in this exploratory study wespeculate that TNF-120572 might be involved in postoperative-fluid retention related to major surgery
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgment
The authors thank Mrs Roberta Frosini for the excellenttechnical assistance
References
[1] E Itobi M Stroud and M Elia ldquoImpact of oedema onrecovery after major abdominal surgery and potential valueof multifrequency bioimpedance measurementsrdquo The BritishJournal of Surgery vol 93 no 3 pp 354ndash361 2006
[2] J A Lowell C Schifferdecker D F Driscoll P N Benottiand B R Bistrian ldquoPostoperative fluid overload not a benignproblemrdquo Critical Care Medicine vol 18 no 7 pp 728ndash7331990
[3] A M Moslashller T Pedersen P-E Svendsen and A EngquistldquoPerioperative risk factors in elective pneumonectomy theimpact of excess fluid balancerdquo European Journal of Anaesthe-siology vol 19 no 1 pp 57ndash62 2002
[4] E Bennett-Guerrero D E Feierman G R Barclay et alldquoPreoperative and intraoperative predictors of postoperativemorbidity poor graft function and early rejection in 190patients undergoing liver transplantationrdquo Archives of Surgeryvol 136 no 10 pp 1177ndash1183 2001
[5] J P Desborough ldquoThe stress response to trauma and surgeryrdquoThe British Journal of Anaesthesia vol 85 no 1 pp 109ndash1172000
[6] H Tsujimoto S Ono T Majima et al ldquoDifferential toll-likereceptor expression after ex vivo lipopolysaccharide exposurein patients with sepsis and following surgical stressrdquo ClinicalImmunology vol 119 no 2 pp 180ndash187 2006
[7] C Evans C Galustian D Kumar et al ldquoImpact of surgeryon immunologic function comparison between minimallyinvasive techniques and conventional laparotomy for surgi-cal resection of colorectal tumorsrdquo The American Journal ofSurgery vol 197 no 2 pp 238ndash245 2009
[8] F Catena L Ansaloni A Avanzolini et al ldquoSystemic cytokineresponse after emergency and elective surgery for colorectalcarcinomardquo International Journal of Colorectal Disease vol 24no 7 pp 803ndash808 2009
[9] R Sarbinowski S Arvidsson M Tylman T Oresland andA Bengtsson ldquoPlasma concentration of procalcitonin andsystemic inflammatory response syndrome after colorectalsurgeryrdquo Acta Anaesthesiologica Scandinavica vol 49 no 2 pp191ndash196 2005
[10] P Sheeran and G M Hall ldquoCytokines in anaesthesiardquo TheBritish Journal of Anaesthesia vol 78 no 2 pp 201ndash219 1997
[11] L Cagini R Capozzi V Tassi et al ldquoFluid and electrolytebalance after major thoracic surgery by bioimpedance andendocrine evaluationrdquo European Journal of Cardio-ThoracicSurgery vol 40 no 2 pp e71ndashe76 2011
[12] T Omland and T Hagve ldquoNatriuretic peptides physiologic andanalytic considerationsrdquo Heart Failure Clinics vol 5 no 4 pp471ndash487 2009
[13] L R Potter A R Yoder D R Flora L K Antos and DM Dickey ldquoNatriuretic peptides their structures receptorsphysiologic functions and therapeutic applicationsrdquo Handbookof Experimental Pharmacology vol 191 pp 341ndash366 2009
[14] T G Flynn M L de Bold and A J de Bold ldquoThe aminoacid sequence of an atrial peptide with potent diuretic andnatriuretic propertiesrdquo Biochemical and Biophysical ResearchCommunications vol 117 no 3 pp 859ndash865 1983
[15] A J de Bold ldquoAtrial natriuretic factor a hormone produced bythe heartrdquo Science vol 230 pp 767ndash770 1985
[16] K Kangawa Y Tawaragi S Oikawa et al ldquoIdentification of rat 120574atrial natriuretic polypeptide and characterization of the cDNAencoding its precursorrdquo Nature vol 312 no 5990 pp 152ndash1551984
[17] T Sudoh K Kangawa N Minamino and H Matsuo ldquoA newnatriuretic peptide in porcine brainrdquo Nature vol 332 no 6159pp 78ndash81 1988
[18] M Kuhn ldquoEndothelial actions of atrial and B-type natriureticpeptidesrdquo The British Journal of Pharmacology vol 166 no 2pp 522ndash531 2012
[19] A J de Bold ldquoCardiac natriuretic peptides gene expression andsecretion in inflammationrdquo Journal of Investigative Medicinevol 57 pp 29ndash32 2009
[20] S P DrsquoSouza M Davis and G F Baxter ldquoAutocrine andparacrine actions of natriuretic peptides in the heartrdquo Pharma-cology andTherapeutics vol 101 no 2 pp 113ndash129 2004
[21] M Brueckmann G Huhle S Lang et al ldquoPrognostic valueof plasma N-terminal pro-brain natriuretic peptide in patientswith severe sepsisrdquoCirculation vol 112 no 4 pp 527ndash534 2005
[22] J Charpentier C E Luyt Y Fulla et al ldquoBrain natriureticpeptide a marker of myocardial dysfunction and prognosisduring severe sepsisrdquo Critical Care Medicine vol 32 no 3 pp660ndash665 2004
[23] R Shor Y Rozenman A Bolshinsky et al ldquoBNP in septicpatients without systolic myocardial dysfunctionrdquo EuropeanJournal of Internal Medicine vol 17 no 8 pp 536ndash540 2006
[24] T Inoue M Kawai T Nakane et al ldquoInfluence of low-gradeinflammation on plasma B-type natriuretic peptide levelsrdquoInternal Medicine vol 49 no 24 pp 2659ndash2668 2010
[25] P Agre L S King M Yasui et al ldquoAquaporin water channelsmdashfrom atomic structure to clinical medicinerdquo Journal of Physiol-ogy vol 542 no 1 pp 3ndash16 2002
[26] P Agre and D Kozono ldquoAquaporin water channels molecularmechanisms for human diseasesrdquo FEBS Letters vol 555 no 1pp 72ndash78 2003
Mediators of Inflammation 13
[27] J M Carbrey and P Agre ldquoDiscovery of the aquaporins anddevelopment of the fieldrdquo Handbook of Experimental Pharma-cology vol 190 pp 3ndash28 2009
[28] O Bloch and G T Manley ldquoThe role of aquaporin-4 in cerebralwater transport and edemardquo Neurosurgical Focus vol 22 no 5article E3 2007
[29] J Li M Xu Q Fan et al ldquoTanshinone IIA ameliorates seawaterexposure-induced lung injury by inhibiting aquaporins (AQP)1 and AQP5 expression in lungrdquo Respiratory Physiology andNeurobiology vol 176 no 1-2 pp 39ndash49 2011
[30] J E Towne K S Harrod C M Krane and A G MenonldquoDecreased expression of aquaporin (AQP)1 and AQP5 inmouse lung after acute viral infectionrdquoThe American Journal ofRespiratory Cell and Molecular Biology vol 22 no 1 pp 34ndash442000
[31] J E Towne C M Krane C J Bachurski and A G MenonldquoTumor necrosis factor-120572 inhibits aquaporin 5 expression inmouse lung epithelial cellsrdquoThe Journal of Biological Chemistryvol 276 no 22 pp 18657ndash18664 2001
[32] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001
[33] K Verhelst I Carpentier and R Beyaert ldquoRegulation of TNF-induced NF-120581B activation by different cytoplasmic ubiquitina-tion eventsrdquo Cytokine and Growth Factor Reviews vol 22 no5-6 pp 277ndash286 2011
[34] L Qian Y Shen J C Chen et al ldquo3D-QSAR and dockingstudies of quinazoline derivatives with the inhibatory activitytoward NF-120581BrdquoQSAR and Combinatorial Science vol 27 no 8pp 984ndash995 2008
[35] P D NrsquoGuessan S Hippenstiel M O Etouem et al ldquoStrepto-coccus pneumoniae induced p38MAPK- andNF-120581B-dependentCOX-2 expression in human lung epitheliumrdquo The AmericanJournal of PhysiologymdashLung Cellular and Molecular Physiologyvol 290 no 6 pp L1131ndashL1138 2006
[36] B Schmeck K Moog J Zahlten et al ldquoStreptococcus pneumo-niae induced c-Jun-N-terminal kinase- andAP-I-dependent IL-8 release by lung epithelial BEAS-2B cellsrdquoRespiratory Researchvol 7 article 98 2006
[37] M GMatera L Calzetta D Passeri et al ldquoEpithelium integrityis crucial for the relaxant activity of brain natriuretic peptide inhuman isolated bronchirdquo The British Journal of Pharmacologyvol 163 no 8 pp 1740ndash1754 2011
[38] R QuirionM Dalpe A de Lean J Gutkowska M Cantin andJ Genest ldquoAtrial natriuretic factor (ANF) binding sites in brainand related structuresrdquoPeptides vol 5 no 6 pp 1167ndash1172 1984
[39] M B Pamnani D L Clough J S Chen W T Link andF J Haddy ldquoEffects of rat atrial extract on sodium transportand blood pressure in the ratrdquo Proceedings of the Society forExperimental Biology and Medicine vol 176 no 2 pp 123ndash1311984
[40] T R Traynor and S M OrsquoGrady ldquoBrain natriuretic peptidestimulates K andCl secretion across porcine distal colon epithe-liumrdquoTheAmerican Journal of PhysiologymdashCell Physiology vol260 no 4 pp C750ndashC755 1991
[41] R Rollın A Mediero M Roldan-Pallares A Fernandez-Cruzand R Fernandez-Durango ldquoNatriuretic peptide system in thehuman retinardquoMolecular Vision vol 10 pp 15ndash22 2004
[42] M Harada H Itoh O Nakagawa et al ldquoSignificance ofventricular myocytes and nonmyocytes interaction during car-diocyte hypertrophy evidence for endothelin-1 as a paracrinehypertrophic factor from cardiac nonmyocytesrdquo Circulationvol 96 no 10 pp 3737ndash3744 1997
[43] K Kuwahara Y Saito M Harada et al ldquoInvolvement ofcardiotrophin-1 in cardiac myocyte-nonmyocyte interactionsduring hypertrophy of rat cardiac myocytes in vitrordquo Circula-tion vol 100 no 10 pp 1116ndash1124 1999
[44] E Harada O Nakagawa M Yoshimura et al ldquoEffect ofinterleukin-1120573 on cardiac hypertrophy and production of natri-uretic peptides in rat cardiocyte culturerdquo Journal of Molecularand Cellular Cardiology vol 31 no 11 pp 1997ndash2006 1999
[45] V Chiurchiu V Izzi F DrsquoAquilio F Carotenuto P Di Nardoand P M Baldini ldquoBrain natriuretic peptide (BNP) regulatesthe production of inflammatory mediators in human THP-1macrophagesrdquo Regulatory Peptides vol 148 no 1ndash3 pp 26ndash322008
[46] N S Holden C F Rider M J Bell et al ldquoEnhancement ofinflammatory mediator release by 1205732-adrenoceptor agonists inairway epithelial cells is reversed by glucocorticoid actionrdquoTheBritish Journal of Pharmacology vol 160 no 2 pp 410ndash4202010
[47] V S Fang C C Juan J G S Won and L T Ho ldquoFurther studyof aldosterone secretion-inhibitory factor and brain natriureticpeptide on cortisol production of Guinea pig zona fasciculatacellsrdquo Chinese Journal of Physiology vol 43 no 4 pp 141ndash1472000
[48] G Hellermann X Kong J Gunnarsdottir et al ldquoMechanismof bronchoprotective effects of a novel natriuretic hormonepeptiderdquo Journal of Allergy and Clinical Immunology vol 113no 1 pp 79ndash85 2004
[49] K K Ma T Ogawa and A J de Bold ldquoSelective upregulationof cardiac brain natriuretic peptide at the transcriptional andtranslational levels by pro-inflammatory cytokines and by con-ditionedmedium derived frommixed lymphocyte reactions viap38 MAP kinaserdquo Journal of Molecular and Cellular Cardiologyvol 36 no 4 pp 505ndash513 2004
[50] T Ogawa J P Veinot M L K de Bold T Georgalis and AJ De Bold ldquoAngiotensin II receptor antagonism reverts theselective cardiac BNP upregulation and secretion observed inmyocarditisrdquo The American Journal of PhysiologymdashHeart andCirculatory Physiology vol 294 no 6 pp H2596ndashH2603 2008
[51] B J Carver E J Plosa A M Stinnett T S Blackwell andL S Prince ldquoInteractions between NF-120581B and SP3 connectinflammatory signaling with reduced FGF-10 expressionrdquo TheJournal of Biological Chemistry vol 288 no 21 pp 15318ndash153252013
[52] C Dong G Wang B Li et al ldquoAnti-asthmatic agents alleviatepulmonary edema by upregulating AQP1 and AQP5 expressionin the lungs of mice with OVA-induced asthmardquo RespiratoryPhysiology and Neurobiology vol 181 no 1 pp 21ndash28 2012
peptides (NPs) family [11] In particular a significant weightgain was found to be correlated with large volumes of fluidsaccumulation on the postoperative day 2 despite a negativeintraoperative fluid balance and peroperative strict fluidrestriction [11] Moreover the patients none of whom devel-oped signs or symptoms of heart failure during the postop-erative period showed immediately after surgery (on day 1)a significant increase in BNP plasma concentration [11] NPsare hormoneparacrine factors that are released by the heartin response to myocardial stretch and overload modulatingbody fluid homeostasis [12 13] BNP secreted from the car-diac ventricles and atrial natriuretic peptide (ANP) secretedfrom the cardiac atria activate the same transmembraneguanylyl cyclase-Anatriuretic peptide receptor-A (NPR-A orNPR-1) [14ndash17] In addition to vasodilation cardiovascularhomeostasis sodium excretion and inhibition of aldosteronesecretion it is becoming increasingly recognized that NPspossess a much broader range of biological activities includ-ing effects on endothelial function and inflammation [1819] The genetic expression and secretion of ANP and BNPhave been studied mainly in the context of cardiac diseasesassociated with neuroendocrine and hemodynamic changes[12 13 20] however it has been pointed out that changes inBNP also occur in a context of an acute inflammatory process[19 21ndash24]
Another family of proteins aquaporins (AQPs) is deeplyinvolved in the physiological response to change of fluidvolume and osmolarity [25 26] AQPs are widely distributedin various tissues throughout the body and facilitate osmot-ically driven water transport across cell membranes [25ndash27]Recently AQPs involvement in edema development has beenpointed out In particular it has been shown that AQP4 isan essential mediator in the formation and resorption ofedema fluid from brain parenchyma [28] and that AQP1 andAQP5 might play an important role in lung edema [29] Inaddition AQP1 and AQP5 expression is decreased in lunginflammation [30 31]
The aim of our work was to investigate the potentialinvolvement of TNF-120572 in the regulation of ANP BNP andtheir receptor NPR-1 as well as AQP1 and AQP5 keymolecules involved in body fluid homeostasis In order toexclude any hemodynamic change able to modulate NPsexpression we carried out an in vitro study in humanbronchial epithelial cells BEAS-2B
2 Materials and Methods
21 Reagents Human TNF-120572 was obtained from Immuno-Tools GmbH (Friesoythe Germany) The Nuclear Factor-120581B (NF-120581B) inhibitors BAY 11ndash7082 and QNZ as wellas the p38 mitogen-activated protein kinases (p38 MAPK)inhibitor SB 203580 the c-Jun N-terminal kinases 12(JNK 12) inhibitor SP 600125 and the extracellular-signal-regulated kinases 12 (ERK 12) inhibitor U-0126 wereobtained from Santa Cruz Biotechnology Inc (HeidelbergGermany) andwere dissolved in dimethyl sulfoxide (DMSO)Human BNP was obtained from Phoenix Europe GmbH(Karlsruhe Germany) Dexamethasone (DEX) was fromSigma-Aldrich (Milan Italy) Rabbit polyclonal antibodies
(Abs) against ANP NPR-1 AQP1 and AQP5 as well as themouse monoclonal Ab against 120573-actin and the appropriateHRP-conjugated secondary Abs were purchased from SantaCruz Biotechnology Inc (Heidelberg Germany) Rabbitmonoclonal Abs against Phospho-I120581B-120572 and Phospho-p38MAPK were purchased from Cell Signaling Technology Inc(Danvers MA)
22 Cell Culture and Drug Treatments Human bronchialepithelial cell line BEAS-2B was purchased from AmericanType Culture Collection (ATCC) and was routinely main-tained at 37∘C in 5 CO
2in RPMI 1640 supplemented
with 10 heat inactivated (1 h at 56∘C) fetal calf serum1X Lglutamine 1mM sodium pyruvate 1X nonessentialamino acids 100 unitsmL of penicillin and 01mgmL ofstreptomycin (Invitrogen Milan Italy) Forty-eight hoursbefore study cells were seeded onto six-well culture dishes at300000 cellswell TNF-120572was dissolved in distilledwater andused at the concentrations of 10 20 and 40 ngmL for 24 hInhibitors BAY 11-7082 QNZ SB 203580 SP 600125 and U-0126 were dissolved in 05 DMSO The anti-inflammatoryglucocorticoid DEX was dissolved in 01 methanol Inindependent experiments BAY 11-7082 QNZ SB 203580 SP600125 and U-0126 and DEX were added to cells 60minbefore TNF-120572 administration at the concentration of 1120583Mfor BAY 11-7082 or DEX and 10 120583M for QNZ SB 203580 SP600125 or U-0126 DMSO or methanol final concentrationsin each assay were 0005 and 0001 respectively Controlcells with DMSO or methanol did not show any significantdifference respect to control cells in RPMI 1640 mediumtherefore all the relative treatments were compared to theselatter controls
23 Cell Viability The effects of TNF-120572 BAY 11-7082 QNZSB 203580 SP 600125 U-0126 DEX and BNP treatmentswere measured with a standard trypan blue uptake assayCell cultures were also examined morphologically via lightmicroscopy
24 RNA Isolation and cDNA Synthesis Total cellular RNAwas isolated using TRIzol Reagent (Invitrogen Milan Italy)according to the manufacturerrsquos instructions and 1 120583g wasreverse transcribed using the RevertAidHMinus First StrandcDNA Synthesis Kit (Fermentas Hanover MD) and randomprimers System (Invitrogen Milan Italy) according to themanufacturerrsquos instructions
25 Quantitative Real Time SYBR Green PCR Analysis Weemployed quantitative Real Time SYBR Green PCR analysis(qRT-PCR) on Mx3000P QPCR Systems (Agilent Tech-nologies Milan Italy) to evaluate the expression of ANP(NM 006172) BNP (NM 002521) NPR-1 (NM 000906)AQP1(NM 198098) and AQP5 (NM 001651) versus ACTB(NM 001101)The sequences of oligonucleotide primers usedfor qRT-PCR and the thermal cycling conditions are sum-marized in Table 1 All primers were designed using BeaconDesigner 4 software (Stratagene La Jolla CA) starting frompublished sequences data supplied by the NCBI database
Mediators of Inflammation 3
Table 1 Primer sequences primer concentrations and qRT-PCR cycling conditions
Gene Primer sequences Primer concentrations qRT-PCR cycling conditions
ANP F 51015840-tcagcccagcccagagag-31015840 200 nM 1 cycle at 95∘C for 10min 40 cycles at 95∘C for 1minR 51015840-gctccaatcctgtccatcctg-31015840 200 nM 60∘C for 30 sec and 72∘C for 30 sec
BNP F 51015840-gagggcaggtgggaagcaaac-31015840 200 nM 1 cycle at 95∘C for 10min 40 cycles at 95∘C for 1minR 51015840-gcaagaagagcaggagcaggag-31015840 200 nM 60∘C for 30 sec and 72∘C for 30 sec
NPR-1 F 51015840-ccctggaggtgctggctttgg-31015840 200 nM 1 cycle at 95∘C for 10min 40 cycles at 95∘C for 1minR 51015840-ctctcaaggctactgggctcaacg-31015840 200 nM 59∘C for 30 sec and 72∘C for 30 sec
AQP1 F 51015840-ttggacacctcctggctattgact-31015840 400 nM 1 cycle at 95∘C for 10min 40 cycles at 95∘C for 1minR 51015840-ccagtggttgctgaagttgtgtgt-31015840 400 nM 60∘C for 30 sec and 72∘C for 30 sec
AQP5 F 51015840-cgctcaacaacaacacaacgc-31015840 200 nM 1 cycle at 95∘C for 10min 40 cycles at 95∘C for 1minR 51015840-ccagtgaagtagattccgacaagg-31015840 200 nM 59∘C for 30 sec and 72∘C for 30 sec
ACTB F 51015840-cactcttccagccttccttcc-31015840 600 nM 1 cycle at 95∘C for 10min 40 cycles at 95∘C for 1minR 51015840-acagcactgtgttggcgtac-31015840 600 nM 59∘C or 60∘C for 30 sec and 72∘C for 30 sec
qRT-PCR quantitative real time polymerase chain reaction F forward R reverse
Reactionswere performed in a total volume of 25120583L contain-ing 250 ng or 500 ng of cDNA 1X Reagent Brilliant II SYBRGreen QPCRMaster Mix and the appropriate concentrationof the specific primers (Medical Milan Italy) Data for com-parative analysis of gene expression were obtained using theΔΔCt method as described in the ABI Prism 7000 sequencedetection system user guide [32] Agarose gel electrophoreticanalysis was used to check the predicted size amplicons forANP (160 bp) BNP (148 bp) NPR-1 (151 bp) AQP1 (98 bp)and AQP5 (180 bp)
26Western Blot Analysis Total protein extracts (40120583g)wereseparated by 12 sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) andblotted onto a nitrocellulosemembrane using iBlot Dry Blotting System (InvitrogenMilan Italy) Nonspecific binding sites were blocked in Roti-Block (Roth GmbH Karlsruhe Germany) for 2 h at roomtemperature The membranes were blotted overnight at 4∘Cwith a 1 100 dilution of the primary specific Abs and washedwith TBSTThe antigen-Ab complex was detected by incuba-tion of the membranes for 2 h at room temperature with theappropriated HRP-conjugated secondary Abs and revealedusing the enhanced chemiluminescence (ECL) system byAmersham Pharmacia Biotech (Uppsala Sweden)
As internal loading controls and for protein expressionnormalizing purpose all membranes were subsequentlystripped of the first antibody in a stripping buffer (100mM2-ME 2 SDS and 625mM Tris-HCl pH 68) for 30minat 50∘C followed by washings with TBST The membraneswere then reprobed with anti-120573-actin antibody followed byincubation with HRP-conjugated Ab
27 BNP Protein Concentration in Culture Medium BNPprotein concentrationwasmeasured in culturemediumusingthe chemiluminescent enzyme immunoassay kit TRIAGEBNP (Biosite Incorporated San Diego USA) The minimumquantity of human BNP detectable with this system is10 pgmL The intraassay and the interassay coefficients ofvariations were 31 and 45 respectively
28 Statistical Analysis Results were expressed as means plusmnSD of three independent experiments The statistical signifi-cance of differences between treated and untreated cells wasassessed by Studentrsquos t-test Differences between groups wereconsidered significant when 119875 lt 005
3 Results
31 TNF-120572 Selectively Modulates ANP BNP and Their Recep-tor NPR-1 Expression in BEAS-2B Cells We firstly demon-strated that BEAS-2B cell line expresses ANP BNP NPR-1AQP1 and AQP5 By qRT-PCR analysis fragments of thepredicted molecular size were generated (Figure 1) To deter-mine whether the expression of ANP BNP and NPR-1 wasaffected by TNF-120572 BEAS-2B cells were treated with 10 20or 40 ngmL TNF-120572 for 24 h ANP mRNA was significantlydecreased about 75 (119875 lt 00001) and about 40 (119875 lt005) of control after treatment with 10 and 20 ngmL TNF-120572 respectively Forty ngmL TNF-120572 induced an upregulation(about 20 of control) of ANP expression even though notstatistically significant (119875 gt 005) (Figure 2(a)) A similartrend was also observed at protein level (Figure 2(b)) BNPmRNA expression was significantly (119875 lt 001) increased(62 of control) since 10 ngmL TNF-120572 treatment maximumstimulation (80 of control) being obtained with 40 ngmL(119875 lt 00001) (Figure 2(c)) Treatment with TNF-120572 at theabove described concentrations also increased BNP proteinsecretion in culture medium (119875 lt 001) (Figure 2(d))NPR-1 gene expression level was dramatically decreased (70ndash80 of control) after TNF-120572 exposure at all the used doses(119875 lt 0001) (Figure 2(e)) paralleled by a decrease of proteinexpression (Figure 2(f))
32 NPs and NPR-1 Modulation by TNF-120572 May RequireActivation of NF-120581B Signaling Pathway in BEAS-2B CellsAs known TNF-120572 regulates numerous genes essential tothe inflammatory process through the activation of mul-tiple signal transduction pathways including NF-120581B [33]Therefore to investigate the possible involvement of NF-120581B signaling pathway in TNF-120572-mediated regulation of NPs
4 Mediators of Inflammation
2 3 4M
242190147
(bp) (bp)M
111110147190
67
5 6
Figure 1 Identification of ANP BNP NPR-1 AQP1 and AQP5transcripts in BEAS-2B cells qReal-Time PCR was performed usinggene specific primers for ANP (lane 2) BNP (lane 3) NPR-1 (lane 4)AQP1 (lane 5) sand AQP5 (lane 6) in BEAS-2B cells Single bands ofthe predicted molecular size for ANP (160 bp) BNP (148 bp) NPR-1(151 bp) AQP1 (98 bp) andAQP5 (180 bp) transcripts were detectedLane M DNA molecular marker
and NPR-1 we used the specific NF-kB inhibitor BAY 11-7082 that diminishes the activation of NF-120581B by preventingphosphorylation of its inhibitory I120581B-120572 protein As shownin Figure 3(a) TNF-120572-induced downregulation of ANP wascompletely reverted by BAY 11-7082 at both mRNA and pro-tein level (Figure 3(b)) suggesting that this response requiresNF-120581B activation pathway Regarding TNF-120572-induced BNPupregulation this was only partially reverted by BAY 11-7082at mRNA level (Figure 3(c)) while was completely revertedat protein level (Figure 3(d)) Conversely TNF-120572-induceddecrement in NPR-1 expression was not affected by BAY 11ndash7082 either at mRNA or protein level (Figures 3(e) and 3(f))
Western blot analysis for the Ser32-phosphorylated I120581B-120572protein proved the biochemistry evidence of the inhibitoryaction of BAY 11-7082 on NF-120581B activity (Figure 3(g)) Tofurther investigate the role of NF-120581B in the modulation ofBNP and NPR-1 in BEAS-2B cells after TNF-120572 treatmentwe employed theNF-120581B-DNA binding inhibitor QNZ whichprevents free NF-120581B from binding to DNA [34] The resultsobtained with such additional inhibitor confirmed the previ-ous findings obtained with BAY 11-7082 In particular TNF-120572-induced BNP up-regulation was not reverted by QNZ atmRNA level (Figure 4(a)) while it was completely reverted atprotein level (Figure 4(b)) Accordingly also TNF-120572-induceddecrement of NPR-1 expression was not affected by QNZeither at mRNA or protein level (Figures 4(c) and 4(d))Western blot analysis for the Ser32-phosphorylated I120581B-120572protein proved the biochemistry evidence of the inhibitoryaction of QNZ on NF-120581B activity (Figure 4(e))
33 BNP and NPR-1 Modulation by TNF-120572 May RequireActivation of MAPKs Signaling Pathway in BEAS-2B CellsBecause TNF-120572 is able to activate multiple signal transduc-tion pathways including MAPKs and because the activationof p38 MAPK and JNK participates in the regulation ofinflammatory processes in bronchial epithelial cells [35 36]we then investigatedMAPKs pathway involvement in TNF-120572modulation of BNP and NPR-1 expression TNF-120572-mediated
up-regulation of BNP was not affected by SB 203580 or SP600125 p38 MAPK and JNK 12 inhibitors respectivelywhile it was completely reverted by the ERK 12 inhibitorU-0126 (Figures 5(a) and 5(b)) suggesting that ERK 12activation pathway is required for BNP TNF-120572-mediatedmodulation Regarding NPR-1 the TNF-120572-mediated down-regulation was completely reverted by the use of all inhibitors(Figures 5(c) and 5(d)) Western blot analysis for theThr180Thr182-phosphorylated form of p-38 MAPK provedthe biochemistry evidence of the inhibitory action of SB203580 on p38 MAPK activity (Figure 5(e))
34 TNF-120572 Decreases AQP1 and AQP5 Expression in BEAS2B Cells We found that TNF-120572 induced a dose dependentdown-regulation of AQP1 expression (119875 lt 001) at mRNA(Figure 6(a)) and protein level (Figure 6(b)) and a dramaticdecrease in AQP5 gene expression level (about 80 ofcontrol) with all the used doses (119875 lt 0001) (Figure 6(c)) par-alleled by a comparable trend at protein level (Figure 6(d))
35 Decreased AQP1 andAQP5 Expression by TNF-120572DoesNotRequire Activation of NF-120581B Signaling Pathway in BEAS-2BCells To examine a possible involvement of NF-120581B in TNF-120572-mediated down-regulation of AQP1 and AQP5 BEAS-2Bcells were incubated with BAY 11-7082 Cotreatment of suchNF-120581B inhibitor with TNF-120572 resulted in a decrease of AQP1expression which was similar to that observed with TNF-120572alone (Figures 7(a) and 7(b)) suggesting that the observedresponse does not require NF-120581B activation Conversely co-treatment of BAY 11-7082 with TNF-120572 affected AQP5 only atprotein level (Figures 7(c) and 7(d))
36 BNP Effect on ANP NPR-1 and AQP1 or AQP5 mRNAExpression in BEAS-2BCells In order to determine a possibledirect involvement of BNP on the gene expression of ANPNPR-1 and AQP1 or AQP5 BEAS-2B cells were treated with001 nM BNP This concentration was previously found tobe accumulated in culture medium after 40 ngmL TNF-120572treatment the dose that induced the maximum effect on theexpression of the studied genes BNP administration induceda significant (119875 lt 005) increase (100 of control) on ANPmRNA level (Figure 8(a)) and did not affectNPR-1 expression(Figure 8(b)) while induced a marked decrease (45 ofcontrol) on AQP1 mRNA level (119875 lt 005) (Figure 8(c))A trend in decreasing was also observed for AQP5 geneexpression even though not statistically significant (119875 gt005) (Figure 8(d))
37 Dexamethasone (DEX) Effect on BNP mRNA and ProteinLevels in BEAS-2B Cells In order to study the capability ofthe anti-inflammatory drug DEX to affect TNF-120572-mediatedup-regulation of BNP mRNA and protein levels BEAS-2Bcells were treated with 1120583MDEX As shown in Figure 9 DEXinduced a down-regulation of BNP expression and proteinlevels alone or in combination with 40 ngmL TNF-120572 (119875 lt001) demonstrating the capability of this glucocorticoid torevert TNF-120572 effects
Mediators of Inflammation 5
0 10 20 40TNF-120572 (ngmL)
AN
P (
of c
ontro
l)
160
140
120
100
80
60
40
20
0
lowast
lowastlowastlowastlowast
(a)
0 10 20 40
ANP
Actin TNF-120572 (ngmL)
(b)
0
50
100
150
200
0 10 20 40
BNP
( o
f con
trol)
TNF-120572 (ngmL)
lowastlowastlowastlowastlowastlowast
lowast
(c)
0
50
100
150
200
0 10 20 40BN
P (
of c
ontro
l)
TNF-120572 (ngmL)
lowastlowast
lowastlowast
lowastlowast
(d)
0 10 20 40TNF-120572 (ngmL)
120
100
80
60
40
20
0
NPR
-1 (
of c
ontro
l)
lowastlowast
lowastlowastlowastlowastlowastlowastlowastlowast
(e)
0 10 20 40
NPR-1
Actin TNF-120572 (ngmL)
(f)
Figure 2 TNF-120572 selectively modulates ANP BNP and NPR-1 expression BEAS-2B cells were treated with 10 20 and 40 ngmL TNF-120572 for24 h ((a) (b)) ANP ((c) (d)) BNP and ((e) (f)) NPR-1 gene expression at mRNA ((a) (c) and (e)) and protein level ((b) (d) and (f))Western blots were obtained by using the specific rabbit polyclonal Abs The blots were stripped of the bound Ab and reprobed with mouseanti-120573-actin to confirm equal loading Western blots are representative of three separate experiments All histograms indicate meansplusmn SD ofthree different cultures each one tested in quadruplicate and expressed as percentage of control (lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 andlowastlowastlowastlowast
119875 lt 00001 versus untreated cells)
4 Discussion
Postoperative-fluid retention is a severe complication fre-quently reported in patients undergoing major surgical pro-cedures [1ndash4] The complex network of molecules involvedin such a severe surgery-induced condition remains poorlyunderstood After major surgery TNF-120572 is one of the mainproinflammatory cytokines initially released in the damagedtissue where it stimulates the production and release of morecytokines responsible for inducing the systemic changesknown as the acute phase response [5 10] Therefore it isreasonable to assume TNF-120572 involvement among the variouscauses of fluid retention and very important to understandthemechanisms underlying its involvement in such an ambit
In the present study we demonstrated for the first time toour knowledge that TNF-120572modulates the expression ofANPBNP NPR-1 AQP1 and AQP5 in human bronchial epithelialcells BEAS-2B via different involvement of NF-120581B andMAPKs signaling pathway activation Here we also providedthe first demonstration that human bronchial epithelial cellsexpress ANP BNP NPR-1 and AQP1 and that BNP is able tomodulate AQP1 expression
The observed expression of NPs and NPR-1 suggestsan autocrine andor paracrine function for these moleculesand indicates important roles for NPs in several biologicalfunctions including regulation of fluid movement across theairway epithelial cells bronchial relaxation [37] vasodilata-tion and pulmonary vascular permeability About the former
6 Mediators of Inflammation
020406080
100120140160
C BAY 10 20 40
AN
P (
of c
ontro
l)
TNF-120572 (ngmL)
TNF-120572TNF-120572 + BAY
lowast
lowastlowastlowastlowast
(a)
ANP
Actin 40
BAY 0 0 10 20 40 10 20minus + +minus minus minus + +
Figure 3 TNF-120572 modulation of NPs and NPR-1 may require activation of NF-120581B pathway BEAS-2B cells were treated with 10 20 and40 ngmL TNF-120572 alone or in combination with NF-120581B inhibitor BAY 11-7082 (1120583M) for 24 h ((a) (b)) ANP ((c) (d)) BNP ((e) (f)) NPR-1gene expression at mRNA ((a) (c) and (e)) and protein level ((b) (d) (f)) and (g) Phospho-I120581B-120572 protein level Western blots were obtainedby using the specific rabbit polyclonal or monoclonal AbsThe blots were stripped of the bound Ab and reprobed with mouse anti-120573-actin toconfirm equal loading Western blots are representative of three separate experiments All histograms indicate means plusmn SD of three differentcultures each of one tested in quadruplicate and expressed as percentage of control (lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowastlowast119875 lt 00001 versus Controls)(∘119875 lt 005 ∘∘119875 lt 001 and ∘∘∘∘119875 lt 00001 versus BAY) C Controls (untreated cells)
process the role of ANP and BNP could be analogous to thathypothesized forNPs in transepithelial ion flux in the choroidplexus [38] in the kidney [39] colon [40] and retina [41]
The observed TNF-120572 capability of selectively regulatingANP BNP and NPR-1 gene expression in BEAS-2B cellline is of particular interest because it demonstrates that
inflammation alters NPs expression levels In particular thesignificant up-regulation of BNP expression and proteinsecretion after TNF-120572 treatment shows that this modulationcan occur independently of hemodynamic influences andthat inflammation should be considered an extracardiaccause of BNP elevation Besides in vitro studies conducted in
Mediators of Inflammation 7
020406080
100120140160180200
C QNZ TNF
BNP
( o
f con
trol)
QNZ + TNF
lowastlowast
∘∘
(a)
0
20
40
60
80
100
120
140
160
180
BNP
( o
f con
trol)
C QNZ TNF QNZ + TNF
lowastlowast
(b)
0
20
40
60
80
100
120
C QNZ TNF QNZ + TNF
∘∘
lowastlowastlowastlowast
lowastlowastlowastlowast
NPR
-1(
of c
ontro
l)
(c)
NPR-1 Actin
0 0QNZ
10 10TNF-120572+minus +minus
(d)
Actin
QNZ TNF-120572 + +
minus ++
minusminus
minus
P-I120581B-120572
(e)
Figure 4 TNF-120572 modulation of protein levels of BNP and NPR-1 may require activation of NF-120581B pathway BEAS-2B cells were treated10 ngmL TNF-120572 alone or in combination with the inhibitor QNZ (10120583M) for 24 h ((a) (b)) BNP ((c) (d)) NPR-1 gene expression at mRNA((a) (c)) and protein ((b) (d)) level and (e) Phospho-I120581B-120572 protein level Western blots were obtained by using the specific rabbit polyclonalor monoclonal AbsThe blots were stripped of the bound Ab and reprobed with mouse anti-120573-actin to confirm equal loading Western blotsare representative of three separate experiments All histograms indicatemeansplusmnSDof three different cultures each one tested in quadruplicateand expressed as percentage of control (lowastlowast119875 lt 001 and lowastlowastlowastlowast119875 lt 00001 versus Controls) (∘∘119875 lt 001 versus QNZ) C Controls (untreatedcells)
rat cardiomyocytes have shown that not only hemodynamicfactors but also neurohumoral factors activated during heartfailure such as angiotensin II endothelin and cytokinescause BNP secretion [42ndash44] In addition in patients withcardiovascular diseases plasma BNP levels have been shownto be also affected by low-grade inflammation [24] and aselective increase inBNPplasma levels has been proposed as ageneral feature of inflammation [19]The lack on any apparentTNF-120572 dose-depending increase in BNP gene expression thatwe observed in our system could be due to an autocrinenegative feedback action mediated by BNP itself followingbinding to the NPR-1 receptor expressed in BEAS-2B cells
The biological role of BNPup-regulation after TNF-120572 treatment remains to be elucidated However since it
has been demonstrated that BNP regulates the productionof major inflammatory molecules such as IL-12 IL-10leukotriene B and prostaglandin E2 in human macrophages[45] and that BEAS-2B are able to release inflammatorymolecules [46] it could be reasonable to assume that theobserved BNP increase after TNF-120572 treatment may stimu-late the production and release of such cytokines also fromhuman epithelial bronchial cells In addition in in vitrostudies a potent BNP inhibitory action on the productionof the glucocorticoid anti-inflammatory hormone cortisolhas been described [47] Therefore BNP could be con-sidered a proinflammatory molecule by inducing cytokinesexpression in target cells and inhibiting the production ofanti-inflammatory hormones Therefore the observed BNP
8 Mediators of Inflammation
0
50
100
150
200
250BN
P (
of c
ontro
l)
C
TNF SB
SB+
TNF SP
SP+
TNF
U-0
126
U-0126+
TNF
lowastlowast
lowastlowast
∘∘
sect
(a)
020406080
100120140160180
BNP
( o
f con
trol)
C
TNF SB
SB+
TNF SP
SP+
TNF
U-0
126
U-0126+
TNF
lowastlowast
lowastlowast
∘∘∘
sectsect
(b)
0
20
40
60
80
100
120
140
160
C
TNF SB
SB+
TNF SP
SP+
TNF
U-0
126
U-0126+
TNF
lowastlowastlowast
lowastlowastlowast
lowast
lowast
sect
NPR
-1(
of c
ontro
l)
(c)
+
SB 203580
+
SP 600125 U-0126
+ActinTNF-120572
NPR-1
minus minus minus minus +
(d)
SB 203580
Actin
P-p38 MAPK
TNF-120572 + +
minus ++
minusminus
minus
(e)
Figure 5 TNF-120572 modulation of BNP and NPR-1 expression may require activation of MAPKs pathway BEAS-2B cells were treated with10 ngmL TNF-120572 alone or in combination with 10 120583M SB 203580 (p38 MAPK inhibitor) SP 600125 (JNK 12 inhibitor) or U-0126 (ERK12 inhibitor) for 24 h Panels show ((a) (b)) BNP ((c) (d)) NPR-1 gene expression at mRNA ((a) (c)) and protein ((b) (d)) level and (e)Phospho-p38 MAPK protein level Western blots were obtained by using the specific rabbit polyclonal or monoclonal Abs The blots werestripped of the bound Ab and reprobed with mouse anti-120573-actin to confirm equal loading Western blots are representative of three separateexperiments All histograms indicate means plusmn SD of three different cultures each one tested in quadruplicate and expressed as percentage ofcontrol (lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 versus Controls) (∙∙119875 lt 001 versus SB) (sect119875 lt 005 sectsect
119875 lt 001 versus SP) (∘∘119875 lt 001∘∘∘
119875 lt 0001 versus U-0126) C Controls (untreated cells)
up-regulation after TNF-120572 treatment could concur in poten-tiating an inflammatory statusThe increased production andrelease of specific cytokines causing systemic changes knownas the acute phase response [5 8 10] induced by TNF-120572 maythus also occur via BNP up-regulation
Recently a BNP rise has been observed in patientsundergoing pulmonary lobectomy for lung cancer [11] Inparticular the patients none of whom developed signs orsymptoms of heart failure during the postoperative period
showed an early significant increase on day 1 immediatelyafter surgery of the plasma concentrations of BNP whichwas followed on the postoperative day 2 by a significantweight gain correlated with large volumes of fluids accumu-lation despite a negative intraoperative fluid balance andperoperative strict fluid restriction [11] We suggest thatsuch an increase could be at least in part explained bythe ability of proinflammatory cytokines such as TNF-120572 toupregulate BNP gene expression and secretion and that this
Mediators of Inflammation 9
0
20
40
60
80
100
120
0 10 20 40TNF-120572 (ngmL)
AQP1
( o
f con
trol)
lowast
lowastlowastlowast
lowastlowastlowast
(a)
Actin 0 10 20 40
AQP1
TNF-120572 (ngmL)
(b)
0
20
40
60
80
100
120
AQP5
( o
f con
trol)
0 10 20 40
TNF-120572 (ngmL)
lowastlowast
lowastlowastlowastlowast
(c)
AQP5
Actin 0 10 20 40TNF-120572 (ngmL)
(d)
Figure 6 TNF-120572 decreases AQP1 and AQP5 expression in BEAS 2B cells BEAS-2B cells were treated with 10 20 and 40 ngmL TNF-120572 for24 h Gene expression of AQP1 ((a) (b)) and AQP5 ((c) (d)) at mRNA ((a) (c)) and protein ((b) (d)) level Western blots were obtained byusing the specific rabbit polyclonal Abs The blots were stripped of the bound Ab and reprobed with mouse anti-120573-actin to confirm equalloading Western blots are representative of three separate experiments All histograms indicate means plusmn SD of three different cultures eachone tested in quadruplicate and expressed as percentage of control (lowast119875 lt 001 lowastlowast119875 lt 0001 and lowastlowastlowast119875 lt 00001 versus untreated cells)
up-regulation could enhance the major surgery-related earlyhyperinflammatory response essential for tissue repair andhost defense [5]
The observed dexamethasone capability to completelyrevert TNF-120572-induced BNP up-regulation suggests that thisanti-inflammatory glucocorticoid may prevent BNP-relatedeffects
Regarding ANP the observed significant down-regula-tion of mRNA and protein levels after 10 20 ngmL TNF-120572treatment suggests that also this condition could affect localinflammation status In fact ANP administration determinesanti-inflammatory effects in airway epithelial cells [48] play-ing important roles in modulating inflammatory responseIn consideration of such ANP anti-inflammatory action wespeculate that the observed TNF-120572-induced ANP down-regulation could concur in enhancing an inflammatory sta-tus Differently from the above reported doses 40 ngmLTNF-120572 administration induced ANP up-regulation eventhough not statistically significant We suggest that this resultmight be due to a direct BNP involvement In fact a markedANP up-regulation was observed after BNP treatment at the
dose accumulated in culture medium after 40 ngmL TNF-120572treatment
The observed selectivemodulation inANP and BNP geneexpression after TNF-120572 treatment in line with other studiesin different cell models [19 49 50] could be related to adifferent regulation mechanism at transcriptional level assuggested by our observation on the different involvementof NF-120581B and MAPKs signaling pathways In particular wedemonstrated that ANP down-regulation TNF-120572-mediatedoccurs via NF-120581B activation pathway whereas BNP modula-tion requires ERK 12 activation pathway NF-120581B involvementin TNF-120572-induced ANP down-regulation is not opposed tothe proinflammatory role of this transcription factor beingANP a molecule that exerts an anti-inflammatory effect inairway epithelial cells [48] Regarding themechanism linkingNF-120581B to the downregulation of ANP gene expression wesuggest that it could be similar to that recently proposedfor Fibroblast Growth Factor 10 (FGF-10) where NF-120581Bactivationmay lead to reduced gene expression by recruitinginhibitory factors to specific gene promoters [51]
10 Mediators of Inflammation
0
20
40
60
80
100
120
C BAY 10 20 40
AQP1
( o
f con
trol)
TNF-120572 (ngmL)
TNF-120572TNF-120572 + BAY
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowast
(a)
BAY
AQP1Actin
0 0 10 20 40 10 20 40TNF-120572 (ngmL)minus + +minus minus minus + +
(b)
0
20
40
60
80
100
120
AQP5
( o
f con
trol)
C BAY 10 20 40
TNF-120572 (ngmL)
TNF-120572TNF-120572 + BAY
∘∘∘∘∘∘
∘∘∘
lowastlowastlowastlowast
lowastlowast
(c)
AQP5Actin
0 0 10 20 40 10 20 40TNF-120572 (ngmL)BAY minus + +minus minus minus + +
(d)
Figure 7 TNF-120572 downregulation of AQP1 and AQP5 expression may require activation of NF-120581B pathwayBEAS-2B cells were treated with10 20 and 40 ngmL TNF-120572 alone or in combination with NF-120581B inhibitor BAY 11-7082 (1 120583M) for 24 h Gene expression of AQP1 ((a) (b))and AQP5 ((c) (d)) at mRNA ((a) (c)) and protein ((b) (d)) level Western blots were obtained by using the specific rabbit polyclonal AbsThe blots were stripped of the bound Ab and reprobed with mouse anti-120573-actin to confirm equal loading Western blots are representative ofthree separate experiments All histograms indicate means plusmn SD of three different cultures each one tested in quadruplicate and expressed aspercentage of control (lowast119875 lt 001 lowastlowast119875 lt 0001 lowastlowastlowast119875 lt 00001 versus Controls and ∘∘∘119875 lt 00001 versus BAY) C Controls (untreated cells)
ANP and BNP biological functions occur through theirbinding to the same NPR-1 [13] Here we demonstratedfor the first time to our knowledge TNF-120572 capability todownregulateNPR-1mRNAandprotein expression in BEAS-2B cells and that this regulation occurs via MAPKs signalingpathway activation The consequence of the complex TNF-120572-induced NPs and NPR-1 observed modulation associatedwith their binding to the same receptor on BEAS-2B cellsmakes it difficult to predict what would be the net resultingeffect both in vitro and in vivo in relation also to the presenceof a possible autocrine regulation by NPs andor to the effectsof different cytokines activated by both BNP and TNF-120572We hypothesize that the TNF-120572-induced downregulation of
NPR-1 paralleled by an up-regulation of BNP could alsolead to a paracrine action of this peptide possibly on themicrovascular endothelium of the lung where NPR-1 isdensely expressed [18] In this context we speculate that theinduction of local modification on BNP or ANP by TNF-120572 inhuman bronchial epithelial cells could alter the barrier per-meability of the pulmonary microcirculation Hence TNF-120572 acting on epithelial cells could also indirectly contributeto endothelial cells barrier dysfunction and permeability byenhancing the local production of proinflammatory BNPand by down-regulating the anti-inflammatory expressionof ANP Besides exogenous synthetic ANP has been shownto protect from endothelial barrier dysfunction in in vivo
Mediators of Inflammation 11
0
50
100
150
200
250
300
0 001
AN
P (
of c
ontro
l)
BNP (nM)
lowast
(a)
0
50
100
150
0 001BNP (nM)
NPR
-1(
of c
ontro
l)
(b)
0
20
40
60
80
100
120
0 001
BNP (nM)
lowast
AQP1
( o
f con
trol)
(c)
0
20
40
60
80
100
120
0 001
BNP (nM)
AQP5
( o
f con
trol)
(d)
Figure 8 BNP effect on ANP NPR-1 AQP1 and AQP5 expressionBEAS-2B cells were treated with 001 nM BNP for 24 h Histograms show(a) ANP (b) NPR-1 (c) AQP1 and (d) AQP5 gene expression at mRNA level All histograms indicate means plusmn SD of three different cultureseach one tested in quadruplicate and expressed as percentage of control (lowast119875 lt 005 versus untreated cells)
020406080
100120140160180200
DEX
BNP
( o
f con
trol)
C TNF-120572 TNF-120572 + DEX
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowast
lowast
lowast
Figure 9 Dexamethasone effect on BNP expression BEAS-2B cellswere treatedwith 1 120583Mdexamethasone (DEX) and 40 ngmLTNF-120572alone or in combination with DEX for 24 h Histograms show BNPgene expression at mRNA (gray bars) and protein (black bars) levelAll histograms indicate means plusmn SD of three different cultures eachone tested in quadruplicate and expressed as percentage of control(lowast119875 lt 001 lowastlowast119875 lt 0001 and lowastlowastlowast119875 lt 00001 versus Controls) CControls (untreated cells)
and in vitro models of lung injury [18] and the therapeu-tical relevance of these experimental observations has beenshown in intensive care patients without heart disease whereintravenous ANP infusion diminished pulmonary vascularpermeability and pulmonary edema [18]
Regarding AQPs we demonstrated that BEAS-2B cellsexpress AQP1 and AQP5 and that TNF-120572 directly down-regulates their expression at mRNA and protein levelsRegarding AQP1 also an additional BNP-mediated TNF-120572involvement can be proposed In fact AQP1 down-regulationwas observed with BNP treatment at the dose accumulatedin culture medium after 40 ngmL TNF-120572 treatment Themechanisms that underlie AQPs regulation are of consider-able interest Several AQPs has recently been demonstratedto undergo complex regulation [27] Our results show thatAQP1 and APQ5 are subject to inflammation regulationadding an important piece of information in this contextOur results are in agreement with other studies describinga marked reduction in the expression levels of AQP1 andAQP5 inmouse lung [30] andmouse lung epithelial cells [31]The biological significance of the observed TNF-120572-inducedAQP1 and AQP5 down-regulation is at present unclear Ithas been recently suggested that the decrease in both AQPsmay play an important role in the development of lung edema[52] which is known to be related with fluid retention Wetherefore speculate that TNF-120572-induced AQP1 and AQP5down-regulation might play a role also in the excess of fluidaccumulation related to major surgical procedures
5 Conclusions
In conclusion our results provided evidence that in humanbronchial epithelial cells BEAS-2B TNF-120572 selectively mod-ulates mRNA and protein expression levels of different
12 Mediators of Inflammation
molecules involved in body fluid homeostasis ANP BNPand their receptor NPR-1 AQP1 and AQP5 Moreoverour data pointed out that the changes specifically affectingNPs expression occur independently from hemodynamicinfluences and that inflammation should be considered anextracardiac cause of BNP elevation adding an importantpiece of information in the novel area of study regarding NPsand inflammation In addition we demonstrated that such amodulation occurs via different involvement of NK-120581B andMAPKs signaling pathways Finally we suggested inflamma-tion among the mechanisms involved in the regulation ofAQP1 and AQP5 Therefore in this exploratory study wespeculate that TNF-120572 might be involved in postoperative-fluid retention related to major surgery
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgment
The authors thank Mrs Roberta Frosini for the excellenttechnical assistance
References
[1] E Itobi M Stroud and M Elia ldquoImpact of oedema onrecovery after major abdominal surgery and potential valueof multifrequency bioimpedance measurementsrdquo The BritishJournal of Surgery vol 93 no 3 pp 354ndash361 2006
[2] J A Lowell C Schifferdecker D F Driscoll P N Benottiand B R Bistrian ldquoPostoperative fluid overload not a benignproblemrdquo Critical Care Medicine vol 18 no 7 pp 728ndash7331990
[3] A M Moslashller T Pedersen P-E Svendsen and A EngquistldquoPerioperative risk factors in elective pneumonectomy theimpact of excess fluid balancerdquo European Journal of Anaesthe-siology vol 19 no 1 pp 57ndash62 2002
[4] E Bennett-Guerrero D E Feierman G R Barclay et alldquoPreoperative and intraoperative predictors of postoperativemorbidity poor graft function and early rejection in 190patients undergoing liver transplantationrdquo Archives of Surgeryvol 136 no 10 pp 1177ndash1183 2001
[5] J P Desborough ldquoThe stress response to trauma and surgeryrdquoThe British Journal of Anaesthesia vol 85 no 1 pp 109ndash1172000
[6] H Tsujimoto S Ono T Majima et al ldquoDifferential toll-likereceptor expression after ex vivo lipopolysaccharide exposurein patients with sepsis and following surgical stressrdquo ClinicalImmunology vol 119 no 2 pp 180ndash187 2006
[7] C Evans C Galustian D Kumar et al ldquoImpact of surgeryon immunologic function comparison between minimallyinvasive techniques and conventional laparotomy for surgi-cal resection of colorectal tumorsrdquo The American Journal ofSurgery vol 197 no 2 pp 238ndash245 2009
[8] F Catena L Ansaloni A Avanzolini et al ldquoSystemic cytokineresponse after emergency and elective surgery for colorectalcarcinomardquo International Journal of Colorectal Disease vol 24no 7 pp 803ndash808 2009
[9] R Sarbinowski S Arvidsson M Tylman T Oresland andA Bengtsson ldquoPlasma concentration of procalcitonin andsystemic inflammatory response syndrome after colorectalsurgeryrdquo Acta Anaesthesiologica Scandinavica vol 49 no 2 pp191ndash196 2005
[10] P Sheeran and G M Hall ldquoCytokines in anaesthesiardquo TheBritish Journal of Anaesthesia vol 78 no 2 pp 201ndash219 1997
[11] L Cagini R Capozzi V Tassi et al ldquoFluid and electrolytebalance after major thoracic surgery by bioimpedance andendocrine evaluationrdquo European Journal of Cardio-ThoracicSurgery vol 40 no 2 pp e71ndashe76 2011
[12] T Omland and T Hagve ldquoNatriuretic peptides physiologic andanalytic considerationsrdquo Heart Failure Clinics vol 5 no 4 pp471ndash487 2009
[13] L R Potter A R Yoder D R Flora L K Antos and DM Dickey ldquoNatriuretic peptides their structures receptorsphysiologic functions and therapeutic applicationsrdquo Handbookof Experimental Pharmacology vol 191 pp 341ndash366 2009
[14] T G Flynn M L de Bold and A J de Bold ldquoThe aminoacid sequence of an atrial peptide with potent diuretic andnatriuretic propertiesrdquo Biochemical and Biophysical ResearchCommunications vol 117 no 3 pp 859ndash865 1983
[15] A J de Bold ldquoAtrial natriuretic factor a hormone produced bythe heartrdquo Science vol 230 pp 767ndash770 1985
[16] K Kangawa Y Tawaragi S Oikawa et al ldquoIdentification of rat 120574atrial natriuretic polypeptide and characterization of the cDNAencoding its precursorrdquo Nature vol 312 no 5990 pp 152ndash1551984
[17] T Sudoh K Kangawa N Minamino and H Matsuo ldquoA newnatriuretic peptide in porcine brainrdquo Nature vol 332 no 6159pp 78ndash81 1988
[18] M Kuhn ldquoEndothelial actions of atrial and B-type natriureticpeptidesrdquo The British Journal of Pharmacology vol 166 no 2pp 522ndash531 2012
[19] A J de Bold ldquoCardiac natriuretic peptides gene expression andsecretion in inflammationrdquo Journal of Investigative Medicinevol 57 pp 29ndash32 2009
[20] S P DrsquoSouza M Davis and G F Baxter ldquoAutocrine andparacrine actions of natriuretic peptides in the heartrdquo Pharma-cology andTherapeutics vol 101 no 2 pp 113ndash129 2004
[21] M Brueckmann G Huhle S Lang et al ldquoPrognostic valueof plasma N-terminal pro-brain natriuretic peptide in patientswith severe sepsisrdquoCirculation vol 112 no 4 pp 527ndash534 2005
[22] J Charpentier C E Luyt Y Fulla et al ldquoBrain natriureticpeptide a marker of myocardial dysfunction and prognosisduring severe sepsisrdquo Critical Care Medicine vol 32 no 3 pp660ndash665 2004
[23] R Shor Y Rozenman A Bolshinsky et al ldquoBNP in septicpatients without systolic myocardial dysfunctionrdquo EuropeanJournal of Internal Medicine vol 17 no 8 pp 536ndash540 2006
[24] T Inoue M Kawai T Nakane et al ldquoInfluence of low-gradeinflammation on plasma B-type natriuretic peptide levelsrdquoInternal Medicine vol 49 no 24 pp 2659ndash2668 2010
[25] P Agre L S King M Yasui et al ldquoAquaporin water channelsmdashfrom atomic structure to clinical medicinerdquo Journal of Physiol-ogy vol 542 no 1 pp 3ndash16 2002
[26] P Agre and D Kozono ldquoAquaporin water channels molecularmechanisms for human diseasesrdquo FEBS Letters vol 555 no 1pp 72ndash78 2003
Mediators of Inflammation 13
[27] J M Carbrey and P Agre ldquoDiscovery of the aquaporins anddevelopment of the fieldrdquo Handbook of Experimental Pharma-cology vol 190 pp 3ndash28 2009
[28] O Bloch and G T Manley ldquoThe role of aquaporin-4 in cerebralwater transport and edemardquo Neurosurgical Focus vol 22 no 5article E3 2007
[29] J Li M Xu Q Fan et al ldquoTanshinone IIA ameliorates seawaterexposure-induced lung injury by inhibiting aquaporins (AQP)1 and AQP5 expression in lungrdquo Respiratory Physiology andNeurobiology vol 176 no 1-2 pp 39ndash49 2011
[30] J E Towne K S Harrod C M Krane and A G MenonldquoDecreased expression of aquaporin (AQP)1 and AQP5 inmouse lung after acute viral infectionrdquoThe American Journal ofRespiratory Cell and Molecular Biology vol 22 no 1 pp 34ndash442000
[31] J E Towne C M Krane C J Bachurski and A G MenonldquoTumor necrosis factor-120572 inhibits aquaporin 5 expression inmouse lung epithelial cellsrdquoThe Journal of Biological Chemistryvol 276 no 22 pp 18657ndash18664 2001
[32] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001
[33] K Verhelst I Carpentier and R Beyaert ldquoRegulation of TNF-induced NF-120581B activation by different cytoplasmic ubiquitina-tion eventsrdquo Cytokine and Growth Factor Reviews vol 22 no5-6 pp 277ndash286 2011
[34] L Qian Y Shen J C Chen et al ldquo3D-QSAR and dockingstudies of quinazoline derivatives with the inhibatory activitytoward NF-120581BrdquoQSAR and Combinatorial Science vol 27 no 8pp 984ndash995 2008
[35] P D NrsquoGuessan S Hippenstiel M O Etouem et al ldquoStrepto-coccus pneumoniae induced p38MAPK- andNF-120581B-dependentCOX-2 expression in human lung epitheliumrdquo The AmericanJournal of PhysiologymdashLung Cellular and Molecular Physiologyvol 290 no 6 pp L1131ndashL1138 2006
[36] B Schmeck K Moog J Zahlten et al ldquoStreptococcus pneumo-niae induced c-Jun-N-terminal kinase- andAP-I-dependent IL-8 release by lung epithelial BEAS-2B cellsrdquoRespiratory Researchvol 7 article 98 2006
[37] M GMatera L Calzetta D Passeri et al ldquoEpithelium integrityis crucial for the relaxant activity of brain natriuretic peptide inhuman isolated bronchirdquo The British Journal of Pharmacologyvol 163 no 8 pp 1740ndash1754 2011
[38] R QuirionM Dalpe A de Lean J Gutkowska M Cantin andJ Genest ldquoAtrial natriuretic factor (ANF) binding sites in brainand related structuresrdquoPeptides vol 5 no 6 pp 1167ndash1172 1984
[39] M B Pamnani D L Clough J S Chen W T Link andF J Haddy ldquoEffects of rat atrial extract on sodium transportand blood pressure in the ratrdquo Proceedings of the Society forExperimental Biology and Medicine vol 176 no 2 pp 123ndash1311984
[40] T R Traynor and S M OrsquoGrady ldquoBrain natriuretic peptidestimulates K andCl secretion across porcine distal colon epithe-liumrdquoTheAmerican Journal of PhysiologymdashCell Physiology vol260 no 4 pp C750ndashC755 1991
[41] R Rollın A Mediero M Roldan-Pallares A Fernandez-Cruzand R Fernandez-Durango ldquoNatriuretic peptide system in thehuman retinardquoMolecular Vision vol 10 pp 15ndash22 2004
[42] M Harada H Itoh O Nakagawa et al ldquoSignificance ofventricular myocytes and nonmyocytes interaction during car-diocyte hypertrophy evidence for endothelin-1 as a paracrinehypertrophic factor from cardiac nonmyocytesrdquo Circulationvol 96 no 10 pp 3737ndash3744 1997
[43] K Kuwahara Y Saito M Harada et al ldquoInvolvement ofcardiotrophin-1 in cardiac myocyte-nonmyocyte interactionsduring hypertrophy of rat cardiac myocytes in vitrordquo Circula-tion vol 100 no 10 pp 1116ndash1124 1999
[44] E Harada O Nakagawa M Yoshimura et al ldquoEffect ofinterleukin-1120573 on cardiac hypertrophy and production of natri-uretic peptides in rat cardiocyte culturerdquo Journal of Molecularand Cellular Cardiology vol 31 no 11 pp 1997ndash2006 1999
[45] V Chiurchiu V Izzi F DrsquoAquilio F Carotenuto P Di Nardoand P M Baldini ldquoBrain natriuretic peptide (BNP) regulatesthe production of inflammatory mediators in human THP-1macrophagesrdquo Regulatory Peptides vol 148 no 1ndash3 pp 26ndash322008
[46] N S Holden C F Rider M J Bell et al ldquoEnhancement ofinflammatory mediator release by 1205732-adrenoceptor agonists inairway epithelial cells is reversed by glucocorticoid actionrdquoTheBritish Journal of Pharmacology vol 160 no 2 pp 410ndash4202010
[47] V S Fang C C Juan J G S Won and L T Ho ldquoFurther studyof aldosterone secretion-inhibitory factor and brain natriureticpeptide on cortisol production of Guinea pig zona fasciculatacellsrdquo Chinese Journal of Physiology vol 43 no 4 pp 141ndash1472000
[48] G Hellermann X Kong J Gunnarsdottir et al ldquoMechanismof bronchoprotective effects of a novel natriuretic hormonepeptiderdquo Journal of Allergy and Clinical Immunology vol 113no 1 pp 79ndash85 2004
[49] K K Ma T Ogawa and A J de Bold ldquoSelective upregulationof cardiac brain natriuretic peptide at the transcriptional andtranslational levels by pro-inflammatory cytokines and by con-ditionedmedium derived frommixed lymphocyte reactions viap38 MAP kinaserdquo Journal of Molecular and Cellular Cardiologyvol 36 no 4 pp 505ndash513 2004
[50] T Ogawa J P Veinot M L K de Bold T Georgalis and AJ De Bold ldquoAngiotensin II receptor antagonism reverts theselective cardiac BNP upregulation and secretion observed inmyocarditisrdquo The American Journal of PhysiologymdashHeart andCirculatory Physiology vol 294 no 6 pp H2596ndashH2603 2008
[51] B J Carver E J Plosa A M Stinnett T S Blackwell andL S Prince ldquoInteractions between NF-120581B and SP3 connectinflammatory signaling with reduced FGF-10 expressionrdquo TheJournal of Biological Chemistry vol 288 no 21 pp 15318ndash153252013
[52] C Dong G Wang B Li et al ldquoAnti-asthmatic agents alleviatepulmonary edema by upregulating AQP1 and AQP5 expressionin the lungs of mice with OVA-induced asthmardquo RespiratoryPhysiology and Neurobiology vol 181 no 1 pp 21ndash28 2012
Table 1 Primer sequences primer concentrations and qRT-PCR cycling conditions
Gene Primer sequences Primer concentrations qRT-PCR cycling conditions
ANP F 51015840-tcagcccagcccagagag-31015840 200 nM 1 cycle at 95∘C for 10min 40 cycles at 95∘C for 1minR 51015840-gctccaatcctgtccatcctg-31015840 200 nM 60∘C for 30 sec and 72∘C for 30 sec
BNP F 51015840-gagggcaggtgggaagcaaac-31015840 200 nM 1 cycle at 95∘C for 10min 40 cycles at 95∘C for 1minR 51015840-gcaagaagagcaggagcaggag-31015840 200 nM 60∘C for 30 sec and 72∘C for 30 sec
NPR-1 F 51015840-ccctggaggtgctggctttgg-31015840 200 nM 1 cycle at 95∘C for 10min 40 cycles at 95∘C for 1minR 51015840-ctctcaaggctactgggctcaacg-31015840 200 nM 59∘C for 30 sec and 72∘C for 30 sec
AQP1 F 51015840-ttggacacctcctggctattgact-31015840 400 nM 1 cycle at 95∘C for 10min 40 cycles at 95∘C for 1minR 51015840-ccagtggttgctgaagttgtgtgt-31015840 400 nM 60∘C for 30 sec and 72∘C for 30 sec
AQP5 F 51015840-cgctcaacaacaacacaacgc-31015840 200 nM 1 cycle at 95∘C for 10min 40 cycles at 95∘C for 1minR 51015840-ccagtgaagtagattccgacaagg-31015840 200 nM 59∘C for 30 sec and 72∘C for 30 sec
ACTB F 51015840-cactcttccagccttccttcc-31015840 600 nM 1 cycle at 95∘C for 10min 40 cycles at 95∘C for 1minR 51015840-acagcactgtgttggcgtac-31015840 600 nM 59∘C or 60∘C for 30 sec and 72∘C for 30 sec
qRT-PCR quantitative real time polymerase chain reaction F forward R reverse
Reactionswere performed in a total volume of 25120583L contain-ing 250 ng or 500 ng of cDNA 1X Reagent Brilliant II SYBRGreen QPCRMaster Mix and the appropriate concentrationof the specific primers (Medical Milan Italy) Data for com-parative analysis of gene expression were obtained using theΔΔCt method as described in the ABI Prism 7000 sequencedetection system user guide [32] Agarose gel electrophoreticanalysis was used to check the predicted size amplicons forANP (160 bp) BNP (148 bp) NPR-1 (151 bp) AQP1 (98 bp)and AQP5 (180 bp)
26Western Blot Analysis Total protein extracts (40120583g)wereseparated by 12 sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) andblotted onto a nitrocellulosemembrane using iBlot Dry Blotting System (InvitrogenMilan Italy) Nonspecific binding sites were blocked in Roti-Block (Roth GmbH Karlsruhe Germany) for 2 h at roomtemperature The membranes were blotted overnight at 4∘Cwith a 1 100 dilution of the primary specific Abs and washedwith TBSTThe antigen-Ab complex was detected by incuba-tion of the membranes for 2 h at room temperature with theappropriated HRP-conjugated secondary Abs and revealedusing the enhanced chemiluminescence (ECL) system byAmersham Pharmacia Biotech (Uppsala Sweden)
As internal loading controls and for protein expressionnormalizing purpose all membranes were subsequentlystripped of the first antibody in a stripping buffer (100mM2-ME 2 SDS and 625mM Tris-HCl pH 68) for 30minat 50∘C followed by washings with TBST The membraneswere then reprobed with anti-120573-actin antibody followed byincubation with HRP-conjugated Ab
27 BNP Protein Concentration in Culture Medium BNPprotein concentrationwasmeasured in culturemediumusingthe chemiluminescent enzyme immunoassay kit TRIAGEBNP (Biosite Incorporated San Diego USA) The minimumquantity of human BNP detectable with this system is10 pgmL The intraassay and the interassay coefficients ofvariations were 31 and 45 respectively
28 Statistical Analysis Results were expressed as means plusmnSD of three independent experiments The statistical signifi-cance of differences between treated and untreated cells wasassessed by Studentrsquos t-test Differences between groups wereconsidered significant when 119875 lt 005
3 Results
31 TNF-120572 Selectively Modulates ANP BNP and Their Recep-tor NPR-1 Expression in BEAS-2B Cells We firstly demon-strated that BEAS-2B cell line expresses ANP BNP NPR-1AQP1 and AQP5 By qRT-PCR analysis fragments of thepredicted molecular size were generated (Figure 1) To deter-mine whether the expression of ANP BNP and NPR-1 wasaffected by TNF-120572 BEAS-2B cells were treated with 10 20or 40 ngmL TNF-120572 for 24 h ANP mRNA was significantlydecreased about 75 (119875 lt 00001) and about 40 (119875 lt005) of control after treatment with 10 and 20 ngmL TNF-120572 respectively Forty ngmL TNF-120572 induced an upregulation(about 20 of control) of ANP expression even though notstatistically significant (119875 gt 005) (Figure 2(a)) A similartrend was also observed at protein level (Figure 2(b)) BNPmRNA expression was significantly (119875 lt 001) increased(62 of control) since 10 ngmL TNF-120572 treatment maximumstimulation (80 of control) being obtained with 40 ngmL(119875 lt 00001) (Figure 2(c)) Treatment with TNF-120572 at theabove described concentrations also increased BNP proteinsecretion in culture medium (119875 lt 001) (Figure 2(d))NPR-1 gene expression level was dramatically decreased (70ndash80 of control) after TNF-120572 exposure at all the used doses(119875 lt 0001) (Figure 2(e)) paralleled by a decrease of proteinexpression (Figure 2(f))
32 NPs and NPR-1 Modulation by TNF-120572 May RequireActivation of NF-120581B Signaling Pathway in BEAS-2B CellsAs known TNF-120572 regulates numerous genes essential tothe inflammatory process through the activation of mul-tiple signal transduction pathways including NF-120581B [33]Therefore to investigate the possible involvement of NF-120581B signaling pathway in TNF-120572-mediated regulation of NPs
4 Mediators of Inflammation
2 3 4M
242190147
(bp) (bp)M
111110147190
67
5 6
Figure 1 Identification of ANP BNP NPR-1 AQP1 and AQP5transcripts in BEAS-2B cells qReal-Time PCR was performed usinggene specific primers for ANP (lane 2) BNP (lane 3) NPR-1 (lane 4)AQP1 (lane 5) sand AQP5 (lane 6) in BEAS-2B cells Single bands ofthe predicted molecular size for ANP (160 bp) BNP (148 bp) NPR-1(151 bp) AQP1 (98 bp) andAQP5 (180 bp) transcripts were detectedLane M DNA molecular marker
and NPR-1 we used the specific NF-kB inhibitor BAY 11-7082 that diminishes the activation of NF-120581B by preventingphosphorylation of its inhibitory I120581B-120572 protein As shownin Figure 3(a) TNF-120572-induced downregulation of ANP wascompletely reverted by BAY 11-7082 at both mRNA and pro-tein level (Figure 3(b)) suggesting that this response requiresNF-120581B activation pathway Regarding TNF-120572-induced BNPupregulation this was only partially reverted by BAY 11-7082at mRNA level (Figure 3(c)) while was completely revertedat protein level (Figure 3(d)) Conversely TNF-120572-induceddecrement in NPR-1 expression was not affected by BAY 11ndash7082 either at mRNA or protein level (Figures 3(e) and 3(f))
Western blot analysis for the Ser32-phosphorylated I120581B-120572protein proved the biochemistry evidence of the inhibitoryaction of BAY 11-7082 on NF-120581B activity (Figure 3(g)) Tofurther investigate the role of NF-120581B in the modulation ofBNP and NPR-1 in BEAS-2B cells after TNF-120572 treatmentwe employed theNF-120581B-DNA binding inhibitor QNZ whichprevents free NF-120581B from binding to DNA [34] The resultsobtained with such additional inhibitor confirmed the previ-ous findings obtained with BAY 11-7082 In particular TNF-120572-induced BNP up-regulation was not reverted by QNZ atmRNA level (Figure 4(a)) while it was completely reverted atprotein level (Figure 4(b)) Accordingly also TNF-120572-induceddecrement of NPR-1 expression was not affected by QNZeither at mRNA or protein level (Figures 4(c) and 4(d))Western blot analysis for the Ser32-phosphorylated I120581B-120572protein proved the biochemistry evidence of the inhibitoryaction of QNZ on NF-120581B activity (Figure 4(e))
33 BNP and NPR-1 Modulation by TNF-120572 May RequireActivation of MAPKs Signaling Pathway in BEAS-2B CellsBecause TNF-120572 is able to activate multiple signal transduc-tion pathways including MAPKs and because the activationof p38 MAPK and JNK participates in the regulation ofinflammatory processes in bronchial epithelial cells [35 36]we then investigatedMAPKs pathway involvement in TNF-120572modulation of BNP and NPR-1 expression TNF-120572-mediated
up-regulation of BNP was not affected by SB 203580 or SP600125 p38 MAPK and JNK 12 inhibitors respectivelywhile it was completely reverted by the ERK 12 inhibitorU-0126 (Figures 5(a) and 5(b)) suggesting that ERK 12activation pathway is required for BNP TNF-120572-mediatedmodulation Regarding NPR-1 the TNF-120572-mediated down-regulation was completely reverted by the use of all inhibitors(Figures 5(c) and 5(d)) Western blot analysis for theThr180Thr182-phosphorylated form of p-38 MAPK provedthe biochemistry evidence of the inhibitory action of SB203580 on p38 MAPK activity (Figure 5(e))
34 TNF-120572 Decreases AQP1 and AQP5 Expression in BEAS2B Cells We found that TNF-120572 induced a dose dependentdown-regulation of AQP1 expression (119875 lt 001) at mRNA(Figure 6(a)) and protein level (Figure 6(b)) and a dramaticdecrease in AQP5 gene expression level (about 80 ofcontrol) with all the used doses (119875 lt 0001) (Figure 6(c)) par-alleled by a comparable trend at protein level (Figure 6(d))
35 Decreased AQP1 andAQP5 Expression by TNF-120572DoesNotRequire Activation of NF-120581B Signaling Pathway in BEAS-2BCells To examine a possible involvement of NF-120581B in TNF-120572-mediated down-regulation of AQP1 and AQP5 BEAS-2Bcells were incubated with BAY 11-7082 Cotreatment of suchNF-120581B inhibitor with TNF-120572 resulted in a decrease of AQP1expression which was similar to that observed with TNF-120572alone (Figures 7(a) and 7(b)) suggesting that the observedresponse does not require NF-120581B activation Conversely co-treatment of BAY 11-7082 with TNF-120572 affected AQP5 only atprotein level (Figures 7(c) and 7(d))
36 BNP Effect on ANP NPR-1 and AQP1 or AQP5 mRNAExpression in BEAS-2BCells In order to determine a possibledirect involvement of BNP on the gene expression of ANPNPR-1 and AQP1 or AQP5 BEAS-2B cells were treated with001 nM BNP This concentration was previously found tobe accumulated in culture medium after 40 ngmL TNF-120572treatment the dose that induced the maximum effect on theexpression of the studied genes BNP administration induceda significant (119875 lt 005) increase (100 of control) on ANPmRNA level (Figure 8(a)) and did not affectNPR-1 expression(Figure 8(b)) while induced a marked decrease (45 ofcontrol) on AQP1 mRNA level (119875 lt 005) (Figure 8(c))A trend in decreasing was also observed for AQP5 geneexpression even though not statistically significant (119875 gt005) (Figure 8(d))
37 Dexamethasone (DEX) Effect on BNP mRNA and ProteinLevels in BEAS-2B Cells In order to study the capability ofthe anti-inflammatory drug DEX to affect TNF-120572-mediatedup-regulation of BNP mRNA and protein levels BEAS-2Bcells were treated with 1120583MDEX As shown in Figure 9 DEXinduced a down-regulation of BNP expression and proteinlevels alone or in combination with 40 ngmL TNF-120572 (119875 lt001) demonstrating the capability of this glucocorticoid torevert TNF-120572 effects
Mediators of Inflammation 5
0 10 20 40TNF-120572 (ngmL)
AN
P (
of c
ontro
l)
160
140
120
100
80
60
40
20
0
lowast
lowastlowastlowastlowast
(a)
0 10 20 40
ANP
Actin TNF-120572 (ngmL)
(b)
0
50
100
150
200
0 10 20 40
BNP
( o
f con
trol)
TNF-120572 (ngmL)
lowastlowastlowastlowastlowastlowast
lowast
(c)
0
50
100
150
200
0 10 20 40BN
P (
of c
ontro
l)
TNF-120572 (ngmL)
lowastlowast
lowastlowast
lowastlowast
(d)
0 10 20 40TNF-120572 (ngmL)
120
100
80
60
40
20
0
NPR
-1 (
of c
ontro
l)
lowastlowast
lowastlowastlowastlowastlowastlowastlowastlowast
(e)
0 10 20 40
NPR-1
Actin TNF-120572 (ngmL)
(f)
Figure 2 TNF-120572 selectively modulates ANP BNP and NPR-1 expression BEAS-2B cells were treated with 10 20 and 40 ngmL TNF-120572 for24 h ((a) (b)) ANP ((c) (d)) BNP and ((e) (f)) NPR-1 gene expression at mRNA ((a) (c) and (e)) and protein level ((b) (d) and (f))Western blots were obtained by using the specific rabbit polyclonal Abs The blots were stripped of the bound Ab and reprobed with mouseanti-120573-actin to confirm equal loading Western blots are representative of three separate experiments All histograms indicate meansplusmn SD ofthree different cultures each one tested in quadruplicate and expressed as percentage of control (lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 andlowastlowastlowastlowast
119875 lt 00001 versus untreated cells)
4 Discussion
Postoperative-fluid retention is a severe complication fre-quently reported in patients undergoing major surgical pro-cedures [1ndash4] The complex network of molecules involvedin such a severe surgery-induced condition remains poorlyunderstood After major surgery TNF-120572 is one of the mainproinflammatory cytokines initially released in the damagedtissue where it stimulates the production and release of morecytokines responsible for inducing the systemic changesknown as the acute phase response [5 10] Therefore it isreasonable to assume TNF-120572 involvement among the variouscauses of fluid retention and very important to understandthemechanisms underlying its involvement in such an ambit
In the present study we demonstrated for the first time toour knowledge that TNF-120572modulates the expression ofANPBNP NPR-1 AQP1 and AQP5 in human bronchial epithelialcells BEAS-2B via different involvement of NF-120581B andMAPKs signaling pathway activation Here we also providedthe first demonstration that human bronchial epithelial cellsexpress ANP BNP NPR-1 and AQP1 and that BNP is able tomodulate AQP1 expression
The observed expression of NPs and NPR-1 suggestsan autocrine andor paracrine function for these moleculesand indicates important roles for NPs in several biologicalfunctions including regulation of fluid movement across theairway epithelial cells bronchial relaxation [37] vasodilata-tion and pulmonary vascular permeability About the former
6 Mediators of Inflammation
020406080
100120140160
C BAY 10 20 40
AN
P (
of c
ontro
l)
TNF-120572 (ngmL)
TNF-120572TNF-120572 + BAY
lowast
lowastlowastlowastlowast
(a)
ANP
Actin 40
BAY 0 0 10 20 40 10 20minus + +minus minus minus + +
Figure 3 TNF-120572 modulation of NPs and NPR-1 may require activation of NF-120581B pathway BEAS-2B cells were treated with 10 20 and40 ngmL TNF-120572 alone or in combination with NF-120581B inhibitor BAY 11-7082 (1120583M) for 24 h ((a) (b)) ANP ((c) (d)) BNP ((e) (f)) NPR-1gene expression at mRNA ((a) (c) and (e)) and protein level ((b) (d) (f)) and (g) Phospho-I120581B-120572 protein level Western blots were obtainedby using the specific rabbit polyclonal or monoclonal AbsThe blots were stripped of the bound Ab and reprobed with mouse anti-120573-actin toconfirm equal loading Western blots are representative of three separate experiments All histograms indicate means plusmn SD of three differentcultures each of one tested in quadruplicate and expressed as percentage of control (lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowastlowast119875 lt 00001 versus Controls)(∘119875 lt 005 ∘∘119875 lt 001 and ∘∘∘∘119875 lt 00001 versus BAY) C Controls (untreated cells)
process the role of ANP and BNP could be analogous to thathypothesized forNPs in transepithelial ion flux in the choroidplexus [38] in the kidney [39] colon [40] and retina [41]
The observed TNF-120572 capability of selectively regulatingANP BNP and NPR-1 gene expression in BEAS-2B cellline is of particular interest because it demonstrates that
inflammation alters NPs expression levels In particular thesignificant up-regulation of BNP expression and proteinsecretion after TNF-120572 treatment shows that this modulationcan occur independently of hemodynamic influences andthat inflammation should be considered an extracardiaccause of BNP elevation Besides in vitro studies conducted in
Mediators of Inflammation 7
020406080
100120140160180200
C QNZ TNF
BNP
( o
f con
trol)
QNZ + TNF
lowastlowast
∘∘
(a)
0
20
40
60
80
100
120
140
160
180
BNP
( o
f con
trol)
C QNZ TNF QNZ + TNF
lowastlowast
(b)
0
20
40
60
80
100
120
C QNZ TNF QNZ + TNF
∘∘
lowastlowastlowastlowast
lowastlowastlowastlowast
NPR
-1(
of c
ontro
l)
(c)
NPR-1 Actin
0 0QNZ
10 10TNF-120572+minus +minus
(d)
Actin
QNZ TNF-120572 + +
minus ++
minusminus
minus
P-I120581B-120572
(e)
Figure 4 TNF-120572 modulation of protein levels of BNP and NPR-1 may require activation of NF-120581B pathway BEAS-2B cells were treated10 ngmL TNF-120572 alone or in combination with the inhibitor QNZ (10120583M) for 24 h ((a) (b)) BNP ((c) (d)) NPR-1 gene expression at mRNA((a) (c)) and protein ((b) (d)) level and (e) Phospho-I120581B-120572 protein level Western blots were obtained by using the specific rabbit polyclonalor monoclonal AbsThe blots were stripped of the bound Ab and reprobed with mouse anti-120573-actin to confirm equal loading Western blotsare representative of three separate experiments All histograms indicatemeansplusmnSDof three different cultures each one tested in quadruplicateand expressed as percentage of control (lowastlowast119875 lt 001 and lowastlowastlowastlowast119875 lt 00001 versus Controls) (∘∘119875 lt 001 versus QNZ) C Controls (untreatedcells)
rat cardiomyocytes have shown that not only hemodynamicfactors but also neurohumoral factors activated during heartfailure such as angiotensin II endothelin and cytokinescause BNP secretion [42ndash44] In addition in patients withcardiovascular diseases plasma BNP levels have been shownto be also affected by low-grade inflammation [24] and aselective increase inBNPplasma levels has been proposed as ageneral feature of inflammation [19]The lack on any apparentTNF-120572 dose-depending increase in BNP gene expression thatwe observed in our system could be due to an autocrinenegative feedback action mediated by BNP itself followingbinding to the NPR-1 receptor expressed in BEAS-2B cells
The biological role of BNPup-regulation after TNF-120572 treatment remains to be elucidated However since it
has been demonstrated that BNP regulates the productionof major inflammatory molecules such as IL-12 IL-10leukotriene B and prostaglandin E2 in human macrophages[45] and that BEAS-2B are able to release inflammatorymolecules [46] it could be reasonable to assume that theobserved BNP increase after TNF-120572 treatment may stimu-late the production and release of such cytokines also fromhuman epithelial bronchial cells In addition in in vitrostudies a potent BNP inhibitory action on the productionof the glucocorticoid anti-inflammatory hormone cortisolhas been described [47] Therefore BNP could be con-sidered a proinflammatory molecule by inducing cytokinesexpression in target cells and inhibiting the production ofanti-inflammatory hormones Therefore the observed BNP
8 Mediators of Inflammation
0
50
100
150
200
250BN
P (
of c
ontro
l)
C
TNF SB
SB+
TNF SP
SP+
TNF
U-0
126
U-0126+
TNF
lowastlowast
lowastlowast
∘∘
sect
(a)
020406080
100120140160180
BNP
( o
f con
trol)
C
TNF SB
SB+
TNF SP
SP+
TNF
U-0
126
U-0126+
TNF
lowastlowast
lowastlowast
∘∘∘
sectsect
(b)
0
20
40
60
80
100
120
140
160
C
TNF SB
SB+
TNF SP
SP+
TNF
U-0
126
U-0126+
TNF
lowastlowastlowast
lowastlowastlowast
lowast
lowast
sect
NPR
-1(
of c
ontro
l)
(c)
+
SB 203580
+
SP 600125 U-0126
+ActinTNF-120572
NPR-1
minus minus minus minus +
(d)
SB 203580
Actin
P-p38 MAPK
TNF-120572 + +
minus ++
minusminus
minus
(e)
Figure 5 TNF-120572 modulation of BNP and NPR-1 expression may require activation of MAPKs pathway BEAS-2B cells were treated with10 ngmL TNF-120572 alone or in combination with 10 120583M SB 203580 (p38 MAPK inhibitor) SP 600125 (JNK 12 inhibitor) or U-0126 (ERK12 inhibitor) for 24 h Panels show ((a) (b)) BNP ((c) (d)) NPR-1 gene expression at mRNA ((a) (c)) and protein ((b) (d)) level and (e)Phospho-p38 MAPK protein level Western blots were obtained by using the specific rabbit polyclonal or monoclonal Abs The blots werestripped of the bound Ab and reprobed with mouse anti-120573-actin to confirm equal loading Western blots are representative of three separateexperiments All histograms indicate means plusmn SD of three different cultures each one tested in quadruplicate and expressed as percentage ofcontrol (lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 versus Controls) (∙∙119875 lt 001 versus SB) (sect119875 lt 005 sectsect
119875 lt 001 versus SP) (∘∘119875 lt 001∘∘∘
119875 lt 0001 versus U-0126) C Controls (untreated cells)
up-regulation after TNF-120572 treatment could concur in poten-tiating an inflammatory statusThe increased production andrelease of specific cytokines causing systemic changes knownas the acute phase response [5 8 10] induced by TNF-120572 maythus also occur via BNP up-regulation
Recently a BNP rise has been observed in patientsundergoing pulmonary lobectomy for lung cancer [11] Inparticular the patients none of whom developed signs orsymptoms of heart failure during the postoperative period
showed an early significant increase on day 1 immediatelyafter surgery of the plasma concentrations of BNP whichwas followed on the postoperative day 2 by a significantweight gain correlated with large volumes of fluids accumu-lation despite a negative intraoperative fluid balance andperoperative strict fluid restriction [11] We suggest thatsuch an increase could be at least in part explained bythe ability of proinflammatory cytokines such as TNF-120572 toupregulate BNP gene expression and secretion and that this
Mediators of Inflammation 9
0
20
40
60
80
100
120
0 10 20 40TNF-120572 (ngmL)
AQP1
( o
f con
trol)
lowast
lowastlowastlowast
lowastlowastlowast
(a)
Actin 0 10 20 40
AQP1
TNF-120572 (ngmL)
(b)
0
20
40
60
80
100
120
AQP5
( o
f con
trol)
0 10 20 40
TNF-120572 (ngmL)
lowastlowast
lowastlowastlowastlowast
(c)
AQP5
Actin 0 10 20 40TNF-120572 (ngmL)
(d)
Figure 6 TNF-120572 decreases AQP1 and AQP5 expression in BEAS 2B cells BEAS-2B cells were treated with 10 20 and 40 ngmL TNF-120572 for24 h Gene expression of AQP1 ((a) (b)) and AQP5 ((c) (d)) at mRNA ((a) (c)) and protein ((b) (d)) level Western blots were obtained byusing the specific rabbit polyclonal Abs The blots were stripped of the bound Ab and reprobed with mouse anti-120573-actin to confirm equalloading Western blots are representative of three separate experiments All histograms indicate means plusmn SD of three different cultures eachone tested in quadruplicate and expressed as percentage of control (lowast119875 lt 001 lowastlowast119875 lt 0001 and lowastlowastlowast119875 lt 00001 versus untreated cells)
up-regulation could enhance the major surgery-related earlyhyperinflammatory response essential for tissue repair andhost defense [5]
The observed dexamethasone capability to completelyrevert TNF-120572-induced BNP up-regulation suggests that thisanti-inflammatory glucocorticoid may prevent BNP-relatedeffects
Regarding ANP the observed significant down-regula-tion of mRNA and protein levels after 10 20 ngmL TNF-120572treatment suggests that also this condition could affect localinflammation status In fact ANP administration determinesanti-inflammatory effects in airway epithelial cells [48] play-ing important roles in modulating inflammatory responseIn consideration of such ANP anti-inflammatory action wespeculate that the observed TNF-120572-induced ANP down-regulation could concur in enhancing an inflammatory sta-tus Differently from the above reported doses 40 ngmLTNF-120572 administration induced ANP up-regulation eventhough not statistically significant We suggest that this resultmight be due to a direct BNP involvement In fact a markedANP up-regulation was observed after BNP treatment at the
dose accumulated in culture medium after 40 ngmL TNF-120572treatment
The observed selectivemodulation inANP and BNP geneexpression after TNF-120572 treatment in line with other studiesin different cell models [19 49 50] could be related to adifferent regulation mechanism at transcriptional level assuggested by our observation on the different involvementof NF-120581B and MAPKs signaling pathways In particular wedemonstrated that ANP down-regulation TNF-120572-mediatedoccurs via NF-120581B activation pathway whereas BNP modula-tion requires ERK 12 activation pathway NF-120581B involvementin TNF-120572-induced ANP down-regulation is not opposed tothe proinflammatory role of this transcription factor beingANP a molecule that exerts an anti-inflammatory effect inairway epithelial cells [48] Regarding themechanism linkingNF-120581B to the downregulation of ANP gene expression wesuggest that it could be similar to that recently proposedfor Fibroblast Growth Factor 10 (FGF-10) where NF-120581Bactivationmay lead to reduced gene expression by recruitinginhibitory factors to specific gene promoters [51]
10 Mediators of Inflammation
0
20
40
60
80
100
120
C BAY 10 20 40
AQP1
( o
f con
trol)
TNF-120572 (ngmL)
TNF-120572TNF-120572 + BAY
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowast
(a)
BAY
AQP1Actin
0 0 10 20 40 10 20 40TNF-120572 (ngmL)minus + +minus minus minus + +
(b)
0
20
40
60
80
100
120
AQP5
( o
f con
trol)
C BAY 10 20 40
TNF-120572 (ngmL)
TNF-120572TNF-120572 + BAY
∘∘∘∘∘∘
∘∘∘
lowastlowastlowastlowast
lowastlowast
(c)
AQP5Actin
0 0 10 20 40 10 20 40TNF-120572 (ngmL)BAY minus + +minus minus minus + +
(d)
Figure 7 TNF-120572 downregulation of AQP1 and AQP5 expression may require activation of NF-120581B pathwayBEAS-2B cells were treated with10 20 and 40 ngmL TNF-120572 alone or in combination with NF-120581B inhibitor BAY 11-7082 (1 120583M) for 24 h Gene expression of AQP1 ((a) (b))and AQP5 ((c) (d)) at mRNA ((a) (c)) and protein ((b) (d)) level Western blots were obtained by using the specific rabbit polyclonal AbsThe blots were stripped of the bound Ab and reprobed with mouse anti-120573-actin to confirm equal loading Western blots are representative ofthree separate experiments All histograms indicate means plusmn SD of three different cultures each one tested in quadruplicate and expressed aspercentage of control (lowast119875 lt 001 lowastlowast119875 lt 0001 lowastlowastlowast119875 lt 00001 versus Controls and ∘∘∘119875 lt 00001 versus BAY) C Controls (untreated cells)
ANP and BNP biological functions occur through theirbinding to the same NPR-1 [13] Here we demonstratedfor the first time to our knowledge TNF-120572 capability todownregulateNPR-1mRNAandprotein expression in BEAS-2B cells and that this regulation occurs via MAPKs signalingpathway activation The consequence of the complex TNF-120572-induced NPs and NPR-1 observed modulation associatedwith their binding to the same receptor on BEAS-2B cellsmakes it difficult to predict what would be the net resultingeffect both in vitro and in vivo in relation also to the presenceof a possible autocrine regulation by NPs andor to the effectsof different cytokines activated by both BNP and TNF-120572We hypothesize that the TNF-120572-induced downregulation of
NPR-1 paralleled by an up-regulation of BNP could alsolead to a paracrine action of this peptide possibly on themicrovascular endothelium of the lung where NPR-1 isdensely expressed [18] In this context we speculate that theinduction of local modification on BNP or ANP by TNF-120572 inhuman bronchial epithelial cells could alter the barrier per-meability of the pulmonary microcirculation Hence TNF-120572 acting on epithelial cells could also indirectly contributeto endothelial cells barrier dysfunction and permeability byenhancing the local production of proinflammatory BNPand by down-regulating the anti-inflammatory expressionof ANP Besides exogenous synthetic ANP has been shownto protect from endothelial barrier dysfunction in in vivo
Mediators of Inflammation 11
0
50
100
150
200
250
300
0 001
AN
P (
of c
ontro
l)
BNP (nM)
lowast
(a)
0
50
100
150
0 001BNP (nM)
NPR
-1(
of c
ontro
l)
(b)
0
20
40
60
80
100
120
0 001
BNP (nM)
lowast
AQP1
( o
f con
trol)
(c)
0
20
40
60
80
100
120
0 001
BNP (nM)
AQP5
( o
f con
trol)
(d)
Figure 8 BNP effect on ANP NPR-1 AQP1 and AQP5 expressionBEAS-2B cells were treated with 001 nM BNP for 24 h Histograms show(a) ANP (b) NPR-1 (c) AQP1 and (d) AQP5 gene expression at mRNA level All histograms indicate means plusmn SD of three different cultureseach one tested in quadruplicate and expressed as percentage of control (lowast119875 lt 005 versus untreated cells)
020406080
100120140160180200
DEX
BNP
( o
f con
trol)
C TNF-120572 TNF-120572 + DEX
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowast
lowast
lowast
Figure 9 Dexamethasone effect on BNP expression BEAS-2B cellswere treatedwith 1 120583Mdexamethasone (DEX) and 40 ngmLTNF-120572alone or in combination with DEX for 24 h Histograms show BNPgene expression at mRNA (gray bars) and protein (black bars) levelAll histograms indicate means plusmn SD of three different cultures eachone tested in quadruplicate and expressed as percentage of control(lowast119875 lt 001 lowastlowast119875 lt 0001 and lowastlowastlowast119875 lt 00001 versus Controls) CControls (untreated cells)
and in vitro models of lung injury [18] and the therapeu-tical relevance of these experimental observations has beenshown in intensive care patients without heart disease whereintravenous ANP infusion diminished pulmonary vascularpermeability and pulmonary edema [18]
Regarding AQPs we demonstrated that BEAS-2B cellsexpress AQP1 and AQP5 and that TNF-120572 directly down-regulates their expression at mRNA and protein levelsRegarding AQP1 also an additional BNP-mediated TNF-120572involvement can be proposed In fact AQP1 down-regulationwas observed with BNP treatment at the dose accumulatedin culture medium after 40 ngmL TNF-120572 treatment Themechanisms that underlie AQPs regulation are of consider-able interest Several AQPs has recently been demonstratedto undergo complex regulation [27] Our results show thatAQP1 and APQ5 are subject to inflammation regulationadding an important piece of information in this contextOur results are in agreement with other studies describinga marked reduction in the expression levels of AQP1 andAQP5 inmouse lung [30] andmouse lung epithelial cells [31]The biological significance of the observed TNF-120572-inducedAQP1 and AQP5 down-regulation is at present unclear Ithas been recently suggested that the decrease in both AQPsmay play an important role in the development of lung edema[52] which is known to be related with fluid retention Wetherefore speculate that TNF-120572-induced AQP1 and AQP5down-regulation might play a role also in the excess of fluidaccumulation related to major surgical procedures
5 Conclusions
In conclusion our results provided evidence that in humanbronchial epithelial cells BEAS-2B TNF-120572 selectively mod-ulates mRNA and protein expression levels of different
12 Mediators of Inflammation
molecules involved in body fluid homeostasis ANP BNPand their receptor NPR-1 AQP1 and AQP5 Moreoverour data pointed out that the changes specifically affectingNPs expression occur independently from hemodynamicinfluences and that inflammation should be considered anextracardiac cause of BNP elevation adding an importantpiece of information in the novel area of study regarding NPsand inflammation In addition we demonstrated that such amodulation occurs via different involvement of NK-120581B andMAPKs signaling pathways Finally we suggested inflamma-tion among the mechanisms involved in the regulation ofAQP1 and AQP5 Therefore in this exploratory study wespeculate that TNF-120572 might be involved in postoperative-fluid retention related to major surgery
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgment
The authors thank Mrs Roberta Frosini for the excellenttechnical assistance
References
[1] E Itobi M Stroud and M Elia ldquoImpact of oedema onrecovery after major abdominal surgery and potential valueof multifrequency bioimpedance measurementsrdquo The BritishJournal of Surgery vol 93 no 3 pp 354ndash361 2006
[2] J A Lowell C Schifferdecker D F Driscoll P N Benottiand B R Bistrian ldquoPostoperative fluid overload not a benignproblemrdquo Critical Care Medicine vol 18 no 7 pp 728ndash7331990
[3] A M Moslashller T Pedersen P-E Svendsen and A EngquistldquoPerioperative risk factors in elective pneumonectomy theimpact of excess fluid balancerdquo European Journal of Anaesthe-siology vol 19 no 1 pp 57ndash62 2002
[4] E Bennett-Guerrero D E Feierman G R Barclay et alldquoPreoperative and intraoperative predictors of postoperativemorbidity poor graft function and early rejection in 190patients undergoing liver transplantationrdquo Archives of Surgeryvol 136 no 10 pp 1177ndash1183 2001
[5] J P Desborough ldquoThe stress response to trauma and surgeryrdquoThe British Journal of Anaesthesia vol 85 no 1 pp 109ndash1172000
[6] H Tsujimoto S Ono T Majima et al ldquoDifferential toll-likereceptor expression after ex vivo lipopolysaccharide exposurein patients with sepsis and following surgical stressrdquo ClinicalImmunology vol 119 no 2 pp 180ndash187 2006
[7] C Evans C Galustian D Kumar et al ldquoImpact of surgeryon immunologic function comparison between minimallyinvasive techniques and conventional laparotomy for surgi-cal resection of colorectal tumorsrdquo The American Journal ofSurgery vol 197 no 2 pp 238ndash245 2009
[8] F Catena L Ansaloni A Avanzolini et al ldquoSystemic cytokineresponse after emergency and elective surgery for colorectalcarcinomardquo International Journal of Colorectal Disease vol 24no 7 pp 803ndash808 2009
[9] R Sarbinowski S Arvidsson M Tylman T Oresland andA Bengtsson ldquoPlasma concentration of procalcitonin andsystemic inflammatory response syndrome after colorectalsurgeryrdquo Acta Anaesthesiologica Scandinavica vol 49 no 2 pp191ndash196 2005
[10] P Sheeran and G M Hall ldquoCytokines in anaesthesiardquo TheBritish Journal of Anaesthesia vol 78 no 2 pp 201ndash219 1997
[11] L Cagini R Capozzi V Tassi et al ldquoFluid and electrolytebalance after major thoracic surgery by bioimpedance andendocrine evaluationrdquo European Journal of Cardio-ThoracicSurgery vol 40 no 2 pp e71ndashe76 2011
[12] T Omland and T Hagve ldquoNatriuretic peptides physiologic andanalytic considerationsrdquo Heart Failure Clinics vol 5 no 4 pp471ndash487 2009
[13] L R Potter A R Yoder D R Flora L K Antos and DM Dickey ldquoNatriuretic peptides their structures receptorsphysiologic functions and therapeutic applicationsrdquo Handbookof Experimental Pharmacology vol 191 pp 341ndash366 2009
[14] T G Flynn M L de Bold and A J de Bold ldquoThe aminoacid sequence of an atrial peptide with potent diuretic andnatriuretic propertiesrdquo Biochemical and Biophysical ResearchCommunications vol 117 no 3 pp 859ndash865 1983
[15] A J de Bold ldquoAtrial natriuretic factor a hormone produced bythe heartrdquo Science vol 230 pp 767ndash770 1985
[16] K Kangawa Y Tawaragi S Oikawa et al ldquoIdentification of rat 120574atrial natriuretic polypeptide and characterization of the cDNAencoding its precursorrdquo Nature vol 312 no 5990 pp 152ndash1551984
[17] T Sudoh K Kangawa N Minamino and H Matsuo ldquoA newnatriuretic peptide in porcine brainrdquo Nature vol 332 no 6159pp 78ndash81 1988
[18] M Kuhn ldquoEndothelial actions of atrial and B-type natriureticpeptidesrdquo The British Journal of Pharmacology vol 166 no 2pp 522ndash531 2012
[19] A J de Bold ldquoCardiac natriuretic peptides gene expression andsecretion in inflammationrdquo Journal of Investigative Medicinevol 57 pp 29ndash32 2009
[20] S P DrsquoSouza M Davis and G F Baxter ldquoAutocrine andparacrine actions of natriuretic peptides in the heartrdquo Pharma-cology andTherapeutics vol 101 no 2 pp 113ndash129 2004
[21] M Brueckmann G Huhle S Lang et al ldquoPrognostic valueof plasma N-terminal pro-brain natriuretic peptide in patientswith severe sepsisrdquoCirculation vol 112 no 4 pp 527ndash534 2005
[22] J Charpentier C E Luyt Y Fulla et al ldquoBrain natriureticpeptide a marker of myocardial dysfunction and prognosisduring severe sepsisrdquo Critical Care Medicine vol 32 no 3 pp660ndash665 2004
[23] R Shor Y Rozenman A Bolshinsky et al ldquoBNP in septicpatients without systolic myocardial dysfunctionrdquo EuropeanJournal of Internal Medicine vol 17 no 8 pp 536ndash540 2006
[24] T Inoue M Kawai T Nakane et al ldquoInfluence of low-gradeinflammation on plasma B-type natriuretic peptide levelsrdquoInternal Medicine vol 49 no 24 pp 2659ndash2668 2010
[25] P Agre L S King M Yasui et al ldquoAquaporin water channelsmdashfrom atomic structure to clinical medicinerdquo Journal of Physiol-ogy vol 542 no 1 pp 3ndash16 2002
[26] P Agre and D Kozono ldquoAquaporin water channels molecularmechanisms for human diseasesrdquo FEBS Letters vol 555 no 1pp 72ndash78 2003
Mediators of Inflammation 13
[27] J M Carbrey and P Agre ldquoDiscovery of the aquaporins anddevelopment of the fieldrdquo Handbook of Experimental Pharma-cology vol 190 pp 3ndash28 2009
[28] O Bloch and G T Manley ldquoThe role of aquaporin-4 in cerebralwater transport and edemardquo Neurosurgical Focus vol 22 no 5article E3 2007
[29] J Li M Xu Q Fan et al ldquoTanshinone IIA ameliorates seawaterexposure-induced lung injury by inhibiting aquaporins (AQP)1 and AQP5 expression in lungrdquo Respiratory Physiology andNeurobiology vol 176 no 1-2 pp 39ndash49 2011
[30] J E Towne K S Harrod C M Krane and A G MenonldquoDecreased expression of aquaporin (AQP)1 and AQP5 inmouse lung after acute viral infectionrdquoThe American Journal ofRespiratory Cell and Molecular Biology vol 22 no 1 pp 34ndash442000
[31] J E Towne C M Krane C J Bachurski and A G MenonldquoTumor necrosis factor-120572 inhibits aquaporin 5 expression inmouse lung epithelial cellsrdquoThe Journal of Biological Chemistryvol 276 no 22 pp 18657ndash18664 2001
[32] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001
[33] K Verhelst I Carpentier and R Beyaert ldquoRegulation of TNF-induced NF-120581B activation by different cytoplasmic ubiquitina-tion eventsrdquo Cytokine and Growth Factor Reviews vol 22 no5-6 pp 277ndash286 2011
[34] L Qian Y Shen J C Chen et al ldquo3D-QSAR and dockingstudies of quinazoline derivatives with the inhibatory activitytoward NF-120581BrdquoQSAR and Combinatorial Science vol 27 no 8pp 984ndash995 2008
[35] P D NrsquoGuessan S Hippenstiel M O Etouem et al ldquoStrepto-coccus pneumoniae induced p38MAPK- andNF-120581B-dependentCOX-2 expression in human lung epitheliumrdquo The AmericanJournal of PhysiologymdashLung Cellular and Molecular Physiologyvol 290 no 6 pp L1131ndashL1138 2006
[36] B Schmeck K Moog J Zahlten et al ldquoStreptococcus pneumo-niae induced c-Jun-N-terminal kinase- andAP-I-dependent IL-8 release by lung epithelial BEAS-2B cellsrdquoRespiratory Researchvol 7 article 98 2006
[37] M GMatera L Calzetta D Passeri et al ldquoEpithelium integrityis crucial for the relaxant activity of brain natriuretic peptide inhuman isolated bronchirdquo The British Journal of Pharmacologyvol 163 no 8 pp 1740ndash1754 2011
[38] R QuirionM Dalpe A de Lean J Gutkowska M Cantin andJ Genest ldquoAtrial natriuretic factor (ANF) binding sites in brainand related structuresrdquoPeptides vol 5 no 6 pp 1167ndash1172 1984
[39] M B Pamnani D L Clough J S Chen W T Link andF J Haddy ldquoEffects of rat atrial extract on sodium transportand blood pressure in the ratrdquo Proceedings of the Society forExperimental Biology and Medicine vol 176 no 2 pp 123ndash1311984
[40] T R Traynor and S M OrsquoGrady ldquoBrain natriuretic peptidestimulates K andCl secretion across porcine distal colon epithe-liumrdquoTheAmerican Journal of PhysiologymdashCell Physiology vol260 no 4 pp C750ndashC755 1991
[41] R Rollın A Mediero M Roldan-Pallares A Fernandez-Cruzand R Fernandez-Durango ldquoNatriuretic peptide system in thehuman retinardquoMolecular Vision vol 10 pp 15ndash22 2004
[42] M Harada H Itoh O Nakagawa et al ldquoSignificance ofventricular myocytes and nonmyocytes interaction during car-diocyte hypertrophy evidence for endothelin-1 as a paracrinehypertrophic factor from cardiac nonmyocytesrdquo Circulationvol 96 no 10 pp 3737ndash3744 1997
[43] K Kuwahara Y Saito M Harada et al ldquoInvolvement ofcardiotrophin-1 in cardiac myocyte-nonmyocyte interactionsduring hypertrophy of rat cardiac myocytes in vitrordquo Circula-tion vol 100 no 10 pp 1116ndash1124 1999
[44] E Harada O Nakagawa M Yoshimura et al ldquoEffect ofinterleukin-1120573 on cardiac hypertrophy and production of natri-uretic peptides in rat cardiocyte culturerdquo Journal of Molecularand Cellular Cardiology vol 31 no 11 pp 1997ndash2006 1999
[45] V Chiurchiu V Izzi F DrsquoAquilio F Carotenuto P Di Nardoand P M Baldini ldquoBrain natriuretic peptide (BNP) regulatesthe production of inflammatory mediators in human THP-1macrophagesrdquo Regulatory Peptides vol 148 no 1ndash3 pp 26ndash322008
[46] N S Holden C F Rider M J Bell et al ldquoEnhancement ofinflammatory mediator release by 1205732-adrenoceptor agonists inairway epithelial cells is reversed by glucocorticoid actionrdquoTheBritish Journal of Pharmacology vol 160 no 2 pp 410ndash4202010
[47] V S Fang C C Juan J G S Won and L T Ho ldquoFurther studyof aldosterone secretion-inhibitory factor and brain natriureticpeptide on cortisol production of Guinea pig zona fasciculatacellsrdquo Chinese Journal of Physiology vol 43 no 4 pp 141ndash1472000
[48] G Hellermann X Kong J Gunnarsdottir et al ldquoMechanismof bronchoprotective effects of a novel natriuretic hormonepeptiderdquo Journal of Allergy and Clinical Immunology vol 113no 1 pp 79ndash85 2004
[49] K K Ma T Ogawa and A J de Bold ldquoSelective upregulationof cardiac brain natriuretic peptide at the transcriptional andtranslational levels by pro-inflammatory cytokines and by con-ditionedmedium derived frommixed lymphocyte reactions viap38 MAP kinaserdquo Journal of Molecular and Cellular Cardiologyvol 36 no 4 pp 505ndash513 2004
[50] T Ogawa J P Veinot M L K de Bold T Georgalis and AJ De Bold ldquoAngiotensin II receptor antagonism reverts theselective cardiac BNP upregulation and secretion observed inmyocarditisrdquo The American Journal of PhysiologymdashHeart andCirculatory Physiology vol 294 no 6 pp H2596ndashH2603 2008
[51] B J Carver E J Plosa A M Stinnett T S Blackwell andL S Prince ldquoInteractions between NF-120581B and SP3 connectinflammatory signaling with reduced FGF-10 expressionrdquo TheJournal of Biological Chemistry vol 288 no 21 pp 15318ndash153252013
[52] C Dong G Wang B Li et al ldquoAnti-asthmatic agents alleviatepulmonary edema by upregulating AQP1 and AQP5 expressionin the lungs of mice with OVA-induced asthmardquo RespiratoryPhysiology and Neurobiology vol 181 no 1 pp 21ndash28 2012
Figure 1 Identification of ANP BNP NPR-1 AQP1 and AQP5transcripts in BEAS-2B cells qReal-Time PCR was performed usinggene specific primers for ANP (lane 2) BNP (lane 3) NPR-1 (lane 4)AQP1 (lane 5) sand AQP5 (lane 6) in BEAS-2B cells Single bands ofthe predicted molecular size for ANP (160 bp) BNP (148 bp) NPR-1(151 bp) AQP1 (98 bp) andAQP5 (180 bp) transcripts were detectedLane M DNA molecular marker
and NPR-1 we used the specific NF-kB inhibitor BAY 11-7082 that diminishes the activation of NF-120581B by preventingphosphorylation of its inhibitory I120581B-120572 protein As shownin Figure 3(a) TNF-120572-induced downregulation of ANP wascompletely reverted by BAY 11-7082 at both mRNA and pro-tein level (Figure 3(b)) suggesting that this response requiresNF-120581B activation pathway Regarding TNF-120572-induced BNPupregulation this was only partially reverted by BAY 11-7082at mRNA level (Figure 3(c)) while was completely revertedat protein level (Figure 3(d)) Conversely TNF-120572-induceddecrement in NPR-1 expression was not affected by BAY 11ndash7082 either at mRNA or protein level (Figures 3(e) and 3(f))
Western blot analysis for the Ser32-phosphorylated I120581B-120572protein proved the biochemistry evidence of the inhibitoryaction of BAY 11-7082 on NF-120581B activity (Figure 3(g)) Tofurther investigate the role of NF-120581B in the modulation ofBNP and NPR-1 in BEAS-2B cells after TNF-120572 treatmentwe employed theNF-120581B-DNA binding inhibitor QNZ whichprevents free NF-120581B from binding to DNA [34] The resultsobtained with such additional inhibitor confirmed the previ-ous findings obtained with BAY 11-7082 In particular TNF-120572-induced BNP up-regulation was not reverted by QNZ atmRNA level (Figure 4(a)) while it was completely reverted atprotein level (Figure 4(b)) Accordingly also TNF-120572-induceddecrement of NPR-1 expression was not affected by QNZeither at mRNA or protein level (Figures 4(c) and 4(d))Western blot analysis for the Ser32-phosphorylated I120581B-120572protein proved the biochemistry evidence of the inhibitoryaction of QNZ on NF-120581B activity (Figure 4(e))
33 BNP and NPR-1 Modulation by TNF-120572 May RequireActivation of MAPKs Signaling Pathway in BEAS-2B CellsBecause TNF-120572 is able to activate multiple signal transduc-tion pathways including MAPKs and because the activationof p38 MAPK and JNK participates in the regulation ofinflammatory processes in bronchial epithelial cells [35 36]we then investigatedMAPKs pathway involvement in TNF-120572modulation of BNP and NPR-1 expression TNF-120572-mediated
up-regulation of BNP was not affected by SB 203580 or SP600125 p38 MAPK and JNK 12 inhibitors respectivelywhile it was completely reverted by the ERK 12 inhibitorU-0126 (Figures 5(a) and 5(b)) suggesting that ERK 12activation pathway is required for BNP TNF-120572-mediatedmodulation Regarding NPR-1 the TNF-120572-mediated down-regulation was completely reverted by the use of all inhibitors(Figures 5(c) and 5(d)) Western blot analysis for theThr180Thr182-phosphorylated form of p-38 MAPK provedthe biochemistry evidence of the inhibitory action of SB203580 on p38 MAPK activity (Figure 5(e))
34 TNF-120572 Decreases AQP1 and AQP5 Expression in BEAS2B Cells We found that TNF-120572 induced a dose dependentdown-regulation of AQP1 expression (119875 lt 001) at mRNA(Figure 6(a)) and protein level (Figure 6(b)) and a dramaticdecrease in AQP5 gene expression level (about 80 ofcontrol) with all the used doses (119875 lt 0001) (Figure 6(c)) par-alleled by a comparable trend at protein level (Figure 6(d))
35 Decreased AQP1 andAQP5 Expression by TNF-120572DoesNotRequire Activation of NF-120581B Signaling Pathway in BEAS-2BCells To examine a possible involvement of NF-120581B in TNF-120572-mediated down-regulation of AQP1 and AQP5 BEAS-2Bcells were incubated with BAY 11-7082 Cotreatment of suchNF-120581B inhibitor with TNF-120572 resulted in a decrease of AQP1expression which was similar to that observed with TNF-120572alone (Figures 7(a) and 7(b)) suggesting that the observedresponse does not require NF-120581B activation Conversely co-treatment of BAY 11-7082 with TNF-120572 affected AQP5 only atprotein level (Figures 7(c) and 7(d))
36 BNP Effect on ANP NPR-1 and AQP1 or AQP5 mRNAExpression in BEAS-2BCells In order to determine a possibledirect involvement of BNP on the gene expression of ANPNPR-1 and AQP1 or AQP5 BEAS-2B cells were treated with001 nM BNP This concentration was previously found tobe accumulated in culture medium after 40 ngmL TNF-120572treatment the dose that induced the maximum effect on theexpression of the studied genes BNP administration induceda significant (119875 lt 005) increase (100 of control) on ANPmRNA level (Figure 8(a)) and did not affectNPR-1 expression(Figure 8(b)) while induced a marked decrease (45 ofcontrol) on AQP1 mRNA level (119875 lt 005) (Figure 8(c))A trend in decreasing was also observed for AQP5 geneexpression even though not statistically significant (119875 gt005) (Figure 8(d))
37 Dexamethasone (DEX) Effect on BNP mRNA and ProteinLevels in BEAS-2B Cells In order to study the capability ofthe anti-inflammatory drug DEX to affect TNF-120572-mediatedup-regulation of BNP mRNA and protein levels BEAS-2Bcells were treated with 1120583MDEX As shown in Figure 9 DEXinduced a down-regulation of BNP expression and proteinlevels alone or in combination with 40 ngmL TNF-120572 (119875 lt001) demonstrating the capability of this glucocorticoid torevert TNF-120572 effects
Mediators of Inflammation 5
0 10 20 40TNF-120572 (ngmL)
AN
P (
of c
ontro
l)
160
140
120
100
80
60
40
20
0
lowast
lowastlowastlowastlowast
(a)
0 10 20 40
ANP
Actin TNF-120572 (ngmL)
(b)
0
50
100
150
200
0 10 20 40
BNP
( o
f con
trol)
TNF-120572 (ngmL)
lowastlowastlowastlowastlowastlowast
lowast
(c)
0
50
100
150
200
0 10 20 40BN
P (
of c
ontro
l)
TNF-120572 (ngmL)
lowastlowast
lowastlowast
lowastlowast
(d)
0 10 20 40TNF-120572 (ngmL)
120
100
80
60
40
20
0
NPR
-1 (
of c
ontro
l)
lowastlowast
lowastlowastlowastlowastlowastlowastlowastlowast
(e)
0 10 20 40
NPR-1
Actin TNF-120572 (ngmL)
(f)
Figure 2 TNF-120572 selectively modulates ANP BNP and NPR-1 expression BEAS-2B cells were treated with 10 20 and 40 ngmL TNF-120572 for24 h ((a) (b)) ANP ((c) (d)) BNP and ((e) (f)) NPR-1 gene expression at mRNA ((a) (c) and (e)) and protein level ((b) (d) and (f))Western blots were obtained by using the specific rabbit polyclonal Abs The blots were stripped of the bound Ab and reprobed with mouseanti-120573-actin to confirm equal loading Western blots are representative of three separate experiments All histograms indicate meansplusmn SD ofthree different cultures each one tested in quadruplicate and expressed as percentage of control (lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 andlowastlowastlowastlowast
119875 lt 00001 versus untreated cells)
4 Discussion
Postoperative-fluid retention is a severe complication fre-quently reported in patients undergoing major surgical pro-cedures [1ndash4] The complex network of molecules involvedin such a severe surgery-induced condition remains poorlyunderstood After major surgery TNF-120572 is one of the mainproinflammatory cytokines initially released in the damagedtissue where it stimulates the production and release of morecytokines responsible for inducing the systemic changesknown as the acute phase response [5 10] Therefore it isreasonable to assume TNF-120572 involvement among the variouscauses of fluid retention and very important to understandthemechanisms underlying its involvement in such an ambit
In the present study we demonstrated for the first time toour knowledge that TNF-120572modulates the expression ofANPBNP NPR-1 AQP1 and AQP5 in human bronchial epithelialcells BEAS-2B via different involvement of NF-120581B andMAPKs signaling pathway activation Here we also providedthe first demonstration that human bronchial epithelial cellsexpress ANP BNP NPR-1 and AQP1 and that BNP is able tomodulate AQP1 expression
The observed expression of NPs and NPR-1 suggestsan autocrine andor paracrine function for these moleculesand indicates important roles for NPs in several biologicalfunctions including regulation of fluid movement across theairway epithelial cells bronchial relaxation [37] vasodilata-tion and pulmonary vascular permeability About the former
6 Mediators of Inflammation
020406080
100120140160
C BAY 10 20 40
AN
P (
of c
ontro
l)
TNF-120572 (ngmL)
TNF-120572TNF-120572 + BAY
lowast
lowastlowastlowastlowast
(a)
ANP
Actin 40
BAY 0 0 10 20 40 10 20minus + +minus minus minus + +
Figure 3 TNF-120572 modulation of NPs and NPR-1 may require activation of NF-120581B pathway BEAS-2B cells were treated with 10 20 and40 ngmL TNF-120572 alone or in combination with NF-120581B inhibitor BAY 11-7082 (1120583M) for 24 h ((a) (b)) ANP ((c) (d)) BNP ((e) (f)) NPR-1gene expression at mRNA ((a) (c) and (e)) and protein level ((b) (d) (f)) and (g) Phospho-I120581B-120572 protein level Western blots were obtainedby using the specific rabbit polyclonal or monoclonal AbsThe blots were stripped of the bound Ab and reprobed with mouse anti-120573-actin toconfirm equal loading Western blots are representative of three separate experiments All histograms indicate means plusmn SD of three differentcultures each of one tested in quadruplicate and expressed as percentage of control (lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowastlowast119875 lt 00001 versus Controls)(∘119875 lt 005 ∘∘119875 lt 001 and ∘∘∘∘119875 lt 00001 versus BAY) C Controls (untreated cells)
process the role of ANP and BNP could be analogous to thathypothesized forNPs in transepithelial ion flux in the choroidplexus [38] in the kidney [39] colon [40] and retina [41]
The observed TNF-120572 capability of selectively regulatingANP BNP and NPR-1 gene expression in BEAS-2B cellline is of particular interest because it demonstrates that
inflammation alters NPs expression levels In particular thesignificant up-regulation of BNP expression and proteinsecretion after TNF-120572 treatment shows that this modulationcan occur independently of hemodynamic influences andthat inflammation should be considered an extracardiaccause of BNP elevation Besides in vitro studies conducted in
Mediators of Inflammation 7
020406080
100120140160180200
C QNZ TNF
BNP
( o
f con
trol)
QNZ + TNF
lowastlowast
∘∘
(a)
0
20
40
60
80
100
120
140
160
180
BNP
( o
f con
trol)
C QNZ TNF QNZ + TNF
lowastlowast
(b)
0
20
40
60
80
100
120
C QNZ TNF QNZ + TNF
∘∘
lowastlowastlowastlowast
lowastlowastlowastlowast
NPR
-1(
of c
ontro
l)
(c)
NPR-1 Actin
0 0QNZ
10 10TNF-120572+minus +minus
(d)
Actin
QNZ TNF-120572 + +
minus ++
minusminus
minus
P-I120581B-120572
(e)
Figure 4 TNF-120572 modulation of protein levels of BNP and NPR-1 may require activation of NF-120581B pathway BEAS-2B cells were treated10 ngmL TNF-120572 alone or in combination with the inhibitor QNZ (10120583M) for 24 h ((a) (b)) BNP ((c) (d)) NPR-1 gene expression at mRNA((a) (c)) and protein ((b) (d)) level and (e) Phospho-I120581B-120572 protein level Western blots were obtained by using the specific rabbit polyclonalor monoclonal AbsThe blots were stripped of the bound Ab and reprobed with mouse anti-120573-actin to confirm equal loading Western blotsare representative of three separate experiments All histograms indicatemeansplusmnSDof three different cultures each one tested in quadruplicateand expressed as percentage of control (lowastlowast119875 lt 001 and lowastlowastlowastlowast119875 lt 00001 versus Controls) (∘∘119875 lt 001 versus QNZ) C Controls (untreatedcells)
rat cardiomyocytes have shown that not only hemodynamicfactors but also neurohumoral factors activated during heartfailure such as angiotensin II endothelin and cytokinescause BNP secretion [42ndash44] In addition in patients withcardiovascular diseases plasma BNP levels have been shownto be also affected by low-grade inflammation [24] and aselective increase inBNPplasma levels has been proposed as ageneral feature of inflammation [19]The lack on any apparentTNF-120572 dose-depending increase in BNP gene expression thatwe observed in our system could be due to an autocrinenegative feedback action mediated by BNP itself followingbinding to the NPR-1 receptor expressed in BEAS-2B cells
The biological role of BNPup-regulation after TNF-120572 treatment remains to be elucidated However since it
has been demonstrated that BNP regulates the productionof major inflammatory molecules such as IL-12 IL-10leukotriene B and prostaglandin E2 in human macrophages[45] and that BEAS-2B are able to release inflammatorymolecules [46] it could be reasonable to assume that theobserved BNP increase after TNF-120572 treatment may stimu-late the production and release of such cytokines also fromhuman epithelial bronchial cells In addition in in vitrostudies a potent BNP inhibitory action on the productionof the glucocorticoid anti-inflammatory hormone cortisolhas been described [47] Therefore BNP could be con-sidered a proinflammatory molecule by inducing cytokinesexpression in target cells and inhibiting the production ofanti-inflammatory hormones Therefore the observed BNP
8 Mediators of Inflammation
0
50
100
150
200
250BN
P (
of c
ontro
l)
C
TNF SB
SB+
TNF SP
SP+
TNF
U-0
126
U-0126+
TNF
lowastlowast
lowastlowast
∘∘
sect
(a)
020406080
100120140160180
BNP
( o
f con
trol)
C
TNF SB
SB+
TNF SP
SP+
TNF
U-0
126
U-0126+
TNF
lowastlowast
lowastlowast
∘∘∘
sectsect
(b)
0
20
40
60
80
100
120
140
160
C
TNF SB
SB+
TNF SP
SP+
TNF
U-0
126
U-0126+
TNF
lowastlowastlowast
lowastlowastlowast
lowast
lowast
sect
NPR
-1(
of c
ontro
l)
(c)
+
SB 203580
+
SP 600125 U-0126
+ActinTNF-120572
NPR-1
minus minus minus minus +
(d)
SB 203580
Actin
P-p38 MAPK
TNF-120572 + +
minus ++
minusminus
minus
(e)
Figure 5 TNF-120572 modulation of BNP and NPR-1 expression may require activation of MAPKs pathway BEAS-2B cells were treated with10 ngmL TNF-120572 alone or in combination with 10 120583M SB 203580 (p38 MAPK inhibitor) SP 600125 (JNK 12 inhibitor) or U-0126 (ERK12 inhibitor) for 24 h Panels show ((a) (b)) BNP ((c) (d)) NPR-1 gene expression at mRNA ((a) (c)) and protein ((b) (d)) level and (e)Phospho-p38 MAPK protein level Western blots were obtained by using the specific rabbit polyclonal or monoclonal Abs The blots werestripped of the bound Ab and reprobed with mouse anti-120573-actin to confirm equal loading Western blots are representative of three separateexperiments All histograms indicate means plusmn SD of three different cultures each one tested in quadruplicate and expressed as percentage ofcontrol (lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 versus Controls) (∙∙119875 lt 001 versus SB) (sect119875 lt 005 sectsect
119875 lt 001 versus SP) (∘∘119875 lt 001∘∘∘
119875 lt 0001 versus U-0126) C Controls (untreated cells)
up-regulation after TNF-120572 treatment could concur in poten-tiating an inflammatory statusThe increased production andrelease of specific cytokines causing systemic changes knownas the acute phase response [5 8 10] induced by TNF-120572 maythus also occur via BNP up-regulation
Recently a BNP rise has been observed in patientsundergoing pulmonary lobectomy for lung cancer [11] Inparticular the patients none of whom developed signs orsymptoms of heart failure during the postoperative period
showed an early significant increase on day 1 immediatelyafter surgery of the plasma concentrations of BNP whichwas followed on the postoperative day 2 by a significantweight gain correlated with large volumes of fluids accumu-lation despite a negative intraoperative fluid balance andperoperative strict fluid restriction [11] We suggest thatsuch an increase could be at least in part explained bythe ability of proinflammatory cytokines such as TNF-120572 toupregulate BNP gene expression and secretion and that this
Mediators of Inflammation 9
0
20
40
60
80
100
120
0 10 20 40TNF-120572 (ngmL)
AQP1
( o
f con
trol)
lowast
lowastlowastlowast
lowastlowastlowast
(a)
Actin 0 10 20 40
AQP1
TNF-120572 (ngmL)
(b)
0
20
40
60
80
100
120
AQP5
( o
f con
trol)
0 10 20 40
TNF-120572 (ngmL)
lowastlowast
lowastlowastlowastlowast
(c)
AQP5
Actin 0 10 20 40TNF-120572 (ngmL)
(d)
Figure 6 TNF-120572 decreases AQP1 and AQP5 expression in BEAS 2B cells BEAS-2B cells were treated with 10 20 and 40 ngmL TNF-120572 for24 h Gene expression of AQP1 ((a) (b)) and AQP5 ((c) (d)) at mRNA ((a) (c)) and protein ((b) (d)) level Western blots were obtained byusing the specific rabbit polyclonal Abs The blots were stripped of the bound Ab and reprobed with mouse anti-120573-actin to confirm equalloading Western blots are representative of three separate experiments All histograms indicate means plusmn SD of three different cultures eachone tested in quadruplicate and expressed as percentage of control (lowast119875 lt 001 lowastlowast119875 lt 0001 and lowastlowastlowast119875 lt 00001 versus untreated cells)
up-regulation could enhance the major surgery-related earlyhyperinflammatory response essential for tissue repair andhost defense [5]
The observed dexamethasone capability to completelyrevert TNF-120572-induced BNP up-regulation suggests that thisanti-inflammatory glucocorticoid may prevent BNP-relatedeffects
Regarding ANP the observed significant down-regula-tion of mRNA and protein levels after 10 20 ngmL TNF-120572treatment suggests that also this condition could affect localinflammation status In fact ANP administration determinesanti-inflammatory effects in airway epithelial cells [48] play-ing important roles in modulating inflammatory responseIn consideration of such ANP anti-inflammatory action wespeculate that the observed TNF-120572-induced ANP down-regulation could concur in enhancing an inflammatory sta-tus Differently from the above reported doses 40 ngmLTNF-120572 administration induced ANP up-regulation eventhough not statistically significant We suggest that this resultmight be due to a direct BNP involvement In fact a markedANP up-regulation was observed after BNP treatment at the
dose accumulated in culture medium after 40 ngmL TNF-120572treatment
The observed selectivemodulation inANP and BNP geneexpression after TNF-120572 treatment in line with other studiesin different cell models [19 49 50] could be related to adifferent regulation mechanism at transcriptional level assuggested by our observation on the different involvementof NF-120581B and MAPKs signaling pathways In particular wedemonstrated that ANP down-regulation TNF-120572-mediatedoccurs via NF-120581B activation pathway whereas BNP modula-tion requires ERK 12 activation pathway NF-120581B involvementin TNF-120572-induced ANP down-regulation is not opposed tothe proinflammatory role of this transcription factor beingANP a molecule that exerts an anti-inflammatory effect inairway epithelial cells [48] Regarding themechanism linkingNF-120581B to the downregulation of ANP gene expression wesuggest that it could be similar to that recently proposedfor Fibroblast Growth Factor 10 (FGF-10) where NF-120581Bactivationmay lead to reduced gene expression by recruitinginhibitory factors to specific gene promoters [51]
10 Mediators of Inflammation
0
20
40
60
80
100
120
C BAY 10 20 40
AQP1
( o
f con
trol)
TNF-120572 (ngmL)
TNF-120572TNF-120572 + BAY
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowast
(a)
BAY
AQP1Actin
0 0 10 20 40 10 20 40TNF-120572 (ngmL)minus + +minus minus minus + +
(b)
0
20
40
60
80
100
120
AQP5
( o
f con
trol)
C BAY 10 20 40
TNF-120572 (ngmL)
TNF-120572TNF-120572 + BAY
∘∘∘∘∘∘
∘∘∘
lowastlowastlowastlowast
lowastlowast
(c)
AQP5Actin
0 0 10 20 40 10 20 40TNF-120572 (ngmL)BAY minus + +minus minus minus + +
(d)
Figure 7 TNF-120572 downregulation of AQP1 and AQP5 expression may require activation of NF-120581B pathwayBEAS-2B cells were treated with10 20 and 40 ngmL TNF-120572 alone or in combination with NF-120581B inhibitor BAY 11-7082 (1 120583M) for 24 h Gene expression of AQP1 ((a) (b))and AQP5 ((c) (d)) at mRNA ((a) (c)) and protein ((b) (d)) level Western blots were obtained by using the specific rabbit polyclonal AbsThe blots were stripped of the bound Ab and reprobed with mouse anti-120573-actin to confirm equal loading Western blots are representative ofthree separate experiments All histograms indicate means plusmn SD of three different cultures each one tested in quadruplicate and expressed aspercentage of control (lowast119875 lt 001 lowastlowast119875 lt 0001 lowastlowastlowast119875 lt 00001 versus Controls and ∘∘∘119875 lt 00001 versus BAY) C Controls (untreated cells)
ANP and BNP biological functions occur through theirbinding to the same NPR-1 [13] Here we demonstratedfor the first time to our knowledge TNF-120572 capability todownregulateNPR-1mRNAandprotein expression in BEAS-2B cells and that this regulation occurs via MAPKs signalingpathway activation The consequence of the complex TNF-120572-induced NPs and NPR-1 observed modulation associatedwith their binding to the same receptor on BEAS-2B cellsmakes it difficult to predict what would be the net resultingeffect both in vitro and in vivo in relation also to the presenceof a possible autocrine regulation by NPs andor to the effectsof different cytokines activated by both BNP and TNF-120572We hypothesize that the TNF-120572-induced downregulation of
NPR-1 paralleled by an up-regulation of BNP could alsolead to a paracrine action of this peptide possibly on themicrovascular endothelium of the lung where NPR-1 isdensely expressed [18] In this context we speculate that theinduction of local modification on BNP or ANP by TNF-120572 inhuman bronchial epithelial cells could alter the barrier per-meability of the pulmonary microcirculation Hence TNF-120572 acting on epithelial cells could also indirectly contributeto endothelial cells barrier dysfunction and permeability byenhancing the local production of proinflammatory BNPand by down-regulating the anti-inflammatory expressionof ANP Besides exogenous synthetic ANP has been shownto protect from endothelial barrier dysfunction in in vivo
Mediators of Inflammation 11
0
50
100
150
200
250
300
0 001
AN
P (
of c
ontro
l)
BNP (nM)
lowast
(a)
0
50
100
150
0 001BNP (nM)
NPR
-1(
of c
ontro
l)
(b)
0
20
40
60
80
100
120
0 001
BNP (nM)
lowast
AQP1
( o
f con
trol)
(c)
0
20
40
60
80
100
120
0 001
BNP (nM)
AQP5
( o
f con
trol)
(d)
Figure 8 BNP effect on ANP NPR-1 AQP1 and AQP5 expressionBEAS-2B cells were treated with 001 nM BNP for 24 h Histograms show(a) ANP (b) NPR-1 (c) AQP1 and (d) AQP5 gene expression at mRNA level All histograms indicate means plusmn SD of three different cultureseach one tested in quadruplicate and expressed as percentage of control (lowast119875 lt 005 versus untreated cells)
020406080
100120140160180200
DEX
BNP
( o
f con
trol)
C TNF-120572 TNF-120572 + DEX
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowast
lowast
lowast
Figure 9 Dexamethasone effect on BNP expression BEAS-2B cellswere treatedwith 1 120583Mdexamethasone (DEX) and 40 ngmLTNF-120572alone or in combination with DEX for 24 h Histograms show BNPgene expression at mRNA (gray bars) and protein (black bars) levelAll histograms indicate means plusmn SD of three different cultures eachone tested in quadruplicate and expressed as percentage of control(lowast119875 lt 001 lowastlowast119875 lt 0001 and lowastlowastlowast119875 lt 00001 versus Controls) CControls (untreated cells)
and in vitro models of lung injury [18] and the therapeu-tical relevance of these experimental observations has beenshown in intensive care patients without heart disease whereintravenous ANP infusion diminished pulmonary vascularpermeability and pulmonary edema [18]
Regarding AQPs we demonstrated that BEAS-2B cellsexpress AQP1 and AQP5 and that TNF-120572 directly down-regulates their expression at mRNA and protein levelsRegarding AQP1 also an additional BNP-mediated TNF-120572involvement can be proposed In fact AQP1 down-regulationwas observed with BNP treatment at the dose accumulatedin culture medium after 40 ngmL TNF-120572 treatment Themechanisms that underlie AQPs regulation are of consider-able interest Several AQPs has recently been demonstratedto undergo complex regulation [27] Our results show thatAQP1 and APQ5 are subject to inflammation regulationadding an important piece of information in this contextOur results are in agreement with other studies describinga marked reduction in the expression levels of AQP1 andAQP5 inmouse lung [30] andmouse lung epithelial cells [31]The biological significance of the observed TNF-120572-inducedAQP1 and AQP5 down-regulation is at present unclear Ithas been recently suggested that the decrease in both AQPsmay play an important role in the development of lung edema[52] which is known to be related with fluid retention Wetherefore speculate that TNF-120572-induced AQP1 and AQP5down-regulation might play a role also in the excess of fluidaccumulation related to major surgical procedures
5 Conclusions
In conclusion our results provided evidence that in humanbronchial epithelial cells BEAS-2B TNF-120572 selectively mod-ulates mRNA and protein expression levels of different
12 Mediators of Inflammation
molecules involved in body fluid homeostasis ANP BNPand their receptor NPR-1 AQP1 and AQP5 Moreoverour data pointed out that the changes specifically affectingNPs expression occur independently from hemodynamicinfluences and that inflammation should be considered anextracardiac cause of BNP elevation adding an importantpiece of information in the novel area of study regarding NPsand inflammation In addition we demonstrated that such amodulation occurs via different involvement of NK-120581B andMAPKs signaling pathways Finally we suggested inflamma-tion among the mechanisms involved in the regulation ofAQP1 and AQP5 Therefore in this exploratory study wespeculate that TNF-120572 might be involved in postoperative-fluid retention related to major surgery
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgment
The authors thank Mrs Roberta Frosini for the excellenttechnical assistance
References
[1] E Itobi M Stroud and M Elia ldquoImpact of oedema onrecovery after major abdominal surgery and potential valueof multifrequency bioimpedance measurementsrdquo The BritishJournal of Surgery vol 93 no 3 pp 354ndash361 2006
[2] J A Lowell C Schifferdecker D F Driscoll P N Benottiand B R Bistrian ldquoPostoperative fluid overload not a benignproblemrdquo Critical Care Medicine vol 18 no 7 pp 728ndash7331990
[3] A M Moslashller T Pedersen P-E Svendsen and A EngquistldquoPerioperative risk factors in elective pneumonectomy theimpact of excess fluid balancerdquo European Journal of Anaesthe-siology vol 19 no 1 pp 57ndash62 2002
[4] E Bennett-Guerrero D E Feierman G R Barclay et alldquoPreoperative and intraoperative predictors of postoperativemorbidity poor graft function and early rejection in 190patients undergoing liver transplantationrdquo Archives of Surgeryvol 136 no 10 pp 1177ndash1183 2001
[5] J P Desborough ldquoThe stress response to trauma and surgeryrdquoThe British Journal of Anaesthesia vol 85 no 1 pp 109ndash1172000
[6] H Tsujimoto S Ono T Majima et al ldquoDifferential toll-likereceptor expression after ex vivo lipopolysaccharide exposurein patients with sepsis and following surgical stressrdquo ClinicalImmunology vol 119 no 2 pp 180ndash187 2006
[7] C Evans C Galustian D Kumar et al ldquoImpact of surgeryon immunologic function comparison between minimallyinvasive techniques and conventional laparotomy for surgi-cal resection of colorectal tumorsrdquo The American Journal ofSurgery vol 197 no 2 pp 238ndash245 2009
[8] F Catena L Ansaloni A Avanzolini et al ldquoSystemic cytokineresponse after emergency and elective surgery for colorectalcarcinomardquo International Journal of Colorectal Disease vol 24no 7 pp 803ndash808 2009
[9] R Sarbinowski S Arvidsson M Tylman T Oresland andA Bengtsson ldquoPlasma concentration of procalcitonin andsystemic inflammatory response syndrome after colorectalsurgeryrdquo Acta Anaesthesiologica Scandinavica vol 49 no 2 pp191ndash196 2005
[10] P Sheeran and G M Hall ldquoCytokines in anaesthesiardquo TheBritish Journal of Anaesthesia vol 78 no 2 pp 201ndash219 1997
[11] L Cagini R Capozzi V Tassi et al ldquoFluid and electrolytebalance after major thoracic surgery by bioimpedance andendocrine evaluationrdquo European Journal of Cardio-ThoracicSurgery vol 40 no 2 pp e71ndashe76 2011
[12] T Omland and T Hagve ldquoNatriuretic peptides physiologic andanalytic considerationsrdquo Heart Failure Clinics vol 5 no 4 pp471ndash487 2009
[13] L R Potter A R Yoder D R Flora L K Antos and DM Dickey ldquoNatriuretic peptides their structures receptorsphysiologic functions and therapeutic applicationsrdquo Handbookof Experimental Pharmacology vol 191 pp 341ndash366 2009
[14] T G Flynn M L de Bold and A J de Bold ldquoThe aminoacid sequence of an atrial peptide with potent diuretic andnatriuretic propertiesrdquo Biochemical and Biophysical ResearchCommunications vol 117 no 3 pp 859ndash865 1983
[15] A J de Bold ldquoAtrial natriuretic factor a hormone produced bythe heartrdquo Science vol 230 pp 767ndash770 1985
[16] K Kangawa Y Tawaragi S Oikawa et al ldquoIdentification of rat 120574atrial natriuretic polypeptide and characterization of the cDNAencoding its precursorrdquo Nature vol 312 no 5990 pp 152ndash1551984
[17] T Sudoh K Kangawa N Minamino and H Matsuo ldquoA newnatriuretic peptide in porcine brainrdquo Nature vol 332 no 6159pp 78ndash81 1988
[18] M Kuhn ldquoEndothelial actions of atrial and B-type natriureticpeptidesrdquo The British Journal of Pharmacology vol 166 no 2pp 522ndash531 2012
[19] A J de Bold ldquoCardiac natriuretic peptides gene expression andsecretion in inflammationrdquo Journal of Investigative Medicinevol 57 pp 29ndash32 2009
[20] S P DrsquoSouza M Davis and G F Baxter ldquoAutocrine andparacrine actions of natriuretic peptides in the heartrdquo Pharma-cology andTherapeutics vol 101 no 2 pp 113ndash129 2004
[21] M Brueckmann G Huhle S Lang et al ldquoPrognostic valueof plasma N-terminal pro-brain natriuretic peptide in patientswith severe sepsisrdquoCirculation vol 112 no 4 pp 527ndash534 2005
[22] J Charpentier C E Luyt Y Fulla et al ldquoBrain natriureticpeptide a marker of myocardial dysfunction and prognosisduring severe sepsisrdquo Critical Care Medicine vol 32 no 3 pp660ndash665 2004
[23] R Shor Y Rozenman A Bolshinsky et al ldquoBNP in septicpatients without systolic myocardial dysfunctionrdquo EuropeanJournal of Internal Medicine vol 17 no 8 pp 536ndash540 2006
[24] T Inoue M Kawai T Nakane et al ldquoInfluence of low-gradeinflammation on plasma B-type natriuretic peptide levelsrdquoInternal Medicine vol 49 no 24 pp 2659ndash2668 2010
[25] P Agre L S King M Yasui et al ldquoAquaporin water channelsmdashfrom atomic structure to clinical medicinerdquo Journal of Physiol-ogy vol 542 no 1 pp 3ndash16 2002
[26] P Agre and D Kozono ldquoAquaporin water channels molecularmechanisms for human diseasesrdquo FEBS Letters vol 555 no 1pp 72ndash78 2003
Mediators of Inflammation 13
[27] J M Carbrey and P Agre ldquoDiscovery of the aquaporins anddevelopment of the fieldrdquo Handbook of Experimental Pharma-cology vol 190 pp 3ndash28 2009
[28] O Bloch and G T Manley ldquoThe role of aquaporin-4 in cerebralwater transport and edemardquo Neurosurgical Focus vol 22 no 5article E3 2007
[29] J Li M Xu Q Fan et al ldquoTanshinone IIA ameliorates seawaterexposure-induced lung injury by inhibiting aquaporins (AQP)1 and AQP5 expression in lungrdquo Respiratory Physiology andNeurobiology vol 176 no 1-2 pp 39ndash49 2011
[30] J E Towne K S Harrod C M Krane and A G MenonldquoDecreased expression of aquaporin (AQP)1 and AQP5 inmouse lung after acute viral infectionrdquoThe American Journal ofRespiratory Cell and Molecular Biology vol 22 no 1 pp 34ndash442000
[31] J E Towne C M Krane C J Bachurski and A G MenonldquoTumor necrosis factor-120572 inhibits aquaporin 5 expression inmouse lung epithelial cellsrdquoThe Journal of Biological Chemistryvol 276 no 22 pp 18657ndash18664 2001
[32] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001
[33] K Verhelst I Carpentier and R Beyaert ldquoRegulation of TNF-induced NF-120581B activation by different cytoplasmic ubiquitina-tion eventsrdquo Cytokine and Growth Factor Reviews vol 22 no5-6 pp 277ndash286 2011
[34] L Qian Y Shen J C Chen et al ldquo3D-QSAR and dockingstudies of quinazoline derivatives with the inhibatory activitytoward NF-120581BrdquoQSAR and Combinatorial Science vol 27 no 8pp 984ndash995 2008
[35] P D NrsquoGuessan S Hippenstiel M O Etouem et al ldquoStrepto-coccus pneumoniae induced p38MAPK- andNF-120581B-dependentCOX-2 expression in human lung epitheliumrdquo The AmericanJournal of PhysiologymdashLung Cellular and Molecular Physiologyvol 290 no 6 pp L1131ndashL1138 2006
[36] B Schmeck K Moog J Zahlten et al ldquoStreptococcus pneumo-niae induced c-Jun-N-terminal kinase- andAP-I-dependent IL-8 release by lung epithelial BEAS-2B cellsrdquoRespiratory Researchvol 7 article 98 2006
[37] M GMatera L Calzetta D Passeri et al ldquoEpithelium integrityis crucial for the relaxant activity of brain natriuretic peptide inhuman isolated bronchirdquo The British Journal of Pharmacologyvol 163 no 8 pp 1740ndash1754 2011
[38] R QuirionM Dalpe A de Lean J Gutkowska M Cantin andJ Genest ldquoAtrial natriuretic factor (ANF) binding sites in brainand related structuresrdquoPeptides vol 5 no 6 pp 1167ndash1172 1984
[39] M B Pamnani D L Clough J S Chen W T Link andF J Haddy ldquoEffects of rat atrial extract on sodium transportand blood pressure in the ratrdquo Proceedings of the Society forExperimental Biology and Medicine vol 176 no 2 pp 123ndash1311984
[40] T R Traynor and S M OrsquoGrady ldquoBrain natriuretic peptidestimulates K andCl secretion across porcine distal colon epithe-liumrdquoTheAmerican Journal of PhysiologymdashCell Physiology vol260 no 4 pp C750ndashC755 1991
[41] R Rollın A Mediero M Roldan-Pallares A Fernandez-Cruzand R Fernandez-Durango ldquoNatriuretic peptide system in thehuman retinardquoMolecular Vision vol 10 pp 15ndash22 2004
[42] M Harada H Itoh O Nakagawa et al ldquoSignificance ofventricular myocytes and nonmyocytes interaction during car-diocyte hypertrophy evidence for endothelin-1 as a paracrinehypertrophic factor from cardiac nonmyocytesrdquo Circulationvol 96 no 10 pp 3737ndash3744 1997
[43] K Kuwahara Y Saito M Harada et al ldquoInvolvement ofcardiotrophin-1 in cardiac myocyte-nonmyocyte interactionsduring hypertrophy of rat cardiac myocytes in vitrordquo Circula-tion vol 100 no 10 pp 1116ndash1124 1999
[44] E Harada O Nakagawa M Yoshimura et al ldquoEffect ofinterleukin-1120573 on cardiac hypertrophy and production of natri-uretic peptides in rat cardiocyte culturerdquo Journal of Molecularand Cellular Cardiology vol 31 no 11 pp 1997ndash2006 1999
[45] V Chiurchiu V Izzi F DrsquoAquilio F Carotenuto P Di Nardoand P M Baldini ldquoBrain natriuretic peptide (BNP) regulatesthe production of inflammatory mediators in human THP-1macrophagesrdquo Regulatory Peptides vol 148 no 1ndash3 pp 26ndash322008
[46] N S Holden C F Rider M J Bell et al ldquoEnhancement ofinflammatory mediator release by 1205732-adrenoceptor agonists inairway epithelial cells is reversed by glucocorticoid actionrdquoTheBritish Journal of Pharmacology vol 160 no 2 pp 410ndash4202010
[47] V S Fang C C Juan J G S Won and L T Ho ldquoFurther studyof aldosterone secretion-inhibitory factor and brain natriureticpeptide on cortisol production of Guinea pig zona fasciculatacellsrdquo Chinese Journal of Physiology vol 43 no 4 pp 141ndash1472000
[48] G Hellermann X Kong J Gunnarsdottir et al ldquoMechanismof bronchoprotective effects of a novel natriuretic hormonepeptiderdquo Journal of Allergy and Clinical Immunology vol 113no 1 pp 79ndash85 2004
[49] K K Ma T Ogawa and A J de Bold ldquoSelective upregulationof cardiac brain natriuretic peptide at the transcriptional andtranslational levels by pro-inflammatory cytokines and by con-ditionedmedium derived frommixed lymphocyte reactions viap38 MAP kinaserdquo Journal of Molecular and Cellular Cardiologyvol 36 no 4 pp 505ndash513 2004
[50] T Ogawa J P Veinot M L K de Bold T Georgalis and AJ De Bold ldquoAngiotensin II receptor antagonism reverts theselective cardiac BNP upregulation and secretion observed inmyocarditisrdquo The American Journal of PhysiologymdashHeart andCirculatory Physiology vol 294 no 6 pp H2596ndashH2603 2008
[51] B J Carver E J Plosa A M Stinnett T S Blackwell andL S Prince ldquoInteractions between NF-120581B and SP3 connectinflammatory signaling with reduced FGF-10 expressionrdquo TheJournal of Biological Chemistry vol 288 no 21 pp 15318ndash153252013
[52] C Dong G Wang B Li et al ldquoAnti-asthmatic agents alleviatepulmonary edema by upregulating AQP1 and AQP5 expressionin the lungs of mice with OVA-induced asthmardquo RespiratoryPhysiology and Neurobiology vol 181 no 1 pp 21ndash28 2012
Figure 2 TNF-120572 selectively modulates ANP BNP and NPR-1 expression BEAS-2B cells were treated with 10 20 and 40 ngmL TNF-120572 for24 h ((a) (b)) ANP ((c) (d)) BNP and ((e) (f)) NPR-1 gene expression at mRNA ((a) (c) and (e)) and protein level ((b) (d) and (f))Western blots were obtained by using the specific rabbit polyclonal Abs The blots were stripped of the bound Ab and reprobed with mouseanti-120573-actin to confirm equal loading Western blots are representative of three separate experiments All histograms indicate meansplusmn SD ofthree different cultures each one tested in quadruplicate and expressed as percentage of control (lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 andlowastlowastlowastlowast
119875 lt 00001 versus untreated cells)
4 Discussion
Postoperative-fluid retention is a severe complication fre-quently reported in patients undergoing major surgical pro-cedures [1ndash4] The complex network of molecules involvedin such a severe surgery-induced condition remains poorlyunderstood After major surgery TNF-120572 is one of the mainproinflammatory cytokines initially released in the damagedtissue where it stimulates the production and release of morecytokines responsible for inducing the systemic changesknown as the acute phase response [5 10] Therefore it isreasonable to assume TNF-120572 involvement among the variouscauses of fluid retention and very important to understandthemechanisms underlying its involvement in such an ambit
In the present study we demonstrated for the first time toour knowledge that TNF-120572modulates the expression ofANPBNP NPR-1 AQP1 and AQP5 in human bronchial epithelialcells BEAS-2B via different involvement of NF-120581B andMAPKs signaling pathway activation Here we also providedthe first demonstration that human bronchial epithelial cellsexpress ANP BNP NPR-1 and AQP1 and that BNP is able tomodulate AQP1 expression
The observed expression of NPs and NPR-1 suggestsan autocrine andor paracrine function for these moleculesand indicates important roles for NPs in several biologicalfunctions including regulation of fluid movement across theairway epithelial cells bronchial relaxation [37] vasodilata-tion and pulmonary vascular permeability About the former
6 Mediators of Inflammation
020406080
100120140160
C BAY 10 20 40
AN
P (
of c
ontro
l)
TNF-120572 (ngmL)
TNF-120572TNF-120572 + BAY
lowast
lowastlowastlowastlowast
(a)
ANP
Actin 40
BAY 0 0 10 20 40 10 20minus + +minus minus minus + +
Figure 3 TNF-120572 modulation of NPs and NPR-1 may require activation of NF-120581B pathway BEAS-2B cells were treated with 10 20 and40 ngmL TNF-120572 alone or in combination with NF-120581B inhibitor BAY 11-7082 (1120583M) for 24 h ((a) (b)) ANP ((c) (d)) BNP ((e) (f)) NPR-1gene expression at mRNA ((a) (c) and (e)) and protein level ((b) (d) (f)) and (g) Phospho-I120581B-120572 protein level Western blots were obtainedby using the specific rabbit polyclonal or monoclonal AbsThe blots were stripped of the bound Ab and reprobed with mouse anti-120573-actin toconfirm equal loading Western blots are representative of three separate experiments All histograms indicate means plusmn SD of three differentcultures each of one tested in quadruplicate and expressed as percentage of control (lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowastlowast119875 lt 00001 versus Controls)(∘119875 lt 005 ∘∘119875 lt 001 and ∘∘∘∘119875 lt 00001 versus BAY) C Controls (untreated cells)
process the role of ANP and BNP could be analogous to thathypothesized forNPs in transepithelial ion flux in the choroidplexus [38] in the kidney [39] colon [40] and retina [41]
The observed TNF-120572 capability of selectively regulatingANP BNP and NPR-1 gene expression in BEAS-2B cellline is of particular interest because it demonstrates that
inflammation alters NPs expression levels In particular thesignificant up-regulation of BNP expression and proteinsecretion after TNF-120572 treatment shows that this modulationcan occur independently of hemodynamic influences andthat inflammation should be considered an extracardiaccause of BNP elevation Besides in vitro studies conducted in
Mediators of Inflammation 7
020406080
100120140160180200
C QNZ TNF
BNP
( o
f con
trol)
QNZ + TNF
lowastlowast
∘∘
(a)
0
20
40
60
80
100
120
140
160
180
BNP
( o
f con
trol)
C QNZ TNF QNZ + TNF
lowastlowast
(b)
0
20
40
60
80
100
120
C QNZ TNF QNZ + TNF
∘∘
lowastlowastlowastlowast
lowastlowastlowastlowast
NPR
-1(
of c
ontro
l)
(c)
NPR-1 Actin
0 0QNZ
10 10TNF-120572+minus +minus
(d)
Actin
QNZ TNF-120572 + +
minus ++
minusminus
minus
P-I120581B-120572
(e)
Figure 4 TNF-120572 modulation of protein levels of BNP and NPR-1 may require activation of NF-120581B pathway BEAS-2B cells were treated10 ngmL TNF-120572 alone or in combination with the inhibitor QNZ (10120583M) for 24 h ((a) (b)) BNP ((c) (d)) NPR-1 gene expression at mRNA((a) (c)) and protein ((b) (d)) level and (e) Phospho-I120581B-120572 protein level Western blots were obtained by using the specific rabbit polyclonalor monoclonal AbsThe blots were stripped of the bound Ab and reprobed with mouse anti-120573-actin to confirm equal loading Western blotsare representative of three separate experiments All histograms indicatemeansplusmnSDof three different cultures each one tested in quadruplicateand expressed as percentage of control (lowastlowast119875 lt 001 and lowastlowastlowastlowast119875 lt 00001 versus Controls) (∘∘119875 lt 001 versus QNZ) C Controls (untreatedcells)
rat cardiomyocytes have shown that not only hemodynamicfactors but also neurohumoral factors activated during heartfailure such as angiotensin II endothelin and cytokinescause BNP secretion [42ndash44] In addition in patients withcardiovascular diseases plasma BNP levels have been shownto be also affected by low-grade inflammation [24] and aselective increase inBNPplasma levels has been proposed as ageneral feature of inflammation [19]The lack on any apparentTNF-120572 dose-depending increase in BNP gene expression thatwe observed in our system could be due to an autocrinenegative feedback action mediated by BNP itself followingbinding to the NPR-1 receptor expressed in BEAS-2B cells
The biological role of BNPup-regulation after TNF-120572 treatment remains to be elucidated However since it
has been demonstrated that BNP regulates the productionof major inflammatory molecules such as IL-12 IL-10leukotriene B and prostaglandin E2 in human macrophages[45] and that BEAS-2B are able to release inflammatorymolecules [46] it could be reasonable to assume that theobserved BNP increase after TNF-120572 treatment may stimu-late the production and release of such cytokines also fromhuman epithelial bronchial cells In addition in in vitrostudies a potent BNP inhibitory action on the productionof the glucocorticoid anti-inflammatory hormone cortisolhas been described [47] Therefore BNP could be con-sidered a proinflammatory molecule by inducing cytokinesexpression in target cells and inhibiting the production ofanti-inflammatory hormones Therefore the observed BNP
8 Mediators of Inflammation
0
50
100
150
200
250BN
P (
of c
ontro
l)
C
TNF SB
SB+
TNF SP
SP+
TNF
U-0
126
U-0126+
TNF
lowastlowast
lowastlowast
∘∘
sect
(a)
020406080
100120140160180
BNP
( o
f con
trol)
C
TNF SB
SB+
TNF SP
SP+
TNF
U-0
126
U-0126+
TNF
lowastlowast
lowastlowast
∘∘∘
sectsect
(b)
0
20
40
60
80
100
120
140
160
C
TNF SB
SB+
TNF SP
SP+
TNF
U-0
126
U-0126+
TNF
lowastlowastlowast
lowastlowastlowast
lowast
lowast
sect
NPR
-1(
of c
ontro
l)
(c)
+
SB 203580
+
SP 600125 U-0126
+ActinTNF-120572
NPR-1
minus minus minus minus +
(d)
SB 203580
Actin
P-p38 MAPK
TNF-120572 + +
minus ++
minusminus
minus
(e)
Figure 5 TNF-120572 modulation of BNP and NPR-1 expression may require activation of MAPKs pathway BEAS-2B cells were treated with10 ngmL TNF-120572 alone or in combination with 10 120583M SB 203580 (p38 MAPK inhibitor) SP 600125 (JNK 12 inhibitor) or U-0126 (ERK12 inhibitor) for 24 h Panels show ((a) (b)) BNP ((c) (d)) NPR-1 gene expression at mRNA ((a) (c)) and protein ((b) (d)) level and (e)Phospho-p38 MAPK protein level Western blots were obtained by using the specific rabbit polyclonal or monoclonal Abs The blots werestripped of the bound Ab and reprobed with mouse anti-120573-actin to confirm equal loading Western blots are representative of three separateexperiments All histograms indicate means plusmn SD of three different cultures each one tested in quadruplicate and expressed as percentage ofcontrol (lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 versus Controls) (∙∙119875 lt 001 versus SB) (sect119875 lt 005 sectsect
119875 lt 001 versus SP) (∘∘119875 lt 001∘∘∘
119875 lt 0001 versus U-0126) C Controls (untreated cells)
up-regulation after TNF-120572 treatment could concur in poten-tiating an inflammatory statusThe increased production andrelease of specific cytokines causing systemic changes knownas the acute phase response [5 8 10] induced by TNF-120572 maythus also occur via BNP up-regulation
Recently a BNP rise has been observed in patientsundergoing pulmonary lobectomy for lung cancer [11] Inparticular the patients none of whom developed signs orsymptoms of heart failure during the postoperative period
showed an early significant increase on day 1 immediatelyafter surgery of the plasma concentrations of BNP whichwas followed on the postoperative day 2 by a significantweight gain correlated with large volumes of fluids accumu-lation despite a negative intraoperative fluid balance andperoperative strict fluid restriction [11] We suggest thatsuch an increase could be at least in part explained bythe ability of proinflammatory cytokines such as TNF-120572 toupregulate BNP gene expression and secretion and that this
Mediators of Inflammation 9
0
20
40
60
80
100
120
0 10 20 40TNF-120572 (ngmL)
AQP1
( o
f con
trol)
lowast
lowastlowastlowast
lowastlowastlowast
(a)
Actin 0 10 20 40
AQP1
TNF-120572 (ngmL)
(b)
0
20
40
60
80
100
120
AQP5
( o
f con
trol)
0 10 20 40
TNF-120572 (ngmL)
lowastlowast
lowastlowastlowastlowast
(c)
AQP5
Actin 0 10 20 40TNF-120572 (ngmL)
(d)
Figure 6 TNF-120572 decreases AQP1 and AQP5 expression in BEAS 2B cells BEAS-2B cells were treated with 10 20 and 40 ngmL TNF-120572 for24 h Gene expression of AQP1 ((a) (b)) and AQP5 ((c) (d)) at mRNA ((a) (c)) and protein ((b) (d)) level Western blots were obtained byusing the specific rabbit polyclonal Abs The blots were stripped of the bound Ab and reprobed with mouse anti-120573-actin to confirm equalloading Western blots are representative of three separate experiments All histograms indicate means plusmn SD of three different cultures eachone tested in quadruplicate and expressed as percentage of control (lowast119875 lt 001 lowastlowast119875 lt 0001 and lowastlowastlowast119875 lt 00001 versus untreated cells)
up-regulation could enhance the major surgery-related earlyhyperinflammatory response essential for tissue repair andhost defense [5]
The observed dexamethasone capability to completelyrevert TNF-120572-induced BNP up-regulation suggests that thisanti-inflammatory glucocorticoid may prevent BNP-relatedeffects
Regarding ANP the observed significant down-regula-tion of mRNA and protein levels after 10 20 ngmL TNF-120572treatment suggests that also this condition could affect localinflammation status In fact ANP administration determinesanti-inflammatory effects in airway epithelial cells [48] play-ing important roles in modulating inflammatory responseIn consideration of such ANP anti-inflammatory action wespeculate that the observed TNF-120572-induced ANP down-regulation could concur in enhancing an inflammatory sta-tus Differently from the above reported doses 40 ngmLTNF-120572 administration induced ANP up-regulation eventhough not statistically significant We suggest that this resultmight be due to a direct BNP involvement In fact a markedANP up-regulation was observed after BNP treatment at the
dose accumulated in culture medium after 40 ngmL TNF-120572treatment
The observed selectivemodulation inANP and BNP geneexpression after TNF-120572 treatment in line with other studiesin different cell models [19 49 50] could be related to adifferent regulation mechanism at transcriptional level assuggested by our observation on the different involvementof NF-120581B and MAPKs signaling pathways In particular wedemonstrated that ANP down-regulation TNF-120572-mediatedoccurs via NF-120581B activation pathway whereas BNP modula-tion requires ERK 12 activation pathway NF-120581B involvementin TNF-120572-induced ANP down-regulation is not opposed tothe proinflammatory role of this transcription factor beingANP a molecule that exerts an anti-inflammatory effect inairway epithelial cells [48] Regarding themechanism linkingNF-120581B to the downregulation of ANP gene expression wesuggest that it could be similar to that recently proposedfor Fibroblast Growth Factor 10 (FGF-10) where NF-120581Bactivationmay lead to reduced gene expression by recruitinginhibitory factors to specific gene promoters [51]
10 Mediators of Inflammation
0
20
40
60
80
100
120
C BAY 10 20 40
AQP1
( o
f con
trol)
TNF-120572 (ngmL)
TNF-120572TNF-120572 + BAY
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowast
(a)
BAY
AQP1Actin
0 0 10 20 40 10 20 40TNF-120572 (ngmL)minus + +minus minus minus + +
(b)
0
20
40
60
80
100
120
AQP5
( o
f con
trol)
C BAY 10 20 40
TNF-120572 (ngmL)
TNF-120572TNF-120572 + BAY
∘∘∘∘∘∘
∘∘∘
lowastlowastlowastlowast
lowastlowast
(c)
AQP5Actin
0 0 10 20 40 10 20 40TNF-120572 (ngmL)BAY minus + +minus minus minus + +
(d)
Figure 7 TNF-120572 downregulation of AQP1 and AQP5 expression may require activation of NF-120581B pathwayBEAS-2B cells were treated with10 20 and 40 ngmL TNF-120572 alone or in combination with NF-120581B inhibitor BAY 11-7082 (1 120583M) for 24 h Gene expression of AQP1 ((a) (b))and AQP5 ((c) (d)) at mRNA ((a) (c)) and protein ((b) (d)) level Western blots were obtained by using the specific rabbit polyclonal AbsThe blots were stripped of the bound Ab and reprobed with mouse anti-120573-actin to confirm equal loading Western blots are representative ofthree separate experiments All histograms indicate means plusmn SD of three different cultures each one tested in quadruplicate and expressed aspercentage of control (lowast119875 lt 001 lowastlowast119875 lt 0001 lowastlowastlowast119875 lt 00001 versus Controls and ∘∘∘119875 lt 00001 versus BAY) C Controls (untreated cells)
ANP and BNP biological functions occur through theirbinding to the same NPR-1 [13] Here we demonstratedfor the first time to our knowledge TNF-120572 capability todownregulateNPR-1mRNAandprotein expression in BEAS-2B cells and that this regulation occurs via MAPKs signalingpathway activation The consequence of the complex TNF-120572-induced NPs and NPR-1 observed modulation associatedwith their binding to the same receptor on BEAS-2B cellsmakes it difficult to predict what would be the net resultingeffect both in vitro and in vivo in relation also to the presenceof a possible autocrine regulation by NPs andor to the effectsof different cytokines activated by both BNP and TNF-120572We hypothesize that the TNF-120572-induced downregulation of
NPR-1 paralleled by an up-regulation of BNP could alsolead to a paracrine action of this peptide possibly on themicrovascular endothelium of the lung where NPR-1 isdensely expressed [18] In this context we speculate that theinduction of local modification on BNP or ANP by TNF-120572 inhuman bronchial epithelial cells could alter the barrier per-meability of the pulmonary microcirculation Hence TNF-120572 acting on epithelial cells could also indirectly contributeto endothelial cells barrier dysfunction and permeability byenhancing the local production of proinflammatory BNPand by down-regulating the anti-inflammatory expressionof ANP Besides exogenous synthetic ANP has been shownto protect from endothelial barrier dysfunction in in vivo
Mediators of Inflammation 11
0
50
100
150
200
250
300
0 001
AN
P (
of c
ontro
l)
BNP (nM)
lowast
(a)
0
50
100
150
0 001BNP (nM)
NPR
-1(
of c
ontro
l)
(b)
0
20
40
60
80
100
120
0 001
BNP (nM)
lowast
AQP1
( o
f con
trol)
(c)
0
20
40
60
80
100
120
0 001
BNP (nM)
AQP5
( o
f con
trol)
(d)
Figure 8 BNP effect on ANP NPR-1 AQP1 and AQP5 expressionBEAS-2B cells were treated with 001 nM BNP for 24 h Histograms show(a) ANP (b) NPR-1 (c) AQP1 and (d) AQP5 gene expression at mRNA level All histograms indicate means plusmn SD of three different cultureseach one tested in quadruplicate and expressed as percentage of control (lowast119875 lt 005 versus untreated cells)
020406080
100120140160180200
DEX
BNP
( o
f con
trol)
C TNF-120572 TNF-120572 + DEX
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowast
lowast
lowast
Figure 9 Dexamethasone effect on BNP expression BEAS-2B cellswere treatedwith 1 120583Mdexamethasone (DEX) and 40 ngmLTNF-120572alone or in combination with DEX for 24 h Histograms show BNPgene expression at mRNA (gray bars) and protein (black bars) levelAll histograms indicate means plusmn SD of three different cultures eachone tested in quadruplicate and expressed as percentage of control(lowast119875 lt 001 lowastlowast119875 lt 0001 and lowastlowastlowast119875 lt 00001 versus Controls) CControls (untreated cells)
and in vitro models of lung injury [18] and the therapeu-tical relevance of these experimental observations has beenshown in intensive care patients without heart disease whereintravenous ANP infusion diminished pulmonary vascularpermeability and pulmonary edema [18]
Regarding AQPs we demonstrated that BEAS-2B cellsexpress AQP1 and AQP5 and that TNF-120572 directly down-regulates their expression at mRNA and protein levelsRegarding AQP1 also an additional BNP-mediated TNF-120572involvement can be proposed In fact AQP1 down-regulationwas observed with BNP treatment at the dose accumulatedin culture medium after 40 ngmL TNF-120572 treatment Themechanisms that underlie AQPs regulation are of consider-able interest Several AQPs has recently been demonstratedto undergo complex regulation [27] Our results show thatAQP1 and APQ5 are subject to inflammation regulationadding an important piece of information in this contextOur results are in agreement with other studies describinga marked reduction in the expression levels of AQP1 andAQP5 inmouse lung [30] andmouse lung epithelial cells [31]The biological significance of the observed TNF-120572-inducedAQP1 and AQP5 down-regulation is at present unclear Ithas been recently suggested that the decrease in both AQPsmay play an important role in the development of lung edema[52] which is known to be related with fluid retention Wetherefore speculate that TNF-120572-induced AQP1 and AQP5down-regulation might play a role also in the excess of fluidaccumulation related to major surgical procedures
5 Conclusions
In conclusion our results provided evidence that in humanbronchial epithelial cells BEAS-2B TNF-120572 selectively mod-ulates mRNA and protein expression levels of different
12 Mediators of Inflammation
molecules involved in body fluid homeostasis ANP BNPand their receptor NPR-1 AQP1 and AQP5 Moreoverour data pointed out that the changes specifically affectingNPs expression occur independently from hemodynamicinfluences and that inflammation should be considered anextracardiac cause of BNP elevation adding an importantpiece of information in the novel area of study regarding NPsand inflammation In addition we demonstrated that such amodulation occurs via different involvement of NK-120581B andMAPKs signaling pathways Finally we suggested inflamma-tion among the mechanisms involved in the regulation ofAQP1 and AQP5 Therefore in this exploratory study wespeculate that TNF-120572 might be involved in postoperative-fluid retention related to major surgery
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgment
The authors thank Mrs Roberta Frosini for the excellenttechnical assistance
References
[1] E Itobi M Stroud and M Elia ldquoImpact of oedema onrecovery after major abdominal surgery and potential valueof multifrequency bioimpedance measurementsrdquo The BritishJournal of Surgery vol 93 no 3 pp 354ndash361 2006
[2] J A Lowell C Schifferdecker D F Driscoll P N Benottiand B R Bistrian ldquoPostoperative fluid overload not a benignproblemrdquo Critical Care Medicine vol 18 no 7 pp 728ndash7331990
[3] A M Moslashller T Pedersen P-E Svendsen and A EngquistldquoPerioperative risk factors in elective pneumonectomy theimpact of excess fluid balancerdquo European Journal of Anaesthe-siology vol 19 no 1 pp 57ndash62 2002
[4] E Bennett-Guerrero D E Feierman G R Barclay et alldquoPreoperative and intraoperative predictors of postoperativemorbidity poor graft function and early rejection in 190patients undergoing liver transplantationrdquo Archives of Surgeryvol 136 no 10 pp 1177ndash1183 2001
[5] J P Desborough ldquoThe stress response to trauma and surgeryrdquoThe British Journal of Anaesthesia vol 85 no 1 pp 109ndash1172000
[6] H Tsujimoto S Ono T Majima et al ldquoDifferential toll-likereceptor expression after ex vivo lipopolysaccharide exposurein patients with sepsis and following surgical stressrdquo ClinicalImmunology vol 119 no 2 pp 180ndash187 2006
[7] C Evans C Galustian D Kumar et al ldquoImpact of surgeryon immunologic function comparison between minimallyinvasive techniques and conventional laparotomy for surgi-cal resection of colorectal tumorsrdquo The American Journal ofSurgery vol 197 no 2 pp 238ndash245 2009
[8] F Catena L Ansaloni A Avanzolini et al ldquoSystemic cytokineresponse after emergency and elective surgery for colorectalcarcinomardquo International Journal of Colorectal Disease vol 24no 7 pp 803ndash808 2009
[9] R Sarbinowski S Arvidsson M Tylman T Oresland andA Bengtsson ldquoPlasma concentration of procalcitonin andsystemic inflammatory response syndrome after colorectalsurgeryrdquo Acta Anaesthesiologica Scandinavica vol 49 no 2 pp191ndash196 2005
[10] P Sheeran and G M Hall ldquoCytokines in anaesthesiardquo TheBritish Journal of Anaesthesia vol 78 no 2 pp 201ndash219 1997
[11] L Cagini R Capozzi V Tassi et al ldquoFluid and electrolytebalance after major thoracic surgery by bioimpedance andendocrine evaluationrdquo European Journal of Cardio-ThoracicSurgery vol 40 no 2 pp e71ndashe76 2011
[12] T Omland and T Hagve ldquoNatriuretic peptides physiologic andanalytic considerationsrdquo Heart Failure Clinics vol 5 no 4 pp471ndash487 2009
[13] L R Potter A R Yoder D R Flora L K Antos and DM Dickey ldquoNatriuretic peptides their structures receptorsphysiologic functions and therapeutic applicationsrdquo Handbookof Experimental Pharmacology vol 191 pp 341ndash366 2009
[14] T G Flynn M L de Bold and A J de Bold ldquoThe aminoacid sequence of an atrial peptide with potent diuretic andnatriuretic propertiesrdquo Biochemical and Biophysical ResearchCommunications vol 117 no 3 pp 859ndash865 1983
[15] A J de Bold ldquoAtrial natriuretic factor a hormone produced bythe heartrdquo Science vol 230 pp 767ndash770 1985
[16] K Kangawa Y Tawaragi S Oikawa et al ldquoIdentification of rat 120574atrial natriuretic polypeptide and characterization of the cDNAencoding its precursorrdquo Nature vol 312 no 5990 pp 152ndash1551984
[17] T Sudoh K Kangawa N Minamino and H Matsuo ldquoA newnatriuretic peptide in porcine brainrdquo Nature vol 332 no 6159pp 78ndash81 1988
[18] M Kuhn ldquoEndothelial actions of atrial and B-type natriureticpeptidesrdquo The British Journal of Pharmacology vol 166 no 2pp 522ndash531 2012
[19] A J de Bold ldquoCardiac natriuretic peptides gene expression andsecretion in inflammationrdquo Journal of Investigative Medicinevol 57 pp 29ndash32 2009
[20] S P DrsquoSouza M Davis and G F Baxter ldquoAutocrine andparacrine actions of natriuretic peptides in the heartrdquo Pharma-cology andTherapeutics vol 101 no 2 pp 113ndash129 2004
[21] M Brueckmann G Huhle S Lang et al ldquoPrognostic valueof plasma N-terminal pro-brain natriuretic peptide in patientswith severe sepsisrdquoCirculation vol 112 no 4 pp 527ndash534 2005
[22] J Charpentier C E Luyt Y Fulla et al ldquoBrain natriureticpeptide a marker of myocardial dysfunction and prognosisduring severe sepsisrdquo Critical Care Medicine vol 32 no 3 pp660ndash665 2004
[23] R Shor Y Rozenman A Bolshinsky et al ldquoBNP in septicpatients without systolic myocardial dysfunctionrdquo EuropeanJournal of Internal Medicine vol 17 no 8 pp 536ndash540 2006
[24] T Inoue M Kawai T Nakane et al ldquoInfluence of low-gradeinflammation on plasma B-type natriuretic peptide levelsrdquoInternal Medicine vol 49 no 24 pp 2659ndash2668 2010
[25] P Agre L S King M Yasui et al ldquoAquaporin water channelsmdashfrom atomic structure to clinical medicinerdquo Journal of Physiol-ogy vol 542 no 1 pp 3ndash16 2002
[26] P Agre and D Kozono ldquoAquaporin water channels molecularmechanisms for human diseasesrdquo FEBS Letters vol 555 no 1pp 72ndash78 2003
Mediators of Inflammation 13
[27] J M Carbrey and P Agre ldquoDiscovery of the aquaporins anddevelopment of the fieldrdquo Handbook of Experimental Pharma-cology vol 190 pp 3ndash28 2009
[28] O Bloch and G T Manley ldquoThe role of aquaporin-4 in cerebralwater transport and edemardquo Neurosurgical Focus vol 22 no 5article E3 2007
[29] J Li M Xu Q Fan et al ldquoTanshinone IIA ameliorates seawaterexposure-induced lung injury by inhibiting aquaporins (AQP)1 and AQP5 expression in lungrdquo Respiratory Physiology andNeurobiology vol 176 no 1-2 pp 39ndash49 2011
[30] J E Towne K S Harrod C M Krane and A G MenonldquoDecreased expression of aquaporin (AQP)1 and AQP5 inmouse lung after acute viral infectionrdquoThe American Journal ofRespiratory Cell and Molecular Biology vol 22 no 1 pp 34ndash442000
[31] J E Towne C M Krane C J Bachurski and A G MenonldquoTumor necrosis factor-120572 inhibits aquaporin 5 expression inmouse lung epithelial cellsrdquoThe Journal of Biological Chemistryvol 276 no 22 pp 18657ndash18664 2001
[32] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001
[33] K Verhelst I Carpentier and R Beyaert ldquoRegulation of TNF-induced NF-120581B activation by different cytoplasmic ubiquitina-tion eventsrdquo Cytokine and Growth Factor Reviews vol 22 no5-6 pp 277ndash286 2011
[34] L Qian Y Shen J C Chen et al ldquo3D-QSAR and dockingstudies of quinazoline derivatives with the inhibatory activitytoward NF-120581BrdquoQSAR and Combinatorial Science vol 27 no 8pp 984ndash995 2008
[35] P D NrsquoGuessan S Hippenstiel M O Etouem et al ldquoStrepto-coccus pneumoniae induced p38MAPK- andNF-120581B-dependentCOX-2 expression in human lung epitheliumrdquo The AmericanJournal of PhysiologymdashLung Cellular and Molecular Physiologyvol 290 no 6 pp L1131ndashL1138 2006
[36] B Schmeck K Moog J Zahlten et al ldquoStreptococcus pneumo-niae induced c-Jun-N-terminal kinase- andAP-I-dependent IL-8 release by lung epithelial BEAS-2B cellsrdquoRespiratory Researchvol 7 article 98 2006
[37] M GMatera L Calzetta D Passeri et al ldquoEpithelium integrityis crucial for the relaxant activity of brain natriuretic peptide inhuman isolated bronchirdquo The British Journal of Pharmacologyvol 163 no 8 pp 1740ndash1754 2011
[38] R QuirionM Dalpe A de Lean J Gutkowska M Cantin andJ Genest ldquoAtrial natriuretic factor (ANF) binding sites in brainand related structuresrdquoPeptides vol 5 no 6 pp 1167ndash1172 1984
[39] M B Pamnani D L Clough J S Chen W T Link andF J Haddy ldquoEffects of rat atrial extract on sodium transportand blood pressure in the ratrdquo Proceedings of the Society forExperimental Biology and Medicine vol 176 no 2 pp 123ndash1311984
[40] T R Traynor and S M OrsquoGrady ldquoBrain natriuretic peptidestimulates K andCl secretion across porcine distal colon epithe-liumrdquoTheAmerican Journal of PhysiologymdashCell Physiology vol260 no 4 pp C750ndashC755 1991
[41] R Rollın A Mediero M Roldan-Pallares A Fernandez-Cruzand R Fernandez-Durango ldquoNatriuretic peptide system in thehuman retinardquoMolecular Vision vol 10 pp 15ndash22 2004
[42] M Harada H Itoh O Nakagawa et al ldquoSignificance ofventricular myocytes and nonmyocytes interaction during car-diocyte hypertrophy evidence for endothelin-1 as a paracrinehypertrophic factor from cardiac nonmyocytesrdquo Circulationvol 96 no 10 pp 3737ndash3744 1997
[43] K Kuwahara Y Saito M Harada et al ldquoInvolvement ofcardiotrophin-1 in cardiac myocyte-nonmyocyte interactionsduring hypertrophy of rat cardiac myocytes in vitrordquo Circula-tion vol 100 no 10 pp 1116ndash1124 1999
[44] E Harada O Nakagawa M Yoshimura et al ldquoEffect ofinterleukin-1120573 on cardiac hypertrophy and production of natri-uretic peptides in rat cardiocyte culturerdquo Journal of Molecularand Cellular Cardiology vol 31 no 11 pp 1997ndash2006 1999
[45] V Chiurchiu V Izzi F DrsquoAquilio F Carotenuto P Di Nardoand P M Baldini ldquoBrain natriuretic peptide (BNP) regulatesthe production of inflammatory mediators in human THP-1macrophagesrdquo Regulatory Peptides vol 148 no 1ndash3 pp 26ndash322008
[46] N S Holden C F Rider M J Bell et al ldquoEnhancement ofinflammatory mediator release by 1205732-adrenoceptor agonists inairway epithelial cells is reversed by glucocorticoid actionrdquoTheBritish Journal of Pharmacology vol 160 no 2 pp 410ndash4202010
[47] V S Fang C C Juan J G S Won and L T Ho ldquoFurther studyof aldosterone secretion-inhibitory factor and brain natriureticpeptide on cortisol production of Guinea pig zona fasciculatacellsrdquo Chinese Journal of Physiology vol 43 no 4 pp 141ndash1472000
[48] G Hellermann X Kong J Gunnarsdottir et al ldquoMechanismof bronchoprotective effects of a novel natriuretic hormonepeptiderdquo Journal of Allergy and Clinical Immunology vol 113no 1 pp 79ndash85 2004
[49] K K Ma T Ogawa and A J de Bold ldquoSelective upregulationof cardiac brain natriuretic peptide at the transcriptional andtranslational levels by pro-inflammatory cytokines and by con-ditionedmedium derived frommixed lymphocyte reactions viap38 MAP kinaserdquo Journal of Molecular and Cellular Cardiologyvol 36 no 4 pp 505ndash513 2004
[50] T Ogawa J P Veinot M L K de Bold T Georgalis and AJ De Bold ldquoAngiotensin II receptor antagonism reverts theselective cardiac BNP upregulation and secretion observed inmyocarditisrdquo The American Journal of PhysiologymdashHeart andCirculatory Physiology vol 294 no 6 pp H2596ndashH2603 2008
[51] B J Carver E J Plosa A M Stinnett T S Blackwell andL S Prince ldquoInteractions between NF-120581B and SP3 connectinflammatory signaling with reduced FGF-10 expressionrdquo TheJournal of Biological Chemistry vol 288 no 21 pp 15318ndash153252013
[52] C Dong G Wang B Li et al ldquoAnti-asthmatic agents alleviatepulmonary edema by upregulating AQP1 and AQP5 expressionin the lungs of mice with OVA-induced asthmardquo RespiratoryPhysiology and Neurobiology vol 181 no 1 pp 21ndash28 2012
Figure 3 TNF-120572 modulation of NPs and NPR-1 may require activation of NF-120581B pathway BEAS-2B cells were treated with 10 20 and40 ngmL TNF-120572 alone or in combination with NF-120581B inhibitor BAY 11-7082 (1120583M) for 24 h ((a) (b)) ANP ((c) (d)) BNP ((e) (f)) NPR-1gene expression at mRNA ((a) (c) and (e)) and protein level ((b) (d) (f)) and (g) Phospho-I120581B-120572 protein level Western blots were obtainedby using the specific rabbit polyclonal or monoclonal AbsThe blots were stripped of the bound Ab and reprobed with mouse anti-120573-actin toconfirm equal loading Western blots are representative of three separate experiments All histograms indicate means plusmn SD of three differentcultures each of one tested in quadruplicate and expressed as percentage of control (lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowastlowast119875 lt 00001 versus Controls)(∘119875 lt 005 ∘∘119875 lt 001 and ∘∘∘∘119875 lt 00001 versus BAY) C Controls (untreated cells)
process the role of ANP and BNP could be analogous to thathypothesized forNPs in transepithelial ion flux in the choroidplexus [38] in the kidney [39] colon [40] and retina [41]
The observed TNF-120572 capability of selectively regulatingANP BNP and NPR-1 gene expression in BEAS-2B cellline is of particular interest because it demonstrates that
inflammation alters NPs expression levels In particular thesignificant up-regulation of BNP expression and proteinsecretion after TNF-120572 treatment shows that this modulationcan occur independently of hemodynamic influences andthat inflammation should be considered an extracardiaccause of BNP elevation Besides in vitro studies conducted in
Mediators of Inflammation 7
020406080
100120140160180200
C QNZ TNF
BNP
( o
f con
trol)
QNZ + TNF
lowastlowast
∘∘
(a)
0
20
40
60
80
100
120
140
160
180
BNP
( o
f con
trol)
C QNZ TNF QNZ + TNF
lowastlowast
(b)
0
20
40
60
80
100
120
C QNZ TNF QNZ + TNF
∘∘
lowastlowastlowastlowast
lowastlowastlowastlowast
NPR
-1(
of c
ontro
l)
(c)
NPR-1 Actin
0 0QNZ
10 10TNF-120572+minus +minus
(d)
Actin
QNZ TNF-120572 + +
minus ++
minusminus
minus
P-I120581B-120572
(e)
Figure 4 TNF-120572 modulation of protein levels of BNP and NPR-1 may require activation of NF-120581B pathway BEAS-2B cells were treated10 ngmL TNF-120572 alone or in combination with the inhibitor QNZ (10120583M) for 24 h ((a) (b)) BNP ((c) (d)) NPR-1 gene expression at mRNA((a) (c)) and protein ((b) (d)) level and (e) Phospho-I120581B-120572 protein level Western blots were obtained by using the specific rabbit polyclonalor monoclonal AbsThe blots were stripped of the bound Ab and reprobed with mouse anti-120573-actin to confirm equal loading Western blotsare representative of three separate experiments All histograms indicatemeansplusmnSDof three different cultures each one tested in quadruplicateand expressed as percentage of control (lowastlowast119875 lt 001 and lowastlowastlowastlowast119875 lt 00001 versus Controls) (∘∘119875 lt 001 versus QNZ) C Controls (untreatedcells)
rat cardiomyocytes have shown that not only hemodynamicfactors but also neurohumoral factors activated during heartfailure such as angiotensin II endothelin and cytokinescause BNP secretion [42ndash44] In addition in patients withcardiovascular diseases plasma BNP levels have been shownto be also affected by low-grade inflammation [24] and aselective increase inBNPplasma levels has been proposed as ageneral feature of inflammation [19]The lack on any apparentTNF-120572 dose-depending increase in BNP gene expression thatwe observed in our system could be due to an autocrinenegative feedback action mediated by BNP itself followingbinding to the NPR-1 receptor expressed in BEAS-2B cells
The biological role of BNPup-regulation after TNF-120572 treatment remains to be elucidated However since it
has been demonstrated that BNP regulates the productionof major inflammatory molecules such as IL-12 IL-10leukotriene B and prostaglandin E2 in human macrophages[45] and that BEAS-2B are able to release inflammatorymolecules [46] it could be reasonable to assume that theobserved BNP increase after TNF-120572 treatment may stimu-late the production and release of such cytokines also fromhuman epithelial bronchial cells In addition in in vitrostudies a potent BNP inhibitory action on the productionof the glucocorticoid anti-inflammatory hormone cortisolhas been described [47] Therefore BNP could be con-sidered a proinflammatory molecule by inducing cytokinesexpression in target cells and inhibiting the production ofanti-inflammatory hormones Therefore the observed BNP
8 Mediators of Inflammation
0
50
100
150
200
250BN
P (
of c
ontro
l)
C
TNF SB
SB+
TNF SP
SP+
TNF
U-0
126
U-0126+
TNF
lowastlowast
lowastlowast
∘∘
sect
(a)
020406080
100120140160180
BNP
( o
f con
trol)
C
TNF SB
SB+
TNF SP
SP+
TNF
U-0
126
U-0126+
TNF
lowastlowast
lowastlowast
∘∘∘
sectsect
(b)
0
20
40
60
80
100
120
140
160
C
TNF SB
SB+
TNF SP
SP+
TNF
U-0
126
U-0126+
TNF
lowastlowastlowast
lowastlowastlowast
lowast
lowast
sect
NPR
-1(
of c
ontro
l)
(c)
+
SB 203580
+
SP 600125 U-0126
+ActinTNF-120572
NPR-1
minus minus minus minus +
(d)
SB 203580
Actin
P-p38 MAPK
TNF-120572 + +
minus ++
minusminus
minus
(e)
Figure 5 TNF-120572 modulation of BNP and NPR-1 expression may require activation of MAPKs pathway BEAS-2B cells were treated with10 ngmL TNF-120572 alone or in combination with 10 120583M SB 203580 (p38 MAPK inhibitor) SP 600125 (JNK 12 inhibitor) or U-0126 (ERK12 inhibitor) for 24 h Panels show ((a) (b)) BNP ((c) (d)) NPR-1 gene expression at mRNA ((a) (c)) and protein ((b) (d)) level and (e)Phospho-p38 MAPK protein level Western blots were obtained by using the specific rabbit polyclonal or monoclonal Abs The blots werestripped of the bound Ab and reprobed with mouse anti-120573-actin to confirm equal loading Western blots are representative of three separateexperiments All histograms indicate means plusmn SD of three different cultures each one tested in quadruplicate and expressed as percentage ofcontrol (lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 versus Controls) (∙∙119875 lt 001 versus SB) (sect119875 lt 005 sectsect
119875 lt 001 versus SP) (∘∘119875 lt 001∘∘∘
119875 lt 0001 versus U-0126) C Controls (untreated cells)
up-regulation after TNF-120572 treatment could concur in poten-tiating an inflammatory statusThe increased production andrelease of specific cytokines causing systemic changes knownas the acute phase response [5 8 10] induced by TNF-120572 maythus also occur via BNP up-regulation
Recently a BNP rise has been observed in patientsundergoing pulmonary lobectomy for lung cancer [11] Inparticular the patients none of whom developed signs orsymptoms of heart failure during the postoperative period
showed an early significant increase on day 1 immediatelyafter surgery of the plasma concentrations of BNP whichwas followed on the postoperative day 2 by a significantweight gain correlated with large volumes of fluids accumu-lation despite a negative intraoperative fluid balance andperoperative strict fluid restriction [11] We suggest thatsuch an increase could be at least in part explained bythe ability of proinflammatory cytokines such as TNF-120572 toupregulate BNP gene expression and secretion and that this
Mediators of Inflammation 9
0
20
40
60
80
100
120
0 10 20 40TNF-120572 (ngmL)
AQP1
( o
f con
trol)
lowast
lowastlowastlowast
lowastlowastlowast
(a)
Actin 0 10 20 40
AQP1
TNF-120572 (ngmL)
(b)
0
20
40
60
80
100
120
AQP5
( o
f con
trol)
0 10 20 40
TNF-120572 (ngmL)
lowastlowast
lowastlowastlowastlowast
(c)
AQP5
Actin 0 10 20 40TNF-120572 (ngmL)
(d)
Figure 6 TNF-120572 decreases AQP1 and AQP5 expression in BEAS 2B cells BEAS-2B cells were treated with 10 20 and 40 ngmL TNF-120572 for24 h Gene expression of AQP1 ((a) (b)) and AQP5 ((c) (d)) at mRNA ((a) (c)) and protein ((b) (d)) level Western blots were obtained byusing the specific rabbit polyclonal Abs The blots were stripped of the bound Ab and reprobed with mouse anti-120573-actin to confirm equalloading Western blots are representative of three separate experiments All histograms indicate means plusmn SD of three different cultures eachone tested in quadruplicate and expressed as percentage of control (lowast119875 lt 001 lowastlowast119875 lt 0001 and lowastlowastlowast119875 lt 00001 versus untreated cells)
up-regulation could enhance the major surgery-related earlyhyperinflammatory response essential for tissue repair andhost defense [5]
The observed dexamethasone capability to completelyrevert TNF-120572-induced BNP up-regulation suggests that thisanti-inflammatory glucocorticoid may prevent BNP-relatedeffects
Regarding ANP the observed significant down-regula-tion of mRNA and protein levels after 10 20 ngmL TNF-120572treatment suggests that also this condition could affect localinflammation status In fact ANP administration determinesanti-inflammatory effects in airway epithelial cells [48] play-ing important roles in modulating inflammatory responseIn consideration of such ANP anti-inflammatory action wespeculate that the observed TNF-120572-induced ANP down-regulation could concur in enhancing an inflammatory sta-tus Differently from the above reported doses 40 ngmLTNF-120572 administration induced ANP up-regulation eventhough not statistically significant We suggest that this resultmight be due to a direct BNP involvement In fact a markedANP up-regulation was observed after BNP treatment at the
dose accumulated in culture medium after 40 ngmL TNF-120572treatment
The observed selectivemodulation inANP and BNP geneexpression after TNF-120572 treatment in line with other studiesin different cell models [19 49 50] could be related to adifferent regulation mechanism at transcriptional level assuggested by our observation on the different involvementof NF-120581B and MAPKs signaling pathways In particular wedemonstrated that ANP down-regulation TNF-120572-mediatedoccurs via NF-120581B activation pathway whereas BNP modula-tion requires ERK 12 activation pathway NF-120581B involvementin TNF-120572-induced ANP down-regulation is not opposed tothe proinflammatory role of this transcription factor beingANP a molecule that exerts an anti-inflammatory effect inairway epithelial cells [48] Regarding themechanism linkingNF-120581B to the downregulation of ANP gene expression wesuggest that it could be similar to that recently proposedfor Fibroblast Growth Factor 10 (FGF-10) where NF-120581Bactivationmay lead to reduced gene expression by recruitinginhibitory factors to specific gene promoters [51]
10 Mediators of Inflammation
0
20
40
60
80
100
120
C BAY 10 20 40
AQP1
( o
f con
trol)
TNF-120572 (ngmL)
TNF-120572TNF-120572 + BAY
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowast
(a)
BAY
AQP1Actin
0 0 10 20 40 10 20 40TNF-120572 (ngmL)minus + +minus minus minus + +
(b)
0
20
40
60
80
100
120
AQP5
( o
f con
trol)
C BAY 10 20 40
TNF-120572 (ngmL)
TNF-120572TNF-120572 + BAY
∘∘∘∘∘∘
∘∘∘
lowastlowastlowastlowast
lowastlowast
(c)
AQP5Actin
0 0 10 20 40 10 20 40TNF-120572 (ngmL)BAY minus + +minus minus minus + +
(d)
Figure 7 TNF-120572 downregulation of AQP1 and AQP5 expression may require activation of NF-120581B pathwayBEAS-2B cells were treated with10 20 and 40 ngmL TNF-120572 alone or in combination with NF-120581B inhibitor BAY 11-7082 (1 120583M) for 24 h Gene expression of AQP1 ((a) (b))and AQP5 ((c) (d)) at mRNA ((a) (c)) and protein ((b) (d)) level Western blots were obtained by using the specific rabbit polyclonal AbsThe blots were stripped of the bound Ab and reprobed with mouse anti-120573-actin to confirm equal loading Western blots are representative ofthree separate experiments All histograms indicate means plusmn SD of three different cultures each one tested in quadruplicate and expressed aspercentage of control (lowast119875 lt 001 lowastlowast119875 lt 0001 lowastlowastlowast119875 lt 00001 versus Controls and ∘∘∘119875 lt 00001 versus BAY) C Controls (untreated cells)
ANP and BNP biological functions occur through theirbinding to the same NPR-1 [13] Here we demonstratedfor the first time to our knowledge TNF-120572 capability todownregulateNPR-1mRNAandprotein expression in BEAS-2B cells and that this regulation occurs via MAPKs signalingpathway activation The consequence of the complex TNF-120572-induced NPs and NPR-1 observed modulation associatedwith their binding to the same receptor on BEAS-2B cellsmakes it difficult to predict what would be the net resultingeffect both in vitro and in vivo in relation also to the presenceof a possible autocrine regulation by NPs andor to the effectsof different cytokines activated by both BNP and TNF-120572We hypothesize that the TNF-120572-induced downregulation of
NPR-1 paralleled by an up-regulation of BNP could alsolead to a paracrine action of this peptide possibly on themicrovascular endothelium of the lung where NPR-1 isdensely expressed [18] In this context we speculate that theinduction of local modification on BNP or ANP by TNF-120572 inhuman bronchial epithelial cells could alter the barrier per-meability of the pulmonary microcirculation Hence TNF-120572 acting on epithelial cells could also indirectly contributeto endothelial cells barrier dysfunction and permeability byenhancing the local production of proinflammatory BNPand by down-regulating the anti-inflammatory expressionof ANP Besides exogenous synthetic ANP has been shownto protect from endothelial barrier dysfunction in in vivo
Mediators of Inflammation 11
0
50
100
150
200
250
300
0 001
AN
P (
of c
ontro
l)
BNP (nM)
lowast
(a)
0
50
100
150
0 001BNP (nM)
NPR
-1(
of c
ontro
l)
(b)
0
20
40
60
80
100
120
0 001
BNP (nM)
lowast
AQP1
( o
f con
trol)
(c)
0
20
40
60
80
100
120
0 001
BNP (nM)
AQP5
( o
f con
trol)
(d)
Figure 8 BNP effect on ANP NPR-1 AQP1 and AQP5 expressionBEAS-2B cells were treated with 001 nM BNP for 24 h Histograms show(a) ANP (b) NPR-1 (c) AQP1 and (d) AQP5 gene expression at mRNA level All histograms indicate means plusmn SD of three different cultureseach one tested in quadruplicate and expressed as percentage of control (lowast119875 lt 005 versus untreated cells)
020406080
100120140160180200
DEX
BNP
( o
f con
trol)
C TNF-120572 TNF-120572 + DEX
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowast
lowast
lowast
Figure 9 Dexamethasone effect on BNP expression BEAS-2B cellswere treatedwith 1 120583Mdexamethasone (DEX) and 40 ngmLTNF-120572alone or in combination with DEX for 24 h Histograms show BNPgene expression at mRNA (gray bars) and protein (black bars) levelAll histograms indicate means plusmn SD of three different cultures eachone tested in quadruplicate and expressed as percentage of control(lowast119875 lt 001 lowastlowast119875 lt 0001 and lowastlowastlowast119875 lt 00001 versus Controls) CControls (untreated cells)
and in vitro models of lung injury [18] and the therapeu-tical relevance of these experimental observations has beenshown in intensive care patients without heart disease whereintravenous ANP infusion diminished pulmonary vascularpermeability and pulmonary edema [18]
Regarding AQPs we demonstrated that BEAS-2B cellsexpress AQP1 and AQP5 and that TNF-120572 directly down-regulates their expression at mRNA and protein levelsRegarding AQP1 also an additional BNP-mediated TNF-120572involvement can be proposed In fact AQP1 down-regulationwas observed with BNP treatment at the dose accumulatedin culture medium after 40 ngmL TNF-120572 treatment Themechanisms that underlie AQPs regulation are of consider-able interest Several AQPs has recently been demonstratedto undergo complex regulation [27] Our results show thatAQP1 and APQ5 are subject to inflammation regulationadding an important piece of information in this contextOur results are in agreement with other studies describinga marked reduction in the expression levels of AQP1 andAQP5 inmouse lung [30] andmouse lung epithelial cells [31]The biological significance of the observed TNF-120572-inducedAQP1 and AQP5 down-regulation is at present unclear Ithas been recently suggested that the decrease in both AQPsmay play an important role in the development of lung edema[52] which is known to be related with fluid retention Wetherefore speculate that TNF-120572-induced AQP1 and AQP5down-regulation might play a role also in the excess of fluidaccumulation related to major surgical procedures
5 Conclusions
In conclusion our results provided evidence that in humanbronchial epithelial cells BEAS-2B TNF-120572 selectively mod-ulates mRNA and protein expression levels of different
12 Mediators of Inflammation
molecules involved in body fluid homeostasis ANP BNPand their receptor NPR-1 AQP1 and AQP5 Moreoverour data pointed out that the changes specifically affectingNPs expression occur independently from hemodynamicinfluences and that inflammation should be considered anextracardiac cause of BNP elevation adding an importantpiece of information in the novel area of study regarding NPsand inflammation In addition we demonstrated that such amodulation occurs via different involvement of NK-120581B andMAPKs signaling pathways Finally we suggested inflamma-tion among the mechanisms involved in the regulation ofAQP1 and AQP5 Therefore in this exploratory study wespeculate that TNF-120572 might be involved in postoperative-fluid retention related to major surgery
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgment
The authors thank Mrs Roberta Frosini for the excellenttechnical assistance
References
[1] E Itobi M Stroud and M Elia ldquoImpact of oedema onrecovery after major abdominal surgery and potential valueof multifrequency bioimpedance measurementsrdquo The BritishJournal of Surgery vol 93 no 3 pp 354ndash361 2006
[2] J A Lowell C Schifferdecker D F Driscoll P N Benottiand B R Bistrian ldquoPostoperative fluid overload not a benignproblemrdquo Critical Care Medicine vol 18 no 7 pp 728ndash7331990
[3] A M Moslashller T Pedersen P-E Svendsen and A EngquistldquoPerioperative risk factors in elective pneumonectomy theimpact of excess fluid balancerdquo European Journal of Anaesthe-siology vol 19 no 1 pp 57ndash62 2002
[4] E Bennett-Guerrero D E Feierman G R Barclay et alldquoPreoperative and intraoperative predictors of postoperativemorbidity poor graft function and early rejection in 190patients undergoing liver transplantationrdquo Archives of Surgeryvol 136 no 10 pp 1177ndash1183 2001
[5] J P Desborough ldquoThe stress response to trauma and surgeryrdquoThe British Journal of Anaesthesia vol 85 no 1 pp 109ndash1172000
[6] H Tsujimoto S Ono T Majima et al ldquoDifferential toll-likereceptor expression after ex vivo lipopolysaccharide exposurein patients with sepsis and following surgical stressrdquo ClinicalImmunology vol 119 no 2 pp 180ndash187 2006
[7] C Evans C Galustian D Kumar et al ldquoImpact of surgeryon immunologic function comparison between minimallyinvasive techniques and conventional laparotomy for surgi-cal resection of colorectal tumorsrdquo The American Journal ofSurgery vol 197 no 2 pp 238ndash245 2009
[8] F Catena L Ansaloni A Avanzolini et al ldquoSystemic cytokineresponse after emergency and elective surgery for colorectalcarcinomardquo International Journal of Colorectal Disease vol 24no 7 pp 803ndash808 2009
[9] R Sarbinowski S Arvidsson M Tylman T Oresland andA Bengtsson ldquoPlasma concentration of procalcitonin andsystemic inflammatory response syndrome after colorectalsurgeryrdquo Acta Anaesthesiologica Scandinavica vol 49 no 2 pp191ndash196 2005
[10] P Sheeran and G M Hall ldquoCytokines in anaesthesiardquo TheBritish Journal of Anaesthesia vol 78 no 2 pp 201ndash219 1997
[11] L Cagini R Capozzi V Tassi et al ldquoFluid and electrolytebalance after major thoracic surgery by bioimpedance andendocrine evaluationrdquo European Journal of Cardio-ThoracicSurgery vol 40 no 2 pp e71ndashe76 2011
[12] T Omland and T Hagve ldquoNatriuretic peptides physiologic andanalytic considerationsrdquo Heart Failure Clinics vol 5 no 4 pp471ndash487 2009
[13] L R Potter A R Yoder D R Flora L K Antos and DM Dickey ldquoNatriuretic peptides their structures receptorsphysiologic functions and therapeutic applicationsrdquo Handbookof Experimental Pharmacology vol 191 pp 341ndash366 2009
[14] T G Flynn M L de Bold and A J de Bold ldquoThe aminoacid sequence of an atrial peptide with potent diuretic andnatriuretic propertiesrdquo Biochemical and Biophysical ResearchCommunications vol 117 no 3 pp 859ndash865 1983
[15] A J de Bold ldquoAtrial natriuretic factor a hormone produced bythe heartrdquo Science vol 230 pp 767ndash770 1985
[16] K Kangawa Y Tawaragi S Oikawa et al ldquoIdentification of rat 120574atrial natriuretic polypeptide and characterization of the cDNAencoding its precursorrdquo Nature vol 312 no 5990 pp 152ndash1551984
[17] T Sudoh K Kangawa N Minamino and H Matsuo ldquoA newnatriuretic peptide in porcine brainrdquo Nature vol 332 no 6159pp 78ndash81 1988
[18] M Kuhn ldquoEndothelial actions of atrial and B-type natriureticpeptidesrdquo The British Journal of Pharmacology vol 166 no 2pp 522ndash531 2012
[19] A J de Bold ldquoCardiac natriuretic peptides gene expression andsecretion in inflammationrdquo Journal of Investigative Medicinevol 57 pp 29ndash32 2009
[20] S P DrsquoSouza M Davis and G F Baxter ldquoAutocrine andparacrine actions of natriuretic peptides in the heartrdquo Pharma-cology andTherapeutics vol 101 no 2 pp 113ndash129 2004
[21] M Brueckmann G Huhle S Lang et al ldquoPrognostic valueof plasma N-terminal pro-brain natriuretic peptide in patientswith severe sepsisrdquoCirculation vol 112 no 4 pp 527ndash534 2005
[22] J Charpentier C E Luyt Y Fulla et al ldquoBrain natriureticpeptide a marker of myocardial dysfunction and prognosisduring severe sepsisrdquo Critical Care Medicine vol 32 no 3 pp660ndash665 2004
[23] R Shor Y Rozenman A Bolshinsky et al ldquoBNP in septicpatients without systolic myocardial dysfunctionrdquo EuropeanJournal of Internal Medicine vol 17 no 8 pp 536ndash540 2006
[24] T Inoue M Kawai T Nakane et al ldquoInfluence of low-gradeinflammation on plasma B-type natriuretic peptide levelsrdquoInternal Medicine vol 49 no 24 pp 2659ndash2668 2010
[25] P Agre L S King M Yasui et al ldquoAquaporin water channelsmdashfrom atomic structure to clinical medicinerdquo Journal of Physiol-ogy vol 542 no 1 pp 3ndash16 2002
[26] P Agre and D Kozono ldquoAquaporin water channels molecularmechanisms for human diseasesrdquo FEBS Letters vol 555 no 1pp 72ndash78 2003
Mediators of Inflammation 13
[27] J M Carbrey and P Agre ldquoDiscovery of the aquaporins anddevelopment of the fieldrdquo Handbook of Experimental Pharma-cology vol 190 pp 3ndash28 2009
[28] O Bloch and G T Manley ldquoThe role of aquaporin-4 in cerebralwater transport and edemardquo Neurosurgical Focus vol 22 no 5article E3 2007
[29] J Li M Xu Q Fan et al ldquoTanshinone IIA ameliorates seawaterexposure-induced lung injury by inhibiting aquaporins (AQP)1 and AQP5 expression in lungrdquo Respiratory Physiology andNeurobiology vol 176 no 1-2 pp 39ndash49 2011
[30] J E Towne K S Harrod C M Krane and A G MenonldquoDecreased expression of aquaporin (AQP)1 and AQP5 inmouse lung after acute viral infectionrdquoThe American Journal ofRespiratory Cell and Molecular Biology vol 22 no 1 pp 34ndash442000
[31] J E Towne C M Krane C J Bachurski and A G MenonldquoTumor necrosis factor-120572 inhibits aquaporin 5 expression inmouse lung epithelial cellsrdquoThe Journal of Biological Chemistryvol 276 no 22 pp 18657ndash18664 2001
[32] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001
[33] K Verhelst I Carpentier and R Beyaert ldquoRegulation of TNF-induced NF-120581B activation by different cytoplasmic ubiquitina-tion eventsrdquo Cytokine and Growth Factor Reviews vol 22 no5-6 pp 277ndash286 2011
[34] L Qian Y Shen J C Chen et al ldquo3D-QSAR and dockingstudies of quinazoline derivatives with the inhibatory activitytoward NF-120581BrdquoQSAR and Combinatorial Science vol 27 no 8pp 984ndash995 2008
[35] P D NrsquoGuessan S Hippenstiel M O Etouem et al ldquoStrepto-coccus pneumoniae induced p38MAPK- andNF-120581B-dependentCOX-2 expression in human lung epitheliumrdquo The AmericanJournal of PhysiologymdashLung Cellular and Molecular Physiologyvol 290 no 6 pp L1131ndashL1138 2006
[36] B Schmeck K Moog J Zahlten et al ldquoStreptococcus pneumo-niae induced c-Jun-N-terminal kinase- andAP-I-dependent IL-8 release by lung epithelial BEAS-2B cellsrdquoRespiratory Researchvol 7 article 98 2006
[37] M GMatera L Calzetta D Passeri et al ldquoEpithelium integrityis crucial for the relaxant activity of brain natriuretic peptide inhuman isolated bronchirdquo The British Journal of Pharmacologyvol 163 no 8 pp 1740ndash1754 2011
[38] R QuirionM Dalpe A de Lean J Gutkowska M Cantin andJ Genest ldquoAtrial natriuretic factor (ANF) binding sites in brainand related structuresrdquoPeptides vol 5 no 6 pp 1167ndash1172 1984
[39] M B Pamnani D L Clough J S Chen W T Link andF J Haddy ldquoEffects of rat atrial extract on sodium transportand blood pressure in the ratrdquo Proceedings of the Society forExperimental Biology and Medicine vol 176 no 2 pp 123ndash1311984
[40] T R Traynor and S M OrsquoGrady ldquoBrain natriuretic peptidestimulates K andCl secretion across porcine distal colon epithe-liumrdquoTheAmerican Journal of PhysiologymdashCell Physiology vol260 no 4 pp C750ndashC755 1991
[41] R Rollın A Mediero M Roldan-Pallares A Fernandez-Cruzand R Fernandez-Durango ldquoNatriuretic peptide system in thehuman retinardquoMolecular Vision vol 10 pp 15ndash22 2004
[42] M Harada H Itoh O Nakagawa et al ldquoSignificance ofventricular myocytes and nonmyocytes interaction during car-diocyte hypertrophy evidence for endothelin-1 as a paracrinehypertrophic factor from cardiac nonmyocytesrdquo Circulationvol 96 no 10 pp 3737ndash3744 1997
[43] K Kuwahara Y Saito M Harada et al ldquoInvolvement ofcardiotrophin-1 in cardiac myocyte-nonmyocyte interactionsduring hypertrophy of rat cardiac myocytes in vitrordquo Circula-tion vol 100 no 10 pp 1116ndash1124 1999
[44] E Harada O Nakagawa M Yoshimura et al ldquoEffect ofinterleukin-1120573 on cardiac hypertrophy and production of natri-uretic peptides in rat cardiocyte culturerdquo Journal of Molecularand Cellular Cardiology vol 31 no 11 pp 1997ndash2006 1999
[45] V Chiurchiu V Izzi F DrsquoAquilio F Carotenuto P Di Nardoand P M Baldini ldquoBrain natriuretic peptide (BNP) regulatesthe production of inflammatory mediators in human THP-1macrophagesrdquo Regulatory Peptides vol 148 no 1ndash3 pp 26ndash322008
[46] N S Holden C F Rider M J Bell et al ldquoEnhancement ofinflammatory mediator release by 1205732-adrenoceptor agonists inairway epithelial cells is reversed by glucocorticoid actionrdquoTheBritish Journal of Pharmacology vol 160 no 2 pp 410ndash4202010
[47] V S Fang C C Juan J G S Won and L T Ho ldquoFurther studyof aldosterone secretion-inhibitory factor and brain natriureticpeptide on cortisol production of Guinea pig zona fasciculatacellsrdquo Chinese Journal of Physiology vol 43 no 4 pp 141ndash1472000
[48] G Hellermann X Kong J Gunnarsdottir et al ldquoMechanismof bronchoprotective effects of a novel natriuretic hormonepeptiderdquo Journal of Allergy and Clinical Immunology vol 113no 1 pp 79ndash85 2004
[49] K K Ma T Ogawa and A J de Bold ldquoSelective upregulationof cardiac brain natriuretic peptide at the transcriptional andtranslational levels by pro-inflammatory cytokines and by con-ditionedmedium derived frommixed lymphocyte reactions viap38 MAP kinaserdquo Journal of Molecular and Cellular Cardiologyvol 36 no 4 pp 505ndash513 2004
[50] T Ogawa J P Veinot M L K de Bold T Georgalis and AJ De Bold ldquoAngiotensin II receptor antagonism reverts theselective cardiac BNP upregulation and secretion observed inmyocarditisrdquo The American Journal of PhysiologymdashHeart andCirculatory Physiology vol 294 no 6 pp H2596ndashH2603 2008
[51] B J Carver E J Plosa A M Stinnett T S Blackwell andL S Prince ldquoInteractions between NF-120581B and SP3 connectinflammatory signaling with reduced FGF-10 expressionrdquo TheJournal of Biological Chemistry vol 288 no 21 pp 15318ndash153252013
[52] C Dong G Wang B Li et al ldquoAnti-asthmatic agents alleviatepulmonary edema by upregulating AQP1 and AQP5 expressionin the lungs of mice with OVA-induced asthmardquo RespiratoryPhysiology and Neurobiology vol 181 no 1 pp 21ndash28 2012
Figure 4 TNF-120572 modulation of protein levels of BNP and NPR-1 may require activation of NF-120581B pathway BEAS-2B cells were treated10 ngmL TNF-120572 alone or in combination with the inhibitor QNZ (10120583M) for 24 h ((a) (b)) BNP ((c) (d)) NPR-1 gene expression at mRNA((a) (c)) and protein ((b) (d)) level and (e) Phospho-I120581B-120572 protein level Western blots were obtained by using the specific rabbit polyclonalor monoclonal AbsThe blots were stripped of the bound Ab and reprobed with mouse anti-120573-actin to confirm equal loading Western blotsare representative of three separate experiments All histograms indicatemeansplusmnSDof three different cultures each one tested in quadruplicateand expressed as percentage of control (lowastlowast119875 lt 001 and lowastlowastlowastlowast119875 lt 00001 versus Controls) (∘∘119875 lt 001 versus QNZ) C Controls (untreatedcells)
rat cardiomyocytes have shown that not only hemodynamicfactors but also neurohumoral factors activated during heartfailure such as angiotensin II endothelin and cytokinescause BNP secretion [42ndash44] In addition in patients withcardiovascular diseases plasma BNP levels have been shownto be also affected by low-grade inflammation [24] and aselective increase inBNPplasma levels has been proposed as ageneral feature of inflammation [19]The lack on any apparentTNF-120572 dose-depending increase in BNP gene expression thatwe observed in our system could be due to an autocrinenegative feedback action mediated by BNP itself followingbinding to the NPR-1 receptor expressed in BEAS-2B cells
The biological role of BNPup-regulation after TNF-120572 treatment remains to be elucidated However since it
has been demonstrated that BNP regulates the productionof major inflammatory molecules such as IL-12 IL-10leukotriene B and prostaglandin E2 in human macrophages[45] and that BEAS-2B are able to release inflammatorymolecules [46] it could be reasonable to assume that theobserved BNP increase after TNF-120572 treatment may stimu-late the production and release of such cytokines also fromhuman epithelial bronchial cells In addition in in vitrostudies a potent BNP inhibitory action on the productionof the glucocorticoid anti-inflammatory hormone cortisolhas been described [47] Therefore BNP could be con-sidered a proinflammatory molecule by inducing cytokinesexpression in target cells and inhibiting the production ofanti-inflammatory hormones Therefore the observed BNP
8 Mediators of Inflammation
0
50
100
150
200
250BN
P (
of c
ontro
l)
C
TNF SB
SB+
TNF SP
SP+
TNF
U-0
126
U-0126+
TNF
lowastlowast
lowastlowast
∘∘
sect
(a)
020406080
100120140160180
BNP
( o
f con
trol)
C
TNF SB
SB+
TNF SP
SP+
TNF
U-0
126
U-0126+
TNF
lowastlowast
lowastlowast
∘∘∘
sectsect
(b)
0
20
40
60
80
100
120
140
160
C
TNF SB
SB+
TNF SP
SP+
TNF
U-0
126
U-0126+
TNF
lowastlowastlowast
lowastlowastlowast
lowast
lowast
sect
NPR
-1(
of c
ontro
l)
(c)
+
SB 203580
+
SP 600125 U-0126
+ActinTNF-120572
NPR-1
minus minus minus minus +
(d)
SB 203580
Actin
P-p38 MAPK
TNF-120572 + +
minus ++
minusminus
minus
(e)
Figure 5 TNF-120572 modulation of BNP and NPR-1 expression may require activation of MAPKs pathway BEAS-2B cells were treated with10 ngmL TNF-120572 alone or in combination with 10 120583M SB 203580 (p38 MAPK inhibitor) SP 600125 (JNK 12 inhibitor) or U-0126 (ERK12 inhibitor) for 24 h Panels show ((a) (b)) BNP ((c) (d)) NPR-1 gene expression at mRNA ((a) (c)) and protein ((b) (d)) level and (e)Phospho-p38 MAPK protein level Western blots were obtained by using the specific rabbit polyclonal or monoclonal Abs The blots werestripped of the bound Ab and reprobed with mouse anti-120573-actin to confirm equal loading Western blots are representative of three separateexperiments All histograms indicate means plusmn SD of three different cultures each one tested in quadruplicate and expressed as percentage ofcontrol (lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 versus Controls) (∙∙119875 lt 001 versus SB) (sect119875 lt 005 sectsect
119875 lt 001 versus SP) (∘∘119875 lt 001∘∘∘
119875 lt 0001 versus U-0126) C Controls (untreated cells)
up-regulation after TNF-120572 treatment could concur in poten-tiating an inflammatory statusThe increased production andrelease of specific cytokines causing systemic changes knownas the acute phase response [5 8 10] induced by TNF-120572 maythus also occur via BNP up-regulation
Recently a BNP rise has been observed in patientsundergoing pulmonary lobectomy for lung cancer [11] Inparticular the patients none of whom developed signs orsymptoms of heart failure during the postoperative period
showed an early significant increase on day 1 immediatelyafter surgery of the plasma concentrations of BNP whichwas followed on the postoperative day 2 by a significantweight gain correlated with large volumes of fluids accumu-lation despite a negative intraoperative fluid balance andperoperative strict fluid restriction [11] We suggest thatsuch an increase could be at least in part explained bythe ability of proinflammatory cytokines such as TNF-120572 toupregulate BNP gene expression and secretion and that this
Mediators of Inflammation 9
0
20
40
60
80
100
120
0 10 20 40TNF-120572 (ngmL)
AQP1
( o
f con
trol)
lowast
lowastlowastlowast
lowastlowastlowast
(a)
Actin 0 10 20 40
AQP1
TNF-120572 (ngmL)
(b)
0
20
40
60
80
100
120
AQP5
( o
f con
trol)
0 10 20 40
TNF-120572 (ngmL)
lowastlowast
lowastlowastlowastlowast
(c)
AQP5
Actin 0 10 20 40TNF-120572 (ngmL)
(d)
Figure 6 TNF-120572 decreases AQP1 and AQP5 expression in BEAS 2B cells BEAS-2B cells were treated with 10 20 and 40 ngmL TNF-120572 for24 h Gene expression of AQP1 ((a) (b)) and AQP5 ((c) (d)) at mRNA ((a) (c)) and protein ((b) (d)) level Western blots were obtained byusing the specific rabbit polyclonal Abs The blots were stripped of the bound Ab and reprobed with mouse anti-120573-actin to confirm equalloading Western blots are representative of three separate experiments All histograms indicate means plusmn SD of three different cultures eachone tested in quadruplicate and expressed as percentage of control (lowast119875 lt 001 lowastlowast119875 lt 0001 and lowastlowastlowast119875 lt 00001 versus untreated cells)
up-regulation could enhance the major surgery-related earlyhyperinflammatory response essential for tissue repair andhost defense [5]
The observed dexamethasone capability to completelyrevert TNF-120572-induced BNP up-regulation suggests that thisanti-inflammatory glucocorticoid may prevent BNP-relatedeffects
Regarding ANP the observed significant down-regula-tion of mRNA and protein levels after 10 20 ngmL TNF-120572treatment suggests that also this condition could affect localinflammation status In fact ANP administration determinesanti-inflammatory effects in airway epithelial cells [48] play-ing important roles in modulating inflammatory responseIn consideration of such ANP anti-inflammatory action wespeculate that the observed TNF-120572-induced ANP down-regulation could concur in enhancing an inflammatory sta-tus Differently from the above reported doses 40 ngmLTNF-120572 administration induced ANP up-regulation eventhough not statistically significant We suggest that this resultmight be due to a direct BNP involvement In fact a markedANP up-regulation was observed after BNP treatment at the
dose accumulated in culture medium after 40 ngmL TNF-120572treatment
The observed selectivemodulation inANP and BNP geneexpression after TNF-120572 treatment in line with other studiesin different cell models [19 49 50] could be related to adifferent regulation mechanism at transcriptional level assuggested by our observation on the different involvementof NF-120581B and MAPKs signaling pathways In particular wedemonstrated that ANP down-regulation TNF-120572-mediatedoccurs via NF-120581B activation pathway whereas BNP modula-tion requires ERK 12 activation pathway NF-120581B involvementin TNF-120572-induced ANP down-regulation is not opposed tothe proinflammatory role of this transcription factor beingANP a molecule that exerts an anti-inflammatory effect inairway epithelial cells [48] Regarding themechanism linkingNF-120581B to the downregulation of ANP gene expression wesuggest that it could be similar to that recently proposedfor Fibroblast Growth Factor 10 (FGF-10) where NF-120581Bactivationmay lead to reduced gene expression by recruitinginhibitory factors to specific gene promoters [51]
10 Mediators of Inflammation
0
20
40
60
80
100
120
C BAY 10 20 40
AQP1
( o
f con
trol)
TNF-120572 (ngmL)
TNF-120572TNF-120572 + BAY
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowast
(a)
BAY
AQP1Actin
0 0 10 20 40 10 20 40TNF-120572 (ngmL)minus + +minus minus minus + +
(b)
0
20
40
60
80
100
120
AQP5
( o
f con
trol)
C BAY 10 20 40
TNF-120572 (ngmL)
TNF-120572TNF-120572 + BAY
∘∘∘∘∘∘
∘∘∘
lowastlowastlowastlowast
lowastlowast
(c)
AQP5Actin
0 0 10 20 40 10 20 40TNF-120572 (ngmL)BAY minus + +minus minus minus + +
(d)
Figure 7 TNF-120572 downregulation of AQP1 and AQP5 expression may require activation of NF-120581B pathwayBEAS-2B cells were treated with10 20 and 40 ngmL TNF-120572 alone or in combination with NF-120581B inhibitor BAY 11-7082 (1 120583M) for 24 h Gene expression of AQP1 ((a) (b))and AQP5 ((c) (d)) at mRNA ((a) (c)) and protein ((b) (d)) level Western blots were obtained by using the specific rabbit polyclonal AbsThe blots were stripped of the bound Ab and reprobed with mouse anti-120573-actin to confirm equal loading Western blots are representative ofthree separate experiments All histograms indicate means plusmn SD of three different cultures each one tested in quadruplicate and expressed aspercentage of control (lowast119875 lt 001 lowastlowast119875 lt 0001 lowastlowastlowast119875 lt 00001 versus Controls and ∘∘∘119875 lt 00001 versus BAY) C Controls (untreated cells)
ANP and BNP biological functions occur through theirbinding to the same NPR-1 [13] Here we demonstratedfor the first time to our knowledge TNF-120572 capability todownregulateNPR-1mRNAandprotein expression in BEAS-2B cells and that this regulation occurs via MAPKs signalingpathway activation The consequence of the complex TNF-120572-induced NPs and NPR-1 observed modulation associatedwith their binding to the same receptor on BEAS-2B cellsmakes it difficult to predict what would be the net resultingeffect both in vitro and in vivo in relation also to the presenceof a possible autocrine regulation by NPs andor to the effectsof different cytokines activated by both BNP and TNF-120572We hypothesize that the TNF-120572-induced downregulation of
NPR-1 paralleled by an up-regulation of BNP could alsolead to a paracrine action of this peptide possibly on themicrovascular endothelium of the lung where NPR-1 isdensely expressed [18] In this context we speculate that theinduction of local modification on BNP or ANP by TNF-120572 inhuman bronchial epithelial cells could alter the barrier per-meability of the pulmonary microcirculation Hence TNF-120572 acting on epithelial cells could also indirectly contributeto endothelial cells barrier dysfunction and permeability byenhancing the local production of proinflammatory BNPand by down-regulating the anti-inflammatory expressionof ANP Besides exogenous synthetic ANP has been shownto protect from endothelial barrier dysfunction in in vivo
Mediators of Inflammation 11
0
50
100
150
200
250
300
0 001
AN
P (
of c
ontro
l)
BNP (nM)
lowast
(a)
0
50
100
150
0 001BNP (nM)
NPR
-1(
of c
ontro
l)
(b)
0
20
40
60
80
100
120
0 001
BNP (nM)
lowast
AQP1
( o
f con
trol)
(c)
0
20
40
60
80
100
120
0 001
BNP (nM)
AQP5
( o
f con
trol)
(d)
Figure 8 BNP effect on ANP NPR-1 AQP1 and AQP5 expressionBEAS-2B cells were treated with 001 nM BNP for 24 h Histograms show(a) ANP (b) NPR-1 (c) AQP1 and (d) AQP5 gene expression at mRNA level All histograms indicate means plusmn SD of three different cultureseach one tested in quadruplicate and expressed as percentage of control (lowast119875 lt 005 versus untreated cells)
020406080
100120140160180200
DEX
BNP
( o
f con
trol)
C TNF-120572 TNF-120572 + DEX
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowast
lowast
lowast
Figure 9 Dexamethasone effect on BNP expression BEAS-2B cellswere treatedwith 1 120583Mdexamethasone (DEX) and 40 ngmLTNF-120572alone or in combination with DEX for 24 h Histograms show BNPgene expression at mRNA (gray bars) and protein (black bars) levelAll histograms indicate means plusmn SD of three different cultures eachone tested in quadruplicate and expressed as percentage of control(lowast119875 lt 001 lowastlowast119875 lt 0001 and lowastlowastlowast119875 lt 00001 versus Controls) CControls (untreated cells)
and in vitro models of lung injury [18] and the therapeu-tical relevance of these experimental observations has beenshown in intensive care patients without heart disease whereintravenous ANP infusion diminished pulmonary vascularpermeability and pulmonary edema [18]
Regarding AQPs we demonstrated that BEAS-2B cellsexpress AQP1 and AQP5 and that TNF-120572 directly down-regulates their expression at mRNA and protein levelsRegarding AQP1 also an additional BNP-mediated TNF-120572involvement can be proposed In fact AQP1 down-regulationwas observed with BNP treatment at the dose accumulatedin culture medium after 40 ngmL TNF-120572 treatment Themechanisms that underlie AQPs regulation are of consider-able interest Several AQPs has recently been demonstratedto undergo complex regulation [27] Our results show thatAQP1 and APQ5 are subject to inflammation regulationadding an important piece of information in this contextOur results are in agreement with other studies describinga marked reduction in the expression levels of AQP1 andAQP5 inmouse lung [30] andmouse lung epithelial cells [31]The biological significance of the observed TNF-120572-inducedAQP1 and AQP5 down-regulation is at present unclear Ithas been recently suggested that the decrease in both AQPsmay play an important role in the development of lung edema[52] which is known to be related with fluid retention Wetherefore speculate that TNF-120572-induced AQP1 and AQP5down-regulation might play a role also in the excess of fluidaccumulation related to major surgical procedures
5 Conclusions
In conclusion our results provided evidence that in humanbronchial epithelial cells BEAS-2B TNF-120572 selectively mod-ulates mRNA and protein expression levels of different
12 Mediators of Inflammation
molecules involved in body fluid homeostasis ANP BNPand their receptor NPR-1 AQP1 and AQP5 Moreoverour data pointed out that the changes specifically affectingNPs expression occur independently from hemodynamicinfluences and that inflammation should be considered anextracardiac cause of BNP elevation adding an importantpiece of information in the novel area of study regarding NPsand inflammation In addition we demonstrated that such amodulation occurs via different involvement of NK-120581B andMAPKs signaling pathways Finally we suggested inflamma-tion among the mechanisms involved in the regulation ofAQP1 and AQP5 Therefore in this exploratory study wespeculate that TNF-120572 might be involved in postoperative-fluid retention related to major surgery
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgment
The authors thank Mrs Roberta Frosini for the excellenttechnical assistance
References
[1] E Itobi M Stroud and M Elia ldquoImpact of oedema onrecovery after major abdominal surgery and potential valueof multifrequency bioimpedance measurementsrdquo The BritishJournal of Surgery vol 93 no 3 pp 354ndash361 2006
[2] J A Lowell C Schifferdecker D F Driscoll P N Benottiand B R Bistrian ldquoPostoperative fluid overload not a benignproblemrdquo Critical Care Medicine vol 18 no 7 pp 728ndash7331990
[3] A M Moslashller T Pedersen P-E Svendsen and A EngquistldquoPerioperative risk factors in elective pneumonectomy theimpact of excess fluid balancerdquo European Journal of Anaesthe-siology vol 19 no 1 pp 57ndash62 2002
[4] E Bennett-Guerrero D E Feierman G R Barclay et alldquoPreoperative and intraoperative predictors of postoperativemorbidity poor graft function and early rejection in 190patients undergoing liver transplantationrdquo Archives of Surgeryvol 136 no 10 pp 1177ndash1183 2001
[5] J P Desborough ldquoThe stress response to trauma and surgeryrdquoThe British Journal of Anaesthesia vol 85 no 1 pp 109ndash1172000
[6] H Tsujimoto S Ono T Majima et al ldquoDifferential toll-likereceptor expression after ex vivo lipopolysaccharide exposurein patients with sepsis and following surgical stressrdquo ClinicalImmunology vol 119 no 2 pp 180ndash187 2006
[7] C Evans C Galustian D Kumar et al ldquoImpact of surgeryon immunologic function comparison between minimallyinvasive techniques and conventional laparotomy for surgi-cal resection of colorectal tumorsrdquo The American Journal ofSurgery vol 197 no 2 pp 238ndash245 2009
[8] F Catena L Ansaloni A Avanzolini et al ldquoSystemic cytokineresponse after emergency and elective surgery for colorectalcarcinomardquo International Journal of Colorectal Disease vol 24no 7 pp 803ndash808 2009
[9] R Sarbinowski S Arvidsson M Tylman T Oresland andA Bengtsson ldquoPlasma concentration of procalcitonin andsystemic inflammatory response syndrome after colorectalsurgeryrdquo Acta Anaesthesiologica Scandinavica vol 49 no 2 pp191ndash196 2005
[10] P Sheeran and G M Hall ldquoCytokines in anaesthesiardquo TheBritish Journal of Anaesthesia vol 78 no 2 pp 201ndash219 1997
[11] L Cagini R Capozzi V Tassi et al ldquoFluid and electrolytebalance after major thoracic surgery by bioimpedance andendocrine evaluationrdquo European Journal of Cardio-ThoracicSurgery vol 40 no 2 pp e71ndashe76 2011
[12] T Omland and T Hagve ldquoNatriuretic peptides physiologic andanalytic considerationsrdquo Heart Failure Clinics vol 5 no 4 pp471ndash487 2009
[13] L R Potter A R Yoder D R Flora L K Antos and DM Dickey ldquoNatriuretic peptides their structures receptorsphysiologic functions and therapeutic applicationsrdquo Handbookof Experimental Pharmacology vol 191 pp 341ndash366 2009
[14] T G Flynn M L de Bold and A J de Bold ldquoThe aminoacid sequence of an atrial peptide with potent diuretic andnatriuretic propertiesrdquo Biochemical and Biophysical ResearchCommunications vol 117 no 3 pp 859ndash865 1983
[15] A J de Bold ldquoAtrial natriuretic factor a hormone produced bythe heartrdquo Science vol 230 pp 767ndash770 1985
[16] K Kangawa Y Tawaragi S Oikawa et al ldquoIdentification of rat 120574atrial natriuretic polypeptide and characterization of the cDNAencoding its precursorrdquo Nature vol 312 no 5990 pp 152ndash1551984
[17] T Sudoh K Kangawa N Minamino and H Matsuo ldquoA newnatriuretic peptide in porcine brainrdquo Nature vol 332 no 6159pp 78ndash81 1988
[18] M Kuhn ldquoEndothelial actions of atrial and B-type natriureticpeptidesrdquo The British Journal of Pharmacology vol 166 no 2pp 522ndash531 2012
[19] A J de Bold ldquoCardiac natriuretic peptides gene expression andsecretion in inflammationrdquo Journal of Investigative Medicinevol 57 pp 29ndash32 2009
[20] S P DrsquoSouza M Davis and G F Baxter ldquoAutocrine andparacrine actions of natriuretic peptides in the heartrdquo Pharma-cology andTherapeutics vol 101 no 2 pp 113ndash129 2004
[21] M Brueckmann G Huhle S Lang et al ldquoPrognostic valueof plasma N-terminal pro-brain natriuretic peptide in patientswith severe sepsisrdquoCirculation vol 112 no 4 pp 527ndash534 2005
[22] J Charpentier C E Luyt Y Fulla et al ldquoBrain natriureticpeptide a marker of myocardial dysfunction and prognosisduring severe sepsisrdquo Critical Care Medicine vol 32 no 3 pp660ndash665 2004
[23] R Shor Y Rozenman A Bolshinsky et al ldquoBNP in septicpatients without systolic myocardial dysfunctionrdquo EuropeanJournal of Internal Medicine vol 17 no 8 pp 536ndash540 2006
[24] T Inoue M Kawai T Nakane et al ldquoInfluence of low-gradeinflammation on plasma B-type natriuretic peptide levelsrdquoInternal Medicine vol 49 no 24 pp 2659ndash2668 2010
[25] P Agre L S King M Yasui et al ldquoAquaporin water channelsmdashfrom atomic structure to clinical medicinerdquo Journal of Physiol-ogy vol 542 no 1 pp 3ndash16 2002
[26] P Agre and D Kozono ldquoAquaporin water channels molecularmechanisms for human diseasesrdquo FEBS Letters vol 555 no 1pp 72ndash78 2003
Mediators of Inflammation 13
[27] J M Carbrey and P Agre ldquoDiscovery of the aquaporins anddevelopment of the fieldrdquo Handbook of Experimental Pharma-cology vol 190 pp 3ndash28 2009
[28] O Bloch and G T Manley ldquoThe role of aquaporin-4 in cerebralwater transport and edemardquo Neurosurgical Focus vol 22 no 5article E3 2007
[29] J Li M Xu Q Fan et al ldquoTanshinone IIA ameliorates seawaterexposure-induced lung injury by inhibiting aquaporins (AQP)1 and AQP5 expression in lungrdquo Respiratory Physiology andNeurobiology vol 176 no 1-2 pp 39ndash49 2011
[30] J E Towne K S Harrod C M Krane and A G MenonldquoDecreased expression of aquaporin (AQP)1 and AQP5 inmouse lung after acute viral infectionrdquoThe American Journal ofRespiratory Cell and Molecular Biology vol 22 no 1 pp 34ndash442000
[31] J E Towne C M Krane C J Bachurski and A G MenonldquoTumor necrosis factor-120572 inhibits aquaporin 5 expression inmouse lung epithelial cellsrdquoThe Journal of Biological Chemistryvol 276 no 22 pp 18657ndash18664 2001
[32] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001
[33] K Verhelst I Carpentier and R Beyaert ldquoRegulation of TNF-induced NF-120581B activation by different cytoplasmic ubiquitina-tion eventsrdquo Cytokine and Growth Factor Reviews vol 22 no5-6 pp 277ndash286 2011
[34] L Qian Y Shen J C Chen et al ldquo3D-QSAR and dockingstudies of quinazoline derivatives with the inhibatory activitytoward NF-120581BrdquoQSAR and Combinatorial Science vol 27 no 8pp 984ndash995 2008
[35] P D NrsquoGuessan S Hippenstiel M O Etouem et al ldquoStrepto-coccus pneumoniae induced p38MAPK- andNF-120581B-dependentCOX-2 expression in human lung epitheliumrdquo The AmericanJournal of PhysiologymdashLung Cellular and Molecular Physiologyvol 290 no 6 pp L1131ndashL1138 2006
[36] B Schmeck K Moog J Zahlten et al ldquoStreptococcus pneumo-niae induced c-Jun-N-terminal kinase- andAP-I-dependent IL-8 release by lung epithelial BEAS-2B cellsrdquoRespiratory Researchvol 7 article 98 2006
[37] M GMatera L Calzetta D Passeri et al ldquoEpithelium integrityis crucial for the relaxant activity of brain natriuretic peptide inhuman isolated bronchirdquo The British Journal of Pharmacologyvol 163 no 8 pp 1740ndash1754 2011
[38] R QuirionM Dalpe A de Lean J Gutkowska M Cantin andJ Genest ldquoAtrial natriuretic factor (ANF) binding sites in brainand related structuresrdquoPeptides vol 5 no 6 pp 1167ndash1172 1984
[39] M B Pamnani D L Clough J S Chen W T Link andF J Haddy ldquoEffects of rat atrial extract on sodium transportand blood pressure in the ratrdquo Proceedings of the Society forExperimental Biology and Medicine vol 176 no 2 pp 123ndash1311984
[40] T R Traynor and S M OrsquoGrady ldquoBrain natriuretic peptidestimulates K andCl secretion across porcine distal colon epithe-liumrdquoTheAmerican Journal of PhysiologymdashCell Physiology vol260 no 4 pp C750ndashC755 1991
[41] R Rollın A Mediero M Roldan-Pallares A Fernandez-Cruzand R Fernandez-Durango ldquoNatriuretic peptide system in thehuman retinardquoMolecular Vision vol 10 pp 15ndash22 2004
[42] M Harada H Itoh O Nakagawa et al ldquoSignificance ofventricular myocytes and nonmyocytes interaction during car-diocyte hypertrophy evidence for endothelin-1 as a paracrinehypertrophic factor from cardiac nonmyocytesrdquo Circulationvol 96 no 10 pp 3737ndash3744 1997
[43] K Kuwahara Y Saito M Harada et al ldquoInvolvement ofcardiotrophin-1 in cardiac myocyte-nonmyocyte interactionsduring hypertrophy of rat cardiac myocytes in vitrordquo Circula-tion vol 100 no 10 pp 1116ndash1124 1999
[44] E Harada O Nakagawa M Yoshimura et al ldquoEffect ofinterleukin-1120573 on cardiac hypertrophy and production of natri-uretic peptides in rat cardiocyte culturerdquo Journal of Molecularand Cellular Cardiology vol 31 no 11 pp 1997ndash2006 1999
[45] V Chiurchiu V Izzi F DrsquoAquilio F Carotenuto P Di Nardoand P M Baldini ldquoBrain natriuretic peptide (BNP) regulatesthe production of inflammatory mediators in human THP-1macrophagesrdquo Regulatory Peptides vol 148 no 1ndash3 pp 26ndash322008
[46] N S Holden C F Rider M J Bell et al ldquoEnhancement ofinflammatory mediator release by 1205732-adrenoceptor agonists inairway epithelial cells is reversed by glucocorticoid actionrdquoTheBritish Journal of Pharmacology vol 160 no 2 pp 410ndash4202010
[47] V S Fang C C Juan J G S Won and L T Ho ldquoFurther studyof aldosterone secretion-inhibitory factor and brain natriureticpeptide on cortisol production of Guinea pig zona fasciculatacellsrdquo Chinese Journal of Physiology vol 43 no 4 pp 141ndash1472000
[48] G Hellermann X Kong J Gunnarsdottir et al ldquoMechanismof bronchoprotective effects of a novel natriuretic hormonepeptiderdquo Journal of Allergy and Clinical Immunology vol 113no 1 pp 79ndash85 2004
[49] K K Ma T Ogawa and A J de Bold ldquoSelective upregulationof cardiac brain natriuretic peptide at the transcriptional andtranslational levels by pro-inflammatory cytokines and by con-ditionedmedium derived frommixed lymphocyte reactions viap38 MAP kinaserdquo Journal of Molecular and Cellular Cardiologyvol 36 no 4 pp 505ndash513 2004
[50] T Ogawa J P Veinot M L K de Bold T Georgalis and AJ De Bold ldquoAngiotensin II receptor antagonism reverts theselective cardiac BNP upregulation and secretion observed inmyocarditisrdquo The American Journal of PhysiologymdashHeart andCirculatory Physiology vol 294 no 6 pp H2596ndashH2603 2008
[51] B J Carver E J Plosa A M Stinnett T S Blackwell andL S Prince ldquoInteractions between NF-120581B and SP3 connectinflammatory signaling with reduced FGF-10 expressionrdquo TheJournal of Biological Chemistry vol 288 no 21 pp 15318ndash153252013
[52] C Dong G Wang B Li et al ldquoAnti-asthmatic agents alleviatepulmonary edema by upregulating AQP1 and AQP5 expressionin the lungs of mice with OVA-induced asthmardquo RespiratoryPhysiology and Neurobiology vol 181 no 1 pp 21ndash28 2012
Figure 5 TNF-120572 modulation of BNP and NPR-1 expression may require activation of MAPKs pathway BEAS-2B cells were treated with10 ngmL TNF-120572 alone or in combination with 10 120583M SB 203580 (p38 MAPK inhibitor) SP 600125 (JNK 12 inhibitor) or U-0126 (ERK12 inhibitor) for 24 h Panels show ((a) (b)) BNP ((c) (d)) NPR-1 gene expression at mRNA ((a) (c)) and protein ((b) (d)) level and (e)Phospho-p38 MAPK protein level Western blots were obtained by using the specific rabbit polyclonal or monoclonal Abs The blots werestripped of the bound Ab and reprobed with mouse anti-120573-actin to confirm equal loading Western blots are representative of three separateexperiments All histograms indicate means plusmn SD of three different cultures each one tested in quadruplicate and expressed as percentage ofcontrol (lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 versus Controls) (∙∙119875 lt 001 versus SB) (sect119875 lt 005 sectsect
119875 lt 001 versus SP) (∘∘119875 lt 001∘∘∘
119875 lt 0001 versus U-0126) C Controls (untreated cells)
up-regulation after TNF-120572 treatment could concur in poten-tiating an inflammatory statusThe increased production andrelease of specific cytokines causing systemic changes knownas the acute phase response [5 8 10] induced by TNF-120572 maythus also occur via BNP up-regulation
Recently a BNP rise has been observed in patientsundergoing pulmonary lobectomy for lung cancer [11] Inparticular the patients none of whom developed signs orsymptoms of heart failure during the postoperative period
showed an early significant increase on day 1 immediatelyafter surgery of the plasma concentrations of BNP whichwas followed on the postoperative day 2 by a significantweight gain correlated with large volumes of fluids accumu-lation despite a negative intraoperative fluid balance andperoperative strict fluid restriction [11] We suggest thatsuch an increase could be at least in part explained bythe ability of proinflammatory cytokines such as TNF-120572 toupregulate BNP gene expression and secretion and that this
Mediators of Inflammation 9
0
20
40
60
80
100
120
0 10 20 40TNF-120572 (ngmL)
AQP1
( o
f con
trol)
lowast
lowastlowastlowast
lowastlowastlowast
(a)
Actin 0 10 20 40
AQP1
TNF-120572 (ngmL)
(b)
0
20
40
60
80
100
120
AQP5
( o
f con
trol)
0 10 20 40
TNF-120572 (ngmL)
lowastlowast
lowastlowastlowastlowast
(c)
AQP5
Actin 0 10 20 40TNF-120572 (ngmL)
(d)
Figure 6 TNF-120572 decreases AQP1 and AQP5 expression in BEAS 2B cells BEAS-2B cells were treated with 10 20 and 40 ngmL TNF-120572 for24 h Gene expression of AQP1 ((a) (b)) and AQP5 ((c) (d)) at mRNA ((a) (c)) and protein ((b) (d)) level Western blots were obtained byusing the specific rabbit polyclonal Abs The blots were stripped of the bound Ab and reprobed with mouse anti-120573-actin to confirm equalloading Western blots are representative of three separate experiments All histograms indicate means plusmn SD of three different cultures eachone tested in quadruplicate and expressed as percentage of control (lowast119875 lt 001 lowastlowast119875 lt 0001 and lowastlowastlowast119875 lt 00001 versus untreated cells)
up-regulation could enhance the major surgery-related earlyhyperinflammatory response essential for tissue repair andhost defense [5]
The observed dexamethasone capability to completelyrevert TNF-120572-induced BNP up-regulation suggests that thisanti-inflammatory glucocorticoid may prevent BNP-relatedeffects
Regarding ANP the observed significant down-regula-tion of mRNA and protein levels after 10 20 ngmL TNF-120572treatment suggests that also this condition could affect localinflammation status In fact ANP administration determinesanti-inflammatory effects in airway epithelial cells [48] play-ing important roles in modulating inflammatory responseIn consideration of such ANP anti-inflammatory action wespeculate that the observed TNF-120572-induced ANP down-regulation could concur in enhancing an inflammatory sta-tus Differently from the above reported doses 40 ngmLTNF-120572 administration induced ANP up-regulation eventhough not statistically significant We suggest that this resultmight be due to a direct BNP involvement In fact a markedANP up-regulation was observed after BNP treatment at the
dose accumulated in culture medium after 40 ngmL TNF-120572treatment
The observed selectivemodulation inANP and BNP geneexpression after TNF-120572 treatment in line with other studiesin different cell models [19 49 50] could be related to adifferent regulation mechanism at transcriptional level assuggested by our observation on the different involvementof NF-120581B and MAPKs signaling pathways In particular wedemonstrated that ANP down-regulation TNF-120572-mediatedoccurs via NF-120581B activation pathway whereas BNP modula-tion requires ERK 12 activation pathway NF-120581B involvementin TNF-120572-induced ANP down-regulation is not opposed tothe proinflammatory role of this transcription factor beingANP a molecule that exerts an anti-inflammatory effect inairway epithelial cells [48] Regarding themechanism linkingNF-120581B to the downregulation of ANP gene expression wesuggest that it could be similar to that recently proposedfor Fibroblast Growth Factor 10 (FGF-10) where NF-120581Bactivationmay lead to reduced gene expression by recruitinginhibitory factors to specific gene promoters [51]
10 Mediators of Inflammation
0
20
40
60
80
100
120
C BAY 10 20 40
AQP1
( o
f con
trol)
TNF-120572 (ngmL)
TNF-120572TNF-120572 + BAY
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowast
(a)
BAY
AQP1Actin
0 0 10 20 40 10 20 40TNF-120572 (ngmL)minus + +minus minus minus + +
(b)
0
20
40
60
80
100
120
AQP5
( o
f con
trol)
C BAY 10 20 40
TNF-120572 (ngmL)
TNF-120572TNF-120572 + BAY
∘∘∘∘∘∘
∘∘∘
lowastlowastlowastlowast
lowastlowast
(c)
AQP5Actin
0 0 10 20 40 10 20 40TNF-120572 (ngmL)BAY minus + +minus minus minus + +
(d)
Figure 7 TNF-120572 downregulation of AQP1 and AQP5 expression may require activation of NF-120581B pathwayBEAS-2B cells were treated with10 20 and 40 ngmL TNF-120572 alone or in combination with NF-120581B inhibitor BAY 11-7082 (1 120583M) for 24 h Gene expression of AQP1 ((a) (b))and AQP5 ((c) (d)) at mRNA ((a) (c)) and protein ((b) (d)) level Western blots were obtained by using the specific rabbit polyclonal AbsThe blots were stripped of the bound Ab and reprobed with mouse anti-120573-actin to confirm equal loading Western blots are representative ofthree separate experiments All histograms indicate means plusmn SD of three different cultures each one tested in quadruplicate and expressed aspercentage of control (lowast119875 lt 001 lowastlowast119875 lt 0001 lowastlowastlowast119875 lt 00001 versus Controls and ∘∘∘119875 lt 00001 versus BAY) C Controls (untreated cells)
ANP and BNP biological functions occur through theirbinding to the same NPR-1 [13] Here we demonstratedfor the first time to our knowledge TNF-120572 capability todownregulateNPR-1mRNAandprotein expression in BEAS-2B cells and that this regulation occurs via MAPKs signalingpathway activation The consequence of the complex TNF-120572-induced NPs and NPR-1 observed modulation associatedwith their binding to the same receptor on BEAS-2B cellsmakes it difficult to predict what would be the net resultingeffect both in vitro and in vivo in relation also to the presenceof a possible autocrine regulation by NPs andor to the effectsof different cytokines activated by both BNP and TNF-120572We hypothesize that the TNF-120572-induced downregulation of
NPR-1 paralleled by an up-regulation of BNP could alsolead to a paracrine action of this peptide possibly on themicrovascular endothelium of the lung where NPR-1 isdensely expressed [18] In this context we speculate that theinduction of local modification on BNP or ANP by TNF-120572 inhuman bronchial epithelial cells could alter the barrier per-meability of the pulmonary microcirculation Hence TNF-120572 acting on epithelial cells could also indirectly contributeto endothelial cells barrier dysfunction and permeability byenhancing the local production of proinflammatory BNPand by down-regulating the anti-inflammatory expressionof ANP Besides exogenous synthetic ANP has been shownto protect from endothelial barrier dysfunction in in vivo
Mediators of Inflammation 11
0
50
100
150
200
250
300
0 001
AN
P (
of c
ontro
l)
BNP (nM)
lowast
(a)
0
50
100
150
0 001BNP (nM)
NPR
-1(
of c
ontro
l)
(b)
0
20
40
60
80
100
120
0 001
BNP (nM)
lowast
AQP1
( o
f con
trol)
(c)
0
20
40
60
80
100
120
0 001
BNP (nM)
AQP5
( o
f con
trol)
(d)
Figure 8 BNP effect on ANP NPR-1 AQP1 and AQP5 expressionBEAS-2B cells were treated with 001 nM BNP for 24 h Histograms show(a) ANP (b) NPR-1 (c) AQP1 and (d) AQP5 gene expression at mRNA level All histograms indicate means plusmn SD of three different cultureseach one tested in quadruplicate and expressed as percentage of control (lowast119875 lt 005 versus untreated cells)
020406080
100120140160180200
DEX
BNP
( o
f con
trol)
C TNF-120572 TNF-120572 + DEX
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowast
lowast
lowast
Figure 9 Dexamethasone effect on BNP expression BEAS-2B cellswere treatedwith 1 120583Mdexamethasone (DEX) and 40 ngmLTNF-120572alone or in combination with DEX for 24 h Histograms show BNPgene expression at mRNA (gray bars) and protein (black bars) levelAll histograms indicate means plusmn SD of three different cultures eachone tested in quadruplicate and expressed as percentage of control(lowast119875 lt 001 lowastlowast119875 lt 0001 and lowastlowastlowast119875 lt 00001 versus Controls) CControls (untreated cells)
and in vitro models of lung injury [18] and the therapeu-tical relevance of these experimental observations has beenshown in intensive care patients without heart disease whereintravenous ANP infusion diminished pulmonary vascularpermeability and pulmonary edema [18]
Regarding AQPs we demonstrated that BEAS-2B cellsexpress AQP1 and AQP5 and that TNF-120572 directly down-regulates their expression at mRNA and protein levelsRegarding AQP1 also an additional BNP-mediated TNF-120572involvement can be proposed In fact AQP1 down-regulationwas observed with BNP treatment at the dose accumulatedin culture medium after 40 ngmL TNF-120572 treatment Themechanisms that underlie AQPs regulation are of consider-able interest Several AQPs has recently been demonstratedto undergo complex regulation [27] Our results show thatAQP1 and APQ5 are subject to inflammation regulationadding an important piece of information in this contextOur results are in agreement with other studies describinga marked reduction in the expression levels of AQP1 andAQP5 inmouse lung [30] andmouse lung epithelial cells [31]The biological significance of the observed TNF-120572-inducedAQP1 and AQP5 down-regulation is at present unclear Ithas been recently suggested that the decrease in both AQPsmay play an important role in the development of lung edema[52] which is known to be related with fluid retention Wetherefore speculate that TNF-120572-induced AQP1 and AQP5down-regulation might play a role also in the excess of fluidaccumulation related to major surgical procedures
5 Conclusions
In conclusion our results provided evidence that in humanbronchial epithelial cells BEAS-2B TNF-120572 selectively mod-ulates mRNA and protein expression levels of different
12 Mediators of Inflammation
molecules involved in body fluid homeostasis ANP BNPand their receptor NPR-1 AQP1 and AQP5 Moreoverour data pointed out that the changes specifically affectingNPs expression occur independently from hemodynamicinfluences and that inflammation should be considered anextracardiac cause of BNP elevation adding an importantpiece of information in the novel area of study regarding NPsand inflammation In addition we demonstrated that such amodulation occurs via different involvement of NK-120581B andMAPKs signaling pathways Finally we suggested inflamma-tion among the mechanisms involved in the regulation ofAQP1 and AQP5 Therefore in this exploratory study wespeculate that TNF-120572 might be involved in postoperative-fluid retention related to major surgery
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgment
The authors thank Mrs Roberta Frosini for the excellenttechnical assistance
References
[1] E Itobi M Stroud and M Elia ldquoImpact of oedema onrecovery after major abdominal surgery and potential valueof multifrequency bioimpedance measurementsrdquo The BritishJournal of Surgery vol 93 no 3 pp 354ndash361 2006
[2] J A Lowell C Schifferdecker D F Driscoll P N Benottiand B R Bistrian ldquoPostoperative fluid overload not a benignproblemrdquo Critical Care Medicine vol 18 no 7 pp 728ndash7331990
[3] A M Moslashller T Pedersen P-E Svendsen and A EngquistldquoPerioperative risk factors in elective pneumonectomy theimpact of excess fluid balancerdquo European Journal of Anaesthe-siology vol 19 no 1 pp 57ndash62 2002
[4] E Bennett-Guerrero D E Feierman G R Barclay et alldquoPreoperative and intraoperative predictors of postoperativemorbidity poor graft function and early rejection in 190patients undergoing liver transplantationrdquo Archives of Surgeryvol 136 no 10 pp 1177ndash1183 2001
[5] J P Desborough ldquoThe stress response to trauma and surgeryrdquoThe British Journal of Anaesthesia vol 85 no 1 pp 109ndash1172000
[6] H Tsujimoto S Ono T Majima et al ldquoDifferential toll-likereceptor expression after ex vivo lipopolysaccharide exposurein patients with sepsis and following surgical stressrdquo ClinicalImmunology vol 119 no 2 pp 180ndash187 2006
[7] C Evans C Galustian D Kumar et al ldquoImpact of surgeryon immunologic function comparison between minimallyinvasive techniques and conventional laparotomy for surgi-cal resection of colorectal tumorsrdquo The American Journal ofSurgery vol 197 no 2 pp 238ndash245 2009
[8] F Catena L Ansaloni A Avanzolini et al ldquoSystemic cytokineresponse after emergency and elective surgery for colorectalcarcinomardquo International Journal of Colorectal Disease vol 24no 7 pp 803ndash808 2009
[9] R Sarbinowski S Arvidsson M Tylman T Oresland andA Bengtsson ldquoPlasma concentration of procalcitonin andsystemic inflammatory response syndrome after colorectalsurgeryrdquo Acta Anaesthesiologica Scandinavica vol 49 no 2 pp191ndash196 2005
[10] P Sheeran and G M Hall ldquoCytokines in anaesthesiardquo TheBritish Journal of Anaesthesia vol 78 no 2 pp 201ndash219 1997
[11] L Cagini R Capozzi V Tassi et al ldquoFluid and electrolytebalance after major thoracic surgery by bioimpedance andendocrine evaluationrdquo European Journal of Cardio-ThoracicSurgery vol 40 no 2 pp e71ndashe76 2011
[12] T Omland and T Hagve ldquoNatriuretic peptides physiologic andanalytic considerationsrdquo Heart Failure Clinics vol 5 no 4 pp471ndash487 2009
[13] L R Potter A R Yoder D R Flora L K Antos and DM Dickey ldquoNatriuretic peptides their structures receptorsphysiologic functions and therapeutic applicationsrdquo Handbookof Experimental Pharmacology vol 191 pp 341ndash366 2009
[14] T G Flynn M L de Bold and A J de Bold ldquoThe aminoacid sequence of an atrial peptide with potent diuretic andnatriuretic propertiesrdquo Biochemical and Biophysical ResearchCommunications vol 117 no 3 pp 859ndash865 1983
[15] A J de Bold ldquoAtrial natriuretic factor a hormone produced bythe heartrdquo Science vol 230 pp 767ndash770 1985
[16] K Kangawa Y Tawaragi S Oikawa et al ldquoIdentification of rat 120574atrial natriuretic polypeptide and characterization of the cDNAencoding its precursorrdquo Nature vol 312 no 5990 pp 152ndash1551984
[17] T Sudoh K Kangawa N Minamino and H Matsuo ldquoA newnatriuretic peptide in porcine brainrdquo Nature vol 332 no 6159pp 78ndash81 1988
[18] M Kuhn ldquoEndothelial actions of atrial and B-type natriureticpeptidesrdquo The British Journal of Pharmacology vol 166 no 2pp 522ndash531 2012
[19] A J de Bold ldquoCardiac natriuretic peptides gene expression andsecretion in inflammationrdquo Journal of Investigative Medicinevol 57 pp 29ndash32 2009
[20] S P DrsquoSouza M Davis and G F Baxter ldquoAutocrine andparacrine actions of natriuretic peptides in the heartrdquo Pharma-cology andTherapeutics vol 101 no 2 pp 113ndash129 2004
[21] M Brueckmann G Huhle S Lang et al ldquoPrognostic valueof plasma N-terminal pro-brain natriuretic peptide in patientswith severe sepsisrdquoCirculation vol 112 no 4 pp 527ndash534 2005
[22] J Charpentier C E Luyt Y Fulla et al ldquoBrain natriureticpeptide a marker of myocardial dysfunction and prognosisduring severe sepsisrdquo Critical Care Medicine vol 32 no 3 pp660ndash665 2004
[23] R Shor Y Rozenman A Bolshinsky et al ldquoBNP in septicpatients without systolic myocardial dysfunctionrdquo EuropeanJournal of Internal Medicine vol 17 no 8 pp 536ndash540 2006
[24] T Inoue M Kawai T Nakane et al ldquoInfluence of low-gradeinflammation on plasma B-type natriuretic peptide levelsrdquoInternal Medicine vol 49 no 24 pp 2659ndash2668 2010
[25] P Agre L S King M Yasui et al ldquoAquaporin water channelsmdashfrom atomic structure to clinical medicinerdquo Journal of Physiol-ogy vol 542 no 1 pp 3ndash16 2002
[26] P Agre and D Kozono ldquoAquaporin water channels molecularmechanisms for human diseasesrdquo FEBS Letters vol 555 no 1pp 72ndash78 2003
Mediators of Inflammation 13
[27] J M Carbrey and P Agre ldquoDiscovery of the aquaporins anddevelopment of the fieldrdquo Handbook of Experimental Pharma-cology vol 190 pp 3ndash28 2009
[28] O Bloch and G T Manley ldquoThe role of aquaporin-4 in cerebralwater transport and edemardquo Neurosurgical Focus vol 22 no 5article E3 2007
[29] J Li M Xu Q Fan et al ldquoTanshinone IIA ameliorates seawaterexposure-induced lung injury by inhibiting aquaporins (AQP)1 and AQP5 expression in lungrdquo Respiratory Physiology andNeurobiology vol 176 no 1-2 pp 39ndash49 2011
[30] J E Towne K S Harrod C M Krane and A G MenonldquoDecreased expression of aquaporin (AQP)1 and AQP5 inmouse lung after acute viral infectionrdquoThe American Journal ofRespiratory Cell and Molecular Biology vol 22 no 1 pp 34ndash442000
[31] J E Towne C M Krane C J Bachurski and A G MenonldquoTumor necrosis factor-120572 inhibits aquaporin 5 expression inmouse lung epithelial cellsrdquoThe Journal of Biological Chemistryvol 276 no 22 pp 18657ndash18664 2001
[32] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001
[33] K Verhelst I Carpentier and R Beyaert ldquoRegulation of TNF-induced NF-120581B activation by different cytoplasmic ubiquitina-tion eventsrdquo Cytokine and Growth Factor Reviews vol 22 no5-6 pp 277ndash286 2011
[34] L Qian Y Shen J C Chen et al ldquo3D-QSAR and dockingstudies of quinazoline derivatives with the inhibatory activitytoward NF-120581BrdquoQSAR and Combinatorial Science vol 27 no 8pp 984ndash995 2008
[35] P D NrsquoGuessan S Hippenstiel M O Etouem et al ldquoStrepto-coccus pneumoniae induced p38MAPK- andNF-120581B-dependentCOX-2 expression in human lung epitheliumrdquo The AmericanJournal of PhysiologymdashLung Cellular and Molecular Physiologyvol 290 no 6 pp L1131ndashL1138 2006
[36] B Schmeck K Moog J Zahlten et al ldquoStreptococcus pneumo-niae induced c-Jun-N-terminal kinase- andAP-I-dependent IL-8 release by lung epithelial BEAS-2B cellsrdquoRespiratory Researchvol 7 article 98 2006
[37] M GMatera L Calzetta D Passeri et al ldquoEpithelium integrityis crucial for the relaxant activity of brain natriuretic peptide inhuman isolated bronchirdquo The British Journal of Pharmacologyvol 163 no 8 pp 1740ndash1754 2011
[38] R QuirionM Dalpe A de Lean J Gutkowska M Cantin andJ Genest ldquoAtrial natriuretic factor (ANF) binding sites in brainand related structuresrdquoPeptides vol 5 no 6 pp 1167ndash1172 1984
[39] M B Pamnani D L Clough J S Chen W T Link andF J Haddy ldquoEffects of rat atrial extract on sodium transportand blood pressure in the ratrdquo Proceedings of the Society forExperimental Biology and Medicine vol 176 no 2 pp 123ndash1311984
[40] T R Traynor and S M OrsquoGrady ldquoBrain natriuretic peptidestimulates K andCl secretion across porcine distal colon epithe-liumrdquoTheAmerican Journal of PhysiologymdashCell Physiology vol260 no 4 pp C750ndashC755 1991
[41] R Rollın A Mediero M Roldan-Pallares A Fernandez-Cruzand R Fernandez-Durango ldquoNatriuretic peptide system in thehuman retinardquoMolecular Vision vol 10 pp 15ndash22 2004
[42] M Harada H Itoh O Nakagawa et al ldquoSignificance ofventricular myocytes and nonmyocytes interaction during car-diocyte hypertrophy evidence for endothelin-1 as a paracrinehypertrophic factor from cardiac nonmyocytesrdquo Circulationvol 96 no 10 pp 3737ndash3744 1997
[43] K Kuwahara Y Saito M Harada et al ldquoInvolvement ofcardiotrophin-1 in cardiac myocyte-nonmyocyte interactionsduring hypertrophy of rat cardiac myocytes in vitrordquo Circula-tion vol 100 no 10 pp 1116ndash1124 1999
[44] E Harada O Nakagawa M Yoshimura et al ldquoEffect ofinterleukin-1120573 on cardiac hypertrophy and production of natri-uretic peptides in rat cardiocyte culturerdquo Journal of Molecularand Cellular Cardiology vol 31 no 11 pp 1997ndash2006 1999
[45] V Chiurchiu V Izzi F DrsquoAquilio F Carotenuto P Di Nardoand P M Baldini ldquoBrain natriuretic peptide (BNP) regulatesthe production of inflammatory mediators in human THP-1macrophagesrdquo Regulatory Peptides vol 148 no 1ndash3 pp 26ndash322008
[46] N S Holden C F Rider M J Bell et al ldquoEnhancement ofinflammatory mediator release by 1205732-adrenoceptor agonists inairway epithelial cells is reversed by glucocorticoid actionrdquoTheBritish Journal of Pharmacology vol 160 no 2 pp 410ndash4202010
[47] V S Fang C C Juan J G S Won and L T Ho ldquoFurther studyof aldosterone secretion-inhibitory factor and brain natriureticpeptide on cortisol production of Guinea pig zona fasciculatacellsrdquo Chinese Journal of Physiology vol 43 no 4 pp 141ndash1472000
[48] G Hellermann X Kong J Gunnarsdottir et al ldquoMechanismof bronchoprotective effects of a novel natriuretic hormonepeptiderdquo Journal of Allergy and Clinical Immunology vol 113no 1 pp 79ndash85 2004
[49] K K Ma T Ogawa and A J de Bold ldquoSelective upregulationof cardiac brain natriuretic peptide at the transcriptional andtranslational levels by pro-inflammatory cytokines and by con-ditionedmedium derived frommixed lymphocyte reactions viap38 MAP kinaserdquo Journal of Molecular and Cellular Cardiologyvol 36 no 4 pp 505ndash513 2004
[50] T Ogawa J P Veinot M L K de Bold T Georgalis and AJ De Bold ldquoAngiotensin II receptor antagonism reverts theselective cardiac BNP upregulation and secretion observed inmyocarditisrdquo The American Journal of PhysiologymdashHeart andCirculatory Physiology vol 294 no 6 pp H2596ndashH2603 2008
[51] B J Carver E J Plosa A M Stinnett T S Blackwell andL S Prince ldquoInteractions between NF-120581B and SP3 connectinflammatory signaling with reduced FGF-10 expressionrdquo TheJournal of Biological Chemistry vol 288 no 21 pp 15318ndash153252013
[52] C Dong G Wang B Li et al ldquoAnti-asthmatic agents alleviatepulmonary edema by upregulating AQP1 and AQP5 expressionin the lungs of mice with OVA-induced asthmardquo RespiratoryPhysiology and Neurobiology vol 181 no 1 pp 21ndash28 2012
Figure 6 TNF-120572 decreases AQP1 and AQP5 expression in BEAS 2B cells BEAS-2B cells were treated with 10 20 and 40 ngmL TNF-120572 for24 h Gene expression of AQP1 ((a) (b)) and AQP5 ((c) (d)) at mRNA ((a) (c)) and protein ((b) (d)) level Western blots were obtained byusing the specific rabbit polyclonal Abs The blots were stripped of the bound Ab and reprobed with mouse anti-120573-actin to confirm equalloading Western blots are representative of three separate experiments All histograms indicate means plusmn SD of three different cultures eachone tested in quadruplicate and expressed as percentage of control (lowast119875 lt 001 lowastlowast119875 lt 0001 and lowastlowastlowast119875 lt 00001 versus untreated cells)
up-regulation could enhance the major surgery-related earlyhyperinflammatory response essential for tissue repair andhost defense [5]
The observed dexamethasone capability to completelyrevert TNF-120572-induced BNP up-regulation suggests that thisanti-inflammatory glucocorticoid may prevent BNP-relatedeffects
Regarding ANP the observed significant down-regula-tion of mRNA and protein levels after 10 20 ngmL TNF-120572treatment suggests that also this condition could affect localinflammation status In fact ANP administration determinesanti-inflammatory effects in airway epithelial cells [48] play-ing important roles in modulating inflammatory responseIn consideration of such ANP anti-inflammatory action wespeculate that the observed TNF-120572-induced ANP down-regulation could concur in enhancing an inflammatory sta-tus Differently from the above reported doses 40 ngmLTNF-120572 administration induced ANP up-regulation eventhough not statistically significant We suggest that this resultmight be due to a direct BNP involvement In fact a markedANP up-regulation was observed after BNP treatment at the
dose accumulated in culture medium after 40 ngmL TNF-120572treatment
The observed selectivemodulation inANP and BNP geneexpression after TNF-120572 treatment in line with other studiesin different cell models [19 49 50] could be related to adifferent regulation mechanism at transcriptional level assuggested by our observation on the different involvementof NF-120581B and MAPKs signaling pathways In particular wedemonstrated that ANP down-regulation TNF-120572-mediatedoccurs via NF-120581B activation pathway whereas BNP modula-tion requires ERK 12 activation pathway NF-120581B involvementin TNF-120572-induced ANP down-regulation is not opposed tothe proinflammatory role of this transcription factor beingANP a molecule that exerts an anti-inflammatory effect inairway epithelial cells [48] Regarding themechanism linkingNF-120581B to the downregulation of ANP gene expression wesuggest that it could be similar to that recently proposedfor Fibroblast Growth Factor 10 (FGF-10) where NF-120581Bactivationmay lead to reduced gene expression by recruitinginhibitory factors to specific gene promoters [51]
10 Mediators of Inflammation
0
20
40
60
80
100
120
C BAY 10 20 40
AQP1
( o
f con
trol)
TNF-120572 (ngmL)
TNF-120572TNF-120572 + BAY
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowast
(a)
BAY
AQP1Actin
0 0 10 20 40 10 20 40TNF-120572 (ngmL)minus + +minus minus minus + +
(b)
0
20
40
60
80
100
120
AQP5
( o
f con
trol)
C BAY 10 20 40
TNF-120572 (ngmL)
TNF-120572TNF-120572 + BAY
∘∘∘∘∘∘
∘∘∘
lowastlowastlowastlowast
lowastlowast
(c)
AQP5Actin
0 0 10 20 40 10 20 40TNF-120572 (ngmL)BAY minus + +minus minus minus + +
(d)
Figure 7 TNF-120572 downregulation of AQP1 and AQP5 expression may require activation of NF-120581B pathwayBEAS-2B cells were treated with10 20 and 40 ngmL TNF-120572 alone or in combination with NF-120581B inhibitor BAY 11-7082 (1 120583M) for 24 h Gene expression of AQP1 ((a) (b))and AQP5 ((c) (d)) at mRNA ((a) (c)) and protein ((b) (d)) level Western blots were obtained by using the specific rabbit polyclonal AbsThe blots were stripped of the bound Ab and reprobed with mouse anti-120573-actin to confirm equal loading Western blots are representative ofthree separate experiments All histograms indicate means plusmn SD of three different cultures each one tested in quadruplicate and expressed aspercentage of control (lowast119875 lt 001 lowastlowast119875 lt 0001 lowastlowastlowast119875 lt 00001 versus Controls and ∘∘∘119875 lt 00001 versus BAY) C Controls (untreated cells)
ANP and BNP biological functions occur through theirbinding to the same NPR-1 [13] Here we demonstratedfor the first time to our knowledge TNF-120572 capability todownregulateNPR-1mRNAandprotein expression in BEAS-2B cells and that this regulation occurs via MAPKs signalingpathway activation The consequence of the complex TNF-120572-induced NPs and NPR-1 observed modulation associatedwith their binding to the same receptor on BEAS-2B cellsmakes it difficult to predict what would be the net resultingeffect both in vitro and in vivo in relation also to the presenceof a possible autocrine regulation by NPs andor to the effectsof different cytokines activated by both BNP and TNF-120572We hypothesize that the TNF-120572-induced downregulation of
NPR-1 paralleled by an up-regulation of BNP could alsolead to a paracrine action of this peptide possibly on themicrovascular endothelium of the lung where NPR-1 isdensely expressed [18] In this context we speculate that theinduction of local modification on BNP or ANP by TNF-120572 inhuman bronchial epithelial cells could alter the barrier per-meability of the pulmonary microcirculation Hence TNF-120572 acting on epithelial cells could also indirectly contributeto endothelial cells barrier dysfunction and permeability byenhancing the local production of proinflammatory BNPand by down-regulating the anti-inflammatory expressionof ANP Besides exogenous synthetic ANP has been shownto protect from endothelial barrier dysfunction in in vivo
Mediators of Inflammation 11
0
50
100
150
200
250
300
0 001
AN
P (
of c
ontro
l)
BNP (nM)
lowast
(a)
0
50
100
150
0 001BNP (nM)
NPR
-1(
of c
ontro
l)
(b)
0
20
40
60
80
100
120
0 001
BNP (nM)
lowast
AQP1
( o
f con
trol)
(c)
0
20
40
60
80
100
120
0 001
BNP (nM)
AQP5
( o
f con
trol)
(d)
Figure 8 BNP effect on ANP NPR-1 AQP1 and AQP5 expressionBEAS-2B cells were treated with 001 nM BNP for 24 h Histograms show(a) ANP (b) NPR-1 (c) AQP1 and (d) AQP5 gene expression at mRNA level All histograms indicate means plusmn SD of three different cultureseach one tested in quadruplicate and expressed as percentage of control (lowast119875 lt 005 versus untreated cells)
020406080
100120140160180200
DEX
BNP
( o
f con
trol)
C TNF-120572 TNF-120572 + DEX
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowast
lowast
lowast
Figure 9 Dexamethasone effect on BNP expression BEAS-2B cellswere treatedwith 1 120583Mdexamethasone (DEX) and 40 ngmLTNF-120572alone or in combination with DEX for 24 h Histograms show BNPgene expression at mRNA (gray bars) and protein (black bars) levelAll histograms indicate means plusmn SD of three different cultures eachone tested in quadruplicate and expressed as percentage of control(lowast119875 lt 001 lowastlowast119875 lt 0001 and lowastlowastlowast119875 lt 00001 versus Controls) CControls (untreated cells)
and in vitro models of lung injury [18] and the therapeu-tical relevance of these experimental observations has beenshown in intensive care patients without heart disease whereintravenous ANP infusion diminished pulmonary vascularpermeability and pulmonary edema [18]
Regarding AQPs we demonstrated that BEAS-2B cellsexpress AQP1 and AQP5 and that TNF-120572 directly down-regulates their expression at mRNA and protein levelsRegarding AQP1 also an additional BNP-mediated TNF-120572involvement can be proposed In fact AQP1 down-regulationwas observed with BNP treatment at the dose accumulatedin culture medium after 40 ngmL TNF-120572 treatment Themechanisms that underlie AQPs regulation are of consider-able interest Several AQPs has recently been demonstratedto undergo complex regulation [27] Our results show thatAQP1 and APQ5 are subject to inflammation regulationadding an important piece of information in this contextOur results are in agreement with other studies describinga marked reduction in the expression levels of AQP1 andAQP5 inmouse lung [30] andmouse lung epithelial cells [31]The biological significance of the observed TNF-120572-inducedAQP1 and AQP5 down-regulation is at present unclear Ithas been recently suggested that the decrease in both AQPsmay play an important role in the development of lung edema[52] which is known to be related with fluid retention Wetherefore speculate that TNF-120572-induced AQP1 and AQP5down-regulation might play a role also in the excess of fluidaccumulation related to major surgical procedures
5 Conclusions
In conclusion our results provided evidence that in humanbronchial epithelial cells BEAS-2B TNF-120572 selectively mod-ulates mRNA and protein expression levels of different
12 Mediators of Inflammation
molecules involved in body fluid homeostasis ANP BNPand their receptor NPR-1 AQP1 and AQP5 Moreoverour data pointed out that the changes specifically affectingNPs expression occur independently from hemodynamicinfluences and that inflammation should be considered anextracardiac cause of BNP elevation adding an importantpiece of information in the novel area of study regarding NPsand inflammation In addition we demonstrated that such amodulation occurs via different involvement of NK-120581B andMAPKs signaling pathways Finally we suggested inflamma-tion among the mechanisms involved in the regulation ofAQP1 and AQP5 Therefore in this exploratory study wespeculate that TNF-120572 might be involved in postoperative-fluid retention related to major surgery
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgment
The authors thank Mrs Roberta Frosini for the excellenttechnical assistance
References
[1] E Itobi M Stroud and M Elia ldquoImpact of oedema onrecovery after major abdominal surgery and potential valueof multifrequency bioimpedance measurementsrdquo The BritishJournal of Surgery vol 93 no 3 pp 354ndash361 2006
[2] J A Lowell C Schifferdecker D F Driscoll P N Benottiand B R Bistrian ldquoPostoperative fluid overload not a benignproblemrdquo Critical Care Medicine vol 18 no 7 pp 728ndash7331990
[3] A M Moslashller T Pedersen P-E Svendsen and A EngquistldquoPerioperative risk factors in elective pneumonectomy theimpact of excess fluid balancerdquo European Journal of Anaesthe-siology vol 19 no 1 pp 57ndash62 2002
[4] E Bennett-Guerrero D E Feierman G R Barclay et alldquoPreoperative and intraoperative predictors of postoperativemorbidity poor graft function and early rejection in 190patients undergoing liver transplantationrdquo Archives of Surgeryvol 136 no 10 pp 1177ndash1183 2001
[5] J P Desborough ldquoThe stress response to trauma and surgeryrdquoThe British Journal of Anaesthesia vol 85 no 1 pp 109ndash1172000
[6] H Tsujimoto S Ono T Majima et al ldquoDifferential toll-likereceptor expression after ex vivo lipopolysaccharide exposurein patients with sepsis and following surgical stressrdquo ClinicalImmunology vol 119 no 2 pp 180ndash187 2006
[7] C Evans C Galustian D Kumar et al ldquoImpact of surgeryon immunologic function comparison between minimallyinvasive techniques and conventional laparotomy for surgi-cal resection of colorectal tumorsrdquo The American Journal ofSurgery vol 197 no 2 pp 238ndash245 2009
[8] F Catena L Ansaloni A Avanzolini et al ldquoSystemic cytokineresponse after emergency and elective surgery for colorectalcarcinomardquo International Journal of Colorectal Disease vol 24no 7 pp 803ndash808 2009
[9] R Sarbinowski S Arvidsson M Tylman T Oresland andA Bengtsson ldquoPlasma concentration of procalcitonin andsystemic inflammatory response syndrome after colorectalsurgeryrdquo Acta Anaesthesiologica Scandinavica vol 49 no 2 pp191ndash196 2005
[10] P Sheeran and G M Hall ldquoCytokines in anaesthesiardquo TheBritish Journal of Anaesthesia vol 78 no 2 pp 201ndash219 1997
[11] L Cagini R Capozzi V Tassi et al ldquoFluid and electrolytebalance after major thoracic surgery by bioimpedance andendocrine evaluationrdquo European Journal of Cardio-ThoracicSurgery vol 40 no 2 pp e71ndashe76 2011
[12] T Omland and T Hagve ldquoNatriuretic peptides physiologic andanalytic considerationsrdquo Heart Failure Clinics vol 5 no 4 pp471ndash487 2009
[13] L R Potter A R Yoder D R Flora L K Antos and DM Dickey ldquoNatriuretic peptides their structures receptorsphysiologic functions and therapeutic applicationsrdquo Handbookof Experimental Pharmacology vol 191 pp 341ndash366 2009
[14] T G Flynn M L de Bold and A J de Bold ldquoThe aminoacid sequence of an atrial peptide with potent diuretic andnatriuretic propertiesrdquo Biochemical and Biophysical ResearchCommunications vol 117 no 3 pp 859ndash865 1983
[15] A J de Bold ldquoAtrial natriuretic factor a hormone produced bythe heartrdquo Science vol 230 pp 767ndash770 1985
[16] K Kangawa Y Tawaragi S Oikawa et al ldquoIdentification of rat 120574atrial natriuretic polypeptide and characterization of the cDNAencoding its precursorrdquo Nature vol 312 no 5990 pp 152ndash1551984
[17] T Sudoh K Kangawa N Minamino and H Matsuo ldquoA newnatriuretic peptide in porcine brainrdquo Nature vol 332 no 6159pp 78ndash81 1988
[18] M Kuhn ldquoEndothelial actions of atrial and B-type natriureticpeptidesrdquo The British Journal of Pharmacology vol 166 no 2pp 522ndash531 2012
[19] A J de Bold ldquoCardiac natriuretic peptides gene expression andsecretion in inflammationrdquo Journal of Investigative Medicinevol 57 pp 29ndash32 2009
[20] S P DrsquoSouza M Davis and G F Baxter ldquoAutocrine andparacrine actions of natriuretic peptides in the heartrdquo Pharma-cology andTherapeutics vol 101 no 2 pp 113ndash129 2004
[21] M Brueckmann G Huhle S Lang et al ldquoPrognostic valueof plasma N-terminal pro-brain natriuretic peptide in patientswith severe sepsisrdquoCirculation vol 112 no 4 pp 527ndash534 2005
[22] J Charpentier C E Luyt Y Fulla et al ldquoBrain natriureticpeptide a marker of myocardial dysfunction and prognosisduring severe sepsisrdquo Critical Care Medicine vol 32 no 3 pp660ndash665 2004
[23] R Shor Y Rozenman A Bolshinsky et al ldquoBNP in septicpatients without systolic myocardial dysfunctionrdquo EuropeanJournal of Internal Medicine vol 17 no 8 pp 536ndash540 2006
[24] T Inoue M Kawai T Nakane et al ldquoInfluence of low-gradeinflammation on plasma B-type natriuretic peptide levelsrdquoInternal Medicine vol 49 no 24 pp 2659ndash2668 2010
[25] P Agre L S King M Yasui et al ldquoAquaporin water channelsmdashfrom atomic structure to clinical medicinerdquo Journal of Physiol-ogy vol 542 no 1 pp 3ndash16 2002
[26] P Agre and D Kozono ldquoAquaporin water channels molecularmechanisms for human diseasesrdquo FEBS Letters vol 555 no 1pp 72ndash78 2003
Mediators of Inflammation 13
[27] J M Carbrey and P Agre ldquoDiscovery of the aquaporins anddevelopment of the fieldrdquo Handbook of Experimental Pharma-cology vol 190 pp 3ndash28 2009
[28] O Bloch and G T Manley ldquoThe role of aquaporin-4 in cerebralwater transport and edemardquo Neurosurgical Focus vol 22 no 5article E3 2007
[29] J Li M Xu Q Fan et al ldquoTanshinone IIA ameliorates seawaterexposure-induced lung injury by inhibiting aquaporins (AQP)1 and AQP5 expression in lungrdquo Respiratory Physiology andNeurobiology vol 176 no 1-2 pp 39ndash49 2011
[30] J E Towne K S Harrod C M Krane and A G MenonldquoDecreased expression of aquaporin (AQP)1 and AQP5 inmouse lung after acute viral infectionrdquoThe American Journal ofRespiratory Cell and Molecular Biology vol 22 no 1 pp 34ndash442000
[31] J E Towne C M Krane C J Bachurski and A G MenonldquoTumor necrosis factor-120572 inhibits aquaporin 5 expression inmouse lung epithelial cellsrdquoThe Journal of Biological Chemistryvol 276 no 22 pp 18657ndash18664 2001
[32] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001
[33] K Verhelst I Carpentier and R Beyaert ldquoRegulation of TNF-induced NF-120581B activation by different cytoplasmic ubiquitina-tion eventsrdquo Cytokine and Growth Factor Reviews vol 22 no5-6 pp 277ndash286 2011
[34] L Qian Y Shen J C Chen et al ldquo3D-QSAR and dockingstudies of quinazoline derivatives with the inhibatory activitytoward NF-120581BrdquoQSAR and Combinatorial Science vol 27 no 8pp 984ndash995 2008
[35] P D NrsquoGuessan S Hippenstiel M O Etouem et al ldquoStrepto-coccus pneumoniae induced p38MAPK- andNF-120581B-dependentCOX-2 expression in human lung epitheliumrdquo The AmericanJournal of PhysiologymdashLung Cellular and Molecular Physiologyvol 290 no 6 pp L1131ndashL1138 2006
[36] B Schmeck K Moog J Zahlten et al ldquoStreptococcus pneumo-niae induced c-Jun-N-terminal kinase- andAP-I-dependent IL-8 release by lung epithelial BEAS-2B cellsrdquoRespiratory Researchvol 7 article 98 2006
[37] M GMatera L Calzetta D Passeri et al ldquoEpithelium integrityis crucial for the relaxant activity of brain natriuretic peptide inhuman isolated bronchirdquo The British Journal of Pharmacologyvol 163 no 8 pp 1740ndash1754 2011
[38] R QuirionM Dalpe A de Lean J Gutkowska M Cantin andJ Genest ldquoAtrial natriuretic factor (ANF) binding sites in brainand related structuresrdquoPeptides vol 5 no 6 pp 1167ndash1172 1984
[39] M B Pamnani D L Clough J S Chen W T Link andF J Haddy ldquoEffects of rat atrial extract on sodium transportand blood pressure in the ratrdquo Proceedings of the Society forExperimental Biology and Medicine vol 176 no 2 pp 123ndash1311984
[40] T R Traynor and S M OrsquoGrady ldquoBrain natriuretic peptidestimulates K andCl secretion across porcine distal colon epithe-liumrdquoTheAmerican Journal of PhysiologymdashCell Physiology vol260 no 4 pp C750ndashC755 1991
[41] R Rollın A Mediero M Roldan-Pallares A Fernandez-Cruzand R Fernandez-Durango ldquoNatriuretic peptide system in thehuman retinardquoMolecular Vision vol 10 pp 15ndash22 2004
[42] M Harada H Itoh O Nakagawa et al ldquoSignificance ofventricular myocytes and nonmyocytes interaction during car-diocyte hypertrophy evidence for endothelin-1 as a paracrinehypertrophic factor from cardiac nonmyocytesrdquo Circulationvol 96 no 10 pp 3737ndash3744 1997
[43] K Kuwahara Y Saito M Harada et al ldquoInvolvement ofcardiotrophin-1 in cardiac myocyte-nonmyocyte interactionsduring hypertrophy of rat cardiac myocytes in vitrordquo Circula-tion vol 100 no 10 pp 1116ndash1124 1999
[44] E Harada O Nakagawa M Yoshimura et al ldquoEffect ofinterleukin-1120573 on cardiac hypertrophy and production of natri-uretic peptides in rat cardiocyte culturerdquo Journal of Molecularand Cellular Cardiology vol 31 no 11 pp 1997ndash2006 1999
[45] V Chiurchiu V Izzi F DrsquoAquilio F Carotenuto P Di Nardoand P M Baldini ldquoBrain natriuretic peptide (BNP) regulatesthe production of inflammatory mediators in human THP-1macrophagesrdquo Regulatory Peptides vol 148 no 1ndash3 pp 26ndash322008
[46] N S Holden C F Rider M J Bell et al ldquoEnhancement ofinflammatory mediator release by 1205732-adrenoceptor agonists inairway epithelial cells is reversed by glucocorticoid actionrdquoTheBritish Journal of Pharmacology vol 160 no 2 pp 410ndash4202010
[47] V S Fang C C Juan J G S Won and L T Ho ldquoFurther studyof aldosterone secretion-inhibitory factor and brain natriureticpeptide on cortisol production of Guinea pig zona fasciculatacellsrdquo Chinese Journal of Physiology vol 43 no 4 pp 141ndash1472000
[48] G Hellermann X Kong J Gunnarsdottir et al ldquoMechanismof bronchoprotective effects of a novel natriuretic hormonepeptiderdquo Journal of Allergy and Clinical Immunology vol 113no 1 pp 79ndash85 2004
[49] K K Ma T Ogawa and A J de Bold ldquoSelective upregulationof cardiac brain natriuretic peptide at the transcriptional andtranslational levels by pro-inflammatory cytokines and by con-ditionedmedium derived frommixed lymphocyte reactions viap38 MAP kinaserdquo Journal of Molecular and Cellular Cardiologyvol 36 no 4 pp 505ndash513 2004
[50] T Ogawa J P Veinot M L K de Bold T Georgalis and AJ De Bold ldquoAngiotensin II receptor antagonism reverts theselective cardiac BNP upregulation and secretion observed inmyocarditisrdquo The American Journal of PhysiologymdashHeart andCirculatory Physiology vol 294 no 6 pp H2596ndashH2603 2008
[51] B J Carver E J Plosa A M Stinnett T S Blackwell andL S Prince ldquoInteractions between NF-120581B and SP3 connectinflammatory signaling with reduced FGF-10 expressionrdquo TheJournal of Biological Chemistry vol 288 no 21 pp 15318ndash153252013
[52] C Dong G Wang B Li et al ldquoAnti-asthmatic agents alleviatepulmonary edema by upregulating AQP1 and AQP5 expressionin the lungs of mice with OVA-induced asthmardquo RespiratoryPhysiology and Neurobiology vol 181 no 1 pp 21ndash28 2012
0 0 10 20 40 10 20 40TNF-120572 (ngmL)minus + +minus minus minus + +
(b)
0
20
40
60
80
100
120
AQP5
( o
f con
trol)
C BAY 10 20 40
TNF-120572 (ngmL)
TNF-120572TNF-120572 + BAY
∘∘∘∘∘∘
∘∘∘
lowastlowastlowastlowast
lowastlowast
(c)
AQP5Actin
0 0 10 20 40 10 20 40TNF-120572 (ngmL)BAY minus + +minus minus minus + +
(d)
Figure 7 TNF-120572 downregulation of AQP1 and AQP5 expression may require activation of NF-120581B pathwayBEAS-2B cells were treated with10 20 and 40 ngmL TNF-120572 alone or in combination with NF-120581B inhibitor BAY 11-7082 (1 120583M) for 24 h Gene expression of AQP1 ((a) (b))and AQP5 ((c) (d)) at mRNA ((a) (c)) and protein ((b) (d)) level Western blots were obtained by using the specific rabbit polyclonal AbsThe blots were stripped of the bound Ab and reprobed with mouse anti-120573-actin to confirm equal loading Western blots are representative ofthree separate experiments All histograms indicate means plusmn SD of three different cultures each one tested in quadruplicate and expressed aspercentage of control (lowast119875 lt 001 lowastlowast119875 lt 0001 lowastlowastlowast119875 lt 00001 versus Controls and ∘∘∘119875 lt 00001 versus BAY) C Controls (untreated cells)
ANP and BNP biological functions occur through theirbinding to the same NPR-1 [13] Here we demonstratedfor the first time to our knowledge TNF-120572 capability todownregulateNPR-1mRNAandprotein expression in BEAS-2B cells and that this regulation occurs via MAPKs signalingpathway activation The consequence of the complex TNF-120572-induced NPs and NPR-1 observed modulation associatedwith their binding to the same receptor on BEAS-2B cellsmakes it difficult to predict what would be the net resultingeffect both in vitro and in vivo in relation also to the presenceof a possible autocrine regulation by NPs andor to the effectsof different cytokines activated by both BNP and TNF-120572We hypothesize that the TNF-120572-induced downregulation of
NPR-1 paralleled by an up-regulation of BNP could alsolead to a paracrine action of this peptide possibly on themicrovascular endothelium of the lung where NPR-1 isdensely expressed [18] In this context we speculate that theinduction of local modification on BNP or ANP by TNF-120572 inhuman bronchial epithelial cells could alter the barrier per-meability of the pulmonary microcirculation Hence TNF-120572 acting on epithelial cells could also indirectly contributeto endothelial cells barrier dysfunction and permeability byenhancing the local production of proinflammatory BNPand by down-regulating the anti-inflammatory expressionof ANP Besides exogenous synthetic ANP has been shownto protect from endothelial barrier dysfunction in in vivo
Mediators of Inflammation 11
0
50
100
150
200
250
300
0 001
AN
P (
of c
ontro
l)
BNP (nM)
lowast
(a)
0
50
100
150
0 001BNP (nM)
NPR
-1(
of c
ontro
l)
(b)
0
20
40
60
80
100
120
0 001
BNP (nM)
lowast
AQP1
( o
f con
trol)
(c)
0
20
40
60
80
100
120
0 001
BNP (nM)
AQP5
( o
f con
trol)
(d)
Figure 8 BNP effect on ANP NPR-1 AQP1 and AQP5 expressionBEAS-2B cells were treated with 001 nM BNP for 24 h Histograms show(a) ANP (b) NPR-1 (c) AQP1 and (d) AQP5 gene expression at mRNA level All histograms indicate means plusmn SD of three different cultureseach one tested in quadruplicate and expressed as percentage of control (lowast119875 lt 005 versus untreated cells)
020406080
100120140160180200
DEX
BNP
( o
f con
trol)
C TNF-120572 TNF-120572 + DEX
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowast
lowast
lowast
Figure 9 Dexamethasone effect on BNP expression BEAS-2B cellswere treatedwith 1 120583Mdexamethasone (DEX) and 40 ngmLTNF-120572alone or in combination with DEX for 24 h Histograms show BNPgene expression at mRNA (gray bars) and protein (black bars) levelAll histograms indicate means plusmn SD of three different cultures eachone tested in quadruplicate and expressed as percentage of control(lowast119875 lt 001 lowastlowast119875 lt 0001 and lowastlowastlowast119875 lt 00001 versus Controls) CControls (untreated cells)
and in vitro models of lung injury [18] and the therapeu-tical relevance of these experimental observations has beenshown in intensive care patients without heart disease whereintravenous ANP infusion diminished pulmonary vascularpermeability and pulmonary edema [18]
Regarding AQPs we demonstrated that BEAS-2B cellsexpress AQP1 and AQP5 and that TNF-120572 directly down-regulates their expression at mRNA and protein levelsRegarding AQP1 also an additional BNP-mediated TNF-120572involvement can be proposed In fact AQP1 down-regulationwas observed with BNP treatment at the dose accumulatedin culture medium after 40 ngmL TNF-120572 treatment Themechanisms that underlie AQPs regulation are of consider-able interest Several AQPs has recently been demonstratedto undergo complex regulation [27] Our results show thatAQP1 and APQ5 are subject to inflammation regulationadding an important piece of information in this contextOur results are in agreement with other studies describinga marked reduction in the expression levels of AQP1 andAQP5 inmouse lung [30] andmouse lung epithelial cells [31]The biological significance of the observed TNF-120572-inducedAQP1 and AQP5 down-regulation is at present unclear Ithas been recently suggested that the decrease in both AQPsmay play an important role in the development of lung edema[52] which is known to be related with fluid retention Wetherefore speculate that TNF-120572-induced AQP1 and AQP5down-regulation might play a role also in the excess of fluidaccumulation related to major surgical procedures
5 Conclusions
In conclusion our results provided evidence that in humanbronchial epithelial cells BEAS-2B TNF-120572 selectively mod-ulates mRNA and protein expression levels of different
12 Mediators of Inflammation
molecules involved in body fluid homeostasis ANP BNPand their receptor NPR-1 AQP1 and AQP5 Moreoverour data pointed out that the changes specifically affectingNPs expression occur independently from hemodynamicinfluences and that inflammation should be considered anextracardiac cause of BNP elevation adding an importantpiece of information in the novel area of study regarding NPsand inflammation In addition we demonstrated that such amodulation occurs via different involvement of NK-120581B andMAPKs signaling pathways Finally we suggested inflamma-tion among the mechanisms involved in the regulation ofAQP1 and AQP5 Therefore in this exploratory study wespeculate that TNF-120572 might be involved in postoperative-fluid retention related to major surgery
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgment
The authors thank Mrs Roberta Frosini for the excellenttechnical assistance
References
[1] E Itobi M Stroud and M Elia ldquoImpact of oedema onrecovery after major abdominal surgery and potential valueof multifrequency bioimpedance measurementsrdquo The BritishJournal of Surgery vol 93 no 3 pp 354ndash361 2006
[2] J A Lowell C Schifferdecker D F Driscoll P N Benottiand B R Bistrian ldquoPostoperative fluid overload not a benignproblemrdquo Critical Care Medicine vol 18 no 7 pp 728ndash7331990
[3] A M Moslashller T Pedersen P-E Svendsen and A EngquistldquoPerioperative risk factors in elective pneumonectomy theimpact of excess fluid balancerdquo European Journal of Anaesthe-siology vol 19 no 1 pp 57ndash62 2002
[4] E Bennett-Guerrero D E Feierman G R Barclay et alldquoPreoperative and intraoperative predictors of postoperativemorbidity poor graft function and early rejection in 190patients undergoing liver transplantationrdquo Archives of Surgeryvol 136 no 10 pp 1177ndash1183 2001
[5] J P Desborough ldquoThe stress response to trauma and surgeryrdquoThe British Journal of Anaesthesia vol 85 no 1 pp 109ndash1172000
[6] H Tsujimoto S Ono T Majima et al ldquoDifferential toll-likereceptor expression after ex vivo lipopolysaccharide exposurein patients with sepsis and following surgical stressrdquo ClinicalImmunology vol 119 no 2 pp 180ndash187 2006
[7] C Evans C Galustian D Kumar et al ldquoImpact of surgeryon immunologic function comparison between minimallyinvasive techniques and conventional laparotomy for surgi-cal resection of colorectal tumorsrdquo The American Journal ofSurgery vol 197 no 2 pp 238ndash245 2009
[8] F Catena L Ansaloni A Avanzolini et al ldquoSystemic cytokineresponse after emergency and elective surgery for colorectalcarcinomardquo International Journal of Colorectal Disease vol 24no 7 pp 803ndash808 2009
[9] R Sarbinowski S Arvidsson M Tylman T Oresland andA Bengtsson ldquoPlasma concentration of procalcitonin andsystemic inflammatory response syndrome after colorectalsurgeryrdquo Acta Anaesthesiologica Scandinavica vol 49 no 2 pp191ndash196 2005
[10] P Sheeran and G M Hall ldquoCytokines in anaesthesiardquo TheBritish Journal of Anaesthesia vol 78 no 2 pp 201ndash219 1997
[11] L Cagini R Capozzi V Tassi et al ldquoFluid and electrolytebalance after major thoracic surgery by bioimpedance andendocrine evaluationrdquo European Journal of Cardio-ThoracicSurgery vol 40 no 2 pp e71ndashe76 2011
[12] T Omland and T Hagve ldquoNatriuretic peptides physiologic andanalytic considerationsrdquo Heart Failure Clinics vol 5 no 4 pp471ndash487 2009
[13] L R Potter A R Yoder D R Flora L K Antos and DM Dickey ldquoNatriuretic peptides their structures receptorsphysiologic functions and therapeutic applicationsrdquo Handbookof Experimental Pharmacology vol 191 pp 341ndash366 2009
[14] T G Flynn M L de Bold and A J de Bold ldquoThe aminoacid sequence of an atrial peptide with potent diuretic andnatriuretic propertiesrdquo Biochemical and Biophysical ResearchCommunications vol 117 no 3 pp 859ndash865 1983
[15] A J de Bold ldquoAtrial natriuretic factor a hormone produced bythe heartrdquo Science vol 230 pp 767ndash770 1985
[16] K Kangawa Y Tawaragi S Oikawa et al ldquoIdentification of rat 120574atrial natriuretic polypeptide and characterization of the cDNAencoding its precursorrdquo Nature vol 312 no 5990 pp 152ndash1551984
[17] T Sudoh K Kangawa N Minamino and H Matsuo ldquoA newnatriuretic peptide in porcine brainrdquo Nature vol 332 no 6159pp 78ndash81 1988
[18] M Kuhn ldquoEndothelial actions of atrial and B-type natriureticpeptidesrdquo The British Journal of Pharmacology vol 166 no 2pp 522ndash531 2012
[19] A J de Bold ldquoCardiac natriuretic peptides gene expression andsecretion in inflammationrdquo Journal of Investigative Medicinevol 57 pp 29ndash32 2009
[20] S P DrsquoSouza M Davis and G F Baxter ldquoAutocrine andparacrine actions of natriuretic peptides in the heartrdquo Pharma-cology andTherapeutics vol 101 no 2 pp 113ndash129 2004
[21] M Brueckmann G Huhle S Lang et al ldquoPrognostic valueof plasma N-terminal pro-brain natriuretic peptide in patientswith severe sepsisrdquoCirculation vol 112 no 4 pp 527ndash534 2005
[22] J Charpentier C E Luyt Y Fulla et al ldquoBrain natriureticpeptide a marker of myocardial dysfunction and prognosisduring severe sepsisrdquo Critical Care Medicine vol 32 no 3 pp660ndash665 2004
[23] R Shor Y Rozenman A Bolshinsky et al ldquoBNP in septicpatients without systolic myocardial dysfunctionrdquo EuropeanJournal of Internal Medicine vol 17 no 8 pp 536ndash540 2006
[24] T Inoue M Kawai T Nakane et al ldquoInfluence of low-gradeinflammation on plasma B-type natriuretic peptide levelsrdquoInternal Medicine vol 49 no 24 pp 2659ndash2668 2010
[25] P Agre L S King M Yasui et al ldquoAquaporin water channelsmdashfrom atomic structure to clinical medicinerdquo Journal of Physiol-ogy vol 542 no 1 pp 3ndash16 2002
[26] P Agre and D Kozono ldquoAquaporin water channels molecularmechanisms for human diseasesrdquo FEBS Letters vol 555 no 1pp 72ndash78 2003
Mediators of Inflammation 13
[27] J M Carbrey and P Agre ldquoDiscovery of the aquaporins anddevelopment of the fieldrdquo Handbook of Experimental Pharma-cology vol 190 pp 3ndash28 2009
[28] O Bloch and G T Manley ldquoThe role of aquaporin-4 in cerebralwater transport and edemardquo Neurosurgical Focus vol 22 no 5article E3 2007
[29] J Li M Xu Q Fan et al ldquoTanshinone IIA ameliorates seawaterexposure-induced lung injury by inhibiting aquaporins (AQP)1 and AQP5 expression in lungrdquo Respiratory Physiology andNeurobiology vol 176 no 1-2 pp 39ndash49 2011
[30] J E Towne K S Harrod C M Krane and A G MenonldquoDecreased expression of aquaporin (AQP)1 and AQP5 inmouse lung after acute viral infectionrdquoThe American Journal ofRespiratory Cell and Molecular Biology vol 22 no 1 pp 34ndash442000
[31] J E Towne C M Krane C J Bachurski and A G MenonldquoTumor necrosis factor-120572 inhibits aquaporin 5 expression inmouse lung epithelial cellsrdquoThe Journal of Biological Chemistryvol 276 no 22 pp 18657ndash18664 2001
[32] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001
[33] K Verhelst I Carpentier and R Beyaert ldquoRegulation of TNF-induced NF-120581B activation by different cytoplasmic ubiquitina-tion eventsrdquo Cytokine and Growth Factor Reviews vol 22 no5-6 pp 277ndash286 2011
[34] L Qian Y Shen J C Chen et al ldquo3D-QSAR and dockingstudies of quinazoline derivatives with the inhibatory activitytoward NF-120581BrdquoQSAR and Combinatorial Science vol 27 no 8pp 984ndash995 2008
[35] P D NrsquoGuessan S Hippenstiel M O Etouem et al ldquoStrepto-coccus pneumoniae induced p38MAPK- andNF-120581B-dependentCOX-2 expression in human lung epitheliumrdquo The AmericanJournal of PhysiologymdashLung Cellular and Molecular Physiologyvol 290 no 6 pp L1131ndashL1138 2006
[36] B Schmeck K Moog J Zahlten et al ldquoStreptococcus pneumo-niae induced c-Jun-N-terminal kinase- andAP-I-dependent IL-8 release by lung epithelial BEAS-2B cellsrdquoRespiratory Researchvol 7 article 98 2006
[37] M GMatera L Calzetta D Passeri et al ldquoEpithelium integrityis crucial for the relaxant activity of brain natriuretic peptide inhuman isolated bronchirdquo The British Journal of Pharmacologyvol 163 no 8 pp 1740ndash1754 2011
[38] R QuirionM Dalpe A de Lean J Gutkowska M Cantin andJ Genest ldquoAtrial natriuretic factor (ANF) binding sites in brainand related structuresrdquoPeptides vol 5 no 6 pp 1167ndash1172 1984
[39] M B Pamnani D L Clough J S Chen W T Link andF J Haddy ldquoEffects of rat atrial extract on sodium transportand blood pressure in the ratrdquo Proceedings of the Society forExperimental Biology and Medicine vol 176 no 2 pp 123ndash1311984
[40] T R Traynor and S M OrsquoGrady ldquoBrain natriuretic peptidestimulates K andCl secretion across porcine distal colon epithe-liumrdquoTheAmerican Journal of PhysiologymdashCell Physiology vol260 no 4 pp C750ndashC755 1991
[41] R Rollın A Mediero M Roldan-Pallares A Fernandez-Cruzand R Fernandez-Durango ldquoNatriuretic peptide system in thehuman retinardquoMolecular Vision vol 10 pp 15ndash22 2004
[42] M Harada H Itoh O Nakagawa et al ldquoSignificance ofventricular myocytes and nonmyocytes interaction during car-diocyte hypertrophy evidence for endothelin-1 as a paracrinehypertrophic factor from cardiac nonmyocytesrdquo Circulationvol 96 no 10 pp 3737ndash3744 1997
[43] K Kuwahara Y Saito M Harada et al ldquoInvolvement ofcardiotrophin-1 in cardiac myocyte-nonmyocyte interactionsduring hypertrophy of rat cardiac myocytes in vitrordquo Circula-tion vol 100 no 10 pp 1116ndash1124 1999
[44] E Harada O Nakagawa M Yoshimura et al ldquoEffect ofinterleukin-1120573 on cardiac hypertrophy and production of natri-uretic peptides in rat cardiocyte culturerdquo Journal of Molecularand Cellular Cardiology vol 31 no 11 pp 1997ndash2006 1999
[45] V Chiurchiu V Izzi F DrsquoAquilio F Carotenuto P Di Nardoand P M Baldini ldquoBrain natriuretic peptide (BNP) regulatesthe production of inflammatory mediators in human THP-1macrophagesrdquo Regulatory Peptides vol 148 no 1ndash3 pp 26ndash322008
[46] N S Holden C F Rider M J Bell et al ldquoEnhancement ofinflammatory mediator release by 1205732-adrenoceptor agonists inairway epithelial cells is reversed by glucocorticoid actionrdquoTheBritish Journal of Pharmacology vol 160 no 2 pp 410ndash4202010
[47] V S Fang C C Juan J G S Won and L T Ho ldquoFurther studyof aldosterone secretion-inhibitory factor and brain natriureticpeptide on cortisol production of Guinea pig zona fasciculatacellsrdquo Chinese Journal of Physiology vol 43 no 4 pp 141ndash1472000
[48] G Hellermann X Kong J Gunnarsdottir et al ldquoMechanismof bronchoprotective effects of a novel natriuretic hormonepeptiderdquo Journal of Allergy and Clinical Immunology vol 113no 1 pp 79ndash85 2004
[49] K K Ma T Ogawa and A J de Bold ldquoSelective upregulationof cardiac brain natriuretic peptide at the transcriptional andtranslational levels by pro-inflammatory cytokines and by con-ditionedmedium derived frommixed lymphocyte reactions viap38 MAP kinaserdquo Journal of Molecular and Cellular Cardiologyvol 36 no 4 pp 505ndash513 2004
[50] T Ogawa J P Veinot M L K de Bold T Georgalis and AJ De Bold ldquoAngiotensin II receptor antagonism reverts theselective cardiac BNP upregulation and secretion observed inmyocarditisrdquo The American Journal of PhysiologymdashHeart andCirculatory Physiology vol 294 no 6 pp H2596ndashH2603 2008
[51] B J Carver E J Plosa A M Stinnett T S Blackwell andL S Prince ldquoInteractions between NF-120581B and SP3 connectinflammatory signaling with reduced FGF-10 expressionrdquo TheJournal of Biological Chemistry vol 288 no 21 pp 15318ndash153252013
[52] C Dong G Wang B Li et al ldquoAnti-asthmatic agents alleviatepulmonary edema by upregulating AQP1 and AQP5 expressionin the lungs of mice with OVA-induced asthmardquo RespiratoryPhysiology and Neurobiology vol 181 no 1 pp 21ndash28 2012
Figure 8 BNP effect on ANP NPR-1 AQP1 and AQP5 expressionBEAS-2B cells were treated with 001 nM BNP for 24 h Histograms show(a) ANP (b) NPR-1 (c) AQP1 and (d) AQP5 gene expression at mRNA level All histograms indicate means plusmn SD of three different cultureseach one tested in quadruplicate and expressed as percentage of control (lowast119875 lt 005 versus untreated cells)
020406080
100120140160180200
DEX
BNP
( o
f con
trol)
C TNF-120572 TNF-120572 + DEX
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowast
lowast
lowast
Figure 9 Dexamethasone effect on BNP expression BEAS-2B cellswere treatedwith 1 120583Mdexamethasone (DEX) and 40 ngmLTNF-120572alone or in combination with DEX for 24 h Histograms show BNPgene expression at mRNA (gray bars) and protein (black bars) levelAll histograms indicate means plusmn SD of three different cultures eachone tested in quadruplicate and expressed as percentage of control(lowast119875 lt 001 lowastlowast119875 lt 0001 and lowastlowastlowast119875 lt 00001 versus Controls) CControls (untreated cells)
and in vitro models of lung injury [18] and the therapeu-tical relevance of these experimental observations has beenshown in intensive care patients without heart disease whereintravenous ANP infusion diminished pulmonary vascularpermeability and pulmonary edema [18]
Regarding AQPs we demonstrated that BEAS-2B cellsexpress AQP1 and AQP5 and that TNF-120572 directly down-regulates their expression at mRNA and protein levelsRegarding AQP1 also an additional BNP-mediated TNF-120572involvement can be proposed In fact AQP1 down-regulationwas observed with BNP treatment at the dose accumulatedin culture medium after 40 ngmL TNF-120572 treatment Themechanisms that underlie AQPs regulation are of consider-able interest Several AQPs has recently been demonstratedto undergo complex regulation [27] Our results show thatAQP1 and APQ5 are subject to inflammation regulationadding an important piece of information in this contextOur results are in agreement with other studies describinga marked reduction in the expression levels of AQP1 andAQP5 inmouse lung [30] andmouse lung epithelial cells [31]The biological significance of the observed TNF-120572-inducedAQP1 and AQP5 down-regulation is at present unclear Ithas been recently suggested that the decrease in both AQPsmay play an important role in the development of lung edema[52] which is known to be related with fluid retention Wetherefore speculate that TNF-120572-induced AQP1 and AQP5down-regulation might play a role also in the excess of fluidaccumulation related to major surgical procedures
5 Conclusions
In conclusion our results provided evidence that in humanbronchial epithelial cells BEAS-2B TNF-120572 selectively mod-ulates mRNA and protein expression levels of different
12 Mediators of Inflammation
molecules involved in body fluid homeostasis ANP BNPand their receptor NPR-1 AQP1 and AQP5 Moreoverour data pointed out that the changes specifically affectingNPs expression occur independently from hemodynamicinfluences and that inflammation should be considered anextracardiac cause of BNP elevation adding an importantpiece of information in the novel area of study regarding NPsand inflammation In addition we demonstrated that such amodulation occurs via different involvement of NK-120581B andMAPKs signaling pathways Finally we suggested inflamma-tion among the mechanisms involved in the regulation ofAQP1 and AQP5 Therefore in this exploratory study wespeculate that TNF-120572 might be involved in postoperative-fluid retention related to major surgery
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgment
The authors thank Mrs Roberta Frosini for the excellenttechnical assistance
References
[1] E Itobi M Stroud and M Elia ldquoImpact of oedema onrecovery after major abdominal surgery and potential valueof multifrequency bioimpedance measurementsrdquo The BritishJournal of Surgery vol 93 no 3 pp 354ndash361 2006
[2] J A Lowell C Schifferdecker D F Driscoll P N Benottiand B R Bistrian ldquoPostoperative fluid overload not a benignproblemrdquo Critical Care Medicine vol 18 no 7 pp 728ndash7331990
[3] A M Moslashller T Pedersen P-E Svendsen and A EngquistldquoPerioperative risk factors in elective pneumonectomy theimpact of excess fluid balancerdquo European Journal of Anaesthe-siology vol 19 no 1 pp 57ndash62 2002
[4] E Bennett-Guerrero D E Feierman G R Barclay et alldquoPreoperative and intraoperative predictors of postoperativemorbidity poor graft function and early rejection in 190patients undergoing liver transplantationrdquo Archives of Surgeryvol 136 no 10 pp 1177ndash1183 2001
[5] J P Desborough ldquoThe stress response to trauma and surgeryrdquoThe British Journal of Anaesthesia vol 85 no 1 pp 109ndash1172000
[6] H Tsujimoto S Ono T Majima et al ldquoDifferential toll-likereceptor expression after ex vivo lipopolysaccharide exposurein patients with sepsis and following surgical stressrdquo ClinicalImmunology vol 119 no 2 pp 180ndash187 2006
[7] C Evans C Galustian D Kumar et al ldquoImpact of surgeryon immunologic function comparison between minimallyinvasive techniques and conventional laparotomy for surgi-cal resection of colorectal tumorsrdquo The American Journal ofSurgery vol 197 no 2 pp 238ndash245 2009
[8] F Catena L Ansaloni A Avanzolini et al ldquoSystemic cytokineresponse after emergency and elective surgery for colorectalcarcinomardquo International Journal of Colorectal Disease vol 24no 7 pp 803ndash808 2009
[9] R Sarbinowski S Arvidsson M Tylman T Oresland andA Bengtsson ldquoPlasma concentration of procalcitonin andsystemic inflammatory response syndrome after colorectalsurgeryrdquo Acta Anaesthesiologica Scandinavica vol 49 no 2 pp191ndash196 2005
[10] P Sheeran and G M Hall ldquoCytokines in anaesthesiardquo TheBritish Journal of Anaesthesia vol 78 no 2 pp 201ndash219 1997
[11] L Cagini R Capozzi V Tassi et al ldquoFluid and electrolytebalance after major thoracic surgery by bioimpedance andendocrine evaluationrdquo European Journal of Cardio-ThoracicSurgery vol 40 no 2 pp e71ndashe76 2011
[12] T Omland and T Hagve ldquoNatriuretic peptides physiologic andanalytic considerationsrdquo Heart Failure Clinics vol 5 no 4 pp471ndash487 2009
[13] L R Potter A R Yoder D R Flora L K Antos and DM Dickey ldquoNatriuretic peptides their structures receptorsphysiologic functions and therapeutic applicationsrdquo Handbookof Experimental Pharmacology vol 191 pp 341ndash366 2009
[14] T G Flynn M L de Bold and A J de Bold ldquoThe aminoacid sequence of an atrial peptide with potent diuretic andnatriuretic propertiesrdquo Biochemical and Biophysical ResearchCommunications vol 117 no 3 pp 859ndash865 1983
[15] A J de Bold ldquoAtrial natriuretic factor a hormone produced bythe heartrdquo Science vol 230 pp 767ndash770 1985
[16] K Kangawa Y Tawaragi S Oikawa et al ldquoIdentification of rat 120574atrial natriuretic polypeptide and characterization of the cDNAencoding its precursorrdquo Nature vol 312 no 5990 pp 152ndash1551984
[17] T Sudoh K Kangawa N Minamino and H Matsuo ldquoA newnatriuretic peptide in porcine brainrdquo Nature vol 332 no 6159pp 78ndash81 1988
[18] M Kuhn ldquoEndothelial actions of atrial and B-type natriureticpeptidesrdquo The British Journal of Pharmacology vol 166 no 2pp 522ndash531 2012
[19] A J de Bold ldquoCardiac natriuretic peptides gene expression andsecretion in inflammationrdquo Journal of Investigative Medicinevol 57 pp 29ndash32 2009
[20] S P DrsquoSouza M Davis and G F Baxter ldquoAutocrine andparacrine actions of natriuretic peptides in the heartrdquo Pharma-cology andTherapeutics vol 101 no 2 pp 113ndash129 2004
[21] M Brueckmann G Huhle S Lang et al ldquoPrognostic valueof plasma N-terminal pro-brain natriuretic peptide in patientswith severe sepsisrdquoCirculation vol 112 no 4 pp 527ndash534 2005
[22] J Charpentier C E Luyt Y Fulla et al ldquoBrain natriureticpeptide a marker of myocardial dysfunction and prognosisduring severe sepsisrdquo Critical Care Medicine vol 32 no 3 pp660ndash665 2004
[23] R Shor Y Rozenman A Bolshinsky et al ldquoBNP in septicpatients without systolic myocardial dysfunctionrdquo EuropeanJournal of Internal Medicine vol 17 no 8 pp 536ndash540 2006
[24] T Inoue M Kawai T Nakane et al ldquoInfluence of low-gradeinflammation on plasma B-type natriuretic peptide levelsrdquoInternal Medicine vol 49 no 24 pp 2659ndash2668 2010
[25] P Agre L S King M Yasui et al ldquoAquaporin water channelsmdashfrom atomic structure to clinical medicinerdquo Journal of Physiol-ogy vol 542 no 1 pp 3ndash16 2002
[26] P Agre and D Kozono ldquoAquaporin water channels molecularmechanisms for human diseasesrdquo FEBS Letters vol 555 no 1pp 72ndash78 2003
Mediators of Inflammation 13
[27] J M Carbrey and P Agre ldquoDiscovery of the aquaporins anddevelopment of the fieldrdquo Handbook of Experimental Pharma-cology vol 190 pp 3ndash28 2009
[28] O Bloch and G T Manley ldquoThe role of aquaporin-4 in cerebralwater transport and edemardquo Neurosurgical Focus vol 22 no 5article E3 2007
[29] J Li M Xu Q Fan et al ldquoTanshinone IIA ameliorates seawaterexposure-induced lung injury by inhibiting aquaporins (AQP)1 and AQP5 expression in lungrdquo Respiratory Physiology andNeurobiology vol 176 no 1-2 pp 39ndash49 2011
[30] J E Towne K S Harrod C M Krane and A G MenonldquoDecreased expression of aquaporin (AQP)1 and AQP5 inmouse lung after acute viral infectionrdquoThe American Journal ofRespiratory Cell and Molecular Biology vol 22 no 1 pp 34ndash442000
[31] J E Towne C M Krane C J Bachurski and A G MenonldquoTumor necrosis factor-120572 inhibits aquaporin 5 expression inmouse lung epithelial cellsrdquoThe Journal of Biological Chemistryvol 276 no 22 pp 18657ndash18664 2001
[32] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001
[33] K Verhelst I Carpentier and R Beyaert ldquoRegulation of TNF-induced NF-120581B activation by different cytoplasmic ubiquitina-tion eventsrdquo Cytokine and Growth Factor Reviews vol 22 no5-6 pp 277ndash286 2011
[34] L Qian Y Shen J C Chen et al ldquo3D-QSAR and dockingstudies of quinazoline derivatives with the inhibatory activitytoward NF-120581BrdquoQSAR and Combinatorial Science vol 27 no 8pp 984ndash995 2008
[35] P D NrsquoGuessan S Hippenstiel M O Etouem et al ldquoStrepto-coccus pneumoniae induced p38MAPK- andNF-120581B-dependentCOX-2 expression in human lung epitheliumrdquo The AmericanJournal of PhysiologymdashLung Cellular and Molecular Physiologyvol 290 no 6 pp L1131ndashL1138 2006
[36] B Schmeck K Moog J Zahlten et al ldquoStreptococcus pneumo-niae induced c-Jun-N-terminal kinase- andAP-I-dependent IL-8 release by lung epithelial BEAS-2B cellsrdquoRespiratory Researchvol 7 article 98 2006
[37] M GMatera L Calzetta D Passeri et al ldquoEpithelium integrityis crucial for the relaxant activity of brain natriuretic peptide inhuman isolated bronchirdquo The British Journal of Pharmacologyvol 163 no 8 pp 1740ndash1754 2011
[38] R QuirionM Dalpe A de Lean J Gutkowska M Cantin andJ Genest ldquoAtrial natriuretic factor (ANF) binding sites in brainand related structuresrdquoPeptides vol 5 no 6 pp 1167ndash1172 1984
[39] M B Pamnani D L Clough J S Chen W T Link andF J Haddy ldquoEffects of rat atrial extract on sodium transportand blood pressure in the ratrdquo Proceedings of the Society forExperimental Biology and Medicine vol 176 no 2 pp 123ndash1311984
[40] T R Traynor and S M OrsquoGrady ldquoBrain natriuretic peptidestimulates K andCl secretion across porcine distal colon epithe-liumrdquoTheAmerican Journal of PhysiologymdashCell Physiology vol260 no 4 pp C750ndashC755 1991
[41] R Rollın A Mediero M Roldan-Pallares A Fernandez-Cruzand R Fernandez-Durango ldquoNatriuretic peptide system in thehuman retinardquoMolecular Vision vol 10 pp 15ndash22 2004
[42] M Harada H Itoh O Nakagawa et al ldquoSignificance ofventricular myocytes and nonmyocytes interaction during car-diocyte hypertrophy evidence for endothelin-1 as a paracrinehypertrophic factor from cardiac nonmyocytesrdquo Circulationvol 96 no 10 pp 3737ndash3744 1997
[43] K Kuwahara Y Saito M Harada et al ldquoInvolvement ofcardiotrophin-1 in cardiac myocyte-nonmyocyte interactionsduring hypertrophy of rat cardiac myocytes in vitrordquo Circula-tion vol 100 no 10 pp 1116ndash1124 1999
[44] E Harada O Nakagawa M Yoshimura et al ldquoEffect ofinterleukin-1120573 on cardiac hypertrophy and production of natri-uretic peptides in rat cardiocyte culturerdquo Journal of Molecularand Cellular Cardiology vol 31 no 11 pp 1997ndash2006 1999
[45] V Chiurchiu V Izzi F DrsquoAquilio F Carotenuto P Di Nardoand P M Baldini ldquoBrain natriuretic peptide (BNP) regulatesthe production of inflammatory mediators in human THP-1macrophagesrdquo Regulatory Peptides vol 148 no 1ndash3 pp 26ndash322008
[46] N S Holden C F Rider M J Bell et al ldquoEnhancement ofinflammatory mediator release by 1205732-adrenoceptor agonists inairway epithelial cells is reversed by glucocorticoid actionrdquoTheBritish Journal of Pharmacology vol 160 no 2 pp 410ndash4202010
[47] V S Fang C C Juan J G S Won and L T Ho ldquoFurther studyof aldosterone secretion-inhibitory factor and brain natriureticpeptide on cortisol production of Guinea pig zona fasciculatacellsrdquo Chinese Journal of Physiology vol 43 no 4 pp 141ndash1472000
[48] G Hellermann X Kong J Gunnarsdottir et al ldquoMechanismof bronchoprotective effects of a novel natriuretic hormonepeptiderdquo Journal of Allergy and Clinical Immunology vol 113no 1 pp 79ndash85 2004
[49] K K Ma T Ogawa and A J de Bold ldquoSelective upregulationof cardiac brain natriuretic peptide at the transcriptional andtranslational levels by pro-inflammatory cytokines and by con-ditionedmedium derived frommixed lymphocyte reactions viap38 MAP kinaserdquo Journal of Molecular and Cellular Cardiologyvol 36 no 4 pp 505ndash513 2004
[50] T Ogawa J P Veinot M L K de Bold T Georgalis and AJ De Bold ldquoAngiotensin II receptor antagonism reverts theselective cardiac BNP upregulation and secretion observed inmyocarditisrdquo The American Journal of PhysiologymdashHeart andCirculatory Physiology vol 294 no 6 pp H2596ndashH2603 2008
[51] B J Carver E J Plosa A M Stinnett T S Blackwell andL S Prince ldquoInteractions between NF-120581B and SP3 connectinflammatory signaling with reduced FGF-10 expressionrdquo TheJournal of Biological Chemistry vol 288 no 21 pp 15318ndash153252013
[52] C Dong G Wang B Li et al ldquoAnti-asthmatic agents alleviatepulmonary edema by upregulating AQP1 and AQP5 expressionin the lungs of mice with OVA-induced asthmardquo RespiratoryPhysiology and Neurobiology vol 181 no 1 pp 21ndash28 2012
molecules involved in body fluid homeostasis ANP BNPand their receptor NPR-1 AQP1 and AQP5 Moreoverour data pointed out that the changes specifically affectingNPs expression occur independently from hemodynamicinfluences and that inflammation should be considered anextracardiac cause of BNP elevation adding an importantpiece of information in the novel area of study regarding NPsand inflammation In addition we demonstrated that such amodulation occurs via different involvement of NK-120581B andMAPKs signaling pathways Finally we suggested inflamma-tion among the mechanisms involved in the regulation ofAQP1 and AQP5 Therefore in this exploratory study wespeculate that TNF-120572 might be involved in postoperative-fluid retention related to major surgery
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgment
The authors thank Mrs Roberta Frosini for the excellenttechnical assistance
References
[1] E Itobi M Stroud and M Elia ldquoImpact of oedema onrecovery after major abdominal surgery and potential valueof multifrequency bioimpedance measurementsrdquo The BritishJournal of Surgery vol 93 no 3 pp 354ndash361 2006
[2] J A Lowell C Schifferdecker D F Driscoll P N Benottiand B R Bistrian ldquoPostoperative fluid overload not a benignproblemrdquo Critical Care Medicine vol 18 no 7 pp 728ndash7331990
[3] A M Moslashller T Pedersen P-E Svendsen and A EngquistldquoPerioperative risk factors in elective pneumonectomy theimpact of excess fluid balancerdquo European Journal of Anaesthe-siology vol 19 no 1 pp 57ndash62 2002
[4] E Bennett-Guerrero D E Feierman G R Barclay et alldquoPreoperative and intraoperative predictors of postoperativemorbidity poor graft function and early rejection in 190patients undergoing liver transplantationrdquo Archives of Surgeryvol 136 no 10 pp 1177ndash1183 2001
[5] J P Desborough ldquoThe stress response to trauma and surgeryrdquoThe British Journal of Anaesthesia vol 85 no 1 pp 109ndash1172000
[6] H Tsujimoto S Ono T Majima et al ldquoDifferential toll-likereceptor expression after ex vivo lipopolysaccharide exposurein patients with sepsis and following surgical stressrdquo ClinicalImmunology vol 119 no 2 pp 180ndash187 2006
[7] C Evans C Galustian D Kumar et al ldquoImpact of surgeryon immunologic function comparison between minimallyinvasive techniques and conventional laparotomy for surgi-cal resection of colorectal tumorsrdquo The American Journal ofSurgery vol 197 no 2 pp 238ndash245 2009
[8] F Catena L Ansaloni A Avanzolini et al ldquoSystemic cytokineresponse after emergency and elective surgery for colorectalcarcinomardquo International Journal of Colorectal Disease vol 24no 7 pp 803ndash808 2009
[9] R Sarbinowski S Arvidsson M Tylman T Oresland andA Bengtsson ldquoPlasma concentration of procalcitonin andsystemic inflammatory response syndrome after colorectalsurgeryrdquo Acta Anaesthesiologica Scandinavica vol 49 no 2 pp191ndash196 2005
[10] P Sheeran and G M Hall ldquoCytokines in anaesthesiardquo TheBritish Journal of Anaesthesia vol 78 no 2 pp 201ndash219 1997
[11] L Cagini R Capozzi V Tassi et al ldquoFluid and electrolytebalance after major thoracic surgery by bioimpedance andendocrine evaluationrdquo European Journal of Cardio-ThoracicSurgery vol 40 no 2 pp e71ndashe76 2011
[12] T Omland and T Hagve ldquoNatriuretic peptides physiologic andanalytic considerationsrdquo Heart Failure Clinics vol 5 no 4 pp471ndash487 2009
[13] L R Potter A R Yoder D R Flora L K Antos and DM Dickey ldquoNatriuretic peptides their structures receptorsphysiologic functions and therapeutic applicationsrdquo Handbookof Experimental Pharmacology vol 191 pp 341ndash366 2009
[14] T G Flynn M L de Bold and A J de Bold ldquoThe aminoacid sequence of an atrial peptide with potent diuretic andnatriuretic propertiesrdquo Biochemical and Biophysical ResearchCommunications vol 117 no 3 pp 859ndash865 1983
[15] A J de Bold ldquoAtrial natriuretic factor a hormone produced bythe heartrdquo Science vol 230 pp 767ndash770 1985
[16] K Kangawa Y Tawaragi S Oikawa et al ldquoIdentification of rat 120574atrial natriuretic polypeptide and characterization of the cDNAencoding its precursorrdquo Nature vol 312 no 5990 pp 152ndash1551984
[17] T Sudoh K Kangawa N Minamino and H Matsuo ldquoA newnatriuretic peptide in porcine brainrdquo Nature vol 332 no 6159pp 78ndash81 1988
[18] M Kuhn ldquoEndothelial actions of atrial and B-type natriureticpeptidesrdquo The British Journal of Pharmacology vol 166 no 2pp 522ndash531 2012
[19] A J de Bold ldquoCardiac natriuretic peptides gene expression andsecretion in inflammationrdquo Journal of Investigative Medicinevol 57 pp 29ndash32 2009
[20] S P DrsquoSouza M Davis and G F Baxter ldquoAutocrine andparacrine actions of natriuretic peptides in the heartrdquo Pharma-cology andTherapeutics vol 101 no 2 pp 113ndash129 2004
[21] M Brueckmann G Huhle S Lang et al ldquoPrognostic valueof plasma N-terminal pro-brain natriuretic peptide in patientswith severe sepsisrdquoCirculation vol 112 no 4 pp 527ndash534 2005
[22] J Charpentier C E Luyt Y Fulla et al ldquoBrain natriureticpeptide a marker of myocardial dysfunction and prognosisduring severe sepsisrdquo Critical Care Medicine vol 32 no 3 pp660ndash665 2004
[23] R Shor Y Rozenman A Bolshinsky et al ldquoBNP in septicpatients without systolic myocardial dysfunctionrdquo EuropeanJournal of Internal Medicine vol 17 no 8 pp 536ndash540 2006
[24] T Inoue M Kawai T Nakane et al ldquoInfluence of low-gradeinflammation on plasma B-type natriuretic peptide levelsrdquoInternal Medicine vol 49 no 24 pp 2659ndash2668 2010
[25] P Agre L S King M Yasui et al ldquoAquaporin water channelsmdashfrom atomic structure to clinical medicinerdquo Journal of Physiol-ogy vol 542 no 1 pp 3ndash16 2002
[26] P Agre and D Kozono ldquoAquaporin water channels molecularmechanisms for human diseasesrdquo FEBS Letters vol 555 no 1pp 72ndash78 2003
Mediators of Inflammation 13
[27] J M Carbrey and P Agre ldquoDiscovery of the aquaporins anddevelopment of the fieldrdquo Handbook of Experimental Pharma-cology vol 190 pp 3ndash28 2009
[28] O Bloch and G T Manley ldquoThe role of aquaporin-4 in cerebralwater transport and edemardquo Neurosurgical Focus vol 22 no 5article E3 2007
[29] J Li M Xu Q Fan et al ldquoTanshinone IIA ameliorates seawaterexposure-induced lung injury by inhibiting aquaporins (AQP)1 and AQP5 expression in lungrdquo Respiratory Physiology andNeurobiology vol 176 no 1-2 pp 39ndash49 2011
[30] J E Towne K S Harrod C M Krane and A G MenonldquoDecreased expression of aquaporin (AQP)1 and AQP5 inmouse lung after acute viral infectionrdquoThe American Journal ofRespiratory Cell and Molecular Biology vol 22 no 1 pp 34ndash442000
[31] J E Towne C M Krane C J Bachurski and A G MenonldquoTumor necrosis factor-120572 inhibits aquaporin 5 expression inmouse lung epithelial cellsrdquoThe Journal of Biological Chemistryvol 276 no 22 pp 18657ndash18664 2001
[32] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001
[33] K Verhelst I Carpentier and R Beyaert ldquoRegulation of TNF-induced NF-120581B activation by different cytoplasmic ubiquitina-tion eventsrdquo Cytokine and Growth Factor Reviews vol 22 no5-6 pp 277ndash286 2011
[34] L Qian Y Shen J C Chen et al ldquo3D-QSAR and dockingstudies of quinazoline derivatives with the inhibatory activitytoward NF-120581BrdquoQSAR and Combinatorial Science vol 27 no 8pp 984ndash995 2008
[35] P D NrsquoGuessan S Hippenstiel M O Etouem et al ldquoStrepto-coccus pneumoniae induced p38MAPK- andNF-120581B-dependentCOX-2 expression in human lung epitheliumrdquo The AmericanJournal of PhysiologymdashLung Cellular and Molecular Physiologyvol 290 no 6 pp L1131ndashL1138 2006
[36] B Schmeck K Moog J Zahlten et al ldquoStreptococcus pneumo-niae induced c-Jun-N-terminal kinase- andAP-I-dependent IL-8 release by lung epithelial BEAS-2B cellsrdquoRespiratory Researchvol 7 article 98 2006
[37] M GMatera L Calzetta D Passeri et al ldquoEpithelium integrityis crucial for the relaxant activity of brain natriuretic peptide inhuman isolated bronchirdquo The British Journal of Pharmacologyvol 163 no 8 pp 1740ndash1754 2011
[38] R QuirionM Dalpe A de Lean J Gutkowska M Cantin andJ Genest ldquoAtrial natriuretic factor (ANF) binding sites in brainand related structuresrdquoPeptides vol 5 no 6 pp 1167ndash1172 1984
[39] M B Pamnani D L Clough J S Chen W T Link andF J Haddy ldquoEffects of rat atrial extract on sodium transportand blood pressure in the ratrdquo Proceedings of the Society forExperimental Biology and Medicine vol 176 no 2 pp 123ndash1311984
[40] T R Traynor and S M OrsquoGrady ldquoBrain natriuretic peptidestimulates K andCl secretion across porcine distal colon epithe-liumrdquoTheAmerican Journal of PhysiologymdashCell Physiology vol260 no 4 pp C750ndashC755 1991
[41] R Rollın A Mediero M Roldan-Pallares A Fernandez-Cruzand R Fernandez-Durango ldquoNatriuretic peptide system in thehuman retinardquoMolecular Vision vol 10 pp 15ndash22 2004
[42] M Harada H Itoh O Nakagawa et al ldquoSignificance ofventricular myocytes and nonmyocytes interaction during car-diocyte hypertrophy evidence for endothelin-1 as a paracrinehypertrophic factor from cardiac nonmyocytesrdquo Circulationvol 96 no 10 pp 3737ndash3744 1997
[43] K Kuwahara Y Saito M Harada et al ldquoInvolvement ofcardiotrophin-1 in cardiac myocyte-nonmyocyte interactionsduring hypertrophy of rat cardiac myocytes in vitrordquo Circula-tion vol 100 no 10 pp 1116ndash1124 1999
[44] E Harada O Nakagawa M Yoshimura et al ldquoEffect ofinterleukin-1120573 on cardiac hypertrophy and production of natri-uretic peptides in rat cardiocyte culturerdquo Journal of Molecularand Cellular Cardiology vol 31 no 11 pp 1997ndash2006 1999
[45] V Chiurchiu V Izzi F DrsquoAquilio F Carotenuto P Di Nardoand P M Baldini ldquoBrain natriuretic peptide (BNP) regulatesthe production of inflammatory mediators in human THP-1macrophagesrdquo Regulatory Peptides vol 148 no 1ndash3 pp 26ndash322008
[46] N S Holden C F Rider M J Bell et al ldquoEnhancement ofinflammatory mediator release by 1205732-adrenoceptor agonists inairway epithelial cells is reversed by glucocorticoid actionrdquoTheBritish Journal of Pharmacology vol 160 no 2 pp 410ndash4202010
[47] V S Fang C C Juan J G S Won and L T Ho ldquoFurther studyof aldosterone secretion-inhibitory factor and brain natriureticpeptide on cortisol production of Guinea pig zona fasciculatacellsrdquo Chinese Journal of Physiology vol 43 no 4 pp 141ndash1472000
[48] G Hellermann X Kong J Gunnarsdottir et al ldquoMechanismof bronchoprotective effects of a novel natriuretic hormonepeptiderdquo Journal of Allergy and Clinical Immunology vol 113no 1 pp 79ndash85 2004
[49] K K Ma T Ogawa and A J de Bold ldquoSelective upregulationof cardiac brain natriuretic peptide at the transcriptional andtranslational levels by pro-inflammatory cytokines and by con-ditionedmedium derived frommixed lymphocyte reactions viap38 MAP kinaserdquo Journal of Molecular and Cellular Cardiologyvol 36 no 4 pp 505ndash513 2004
[50] T Ogawa J P Veinot M L K de Bold T Georgalis and AJ De Bold ldquoAngiotensin II receptor antagonism reverts theselective cardiac BNP upregulation and secretion observed inmyocarditisrdquo The American Journal of PhysiologymdashHeart andCirculatory Physiology vol 294 no 6 pp H2596ndashH2603 2008
[51] B J Carver E J Plosa A M Stinnett T S Blackwell andL S Prince ldquoInteractions between NF-120581B and SP3 connectinflammatory signaling with reduced FGF-10 expressionrdquo TheJournal of Biological Chemistry vol 288 no 21 pp 15318ndash153252013
[52] C Dong G Wang B Li et al ldquoAnti-asthmatic agents alleviatepulmonary edema by upregulating AQP1 and AQP5 expressionin the lungs of mice with OVA-induced asthmardquo RespiratoryPhysiology and Neurobiology vol 181 no 1 pp 21ndash28 2012
[27] J M Carbrey and P Agre ldquoDiscovery of the aquaporins anddevelopment of the fieldrdquo Handbook of Experimental Pharma-cology vol 190 pp 3ndash28 2009
[28] O Bloch and G T Manley ldquoThe role of aquaporin-4 in cerebralwater transport and edemardquo Neurosurgical Focus vol 22 no 5article E3 2007
[29] J Li M Xu Q Fan et al ldquoTanshinone IIA ameliorates seawaterexposure-induced lung injury by inhibiting aquaporins (AQP)1 and AQP5 expression in lungrdquo Respiratory Physiology andNeurobiology vol 176 no 1-2 pp 39ndash49 2011
[30] J E Towne K S Harrod C M Krane and A G MenonldquoDecreased expression of aquaporin (AQP)1 and AQP5 inmouse lung after acute viral infectionrdquoThe American Journal ofRespiratory Cell and Molecular Biology vol 22 no 1 pp 34ndash442000
[31] J E Towne C M Krane C J Bachurski and A G MenonldquoTumor necrosis factor-120572 inhibits aquaporin 5 expression inmouse lung epithelial cellsrdquoThe Journal of Biological Chemistryvol 276 no 22 pp 18657ndash18664 2001
[32] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001
[33] K Verhelst I Carpentier and R Beyaert ldquoRegulation of TNF-induced NF-120581B activation by different cytoplasmic ubiquitina-tion eventsrdquo Cytokine and Growth Factor Reviews vol 22 no5-6 pp 277ndash286 2011
[34] L Qian Y Shen J C Chen et al ldquo3D-QSAR and dockingstudies of quinazoline derivatives with the inhibatory activitytoward NF-120581BrdquoQSAR and Combinatorial Science vol 27 no 8pp 984ndash995 2008
[35] P D NrsquoGuessan S Hippenstiel M O Etouem et al ldquoStrepto-coccus pneumoniae induced p38MAPK- andNF-120581B-dependentCOX-2 expression in human lung epitheliumrdquo The AmericanJournal of PhysiologymdashLung Cellular and Molecular Physiologyvol 290 no 6 pp L1131ndashL1138 2006
[36] B Schmeck K Moog J Zahlten et al ldquoStreptococcus pneumo-niae induced c-Jun-N-terminal kinase- andAP-I-dependent IL-8 release by lung epithelial BEAS-2B cellsrdquoRespiratory Researchvol 7 article 98 2006
[37] M GMatera L Calzetta D Passeri et al ldquoEpithelium integrityis crucial for the relaxant activity of brain natriuretic peptide inhuman isolated bronchirdquo The British Journal of Pharmacologyvol 163 no 8 pp 1740ndash1754 2011
[38] R QuirionM Dalpe A de Lean J Gutkowska M Cantin andJ Genest ldquoAtrial natriuretic factor (ANF) binding sites in brainand related structuresrdquoPeptides vol 5 no 6 pp 1167ndash1172 1984
[39] M B Pamnani D L Clough J S Chen W T Link andF J Haddy ldquoEffects of rat atrial extract on sodium transportand blood pressure in the ratrdquo Proceedings of the Society forExperimental Biology and Medicine vol 176 no 2 pp 123ndash1311984
[40] T R Traynor and S M OrsquoGrady ldquoBrain natriuretic peptidestimulates K andCl secretion across porcine distal colon epithe-liumrdquoTheAmerican Journal of PhysiologymdashCell Physiology vol260 no 4 pp C750ndashC755 1991
[41] R Rollın A Mediero M Roldan-Pallares A Fernandez-Cruzand R Fernandez-Durango ldquoNatriuretic peptide system in thehuman retinardquoMolecular Vision vol 10 pp 15ndash22 2004
[42] M Harada H Itoh O Nakagawa et al ldquoSignificance ofventricular myocytes and nonmyocytes interaction during car-diocyte hypertrophy evidence for endothelin-1 as a paracrinehypertrophic factor from cardiac nonmyocytesrdquo Circulationvol 96 no 10 pp 3737ndash3744 1997
[43] K Kuwahara Y Saito M Harada et al ldquoInvolvement ofcardiotrophin-1 in cardiac myocyte-nonmyocyte interactionsduring hypertrophy of rat cardiac myocytes in vitrordquo Circula-tion vol 100 no 10 pp 1116ndash1124 1999
[44] E Harada O Nakagawa M Yoshimura et al ldquoEffect ofinterleukin-1120573 on cardiac hypertrophy and production of natri-uretic peptides in rat cardiocyte culturerdquo Journal of Molecularand Cellular Cardiology vol 31 no 11 pp 1997ndash2006 1999
[45] V Chiurchiu V Izzi F DrsquoAquilio F Carotenuto P Di Nardoand P M Baldini ldquoBrain natriuretic peptide (BNP) regulatesthe production of inflammatory mediators in human THP-1macrophagesrdquo Regulatory Peptides vol 148 no 1ndash3 pp 26ndash322008
[46] N S Holden C F Rider M J Bell et al ldquoEnhancement ofinflammatory mediator release by 1205732-adrenoceptor agonists inairway epithelial cells is reversed by glucocorticoid actionrdquoTheBritish Journal of Pharmacology vol 160 no 2 pp 410ndash4202010
[47] V S Fang C C Juan J G S Won and L T Ho ldquoFurther studyof aldosterone secretion-inhibitory factor and brain natriureticpeptide on cortisol production of Guinea pig zona fasciculatacellsrdquo Chinese Journal of Physiology vol 43 no 4 pp 141ndash1472000
[48] G Hellermann X Kong J Gunnarsdottir et al ldquoMechanismof bronchoprotective effects of a novel natriuretic hormonepeptiderdquo Journal of Allergy and Clinical Immunology vol 113no 1 pp 79ndash85 2004
[49] K K Ma T Ogawa and A J de Bold ldquoSelective upregulationof cardiac brain natriuretic peptide at the transcriptional andtranslational levels by pro-inflammatory cytokines and by con-ditionedmedium derived frommixed lymphocyte reactions viap38 MAP kinaserdquo Journal of Molecular and Cellular Cardiologyvol 36 no 4 pp 505ndash513 2004
[50] T Ogawa J P Veinot M L K de Bold T Georgalis and AJ De Bold ldquoAngiotensin II receptor antagonism reverts theselective cardiac BNP upregulation and secretion observed inmyocarditisrdquo The American Journal of PhysiologymdashHeart andCirculatory Physiology vol 294 no 6 pp H2596ndashH2603 2008
[51] B J Carver E J Plosa A M Stinnett T S Blackwell andL S Prince ldquoInteractions between NF-120581B and SP3 connectinflammatory signaling with reduced FGF-10 expressionrdquo TheJournal of Biological Chemistry vol 288 no 21 pp 15318ndash153252013
[52] C Dong G Wang B Li et al ldquoAnti-asthmatic agents alleviatepulmonary edema by upregulating AQP1 and AQP5 expressionin the lungs of mice with OVA-induced asthmardquo RespiratoryPhysiology and Neurobiology vol 181 no 1 pp 21ndash28 2012
