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This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution
and sharing with colleagues.
Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information
regarding Elsevier’s archiving and manuscript policies areencouraged to visit:
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Immunobiology
j o ur nal homep ag e: www.elsev ier .com/ locate / imbio
Catecholamine production is differently regulated in splenic T- and B-cellsfollowing stress exposure
Marcela Laukovaa,c,∗, Peter Vargovica, Miroslav Vlceka, Katarina Lejavovaa, Sona Hudecovab,Olga Krizanovab, Richard Kvetnanskya
a Institute of Experimental Endocrinology, Slovak Academy of Sciences, Vlarska 3, 83306 Bratislava, Slovakiab Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Vlarska 5, 83334 Bratislava, Slovakiac New York Medical College, 95 Grasslands Rd., BSB 127, Valhalla 10595, NY, USA
a r t i c l e i n f o
Article history:Received 1 August 2012Accepted 27 August 2012
Keywords:ApoptosisCatecholaminesCytokinesImmobilization stressT- and B-cellsSpleen
a b s t r a c t
Objectives: Stress is accompanied also by a rise in splenic catecholamines (CAs). However, indicationsabout endogenous CA production in the spleen exist but there are no data about the cellular sourceof this production and possible modification by stress. Therefore, our aim was to investigate whethersplenic T- and B-cells are one of main sources in the spleen expressing tyrosine hydroxylase (TH), enzymecrucial for CA biosynthesis, and phenylethanolamine N-methyltransferase (PNMT) which is necessaryfor epinephrine production. We also investigated whether stress is able to modify expression of bothenzymes and CA levels within these cell fractions as well as tried to explain functional consequences ofchanges observed.Results: T-cells contain higher levels of TH mRNA than B-cells although protein levels appeared similar.On contrary, the PNMT mRNA and protein were higher in B-cells, which appeared to be the main sourceof PNMT in the spleen. T-cells increased TH and PNMT expression after acute stress while similar rise wasobserved in B-cells after repeated stress, most probably as a consequence of higher CA turnover in bothcell populations. The rise in TH and PNMT was accompanied by an elevation of Bax/Bcl-2 mRNA ratio,number of apoptotic cells and also by a decline of IFN-� mRNA in both cell types. Reduction of IL-2 andIL-4 mRNA was also observed in B-cells.Conclusion: Stress-induced stimulation of endogenous CA biosynthesis in lymphocytes is dependent onthe type of lymphocyte population and duration of stressor and leads to attenuated IFN-� expression andinduction of apoptosis. These changes might contribute to dysregulation of specific immune functionsinvolving T- and B-cells and may decrease the ability to cope with intracellular agents following stresssituations.
Tyrosine hydroxylase (TH, EC 1.14.16.2, UniProt ID: P04177) isthe first and rate limiting enzyme in catecholamine (CA) biosyn-thesis. On the other hand, the final step in the production of CAs– synthesis of epinephrine – is catalyzed by phenylethanolamineN-methyltransferase (PNMT, E.C. 2.1.1.28, UniProt ID: P10937).Besides the adrenal medulla, which contains the highest geneexpression and affinity of TH and PNMT, activity and expressionof these enzymes were also found in neurons of central neuralsystem (Nagatsu 1991) and other non-neural peripheral tissues
∗ Corresponding author at: Institute of Experimental Endocrinology, SlovakAcademy of Sciences, Vlarska 3, 83306 Bratislava, Slovakia. Tel.: +421 904 021 260.
(Hadjiconstantinou et al. 1983; Krizanova et al. 2001; Ziegleret al. 1998) including immune organs, such as thymus and spleen(Andreassi et al. 1998; Jelokova et al. 2002; Warthan et al. 2002).
During the stress exposure, CAs are released mainly from sym-pathetic nerves and taken up from the circulation, hence theirlevels increase rapidly in immune organs, e.g. spleen (Laukovaet al. 2010). Chemical sympathectomy decreased TH activity in thistissue what confirmed predominantly neuronal origin of CA pro-duction (Kawamura et al. 1999). Stress and release of CAs withinthe spleen subsequently regulate many functions of the immunesystem, like production of cytokines (Curtin et al. 2009a; Laukovaet al. 2010), proliferation (Azpiroz et al. 1999), apoptosis of spleniccells (Haberfeld et al. 1999), changes in leukocyte subsets (Avitsuret al. 2005; O’Donnell et al. 2009), splenic macrophage phagocyto-sis (Roy and Rai 2004) and NK cell cytotoxicity (Shimizu et al. 1996)as well as antibody response (Kennedy et al. 2005; Sheridan et al.1998). Psychological stress or in vivo administration of L-DOPA or
M. Laukova et al. / Immunobiology 218 (2013) 780– 789 781
dopamine reduced production of IFN-� and the number of splenicIFN-� producing cells (Carr et al. 2003; Curtin et al. 2009b).
Nevertheless, besides sympathetic innervation, immune organspossess also other cell types producing CAs. This is supported bystudies, where small amount of TH and PNMT mRNA were detectedin thymus and spleen (Andreassi et al. 1998; Jelokova et al. 2002;Kubovcakova et al. 2001; Warthan et al. 2002). Moreover, chronicindividual housing-induced stress attenuated expression of theseenzymes within the spleen (Gavrilovic et al. 2010).
However, several studies have already demonstrated TH expres-sion in various immune cells of human peripheral blood (Mussoet al. 1996; Qiu et al. 2005). Elevation of TH mRNA and CA secretionwas detected in concavalin-A activated lymphocytes isolated fromrat lymph nodes (Qiu et al. 2005) as well as in lipopolysacharideactivated rat peripheral neutrophils and alveolar macrophages(Flierl et al. 2007). Flierl et al. (2009) observed exaggerated THgene expression in neutrophils and macrophages from adrenalec-tomized animals together with the rise of inflammatory response.Increased CA production has been subsequently observed togetherwith a rise in the apoptosis of immune cells and also senescence(Bergquist et al. 1997; Wolkowitz et al. 2011).
Spleen is an organ residing many types of non-immune andimmune cells, including high number of phagocytes and lympho-cytes. Despite this fact, only few studies deal with localization ofendogenously produced TH and PNMT within this tissue. WhileTsao et al. (1998) observed TH protein in enriched murine splenic T-and B-cell lineages using flow cytometry, small amounts of PNMTmRNA and activity was localized only in the marginal zone of thewhite pulp (Jelokova et al. 2002; Warthan et al. 2002). Neverthe-less, the main immune cell type generating TH and PNMT in thespleen has not been clearly elucidated yet.
Moreover, besides innate immune cells (neutrophils, mono-cytes/macrophages), almost no studies exist dealing with endoge-nous CA production in adaptive immune cell populations, such asT- and B-cells. There is also no evidence whether stress per se mayaffect this CA biosynthesis in both lymphocyte populations, sim-ilarly as in other peripheral, non-immune tissues (Kubovcakovaet al. 2006; Kvetnansky et al. 2004; Tillinger et al. 2006).
Therefore, our aims were: (1) to investigate whether T- andB-cells are the potential source of TH and PNMT gene expres-sion within the spleen, (2) to evaluate changes in CA biosynthesisapparatus in splenic T- and B-cells after single and repeated stressexposure and (3) tried to correlate alterations observed in CAbiosynthesis with induction of apoptosis and specific cytokine pro-duction.
Materials and methods
Animals
Male Sprague-Dawley rats (cca 300 g, Charles River, Suzfeld,Germany) were used for our experiments. Rats were housed incontrolled environment (22 ± 2 ◦C, 12 h light/dark cycle). Food andwater were available ad libitum. The Ethical Committee of theInstitute of Experimental Endocrinology (SAS, Bratislava, Slovakia)approved all the experimental procedures with the animals usedin this study in protocol No. RO-2804/07-221/3.
Stress protocol
Immobilization stress (IMO) was performed as originallydescribed by Kvetnansky and Mikulaj (1970) by taping the limbs ofanimals with surgical tape and restricting the motion of the headon an immobilization board. This stress is a potent experimentalstressor because combining physical and psychological stimuli
which activate both sympathoneural and adrenomedullary system.IMO is manifested by the rapid synthesis and release of central andperipheral CAs, as well as stimulation of the HPA axis (Kvetnanskyet al. 2009).
For a single IMO (1 × IMO), rats were immobilized once for 2 h.For repeated IMO, they were immobilized 2 h daily for 7 consecu-tive days (7 × IMO). The IMO was performed at the same time ofthe day (between 8 am and noon). Rats were sacrificed 3 h aftertermination of IMO. The 3-h interval of rest was chosen becausethe highest expression of CA producing enzymes was found pre-viously in various tissues 3 h following IMO (Jelokova et al. 2002;Kubovcakova et al. 2006; Kvetnansky et al. 2004; Kvetnansky et al.2004, 2006; Tillinger et al. 2006). Control animals were not exposedto stress and were sacrificed immediately after removal from theirhome cages.
Isolation of T- and B-cells from rat spleen
After sacrifice, the spleen was removed and gently homogenizedin PBS solution by GentleMax homogenizator (Mylteni Biotech,Germany). Cell suspension was devoid of cell debris by passingthrough the nylon mesh (Mylteni Biotech). Red blood cells, plateletsand polynuclear cells were removed by Ficoll (Sigma Aldrich) gradi-ent centrifugation at 400 × g for 30 min. Isolation of T- and B-cells bymagnetically labeled antibodies (Mylteni Biotech) as well as eval-uation of purity of each fraction was performed by real time PCR asoriginally described (Laukova et al. 2012).
RNA isolation and reverse transcription
Total RNA was isolated from each fraction using TRI Reagentmethod described in the manufacturer’s protocol (MRC Ltd.).The purity and integrity of isolated RNAs was evaluated ona GeneQuant Pro spectrophotometer (Amersham Biosciences).Reverse transcription was performed using 1.5 �g of total RNAs andReady-To-Go You First-Strand Beads with pd (N6) primer (Amer-sham Biosciences), according to manufacturer’s protocol.
Taqman and Sybr green real time PCR analysis
The gene expression of TH and PNMT was evaluated by realtime PCR using Taqman probes. The PCR amplification and detec-tion was carried out using 20% of the reverse transcription product,Probe/ROX Master Mix (Fermentas, Germany) and Taqman probes(Applied Biosystems): Rn00562500 m1 (TH) and Rn01495588 m1(PNMT), in a final volume of 20 �l. Each cycle consisted of 15 sat 95 ◦C, 1 min at 60 ◦C for 40 cycles after the initial activatingstep for 2 min at 50 ◦C and a denaturing step for 10 min at 95 ◦C.Absolute mRNA levels of TH and PNMT were evaluated in spleenhomogenate, T- and B-cells as described previously (Laukova et al.2012; Vargovic et al. 2011). The rest of the genes were analyzedby Sybr green real time PCR as originally described (Laukova et al.2012). Primers, annealing temperatures and the molecular weightof gene fragments are reported in Table 1. The copy number of thetarget genes was normalized to 18S mRNA ribosomal subunit as anendogenous reference, which was not affected by stress exposure.The fold change of controls was set at 1, and normalized fold changeof genes was calculated.
Western blot analysis
Each fraction of T- and B-cells was homogenized in solu-tion containing 0.05 M potassium phosphate (Sigma Aldrich), pH6.65, 0.2% Triton X-100 (Sigma Aldrich) and 0.5 mM PMSF (RocheDiagnostics). Homogenates were incubated on ice for 1 h andthen centrifuged at 10,000 × g for 10 min at 4 ◦C to remove cell
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Table 1Primer sequences and molecular weight of genes analyzed by Sybr green real timePCR. Annealing was performed at 60 ◦C for 30 s.
debris. The supernatant was used to determine protein concentra-tion by bicinchoninic acid (BCA) Protein Assay (Thermo Scientific,Rockford, IL, USA). Ten percent SDS-PAGE gels were used to sepa-rate 80 �g of protein. Glyceraldehyde-3-phosphate dehydrogenase(GAPDH) was used as a loading control. TH, PNMT and GAPDH pro-teins were determined by overnight incubation of the membranein primary antibody. Following three washes in TBST for 10 mineach, membranes were incubated for 1 h at room temperature withhorseradish peroxidase labeled corresponding secondary anti-mouse or anti-rabbit antibody (Sigma–Aldrich Inc., USA) diluted1:10,000. Bands were visualized by Femto Western blotting detec-tion system (Pierce). Optical density of individual bands (o.d/mm2)was analyzed by PC BASE 2.08e software (Raytest, Inc.).
Because of low quality signal for PNMT, evaluation by moresensitive infrared imaging system was performed. After blockingthe membrane, infrared labeled secondary antibodies – goat anti-mouse IRDye 800CW and goat anti-rabbit IRDye 680LT (both fromLi-Cor Biosciences Lincoln, NE) – were added to bind to PNMTand GAPDH primary antibody. The bound complex was detectedusing the Odyssey Infrared Imaging System (Li-Cor). The imageswere analyzed using the Odyssey Application Software, version 1.2(Li-Cor) to obtain the integrated intensities. Numerical data wereanalyzed using GraphPad Prism 4.0 (GraphPad Software, Inc.).
Detection of apoptosis with Annexin-V-Fluos
Apoptosis and necrosis was determined as described previously(Hudecova et al. 2011). Briefly, T- and B-cells were washed withphosphate buffer saline (PBS, pH 7.4), pelleted 200 × g for 5 min,resuspended in 200 �l of Annexin-V-Fluos labeling solution andincubated at room temperature for 20 min in dark. Labeling solu-tion included incubation buffer with 10 mM HEPES/NaOH pH 7.4,140 mM NaCl and 5 mM CaCl2, 2 �l of Annexin-V-Fluos (RocheDiagnostics), and 0.02 �g Propidium iodide. After the incubationwas completed, cells were washed with 1 ml of PBS, pelleted at200 × g for 5 min, suspended in 300 �l of PBS and measured on BD
Accuri® C6 flow cytometer (BD Accuri Cytometers Ann Harbor, MI,USA).
Catecholamine determination
Catecholamine concentration in spleen and T- and B-cell frac-tions was determined using 3-CAT Research RIA kits (LaborDiagnostica Nord, Nordhorn, Germany) according to the man-ufacturer’s protocol, as described previously (Vargovic et al.2011). Concentrations were normalized to protein determinedin homogenate by BCA Protein Assay (Thermo). Values wereexpressed as pg of catecholamine to mg of protein.
Statistical analysis
Each value represents the average of 5–6 animals, from whichspleens were extirpated and both T- and B-cells were isolated.Results are presented as means ± SEM. Statistical differencesamong groups were determined by one-way analysis of variance(ANOVA) followed by the Bonferonni post hoc test or by unpairedt-test (SigmaStat, version 3.1, Systat Software, Inc., USA). Values of*p < 0.05, **p < 0.01, ***p < 0.001 defined the statistical significancevs. control group and values of #p < 0.05, ##p < 0.01, ###p < 0.001defined the significance of repeatedly stressed vs. acutely stressedgroups.
Results
Catecholamine levels in spleen following stress
We have already reported that catecholamine (CA) levels areelevated in the spleen following stress exposure (Laukova et al.2010). To demonstrate that our stress model was effective inelevation CA levels even in this experiment, we measured theirconcentration after single and repeated immobilization stress inthe spleen. While single IMO significantly elevated dopamine(p < 0.001, Fig. 1) and norepinephrine levels (p < 0.05, Fig. 1),epinephrine concentration was markedly increased after repeatedIMO only (p < 0.05, Fig. 1).
TH and PNMT mRNA quantification in splenic T- and B-cells
Spleen possesses own genetic apparatus to produce CAs(Jelokova et al. 2002; Warthan et al. 2002). However, exact cell typeof this endogenous CA biosynthesis is not sufficiently described.Since immune cells are able to produce CAs (Flierl et al. 2007; Mussoet al. 1996; Qiu et al. 2005), we tried to investigate whether splenicT- and B-cells could be the main source expressing TH and PNMTin this tissue. Gene expression analysis showed that splenic T- andB-cells express CA producing enzymes (Fig. 2). The TH mRNA wassignificantly higher in T-cell fraction compared to B-cells (p < 0.001,Fig. 2B) although protein level did not differ between both cell pop-ulations (p > 0.05, Fig. 2D). However, T-cells do not appear to bethe only source of TH, since TH mRNA and protein were markedlyhigher in splenic tissue homogenate (p < 0.001, Fig. 2B and D). Oncontrary, PNMT mRNA and protein were higher in B-cells than inT-cells (p < 0.05, Fig. 2C and E). Although PNMT mRNA was higher inB-cells compared to spleen (p < 0.05, Fig. 2C) and appeared to be themain source of PNMT mRNA within this tissue, the PNMT proteinwas higher in spleen than in lymphocytes (p < 0.001, Fig. 2E).
Catecholamine levels and TH and PNMT expression in splenic T-and B-cells following stress exposure
There is indication that immune cells, particularly phagocytes,increase TH gene expression following adrenalectomy (Flierl et al.
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Fig. 1. Catecholamine levels in the rat spleen after single and repeated immobilization. Dopamine and norepinephrine levels were increased following single immobilization(1 × IMO, 2 h) and returned to the baseline after repeated IMO (7 × IMO, 2 h daily). Epinephrine levels raised significantly after repeated IMO only. Each column represents themean ± SEM and represents an average of 5–6 animals. Statistical significance of IMO versus the control group: *p < 0.05, ***p < 0.001, between 1 × IMO and 7 × IMO #p < 0.05and ###p < 0.001.
Fig. 2. Distribution of intracellular catecholamines and TH and PNMT mRNA in T- and B-cell fractions isolated from the rat spleen. Dopamine level (A) was similar inboth lymphocyte fractions and the spleen. Norepinephrine level was much higher in the spleen compared to T- and B-cells, while the opposite observation was found forepinephrine level. Tyrosine hydroxylase mRNA (B) and phenylethanolamine N-methyltransferase mRNA (C) in each fraction. The TH gene expression was significantly higherin T- compared to B-cells. B-cells expressed higher level of PNMT mRNA than T-cells and appeared to be the main source of PNMT gene expression in the spleen. TH protein(D) and PNMT protein level (E) in each fraction. The signal for both enzymes was still highest in whole spleen than in lymphocytes. Although TH mRNA was higher in T- thanin B-cells, this change was not pronounced in protein level. On the other hand, PNMT protein was higher in B-cells and corresponded with differences in mRNA level. T–T-cellfraction, B–B-cell fraction, spleen–spleen homogenate. Statistical significance between spleen and T- and B-cells *p < 0.05, ***p < 0.001 and between T- and B-cells: +p < 0.05,+++p < 0.001.
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2009) suggesting that regulation of endogenous CA production inimmune cells might be under regulation of adrenal hormones, espe-cially stress hormones. Based on this fact we tried to investigatewhether exposure to a single and repeated immobilization stresswith subsequent release of adrenal CAs and glucocorticoids maymodulate intracellular production of CAs within splenic T- and B-cell.
In T-cells, single IMO (1 × IMO) elevated dopamine (p < 0.05,Fig. 3A), while epinephrine level was declined (p < 0.001, Fig. 3A).Simultaneously, TH and PNMT proteins were exaggerated (p < 0.01,Fig. 3B). Repeated stress exposure (7 × IMO) increased levels ofall three CAs in T-cells (p < 0.05, Fig. 3A), while reduced TH(p < 0.01, Fig. 3B) and PNMT expression (p < 0.05, Fig. 3B) comparedto 1 × IMO. Nevertheless, PNMT protein remained still increased(p < 0.05, Fig. 3B) following repeated IMO compared to TH, whichalready reached the baseline (Fig. 3B).
In B-cells, single IMO induced a drop in norepinephrineand epinephrine (p < 0.05, Fig. 4A), while only epinephrine levelremained significantly decreased after repeated IMO (p < 0.05,Fig. 4A). No changes were observed in TH and PNMT expression fol-lowing single IMO (p > 0.05, Fig. 4B). However, expression of bothenzymes raised after repeated IMO (p < 0.05, Fig. 4B).
Expression of VMAT1 in splenic T- and B-cells
Vesicular monoamine transporter 1 (VMAT1) plays an impor-tant role in the transportation of newly synthesized CAs into storagevesicles (Kvetnansky et al. 2009). Hence, changes in its expressionmight indicate the potential capacity to produce intracellular CAs.In T-cells, VMAT1 mRNA was increased after single IMO (p < 0.001,Fig. 5A) and remained elevated after repeated IMO (p < 0.05, Fig. 5A),though in lesser extent compared to single exposure. In B-cells,VMAT1 mRNA declined after single (p < 0.001, Fig. 5B), while raisedafter repeated IMO (p < 0.05, Fig. 5B). These changes appear to corre-spond with the rise in TH and PNMT expression. In addition, VMAT1mRNA positively correlated with norepinephrine levels in both celltypes (Fig. 6) confirming the stimulation of CA production.
Gene expression of apoptotic and anti-apoptotic markers andcytokines in splenic T- and B-cells following stress
Endogenously produced CAs have been shown to regulatesenescence and apoptosis of immune cells as well as transcriptionprocesses (Bergquist et al. 1997; Wolkowitz et al. 2011). In addition,exposure to stress may evoke a shift towards Th2 immune responseand/or immunosuppression. From among all cytokines, IFN-�, IL-2and IL-4 produced by lymphocytes play a crucial role in this Th2polarization (Calcagni and Elenkov 2006; Kim et al. 2011). Sincebidirectional communication between T- and B-cells via cytokinesis crucial in this process (Harris et al. 2000; Johansson-Lindbomand Borrebaeck 2002), we evaluated apoptotic markers as well asexpression of all three cytokines in T- and B-cells after stress.
Simultaneously, single IMO induced the Bax/Bcl-2 mRNA ratioin T-cells (p < 0.05, Fig. 7A) with no significant changes in executivecaspase 3 mRNA or percentage of annexin positive cells (p > 0.05,Fig. 7A). No changes were found in mRNA of all three apoptoticmarkers after repeated IMO in T-cells. In B-cells, single IMO didnot affect the gene expression of any apoptotic protein investi-gated either number of annexin positive cells (p > 0.05, Fig. 7B),while both, caspase 3 and Bax/Bcl-2 mRNA were induced afterrepeated IMO (p < 0.001, Fig. 7B). Increased apoptosis was con-firmed with Annexin-V-Fluos assay, where we observed an increasein the amount of apoptotic (but not necrotic) cells after repeatedimmobilization in B-cells only (p < 0.001, Fig. 7B).
On the contrary, IFN-� gene expression negatively correlatedwith increased expression of TH and PNMT in both lymphocyte
fractions. IFN-� mRNA declined after single IMO (p < 0.01, Fig. 8A)in T-cells and returned to the baseline following repeated IMO(p > 0.05, Fig. 8A). In B-cells, IFN-� mRNA significantly decreasedafter both, single and repeated IMO (p < 0.001, Fig. 8B). Gene expres-sion of IL-2 and IL-4 were not affected by stress in T-cells (p > 0.05,Fig. 8A), while both of them declined in B-cells after single andrepeated IMO in the same manner as IFN-� (p < 0.001, Fig. 8B).
Discussion
Stress is considered a factor contributing not only to cardio-vascular and metabolic, but also to immune diseases (Elenkov andChrousos 2006; Vanitallie 2002). Nevertheless, the effect of stresson the immune system is dependent on many factors, such asthe type, intensity, duration and frequency of stressor as well asthe immune cell type, microenvironment, in which immune cellsreside and different conditions (Dhabhar 2009; Sanders and Straub2002). However, the mechanism by which stress might initiatethese changes is not clearly appointed. Stress is accompanied bya rapid rise of plasma and tissue catecholamines (CAs), such thosein the spleen (Jelokova et al. 2002; Laukova et al. 2010). Neverthe-less, unchanged levels of DA and NA within this tissue followingrepeated stress indicate their higher release from storage in sym-pathetic nerve terminals (Dronjak and Gavrilovic 2006; Kennedyet al. 2005).
General mechanism, by which exogenous CAs act, involves stim-ulation of adrenergic receptors and modulation of many immunefunctions like splenic cytokine production (Laukova et al. 2010),proliferation and apoptosis of splenic cells (Azpiroz et al. 1999;Haberfeld et al. 1999), reduction of splenic B- and NK-cells, anda concomitant increase in T-cells (O’Donnell et al. 2009; Sheridanet al. 1998). Second mechanism includes up-take of exogenous CAsby splenic noradrenergic varicosities using norepinephrine (NET)or dopamine transporter (DAT, Basu et al. 1993) or direct up-takeby immune cells, such as by lymphocytes via DAT (Amenta et al.2001).
There is evidence that besides circulating CAs immune organsand peripheral immune cells are able to produce endogenous CAs(Bergquist et al. 1994; Warthan et al. 2002). We clearly showedthat besides monocyte/macrophages, neutrophils and mast cells(Flierl et al. 2007; Freeman et al. 2001), splenic T- and B-cellsare one of sources expressing tyrosine hydroxylase (TH), whileB-cells are probably the main source of phenylethanolamine N-methyltransferase (PNMT) mRNA within the rat spleen. Althoughprotein levels for both enzymes were still higher in the spleenthan in lymphocytes, it reflects not only the TH and PNMT pro-tein derived from immune cells but even from local ganglia andsympathetic innervation (Kawamura et al. 1999; Kubovcakovaet al. 2006). Despite changes in TH and PNMT expression betweenT- and B-cells, we have found similar levels of dopamine andepinephrine in both cell types of unstressed rats. These discrep-ancies might be explained by different stability, transcriptionand translation rate of TH and PNMT as well as their activitybetween both cell populations, similarly as in other non-immunecells and tissues (Lenartowski and Goc 2011; Nakashima et al.2007; Panayotacopoulou et al. 2005). It might also reflect differentturnover of CAs within specific cell type.
It has been also shown that TH expression and CA secretionare markedly exaggerated following antigen stimulation in cir-culating immune cells or cells residing within lymph nodes andalveoli (Flierl et al. 2007; Qiu et al. 2005). However, Flierl et al.(2009) observed increased TH gene expression in neutrophils andmacrophages from adrenalectomized animals suggesting thatnot only activation but also hormonal stimuli, especially stress
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Fig. 3. Catecholamine levels and expression of tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) in splenic T-cells after immobilization (IMO)stress. Single IMO (1 × IMO, 2 h) elevated dopamine level in T-cells, while epinephrine level was declined (A). Simultaneously, TH and PNMT expression was exaggerated (B).Repeated stress exposure (7 × IMO, 2 h daily) increased levels of all three catecholamines in T-cells (A) while TH and PNMT expression was reduced compared to 1 × IMO eventhough PNMT remained still increased compared to TH which already reached the baseline (B). Each column represents the mean ± SEM and represents an average of 5-6independent cell fractions, each isolated from 1 animal sacrificed at the same day. Statistical significance of IMO versus the control group: *p < 0.05, **p < 0.01 and ***p < 0.001,and between 1 × IMO and 7 × IMO #p < 0.05 and ##p < 0.01.
hormones, might affect endogenous CA production within immunecells.
We have also confirmed that mediators of stress response areable to initiate CA biosynthesis in lymphocytes, respectively. Dura-tion of stress stimulus seems to be important factor inducingexpression of both, TH and PNMT and subsequent CA production indifferent populations of lymphocytes. We previously reported thatT-cells react more sensitive to acute stress and increase expressionof all three �-ARs already after 2-h immobilization (IMO) stress(Laukova et al. 2012). In experiment presented here, T-cells alsoresponded to acute stress by elevation of TH and PNMT expression.Because epinephrine level was reduced, it points to elevated CAturnover and a need for additional epinephrine production withinthis cell type. However, the adaptation and reduction of TH andPNMT was observed after repeated stress, most probably due tohigher CA accumulation within this cell type and a negative feed-back loop (Fig. 3). On the contrary, B-cells did not respond by a risein CA biosynthesis until repeated IMO when TH and PNMT expres-sion rose in a similar manner as in T-cells, together with a decreaseof intracellular CAs. We suggest the discrepancy between CA lev-els and TH and PNMT expression might be due to stress-inducedCA depletion and hence by a need for CA production, inducedby repeated stress. Moreover, expression of vesicular monoaminetransporter 1 (VMAT1), which plays an important role in the trans-port of newly synthesized CAs into vesicles, positively correlatedwith norepinephrine levels in both T- and B-cells (Fig. 6) what mightsuggest increased capacity for intracellular CA production. Positive
correlation between TH expression and dopamine (r = 0.55895,N = 23, p = 0.0056) in T-cells are in accordance with this claim(not shown). This observation is the first one reporting differentresponse of CA biosynthesis in different lymphocyte populationsdepending on duration of stressor.
Endogenous CAs might further initiate intracellular oxidationand apoptosis (Brown et al. 2003) or might be transported to thenucleus, interact with nuclear receptors and modulate transcrip-tion processes, respectively (Bergquist et al. 2000). On the otherhand, they might be stored into vesicles by vesicular monoaminetransporters (VMAT, Amenta et al. 2001; Flierl et al. 2007; Marinoet al. 1999) or degraded by monoamine oxidase (MAO) andcatechol-O-methyltransferase (COMT, Bidart et al. 1983; Flierl et al.2007; Marino et al. 1999). In our experiments, CA levels withinboth cells populations were declined in most cases following stresscontrary to the spleen homogenate. COMT expression was alsonot affected by stress exposure in both types of lymphocytes (notshown). Even more, we did not find any gene expression for DATor NET in isolated splenic T- or B-cells (not shown). Based on thesefacts, we can propose rather endogenous CA production and secre-tion than re-uptake of CAs occur in splenic T- and B-cells afterstress.
Previous studies demonstrated involvement of endogenous CAsin apoptosis and senescence by induction of Bax and caspase 3and inhibition of Bcl-2 transcription in whole lymphocyte frac-tion (Bergquist et al. 1997; Freeman et al. 2001; Jiang et al. 2006).We observed simultaneous rise in Bax/Bcl-2 mRNA as well as
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Fig. 4. Catecholamine levels and expression of tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) in splenic B-cells after immobilization(IMO) stress. Single IMO (1 × IMO, 2 h) induced a drop of norepinephrine and epinephrine, while only epinephrine level remained significantly decreased after repeated IMO(7 × IMO, 2 h daily) (A). No changes were observed in TH and PNMT expression following single IMO (B). However, protein level of both enzymes was raised after repeatedIMO. Statistical significance of IMO versus the control group: *p < 0.05, **p < 0.01 and between 1 × IMO and 7 × IMO #p < 0.05 and ###p < 0.001.
annexin positive apoptotic cells, and in B-cells also caspase 3 mRNA,together with the rise in CA synthesis. However, these changesmight be transient or reversible, especially in T-cells, as we didnot find any changes in caspase 3 mRNA as well as no significantdifference in percentage of apoptotic cells. This could be partiallyexplained by higher anti-oxidative capacity of specific T-cell sub-populations, such as high amounts of thiols in regulatory T-cells,which might protect these cells from high concentration of endoge-nous CAs, CA oxidation and cell apoptosis (Cosentino et al. 2009).Most probably, this mechanism serves an autoregulatory role.
Besides regulation of apoptosis, the indications about involve-ment of intracellular CAs in regulation of cytokine production in
immune cells exist (Flierl et al. 2007, 2009; Jiang et al. 2006). Thereis also evidence that stress might suppress and/or shift the immuneresponse towards Th2-mediated humoral immune reaction. IL-2and IFN-� are considered a Th1 cytokines, while IL-4 induces theTh2 response. Additionally, IL-2 and IFN-� production is usuallydecreased, while that of IL-4 is increased following stress expo-sure (Calcagni and Elenkov 2006; Kim et al. 2011; Kubera et al.1998; Marshall and Agarwal 2000). Nevertheless, in certain localresponses and under certain conditions, stress hormones may actu-ally facilitate cell-mediated response and inflammation (Calcagniand Elenkov 2006). These changes may favor many immune dis-eases. For example, locally produced CAs have been shown to have
VMAT1 mRNA
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Fig. 5. Gene expression of vesicular monoamine transporter 1 (VMAT1) in T- and B-cells. In T-cells, VMAT1 mRNA was increased after single IMO (1 × IMO, 2 h) and remainedelevated after repeated IMO (7 × IMO, 2 h daily), though in lesser extent compared to 1 × IMO (A). In B-cells, VMAT1 mRNA declined after single, while rose after 7 × IMO(B). Changes in VMAT1 positively correlated with changes found in TH and PNMT expression in both cell fractions (Figs. 3 and 4). Each column represents the mean ± SEMand represents an average of 5-6 independent cell fractions, each isolated from 1 animal sacrificed at the same day. Statistical significance of IMO versus the control group:*p < 0.05, ***p < 0.001 and between 1 × IMO and 7 × IMO #p < 0.05, ###p < 0.001.
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Fig. 6. Correlation of norepinephrine with VMAT 1 expression. Linear regression analysis revealed positive relationship between norepinephrine level and VMAT 1 mRNA inboth, T- and B-cells.
strong anti-inflammatory effects in vivo and in vitro in cells of syn-ovial tissue of rheumatic patients (Capellino et al. 2012). Cosentinoet al. (2005) clearly demonstrated negative correlation betweenendogenous CA production, TH mRNA and IFN-� in peripheral bloodmononuclear cells from multiple sclerosis patients. Even more, psy-chological stress or in vivo administration of dopamine reducedproduction of IFN-� and the number of splenic IFN-� producingcells (Carr et al. 2003; Curtin et al. 2009b). Similarly, we haveconfirmed that the rise of TH and PNMT expression and henceendogenous CA production in T- and B-cells attenuate the levelof IFN-� mRNA during stress. In addition, we have also observed
down-regulation of IL-2 and IL-4 after single and repeated IMOin B-cell fraction. It is very interesting that decreased epinephrinelevels in both lymphocyte populations were accompanied by sim-ilar drop of IFN-� mRNA and in B-cells also IL-2 and IL-4 mRNAfollowing stress what might point to functional importance ofendogenously produced epinephrine within lymphocytes. Sincebidirectional crosstalk between immune cells is needed for appro-priate immune response, we suggest that apoptosis and a declinein cytokine expression in B-cells after stress might be related toless effective activation of some T-cell subpopulation (Johansson-Lindbom and Borrebaeck 2002; Linton et al. 2000; Linton et al. 2000,
Fig. 7. Gene expression of pro-apoptotic (Bax, caspase 3) and anti-apoptotic proteins (Bcl-2) and percentage of apoptotic (annexin positive) T- and B-cells after stressexposure. Single immobilization (1 × IMO, 2 h) induced the Bax/Bcl-2 mRNA ratio and tended to increase the percentage of annexin positive T-cells, while no changes werefound in caspase 3 mRNA. After repeated IMO (7 × IMO, 2 h daily) no changes were detected in T-cells (A). Bax/Bcl-2 mRNA ratio and caspase 3 mRNA were not affectedin B-cells by single IMO, while repeated IMO induced caspase 3, Bax/Bcl-2 mRNA ratio and raised the number of annexin positive cells (B). Each column represents themean ± SEM and represents an average of 5-6 independent cell fractions, each isolated from 1 animal sacrificed at the same day. Statistical significance of IMO versus thecontrol group: *p < 0.05 and ***p < 0.001.
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Fig. 8. The gene expression of interferon-gamma (IFN-�), interleukin-2 (IL-2) and interleukin-4 (IL-4) in T- and B-cells following stress. In T-cells, IFN-� mRNA declined aftersingle immobilization (1 × IMO, 2 h) and returned to the baseline following repeated IMO (7 × IMO, 2 h daily) while IL-2 and IL-4 were not affected by stress (A). In B-cells,IFN-�, IL-2 and IL-4 mRNA decreased after both, single and repeated IMO (B). Each column represents the mean ± SEM and represents an average of 5-6 independent cellfractions, each isolated from 1 animal sacrificed at the same day. Statistical significance of IMO versus the control group: **p < 0.01 and ***p < 0.001.
2003; Scott et al. 1986), attenuation of antibody production andreduced protection against microbial infection as well as healingprocesses what is a typical phenomenon in individuals subjectedto repeated stress (Dhabhar 2009).
Taken together, CA biosynthesis is initiated in splenic T- and B-cells following stress but this stimulation is highly dependent onduration of a stressor. Intracellular CA production is most proba-bly associated with stimulation of apoptotic machinery as well asa drop in IFN-� mRNA in both cell populations. However, a risein endogenous CAs after prolonged stress induces rather immuno-supression of B-cells within the spleen. We might suggest that allthese changes could attenuate ability to activate macrophages, pro-duction of antibodies, decrease resistance to intracellular agents,elimination of bacterial invasion and healing processes (Boehmet al. 1997; Gjessing et al. 2011) following chronic stress (Dhabhar2009). Nevertheless, we cannot exclude the alterations observedmight vary and have slightly different impact on specific subpop-ulation of T- and B-cells. This issue has to be elucidated in furtherstudies as well as the physiological significance of endogenouslyproduced CAs within lymphocytes during stress, their effect onimmune response and whole organism.
Conflict of interest
Authors declare no conflicts of interests.
Acknowledgments
This work was supported by VEGA 02/0188/09, 2/0036/11 and2/0049/10.
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