Production of Reactive Oxygen Intermediates byHuman Macrophages Exposed to Soot Particlesand Asbestos Fibers and Increase in NF-kappa Bp50/p105 mRNA
R. Oettinger, K. Drumm, M. Knorst, P. Krinyak, R. Smolarski, and K. Kienast
III rd Department of Internal Medicine, Division of Pneumology (Head: Prof. Ch. Huber), JohannesGutenberg University Hospital Mainz, Germany
Abstract. Alveolar macrophages (AM) play a decisive role in the immunologicdefense system of the lung and in inflammatory pulmonary pathomechanisms. AMand blood monocytes (BM) were exposed to Chrysotile B, Soot FR 101, and Printex90 (P 90). We evaluated the reactive oxygen intermediate (ROI) release of AM andBM after particle exposure. ROI release was measured by chemiluminescence.Thirty-minute exposure caused a significant (up to 2.5-fold) increase in ROI releaseof AM (100 mg/106 cells) compared with control experiments (p < 0.01). Identicalexposure conditions for BM resulted in a similar reaction pattern (maximum 2.2-fold increase in ROI release;p < 0.05). After a 90-min particle exposure at con-centrations of 10 and 100mg/106 cells, we investigated the steady-state level ofp50/p105 mRNA encoding for the precursor protein of the p50 subunit of nuclearfactor kappa B (NF-kB) by semiquantitative reverse transcription–polymerase chainreaction. One hundredmg Chrysotile B, FR 101, or P 90 induced a significantmaximum 4.0-fold up-regulation of NF-kB gene expression in AM and a 3.3-foldup-regulation in BM (p < 0.05). The addition of superoxide dismutase (200 U/ml)to particle- and fiber-exposed macrophages resulted in inhibition of ROI release anda decrease in NF-kB mRNA expression (70%). NF-kB is an important transcriptionfactor involved in the regulation of numerous genes (e.g., for inflammatory cyto-kines, and cytokine receptors). These cytokines are supposed to be involved ininflammatory pathomechanisms in bronchial epithelial cells, which result, for ex-ample, in chronic obstructive pulmonary disease. Our results suggest that particle-
Offprint requests to:PD. Dr. med. Dipl. Chem. Klaus Kienast, Division of Pneumology, IIIrd Depart-ment of Internal Medicine, University Hospital Mainz, Langenbeckstr. 1, 55131 Mainz, Germany
induced ROI release is associated with an increase in NF-kB (p50/p105) mRNAsteady-state level.
Key words: Alveolar Macrophages—Fibers—Particles—Reactive oxygen interme-diates—NF-kappa B.
Introduction
Soot particles from indoor fireplaces and Chrysotile B fibers (e.g., from insulatingmaterials of buildings) represent indoor air pollutants that are able to induce airway andlung parenchymal injury [13, 17, 25, 27].
Air pollutant exposure activates alveolar macrophages (AM) in the alveolar layercovering the surface of the distal respiratory tract. AM play a decisive role in immu-nologic defense mechanisms of the lung and in inflammatory pulmonary pathomecha-nisms [12, 37]. Activated AM are able to release proinflammatory mediators (e.g.,cytokines) and toxic oxygen radical products reactive oxygen intermediates (ROI) [20,21]. Increased ROI release can induce proliferation of fibroblasts and inflammatorymechanisms in the lung [5, 29, 38]. ROI are known to be involved in the regulation oftranscription factors, which have an important function in the cytokine network [30,34–36]. Transcription factors initiate the transcription of specific genes by binding totheir promoter region. Nuclear factor kappa B (NF-kB) is an important transcriptionfactor activated by ROI-dependent mechanisms [30, 33–36]. The heterodimer NF-kBconsists of a p50 and a p65 subunit [2]. In its inactive form NF-kB is bound to theinhibitory protein I-kappa B (I-kB) located in the cytosol. The p65 subunit is respon-sible for binding I-kB [2]. The dissociation of I-kB from NF-kB results in the trans-location of activated NF-kB in the nucleus and in binding of the p50 subunit to thetarget DNA [2]. The subunit p50 is processed from the precursor protein p105, whichlacks DNA-binding activity [16, 23].
NF-kB is a transcription factor involved in the regulation of expression of numer-ous genes encoding for cytokines, cytokine receptors, and major histocompatibilityantigens [16, 26]. Proinflammatory cytokines are important mediators in pulmonaryinflammation [22]. In this study we analyzed a possible regulation mechanism ofpulmonary inflammatory mediators during particle exposure. We therefore investigatedROI release and gene expression of NF-kB from human AM and BM during particleexposure in vitro.
Material and Methods
Patients
The study population consisted of 7 patients (1 female and 6 male patients) mean age 66 ± 12 y, with anoperated bronchial carcinoma (2 small cell, 5 non-small cell bronchial carcinomas) undergoing bronchoal-veolar lavage (BAL) during restaging bronchoscopy. All patients were nonsmokers. None of the patients
344 R. Oettinger et al.
showed signs of acute bronchitis or pneumonia. No patient received corticosteroids, antibiotics or cytostaticsat the time of the investigation.
BM were obtained from 5 healthy male and 2 female probands, with a mean age of 26 ± 4 y.
BAL and Cell Separation
BAL was performed by means of flexible fiberoptic bronchoscopy at a total volume of 200 ml 0.9% sterilesaline in four 50-ml aliquots as described elsewhere [45]. BAL was carried out in areas of the lung notaffected by the tumor. The cells were washed three times in RPMI 1640 (Seromed) by centrifugation (500× g, 4°C, 10 min). Pure AM were prepared by means of plastic adherence [19]. Differential cell counts wereperformed with Wright’s stained cytocentrifuge preparations. Preparations with AM less than 98% werediscarded.
Blood was obtained by venipuncture and peripheral blood mononuclear cells (PBMNC) were isolatedwith Ficoll-Hypaque density gradient centrifugation [7]. The percentage of BM in PBMNC was determinedby Giemsa staining (i.e., 34 ± 9%). Pure monocytes were prepared from PBMNC by use of their plasticadherence on cell culture clusters (Costar) and washed three times in RPMI 1640 after incubation inhumidified air (37°C, 5% CO2) in RPMI 1640 (+5% fetal calf serum [FCS]) for 45 min).
Description of the Tested Particles and Fibers
The asbestos fiber Chrysotile B (purchased from Fraunhofer Institut Munich, Germany) is a magnesiumsilicate with a low iron content [13]. The length of the fibers used was 1–5mm. The soot particles FR 101and Printex 90 (P 90) (both purchased from Degussa, Germany) are amorphic carbons with differentstructures and properties. Soot FR 101 (specific surface4 20 m2/g; particle size <95 nm) has a coarsestructure and the ability to adsorb polycyclic and other carbons. P 90 is a cleaned soot formed by controlledcombustions. It consists of defined granules (specific surface4 300 m2/g; particle size4 14 nm) with lowabsorbance rates. P 90 particles are predominantly loaded with metallic components (iron <100 ppm, lead<50 ppm, selenium <10 ppm, arsenic <10 ppm, zinc <10 ppm) [14, 15]. Endotoxin contamination of theparticles used in these experiments could be excluded because of the described combustion process andsterile handling.
Exposure to Particles
AM and BM were exposed to Chrysotile B, FR 101, and P 90 at concentrations of 10 and 100mg/106 cells,30 min for detection of ROI release and 90 min for performance of reverse transcription-polymerase chainreaction (RT-PCR). Exposure to the particles was performed with a suspension culture system. Afterdetaching the cells from the plastic dishes by adding 0.05% trypsin/0.02% ethylenediaminetetraacetic acid(EDTA) 8 h before exposure, they were incubated in serum-free culture medium RPMI 1640 and exposedto the particles and fibers in siliconized tubes in humidified air (5% CO2, 37°C) under continuous shaking.Unexposed control experiments were performed under identical conditions. To inhibit the biologic effects ofparticle or fiber-induced ROI release, 200 U/ml superoxide dismutase (SOD) and the particles or fibers wereadded to the culture media for all experiments at the same time.
Chemiluminescence Assay
The chemiluminescence (CL) assay described by Trush and coworkers [41] was used for ROI releasemeasurement. The reaction mixture consisting of 100ml freshly prepared luminol solution (10 mM luminolin dimethylsulfoxide) was diluted 1:50 in RPMI 1640, 100ml RPMI 1640, and a 200-ml cell suspension(containing 106 cells). The particle-exposed cell suspension was pipetted into the prewarmed (37°C) reactionmixture. The addition of the cells marked the beginning of the reaction period. Particles and fibers were notremoved from the cell suspension after exposure because they did not quench the CL signal. CL was
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measured over 50 min at 37°C with a Packard Luminometer (Packard Instruments, Frankfurt, Germany). Theintegral of the curve was detected. The data are presented as counts per minute per 200,000 cells.
Detection of mRNA of NF-kB Expressed by Human AM and BM
After a 90-min exposure of AM and BM to Chrysotile B, FR 101, and P 90 at concentrations of 10 and 100mg/106 cells, the steady-state level of the p50/p105 mRNA—encoding for p105, the precursor protein of thep50 subunit of NF-kB—was investigated by semiquantitative reverse RT-PCR withb-actin as endogenousexternal standard and specific p50/p105 primers. Oligonucleotide primers were synthesized by PharmaciaBiotech. The following sequences of primers were used:
Total RNA was extracted by use of the phenol-chloroform extraction method described by Chomzsinsky andSacchi [9]. cDNA was synthesized from 0.1mg total RNA (as template) using reverse transcriptase (MuLVreverse transcriptase, Perkin Elmer) and an oligo-dT primer (n 4 18). For PCR [32], 5-ml aliquots wereamplified for up to 35 cycles for NF-kB and for up to 24 cycles forb-actin in a 50-ml reaction mixturecontaining 200 mM deoxynucleoside triphosphate (dNTP) mix, 40 pmol primers, 1.5 mM MgCl2, 5 mltenfold PCR buffer (containing 500 mM KCl, 100 mM Tris-HCl [pH 8.3]), and 1 U thermus aquaticus (Taq)DNA polymerase. Each cycle consisted of 45-s denaturation at 95°C, 1-min annealing at 55°C, and 1-minextension at 72°C. For all experiments, control PCR without cDNA was performed to exclude contamination.Ten microliters of the reaction product was analyzed on a 1.5% agarose gel in Tris-borate-EDTA buffercontaining 0.5mg/ml ethidium bromide. The length of the 351-bp fragment reversed transcribed and am-plified by PCR was verified with a molecular weight marker (Boehringer Mannheim, Germany). To avoidthe amplification of a genomic DNA fragment, primers separated by an intronic sequence (intron 10) in thegenome were initially selected. Quantitative evaluation was performed by densitometric scanning.
Cytotoxicity
Cell vitality was checked by trypan blue exclusion and lactate-dehydrogenase quantification (LDH, Boe-hringer Mannheim, Germany). LDH assay was performed in serum-free RPMI 1640 after particle or fiberexposure at concentrations of 10 and 100mg/106 cells for up to 90 min. The average absorbance values oftriplicates were calculated. The following formula was used for calculation to determine the percentagecytotoxicity:
Cytotoxicity (%) =Experiment value− Low control
High control− Low control× 100
Low control 4 Assay medium + CellsHigh control4 Assay medium (+2% Triton X-100) + Cells
Statistical Analysis
The paired and two-tailedt test was used to determine the significance of differences between the valuesrecorded for ROI release or p50/p105 mRNA steady-state level of control exposures and the soot particle orasbestos fiber concentrations.P values ofp < 0.05 were considered significant. Semiquantitative analysis ofPCR products: The raw scanning data were transformed into percentages of the control value for eachexperiment to adjust for intraexperimental variations. All results were adjusted forb-actin as the standard fortotal RNA loading. The data are expressed as mean ± S.E.M.
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Results
Reactive Oxygen Intermediates
We measured ROI release of AM and BM after exposure to Chrysotile B, FR 101, andP 90 at concentrations of 10 and 100mg/106 cells. The CL assay was used for ROImeasurement. ROI accumulation of AM after exposure to different particle concen-trations was almost completely inhibited by 200 U/ml SOD. For example, 30-minChrysotile B exposure (100mg/106 cells) resulted in 76,633 ± 10,834 cpm comparedwith 28,144 ± 4,322 cpm under similar Chrysotile B exposure conditions plus 200U/ml SOD and 30,187 ± 4,821 cpm after control exposure without Chrysotile B.
Thirty-minute exposure of AM to Chrysotile B induced a maximum 2.5-foldincrease in ROI release (p < 0.01, Fig. 1). Exposure of AM to FR 101 over the sameperiod resulted in a nonsignificant 1.4-fold increase in ROI release (p > 0.05). Exposureto P 90 resulted in a 2.1-fold increase in ROI release (p < 0.05). A similar reactionpattern was observed for BM. Chrysotile B also induced a maximum increase in ROIrelease (2.2-fold;p < 0.05).
NF-kB (p50/p105) mRNA
Steady-state level of p50/p105 mRNA in AM and BM was assessed by semiquantita-tive RT-PCR withb-actin as endogenous external standard after 90 min of ChrysotileB, FR 101, and P 90 exposure (at concentrations of 10 and 100mg/106 cells; Fig. 2).
Fig. 1. ROI release of human AM exposed for 30 min to Chrysotile B, FR 101, and Printex 90 at concen-trations of 10 and 100mg/106 cells compared with control exposure. Data represent means and S.E.M.(n 4 7).
Reactive Oxygen Intermediate Production 347
Chrysotile B induced a significant 3.4-fold (10mg) and 3.5-fold (100mg) up-regulation in AM (p < 0.05; Fig. 3) and a maximum 2.9-fold up-regulation in BM(p < 0.05; Fig. 4) of p50/p105 mRNA compared with control exposure without par-ticles. A 2.8-fold (p < 0.05) and 4.4-fold (p > 0.05) up-regulation was detected in AMafter 10 mg and 100mg FR 101 exposure, respectively. BM showed a maximum3.3-fold up-regulation after FR 101 exposure (p < 0.05); 10mg P 90 caused a 2.0-fold(p > 0.05) and 100mg a 4.0-fold (p < 0.05) up-regulation in AM and a maximum3.2-fold up-regulation in BM (p > 0.05).
In the presence of SOD (200 U/ml) the particle-induced (100mg/106 cells) in-crease in the NF-kB p50/p105 mRNA steady-state level was reduced (Fig. 5). TheNF-kB p50/p105 mRNA expression recorded after the addition of SOD ranged at 36%for Chrysotile B, 28% for FR 101, and 36% for Printex 90 (Fig. 6).
Cytotoxicity
Ninety-minute exposure of AM and BM to Chrysotile B, FR 101, and P 90 at con-centrations of 10 and 100mg/106 cells induced a low cytotoxic effect. Particle exposureat a concentration of 100mg/106 cells induced increases in LDH release by AM andBM of up to 7% compared with control exposures (data not shown).
Discussion
The epithelial lining fluid harbors inflammatory cells, predominantly AM, in the distalrespiratory tract [12]. After physiologic activation, macrophages undergo a “respiratoryburst” with consecutive ROI release mediated by the reduced NADPH oxidase system[1, 12]. The exaggerated ROI production observed in acute and chronic pulmonarydiseases may be a causal factor in the development of inflammation in the lung [10,42]. The used in vitro system realizes the exposure of AM to indoor relevant particles
Fig. 2. RT-PCR of p50/p105 mRNA of AM exposed to Chrysotile B (C), FR 101 (FR), and Printex 90 (P)at concentrations of 10 and 100mg/106 cells for 90 min. Lipopolysaccharide (LPS, [100mg/ml]) was usedas positive control. Control exposure without particles and positive control were performed twice in eachexperiment.b-Actin was used as an endogenous external standard for total RNA loading.
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for evaluation of the ROI production and NF-kB specific mRNA expression by thesecells.
On one hand, AM are important representative cells of nonspecific cellular an-swers like the “respiratory burst.” On the other hand, they play an important role in theinitiation of specific pulmonary immunologic mechanisms [43, 44]. Mediators such ascytokines are required to facilitate coordination of the immunologic network. The denovo synthesis of these cytokines is regulated by transcription factors. NF-kB is re-sponsible for the initiation of the transcriptional pathway of some inflammatory cyto-kines [3, 26]. Therefore NF-kB plays a key role in pulmonary inflammation.
Our results demonstrate that exposure of AM and BM to Chrysotile B, FR 101, andP 90 induces cell activation with subsequent ROI production. In our experimentsmaximum ROI releases were measured after a 30-min exposure time. Exposure periodslonger than 30 min resulted in similar ROI releases by AM and BM, which generallydecreased after 6 h [24]. Thirty-minute exposure to Chrysotile B, FR 101, and P 90 atconcentrations of 10 and 100mg/106 cells caused a maximum 2.5-fold significantincrease in ROI release of AM compared with control experiments (p < 0.01). Weobserved similar ROI releases for BM exposures. The accumulation of ROI, especially
Fig. 3. Induction of synthesis of p50/p105 mRNA in human AM exposed to Chrysotile B (C), FR 101 (FR),and Printex 90 (P) at concentrations of 10 and 100mg/106 cells. Macrophages were exposed for 90 min. LPS[100 mg/ml] was used as positive control. Data adjusted for total RNA loading withb-actin. Data representmeans and S.E.M. (n 4 5). Significant results (p < 0.05) are determined for LPS, C 10, C 100, FR 10, andP 100.
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of superoxide anions, after exposure to different particle concentrations was almostcompletely inhibited by 200 U/ml SOD.
We further analyzed the steady-state level of p50/p105 mRNA by performingsemiquantitative RT-PCR after Chrysotile B, FR 101, and P 90 exposure at concen-trations of 10 and 100mg/106 cells of AM and BM for 90 min. We measured an up to4.0-fold significant up-regulation of p50/p105 mRNA in AM and an up to 3.3-foldsignificant up-regulation in BM for all tested particles and fibers (p < 0.05). Antioxi-dative substances like SOD are able to decrease particle- and fiber-induced NF-kBp50/p105 mRNA expression. A maximum inhibitory effect of 72% was noted on theNF-kB p50/p105 mRNA steady-state level.
The results obtained by this study suggest that the membrane-bound NADPHoxidase system is triggered during short-term (30-min) particle exposure of AM andBM, leading to enhancement of ROI production. These radicals are involved directlyin pulmonary inflammatory pathomechanisms causing cell damage or indirectly by theactivation of transcription factors and inflammatory mediators such as inflammatory
Fig. 4. p50/p105 mRNA expression in BM exposed to Chrysotile B (C), FR 101 (FR), and Printex 90 (P)at concentrations of 10 and 100mg/106 cells for 90 min. Data adjusted for total RNA loading withb-actin.Data represent means and S.E.M. (n 4 6). Significant results (p < 0.05) are determined for LPS, C 100, andFR 100.
350 R. Oettinger et al.
cytokines [20, 37]. Superoxide cannot only set up a chain reaction of chemical de-struction in the body and destroy invading microorganisms but it also has an importantfunction in the immune response (e.g., by means of NF-kB activation) [4]. SODprevents accumulation of superoxide. Therefore SOD is also involved in diminishingperoxynitrite formation as superoxide rapidly scavenges nitrogen to the injurious per-oxynitrite anion [8]. Peroxynitrite anions have recently been implicated in the inacti-vation of many enzymes, such as manganese SOD, leading to a depletion of cellularantioxidants [46].
NF-kB is activated by diverse stimuli such as viral infection, lipopolysaccharidesand cytokines (e.g., TNF-a, IL-1) [3]. Many of these stimuli elicit oxidative stress,suggesting an important role of ROI in the pathway of NF-kB activation [35]. Experi-ments using detoxifying antioxidants have demonstrated a decrease in cellular ROIconcentration and the suppression of NF-kB activation [6, 28].
The activation of NF-kB represents a highly regulated process. At the posttran-scriptional level the regulation takes place in the cytosol. Different investigators haveshown that ROI cause dissociation of NF-kB from I-kB in the cytosol [30, 33]. Thedissociation of NF-kB and I-kB leads to a translocation of active NF-kB into thenucleus. Several pathways have been discussed for the regulation of p50/p105 mRNAexpression at the transcriptional level [11, 40]. Tan et al reported a regulation ofp50/p105 gene expression in mouse intestine after tumor necrosis foster-a and PAFstimulation [39]. In this study we investigated the p50/p105 mRNA steady-state levelin human AM and BM after exposure to Chrysotile B, Soot FR 101, and Printex 90.The increase in ROI release, in p50/p105 mRNA steady-state level and inhibition of theparticle-induced increase in p50/p105 mRNA by SOD, suggests that ROI release andNF-kB p50/p105 mRNA expression are associated. Further studies should clarifywhether ROI not only play a major role in the regulation of NF-kB at the posttran-scriptional level [30, 33–36] but also at the gene expression level of NF-kB.
Fig. 5. RT-PCR of NF-kB (p50/p105) mRNA of AM exposed to Chrysotile B (C), FR 101 (FR), and Printex90 (P) at the concentrations of 100mg/106 cells for 90 min. The addition of SOD (200 U/ml) reduced theparticle- or fiber-induced p50/p105 mRNA expression. Control exposures were performed without particles.
Reactive Oxygen Intermediate Production 351
Chrysotile B, FR 101, and P 90 are relevant indoor particles. FR 101 and P 90 bothhave a threshold limit value of 3.5 mg/m3 [14, 15, 31]. Animal experiments andepidemiologic studies indicate that comparable particles like diesel exhaust are impor-tant factors in inflammatory pulmonary processes [18]. Our in vitro experiments sug-gest a possible pathomechanism responsible for inducing inflammatory processes in thelung. The results of this study support the hypothesis that particle-induced ROI releaseis associated with augmented NF-kB mRNA expression and may lead to modificationsin the cytokine network of the lung.
Acknowledgment.This study was supported by the German Ministry of Research and Technology (07INR09 3).
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Fig. 6. RT-PCR of NF-kB (p50/p105) mRNA of AM exposed to Chrysotile B (C), FR 101 (FR), and Printex90 (P) at the concentrations of 100mg/106 cells for 90 min. The addition of SOD (200 U/ml) reduced theparticle- or fiber-induced p50/p105 mRNA expression. Control exposures were performed without particles.Data adjusted for total RNA loading withb-actin. Data represent means and S.E.M. (n 4 4).
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