help button home button
AJRCMB
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Garn, H.
Right arrow Articles by Gemsa, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Garn, H.
Right arrow Articles by Gemsa, D.
American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 386-396, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.4888

Shift Toward an Alternatively Activated Macrophage Response in Lungs of NO2-Exposed Rats

Holger Garn*, Anette Siese*, Sabine Stumpf, Peter J. Barth, Bernd Müller and Diethard Gemsa

Institute of Immunology, Department of Pathology, and Department of Internal Medicine, Laboratory of Lung Cell Biology, Philipps University Marburg, Marburg, Germany

Address correspondence to: Dr. Holger Garn, Institute of Immunology, Philipps University Marburg, Robert-Koch-Str. 17, D-35037 Marburg, Germany. E-mail: garn{at}mailer.uni-marburg.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Inflammatory mechanisms are thought to play an important role in the pathogenesis of acute and chronic obstructive pulmonary diseases. In a rat inhalation model using continuous exposure to 10 ppm nitrogen dioxide for 1, 3, and 20 d, we investigated the inflammatory response with particular focus on the activation state of alveolar macrophages. Whereas the number of inflammatory cells and total protein concentration were increased in the bronchoalveolar lavage (BAL), the amount of the proinflammatory cytokine tumor necrosis factor-{alpha} was markedly reduced with increasing exposure time. In contrast, interleukin (IL)-10 and IL-6 were found at elevated levels and intracellular amounts of suppressor of cytokine signaling-3 protein increased in BAL cells. Upon in vitro lipopolysaccharide stimulation, BAL cells revealed reduced capability to produce the proinflammatory mediators tumor necrosis factor-{alpha}, IL-1ß, and nitric oxide, but showed markedly increased transcription and protein release for IL-10. In addition, elevated levels of IL-6, scavenger receptor B, and suppressor of cytokine signaling-3 mRNA were detected in BAL cells from exposed animals. Analyses of highly purified alveolar macrophages indicated that changes in the activation state of these cells were responsible for the observed effects. In conclusion, a priming toward development of the alternatively activated macrophage phenotype occurred in the lungs of rats following nitrogen dioxide inhalation.

Abbreviations: alveolar macrophages, AMs • bronchoalveolar lavage, BAL • chronic obstructive pulmonary disease, COPD • enzyme-linked immunosorbent assay, ELISA • fluorescence-activated cell sorter, FACS • fetal calf serum, FCS • fluorescein isothiocyanate, FITC • hematoxylin and eosin, H&E • interferon-{gamma}, IFN-{gamma} • interleukin, IL • lipopolysaccharide, LPS • phosphate-buffered saline, PBS • phycoerythrin, PE • reverse transcriptase–polymerase chain reaction, RT-PCR • suppressor of cytokine signaling, SOCS • scavenger receptor B, SR-B • transforming growth factor-ß, TGF-ß • tumor necrosis factor-{alpha}, TNF-{alpha}


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The classical concept of macrophage activation proceeds from an interaction of macrophages with inflammatory agents like lipopolysaccharide (LPS) or necrotic cell material and endogenous priming cytokines such as interferon (IFN)-{gamma}. Such activated macrophages produce a multiplicity of proinflammatory cytokines, reactive oxygen and nitrogen species, proteases, etc., which maintain and further perpetuate the inflammatory process (1). With some delay, macrophages also release antiinflammatory mediators such as interleukin (IL)-10, which, in an autocrine or paracrine way, counteract macrophage activation and suppress the production of inflammatory mediators. The balance of pro- and antiinflammatory factors determines the degree of macrophage activation (2). Recent investigations showed that antiinflammatory agents such as IL-10, IL-4, transforming growth factor (TGF)-ß, and glucocorticoids may not only exert inhibitory actions but may also increase or even induce expression of particular proteins that activate macrophages in a noninflammatory manner (3–5). Among them are IL-10, IL-1 receptor antagonist, several chemokines (e.g., alternative activation-associated chemokine-1 [AMAC-1]), and certain cell surface receptors such as mannose receptor, scavenger receptors, ß-glucan receptor, and CD163 (1, 4, 6–8). These findings led to the new concept of alternative macrophage activation which complements the concept of classical (inflammatory) macrophage activation (1, 3).

In vivo exposure of rats to the oxidant nitrogen dioxide (NO2) has been shown to induce lung tissue inflammation, vascular alterations, alveolar damage, and finally, development of emphysema (9–11). Tissue inflammation is characterized by an infiltration of neutrophil granulocytes and macrophages (12), and both are supposed to play a central role in the inflammatory reaction following NO2 inhalation. However, the mechanism of macrophage activation is poorly understood so far. In vitro exposure of alveolar macrophages to NO2 has been shown to impair macrophage functions by reducing the release of mediators of the classical activation pathway (13, 14). Erroi and coworkers have shown that in vivo exposure to NO2 reduced the production of tumor necrosis factor (TNF)-{alpha} and IL-6 by alveolar macrophages following in vitro endotoxin stimulation (15), and NO2 inhalation has been demonstrated to repress inflammatory cell activation and particle clearance following intratracheal silica particle application in mice (16). These data indicated that classical activation of macrophages was impaired following NO2 exposure. In the present study, we show that NO2 exposure of rats led to functional macrophage alterations that are characteristic of the state of alternative activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal Exposure
Male Fischer 344 rats with a body weight of ~ 120 g were purchased from Charles River Wiga (Sulzfeld, Germany). Animals were maintained in wire cages at temperatures of 18–22°C in a 12–12 h light-dark cycle, and given food and water ad libitum.

Animals in the exposure groups were continuously exposed to 10 ppm NO2 for either 24 h, 3 d, or 20 d. For exposure, cages with a maximum of four animals were placed into air-tight chambers equipped with in- and outlet for the gas mixture and a ventilator to ensure equal distribution of the gas atmosphere throughout the chamber. The volume of the exposure boxes were 60 liters and the gas flow was to 15 liters/min. NO2 (Messer-Griesheim, Duisburg, Germany) concentration was adjusted to 10 ppm by mixing with compressed air and was measured at least twice a day using a NO2-sensitive electrochemical element (ECS 102–1; MPSensor Systems, Munich, Germany). Animal housing conditions and NO2 exposure met German and International Guidelines.

Bronchoalveolar Lavage
Animals were anesthetized by intraperitoneal application of sodium pentobarbital (Narcoren, 100 mg/kg body weight; Merial GmbH, Hallbergmoos, Germany) mixed with 100 IU heparin (LiqueminN; Roche, Basel, Switzerland). The trachea was cannulated, and after thoracotomy the lung was perfused via the pulmonary artery with prewarmed (37°C) perfusion buffer (phosphate-buffered saline [PBS] + Ca2+/Mg2+ supplemented with 10 mM Hepes, 50 µg/ml gentamicin, and 10 U/ml penicillin, pH 7.4) until it became white. Subsequently, lung and heart were removed en bloc. The lung was lavaged extracorporeally six times with 8 ml lavage buffer (Ca2+/Mg2+-free PBS with 10 mM Hepes, 0.2 mM EGTA, 50 µg/ml gentamicin, and 10 U/ml penicillin, pH 7.4) which was allowed to passively run out after each instillation while gently massaging the lung. The bronchoalveolar lavage (BAL) fluid was centrifuged at 300 x g for 10 min at 4°C to obtain alveolar cells and cell-free lavage.

Cells were washed twice in Ca2+/Mg2+-free PBS using the conditions given above, and were finally suspended in 5 ml PBS. The total number of living cells was determined using the CASY1 Cell Counting System (Schärfe Systems, Reutlingen, Germany).

Cell-free lavage was immediately supplemented with Complete protease inhibitor cocktail (1/4 tablet per 15 ml lavage; Roche, Mannheim, Germany) and stored at –20°C until use.

Total BAL protein was quantitated using BCA protein assay kit (Pierce, St. Augustin, Germany) according to manufacturer's instructions.

Preparation of Purified Alveolar Macrophage Populations by Magnetic Bead Separation
To ensure that the observed effects were indeed caused by macrophages, in some experiments highly purified alveolar macrophages (AMs) were prepared. To remove neutrophils and T cells, BAL cells were separated using the MACS magnetic cell sorting system (Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, BAL cells were resuspended in 5 ml MACS-buffer (PBS without Ca2+/Mg2+ + 2 mM EDTA + 0.5% bovine serum albumin) and subsequently filtered through 75- and 30-µm filters to remove cell clumps. After washing and resuspension in 5 ml MACS-buffer, 10 µl of HIS-48-biotin (labels rat neutrophile granulocytes; Pharmingen, Hamburg, Germany) antibody solution were added. Cell suspensions were incubated at 4°C on a roller shaker for 20 min and washed twice in MACS-buffer. Subsequently, cells were suspended in 80 µl MACS-buffer and 10 µl streptavidin-beads and 10 µl rat pan T cell beads (both from Miltenyi) were added. After another 20 min of incubation, cells were washed and finally suspended in 0.5 ml MACS-buffer and applied to prepared MACS-MS columns that were placed in an OctoMACS separation unit. Subsequently, the columns were washed three times with 0.5 ml MACS-buffer. Cells in the pooled flow throughs were washed and suspended in 5 ml RPMI 1640 (Linaris, Bettingen, Germany) supplemented with 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 1 x nonessential amino acids, 100 U/ml penicillin, and 100 µg/ml streptomycin (all purchased from Life Technologies, Gaithersburg, MD), and 1% fetal calf serum (FCS; Biochrom, Berlin, Germany). Purity was analyzed by fluorescence-activated cell sorter (FACS) analysis and yielded > 99 % macrophages in each preparation.

Lung Histology
Excised lungs were cannulated via the stem bronchi and filled with a 4% formaldehyde solution at a constant pressure of 20 cm H2O, and subsequently submersed in the same solution for at least 24 h. After fixation, lungs were sliced by a single sagittal section. Lung tissue was embedded in paraffin and cut into sections of 5 µm thickness. Slices were dewaxed in xylene, rehydrated in graded alcohol solutions, and stained with hematoxylin and eosin (H&E).

FACS Analysis
Differential cell analysis was performed by staining with antibodies to specific cell surface antigens and subsequent flow cytometric analysis as described in detail elsewhere (17). The following primary antibodies (all reagents were purchased from Pharmingen) with the given specificities and labels were used: OX-33-phycoerythrin (PE; CD45RA, B cells), R-73-PE ({alpha}/ß T cell receptor), V65-fluorescein isothiocyanate (FITC; {gamma}/{delta} T cell receptor), 10/78-PE (NKR-P1A = CD161, natural killer cells), His-48-biotin, and 1C7 (macrophages).

Cells were suspended in FACS buffer (PBS supplemented with 1% FCS and 0.1% sodium azide) at a concentration of 2 x 106 cells/ml. Fifty microliters of the appropriately diluted primary antibody were added to 250 µl of the BAL cell suspensions. Following an incubation at 4°C for 30 min, unbound antibodies were removed by three washing steps, including addition of 750 µl FACS buffer and centrifugation at 300 x g for 2 min at 4°C. A second incubation step was necessary for samples labeled with unconjugated or biotinylated primary antibodies. Therefore, 20 µl of FITC-conjugated goat anti-mouse antibody or FITC-conjugated streptavidin were applied to the respective cell suspensions, incubated for 30 min at 4°C, and washed three times. Stained cells were finally suspended in 250 µl FACS fixation buffer (FACS buffer plus 1% formaldehyde), and 250 µl of azide free Diluid (J.T. Baker B.V., Deventer, The Netherlands) were added before FACS analysis. Appropriate controls were performed to ensure the specificity of the labeling reactions including use of irrelevant isotype control immunoglobulins and omission of key reagents.

Flow cytometric analysis of stained cells was performed using a FACScan (Becton Dickinson, Heidelberg, Germany). A forward scatter life gate was set and 5,000 events were measured for each sample using FACScan Plus software. Data analysis was performed with the PC-compatible FlowMate software (Dako A/S, Glostrup, Denmark).

In Vitro Stimulation of BAL Cells
BAL cells were washed twice in Ca2+/Mg2+-free PBS using the above-mentioned conditions, and were finally suspended in supplemented RPMI 1640. The number of living cells was determined using the CASY1 Cell Counting System. BAL cells or purified AMs were incubated at a final concentration of 1 x 106 cells/ml in 48-well cell culture plates (Costar, Corning, NY) at a total volume of 250 µl. Cell cultures were performed in the absence or presence of LPS from Escherichia coli O127:B8 (Difco Laboratories, Chicago, MI) at 37°C in a humid atmosphere containing 5% CO2. Cells were allowed to adhere to the culture plate surface for ~ 1 h before LPS (100 ng/ml) was added. For reverse transcriptase–polymerase chain reaction (RT-PCR) analyses, cells were harvested 6 h after stimulation and lysed with Trizol Reagent (Life Technologies). Cell culture supernatants were collected after 24 h of culture, and stored until use for mediator quantitation at –20°C.

Mediator Quantitation in the Cell-Free BAL and Culture Supernatants
IL-1ß and IL-6 were quantitated in the BAL fluid and in cell culture supernatants with commercially available enzyme-linked immunosorbent assays (ELISAs) to rat IL-1ß obtained from Biosource (Nivelles, Belgium) or rat IL-6 purchased from R&D (Wiesbaden, Germany). BAL fluid TNF-{alpha} was measured using an ultrasensitive ELISA system (Biosource).

Cell culture supernatant TNF-{alpha} and IL-10 were measured with rat specific ELISAs using matched antibody pairs with monoclonal capture and biotinylated detection antibodies and recombinant cytokines (all purchased from Pharmingen) as standards. ELISAs were performed according to a recently described protocol (18) using peroxidase-labeled streptavidine (Roche) and o-phenylendiamine (Sigma, Deisenhofen, Germany) as substrate.

NO was measured as nitrite as follows: 50 µl of the supernatants were incubated with an equal volume of Griess reagent (1 vol 0.1% napthylethylenediamine plus 1 vol 1% sulfanilamide, 5% conc. H3PO4) for 10 min at room temperature. Thereafter, the absorbance of the samples was measured at 550 nm using a Dynatech microplate reader MR7000 (Dynatech Laboratories, Denkendorf, Germany). Nitrite concentration was determined by use of a dilution series of sodium nitrite in supplemented cell culture medium as standard.

Immunodetection of SOCS-3
SOCS-3 protein was analyzed within BAL cells by Western blotting. Total cellular protein was prepared using the M-PER Mammalian Protein Extraction Reagent (Pierce). Protein content of the samples was quantitated with BCA protein assay kit (Pierce). For each sample, 12 µg total protein were separated on NuPage Novex Bis-Tris Gels (Invitrogen, Groningen, The Netherlands) and subsequently blotted onto PVDF-membranes (Immobilon-P; Millipore Corporation, Bedford, MA). Membranes were blocked over-night with Roti-Block (Carl Roth GmbH and Co., Karlsruhe, Germany) at 4°C and incubated with a polyclonal SOCS-3–specific antibody (SC-9023, 0.8 µg/ml in Roti-Block; Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h at room temperature. After washing, bound primary antibody was detected using an alkaline phosphatase–labeled goat anti-rabbit antibody (1 µg/ml, 1 h at room temperature; Roche) and visualized with Sigma Fast BCIP/NBT (Sigma) as substrate.

RT-PCR
Total RNA from cultured BAL cells or purified AMs was obtained by lysis of cells using the Trizol Reagent (Life Technologies) followed by RNA extraction according to the manufacturer's protocol. RNA quality and quantity was determined by spectrophotometry at 260 and 280 nm, and by RiboGreen RNA Quantitation Kit (Molecular Probes, Leiden, The Netherlands).

To eliminate contaminating DNA, 1 µg of total RNA of each sample was treated with DNase I (Life Technologies) and subsequently reverse-transcribed into first-strand cDNA using an oligo(dT)20 primer (MWG Biotech, Ebersberg, Germany) and Omniscript Reverse Transcriptase (Qiagen, Hilden, Germany). All procedures were performed according to the supplier's protocols.

PCR reactions were performed as heat-soaked PCRs using the HotStarTaq Master Mix (Qiagen). Therefore, 1 µl of first-strand cDNA was mixed with 25 µl HotStarTaq Master Mix (containing HotStarTaq Polymerase, dNTPs, and an appropriate reaction buffer), 1 µl of each sense and antisense primer (50 pmol/µl), and 22 µl double-distilled water to yield a final volume of 50 µl per reaction. The samples were subjected to 95°C for 15 min to effectively denature DNA-RNA hybrids and to activate the Taq polymerase. Subsequently, 22–30 PCR cycles (denaturation for 1 min at 94°C, primer annealing for 1 min at optimal temperature, extension at 72°C for 1 min) were performed depending on the abundance of the respective mRNA in the samples. An extension step at 72°C for 10 min was finally added to ensure that all PCR fragments were completely double-stranded. PCR reactions were performed in a TouchDown thermal cycler (Hybaid, Heidelberg, Germany). All products were obtained from the linear phase of the PCR reaction as tested in initial experiments.

We used the following primer pairs for the amplification of the respective gene products at the given annealing temperatures resulting in fragments with the indicated sizes: TNF-{alpha} (fragment size, 296 bp; annealing temperature, 62°C; sense primer, 5'-TCCCAAATGGGCTCCCTCTC-3'; antisense primer, 5'-AAATGGCAAACCGGCTGACG-3'), IL-1ß (fragment size, 505 bp; annealing temperature, 60°C; sense primer, 5'-GCCCGTCCTCTGTGACTCGT-3'; antisense primer, 5'-GGAAGACACGGGTTCCATGG-3'), iNOS (fragment size, 414 bp; annealing temperature, 60°C; sense primer, 5'-TGACCATGGAGCATCCCAAG-3'; antisense primer, 5'-GAGGGACCAGCCAAATCCAG-3'), IL-10 (fragment size, 364 bp; annealing temperature, 60°C; sense primer, 5'-TTGAACCACCCGGCGTCTAC-3'; antisense primer, 5'-TGTGGCCAGCCTTAGGATCG-3'), IL-6 (fragment size, 496 bp; annealing temperature, 60°C; sense primer, 5'-CCACTTCACAAGTCGGAGGC-3'; antisense primer, 5'-CTAGGTTTGCCGAGTAGACC-3'), Scavenger receptor B (fragment size, 295 bp; annealing temperature, 60°C; sense primer, 5'-CCAGCGGGCCTTTATGAACC-3'; antisense primer, 5'-GAATGGTGCCCACATCTGCC-3'), SOCS-3 (fragment size, 551 bp; annealing temperature, 65°C; sense primer, 5'-CGCGGGCACCTTTCTTATCC-3'; antisense primer, 5'-TTGTGCCATGTGCCTCGGAG-3'), and ribosomal protein L32 (fragment size, 290 bp; annealing temperature, 62°C; 5'-AAGCGAAACTGGCGGAAACC-3'; antisense primer, 5'-CTGGCGTTGGGATTGGTGAC-3') as control housekeeping gene.

Resulting PCR products were separated on 1% agarose gels (Life Technologies) with 1x Tris-acetate-EDTA as running buffer. Gels were subsequently visualized by staining with ethidium bromide and documented with a digital gel documentation system (Vilbert-Lourmat, Marne la Vallée, France).

Statistics
All data were calculated and expressed as means ± SEM. After passing normality and equal variance test, data were analyzed for significance with one-way ANOVA and pairwise multiple comparison procedures using Student-Newman-Keuls method.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The Inflammatory Response
Inflammatory processes are usually accompanied by an influx of leukocytes and vascular leakage resulting in increased tissue concentrations of plasma proteins. Therefore, we initially investigated the total number of leukocytes and total protein content in the BAL of NO2-exposed Fischer 344 rats, and analyzed lung histology.

Increased total cell numbers were already found after 1 d of NO2 exposure; these were further elevated following 3 d of exposure, and remained at elevated levels for up to 20 d (Table 1). A similar time course was observed for total BAL protein (Table 1). Significantly increased total protein levels were observed after 1 d of exposure which increased after extended NO2 treatment for 3 or 20 d.


View this table:
[in this window]
[in a new window]
  		TABLE 1 Total cell numbers and total protein content in the BAL of rats after 0, 1, 3, and 20 d exposure to 10 ppm NO2

 
Light microscopy showed no qualitative differences between controls and lung tissue of rat exposed to 10 ppm NO2 for 1 d (Figures 1A and 1B). After exposure for 3 d, the epithelium became hyperplastic, the stroma of the bronchioli disclosed a patchy inflammatory infiltration which was composed of mononuclear cells and a few neutrophil granulocytes. The extent and density of the inflammatory infiltrate was reduced after 20 d of exposure, whereas no difference in the degree of epithelial hyperplasia between the 3 d and 20 d exposure groups was noted (Figure 1).



View larger version (88K):
[in this window]
[in a new window]
  		Figure 1. Lung histology of rats exposed to 10 ppm NO2 for 1 (B), 3 (C), or 20 d (D), in comparison with untreated controls (A). Shown are H&E-stained paraffin sections. Exposure to 10 ppm NO2 for 1 d did not yield any qualitative alterations of the lung tissue (B). Following exposure for 3 d, the epithelium showed hyperplasia (C), which persisted and was also detected after exposure for 20 d (D). The 3-d exposure group disclosed patchy interstitial inflammatory infiltrates which were less intense in the 20-d exposure group.

 
As shown by FACS analyses of BAL cells, the early increase in total cell number was caused by an immediate influx of macrophages and neutrophil granulocytes. For both inflammatory cell populations, the maximum was observed at Day 3 of NO2 exposure. The number of neutrophils declined thereafter (Figure 2B), whereas macrophage numbers stayed at elevated levels for 20 d (Figure 2A). At Day 3 of treatment, a significant number of lymphocytes was detected which almost exclusively consisted of {alpha}/ß T cells (Figure 2C). Despite the general increase in the number of all BAL cell populations at Day 3, T lymphocytes accounted for only 8.1% of all BAL cells at this time point. In all animals tested, B lymphocytes, natural killer cells, and {gamma}/{delta} T cells were present at levels below 1 % of the total BAL cell population.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 2. Absolute numbers of macrophages (A), neutrophils (B), and T lymphocytes (C) in bronchoalveolar lavages of rats exposed to 10 ppm NO2 for different time periods. Relative portions of the respective cell populations in the bronchoalveolar lavage (given as numbers in the bars) were determined by FACS analysis, and absolute numbers were calculated with use of the total cell numbers obtained. Results are the mean ± SEM of at least six rats per group. *Significant difference to untreated controls at P < 0.05.

 
Cytokines in the BAL
Because proinflammatory cytokines such as IL-1ß and TNF-{alpha} are thought to induce and maintain inflammatory processes, we measured their content in the BAL of NO2-exposed animals. Surprisingly, only slightly but not significantly increased amounts of IL-1ß were observed after 1 d of exposure, and no increase of TNF-{alpha} was found at this early inflammatory stage (Figures 3A and 3B). At later time points, when typical signs of inflammation such as mononuclear cell infiltration and increased BAL protein content became apparent, IL-1ß remained at control animal levels (Figure 3A). Unexpectedly, TNF-{alpha} levels were decreased and after 20 d of exposure; almost no immunodetectable amounts of TNF-{alpha} were found in the lavage fluid (Figure 3B).



View larger version (38K):
[in this window]
[in a new window]
  		Figure 3. IL-1ß (A), TNF-{alpha} (B), IL-10 (C), and IL-6 (D) in the BAL of rats following exposure to 10 ppm NO2 for different time periods. Lungs of rats were extracorporeally lavaged and cytokines were quantitated by ELISA. Results are the mean ± SEM of at least 10 rats per group. *Significant difference to untreated controls at P < 0.05.

 
Because antiinflammatory cytokines such as IL-10 may drive the macrophage response into the alternative pathway of activation (1), we examined the time course of IL-10 concentrations in the BAL of NO2-exposed animals. Interestingly, even control animals showed high levels of IL-10 within the BAL fluid which were further elevated after 1 d of exposure. At 3 d of exposure, IL-10 concentrations were similar to control animals and then declined in animals exposed for 20 d (Figure 3C). In contrast, IL-6, which has been shown to exert rather antiinflammatory properties within the lung, was only detectable in very low amounts in the BAL of control animals. Exposure to NO2 resulted in an increase of IL-6 concentrations in the BAL with a peak at 3 d of exposure (Figure 3D). However, due to the low IL-6 levels that exceeded only poorly the detection limit of the available ELISA systems, and due to interindividual differences, these results were not significant.

Immunodetection of SOCS-3
Both IL-10 and IL-6 may induce transcription and translation of the intracellular regulatory molecules of the SOCS family. We analyzed the intracellular protein expression of SOCS-3 in alveolar cells obtained from animals of the different stages of NO2 exposure using Western blotting. As shown in Figure 4, a slight constitutive expression was detected in alveolar cells of animals of the control group (0 d) which was not affected by exposure to NO2 for 1 d. Exposure of animals for 3 d, however, resulted in a markedly increased content of SOCS-3 protein in alveolar cells. After 20 d, SOCS-3 expression was still higher than in cells from control rats.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 4. Immunodetection (Western blot) of SOCS-3 protein in BAL cells from rats exposed to NO2 for 1, 3, and 20 d in comparison with untreated controls (0 d). Following centrifugation of the lavage fluid, cells were lysed to obtain total cellular protein. Proteins were electrophoretically separated using SDS-PAGE and blotted onto a PVDF membrane. Finally, SOCS-3 was detected using an antibody-sandwich technique and a peroxidase substrate. Representative Western blots of two rats per time point are shown.

 
Mediator Release and mRNA Expression of In Vitro Stimulated BAL Cells
To further define the activation state of alveolar cells, BAL cells from differently NO2-exposed animals were cultured in vitro and stimulated with 100 ng/ml LPS. We analyzed the production of pro- and antiinflammatory mediators in the culture supernatants after 24 h culture, and additionally studied the expression of the respective mRNAs 6 h after onset of in vitro culture.

The longer the rats had been exposed to NO2, the more the alveolar cells lost the capability to release proinflammatory mediators upon LPS stimulation (Figures 5A–5C). For TNF-{alpha}, a significant decrease was observed after exposure to NO2 for 3 and 20 d (Figure 5A). For IL-1ß, its LPS-induced production was significantly diminished after 1 and 3 d of exposure, and was finally reduced by ~ 80% after NO2 exposure for 20 d (Figure 5B). In addition, NO production showed a decrease after 20 d of NO2 exposure (Figure 5C). In striking contrast, a 5-fold increase of LPS-induced IL-10 production was found in cells from animals exposed to NO2 for 3 d and also for 20 d (Figure 5D).



View larger version (40K):
[in this window]
[in a new window]
  		Figure 5. Release of TNF-{alpha} (A), IL-1ß (B), NO (C), and IL-10 (D) by in vitro stimulated bronchoalveolar cells from NO2-exposed rats. Cells were obtained from the lungs of animals after the indicated times of NO2 exposure and cultured in the presence of 100 ng/ml LPS. Cytokine content was measured in 24-h culture supernatants by ELISA. Results are the mean ± SEM of at least 10 individually analyzed rats per group. *Significant difference to LPS-stimulated cells of untreated controls at P < 0.05.

 
Further molecular analyses revealed that the increased production of IL-10 by alveolar macrophages from 3 and 20 d exposed animals corresponded to a marked increase in the transcriptional activity. As shown in Figure 6, mRNA amounts for IL-10 were significantly elevated in LPS-stimulated BAL cells from animals that were exposed to NO2 for 3 and 20 d. Similar results were obtained for IL-6 which also showed markedly elevated mRNA levels at Days 3 and 20 after exposure. iNOS mRNA expression was slightly increased at Day 3 after exposure, IL-1ß mRNA remained unchanged, but TNF-{alpha} mRNA expression was markedly depressed after 20 d of NO2 exposure. In addition, scavenger receptor B expression, which is indicative for alternative macrophage activation (7), was also increased in LPS-stimulated AMs of animals that had been exposed to NO2 for 3 d. SOCS-3 expression was elevated in cells from 1 and 3 d exposed animals. In contrast to the expression of IL-10 and IL-6, scavenger receptor B and SOCS-3 mRNA levels were decreased to control cell levels after 20 d of NO2 exposure (Figure 6).




View larger version (128K):
[in this window]
[in a new window]
  		Figure 6. Detection of TNF-{alpha}, IL-1ß, iNOS, IL-10, IL-6, Scavenger Receptor B (SR-B), SOCS-3, and L32 mRNAs in in vitro stimulated bronchoalveolar cells from NO2-exposed rats. Cells were obtained from the lungs of animals after the indicated times of NO2 exposure and cultured in the presence of 100 ng/ml LPS. After 6 h, total RNA was prepared, mRNAs were reverse-transcribed, and primary cDNAs were amplified by PCR using 30 cycles (TNF-{alpha}, IL-10, SR-B), 28 cycles (iNOS), 25 cycles (IL-6, SOCS-3), 22 cycles (L32), or 20 cycles (IL-1ß). Shown are ethidium bromide–stained agarose gels of three individual animals per group and respective densitometric analyses. Densitometric results are expressed as mean ± SEM of six animals per group. * Significant difference to specific mRNA expression in cells of untreated controls at P < 0.05. L32 serves as control housekeeping gene. (Figure 6 continued on next page.)

 
Cytokine Gene Expression in Purified Alveolar Macrophages
To ensure that the observed effects were due to macrophage activation and not the result of changes in the cellular composition of the BAL cell population, we purified AMs and analyzed the mRNA expression for TNF-{alpha} and IL-10. AMs were prepared free of contaminating neutrophils and T cells using MACS technology, finally resulting in cell populations containing > 99 % macrophages as determined by FACS analysis. Resulting cells were either directly lysed in Trizol reagent (Figure 7A) or cultured in the presence of 100 ng/ml LPS (Figure 7B). As shown in Figure 7, in unstimulated AMs a significant decrease in the TNF-{alpha} mRNA expression was observed in cells from animals that had been exposed to NO2 for 3 d. In contrast, IL-10 mRNA expression was markedly increased. LPS stimulation led to a stronger gene expression for both cytokines, as indicated by the lower cycle numbers that were necessary to obtain a comparative signal, but the pattern of decreased TNF-{alpha} and increased IL-10 expression persisted. Unfortunately, due to loss of viability of AMs from 20 d exposed animals during the purification procedure, these cells could not be analyzed. The results indicate that changes in macrophage activation were indeed responsible for the different cytokine patterns in the BAL cell population.



View larger version (43K):
[in this window]
[in a new window]
  		Figure 7. TNF-{alpha}, IL-10, and L32 mRNA expression in MACS-purified alveolar macrophages from NO2-exposed rats. Cells were obtained by bronchoalveolar lavage and subsequently purified by MACS to remove contaminating neutrophils and T cells. Cells were either directly analyzed for cytokine mRNA expression (A) or after in vitro culture for 6 h in the presence of 100 ng/ml LPS (B). Therefore, total RNA was prepared, mRNAs were reverse-transcribed, and primary cDNAs were amplified by PCR using 30 cycles (A) or 25 cycles (B) for TNF-{alpha} PCR, 35 cycles (A), or 30 cycles (B) for IL-10 PCR, and 22 cycles for L32 PCR (A and B). Shown are ethidium bromide–stained agarose gels and the respective densitometric analyses of three individual animals per group. Densitometric results are expressed as mean ± SEM and significant differences (P < 0.05) to mRNA from cells of untreated control animals are indicated by an asterisk.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Because inflammatory processes are regulated by pro- and antiinflammatory mediators (1), a tight control is necessary to ensure a regular course of the inflammatory response. Disturbances in the balance of pro- and antiinflammatory regulators may therefore be responsible for the development of chronic inflammatory diseases such as chronic obstructive pulmonary disease (COPD) (19). Macrophages are involved in initiation, maintenance, and termination of inflammatory reactions by infiltrating the affected tissue and releasing pro- and antiinflammatory cytokines and mediators (20, 21). Whether pro- or antiinflammatory actions prevail depends on the activation state of macrophages. A classical activation of macrophages promotes the release of proinflammatory cytokines such as IL-1ß and TNF-{alpha}, whereas an alternative activation leads to production of antiinflammatory cytokines such as IL-10 and IL-1 receptor antagonist (1, 3).

It is known that, compared with macrophages obtained from other tissues, alveolar macrophages are less potent in the production of inflammatory mediators and antigen presentation (22, 23). Alveolar macrophages are located in direct contact to all pathogens present in the inhaled air which would lead, in the case of classical activation, to permanent inflammation within the lung tissue. High basal levels of the antiinflammatory cytokine IL-10 in the alveolar environment, such as shown by our results in Figure 3C, may contribute to the downregulation of alveolar macrophages. Thus, the balance is directed toward antiinflammation in the normal lung, which, however, may change in chronic inflammatory conditions. The sequence of events from the onset of an inflammatory reaction to the development of the chronic disease is poorly understood (24). Therefore, kinetic studies involving early as well as later stages of the inflammatory process should be particularly suitable to unravel the steps leading to chronicity, an analysis which could only be done in animal models. Inhalation of NO2 by rats has been shown to provide a useful model for the investigation of several stages of the development of acute and chronic obstructive pulmonary disorders. Whereas exposure for 1 or 3 d induced an acute inflammatory response, exposure for 20 d resulted in a chronic state, and emphysema became clearly apparent at Day 25 of exposure (9, 10).

Infiltration of the affected tissue by leukocytes and vascular leakage are main features of inflammatory reactions which can easily be studied in the lung by BAL (21). As shown in Table 1, we detected increasing total protein content and total cell numbers with different kinetics for neutrophil granulocytes and macrophages (Figure 2) in the BAL of NO2-exposed animals. Interestingly, elevated T lymphocyte counts were only detected in animals exposed to NO2 for 3 d. Whether enhanced lymphocyte numbers represent an epiphenomenon due to simultaneous attraction of lymphocytes and macrophages by cross-reacting CC-chemokines, or whether T lymphocytes are causally involved in the progression of the inflammatory process, e.g., by release of IFN-{gamma} as suggested by Wang and coworkers (25), remains to be studied. Together with the histologic observations shown in Figure 1, these data clearly indicate that an inflammatory response was initiated by the exposure of rats to NO2.

Interestingly, the development of a typical inflammation was not at all paralleled by proinflammatory cytokine concentrations in the BAL (Figure 3). We expected a long-lasting increase of TNF-{alpha} and IL-1ß (20) in the BAL of exposed animals. However, with the exception of a slight increase of IL-1ß at Day 1 of exposure, the concentration of both cytokines decreased to or below the level of untreated control animals with increasing exposure time (Figure 3). This effect was even more surprising when taking into account the elevated number of macrophages after long-term NO2 exposure and their potential to produce IL-1ß and TNF-{alpha}. These data are in accordance with observations in humans, where significantly reduced amounts of the proinflammatory cytokine TNF-{alpha} were found in the BAL of heavy smokers (26). In the present study, we demonstrate that in vitro stimulated alveolar macrophages from NO2-exposed rats showed a significantly reduced capability to produce TNF-{alpha} that was caused by markedly decreased transcriptional activity. Our findings confirm observations by Erroi and colleagues (15), who also found decreased TNF-{alpha} production by macrophages from NO2-exposed rats. In addition, similar results were described after in vitro exposure of mouse macrophages to NO2 (13), suggesting that NO2 exerts a direct effect on macrophage TNF-{alpha} expression. On the basis of these findings, we hypothesized that a shift toward an alternative macrophage activation may have occurred.

Along this hypothesis, we examined IL-10 and found that alveolar macrophages from untreated controls poorly transcribed and released IL-10 even upon in vitro stimulation with LPS (Figure 5), which is in accordance with data by Salez and coworkers (27). However, when BAL cells of animals that had been exposed to NO2 for 3 or 20 d were in vitro stimulated with LPS, a significant transcription and protein release was observed for IL-10. Similar results were obtained for IL-6 at the mRNA level. Whereas IL-10 is generally accepted as antiinflammatory cytokine, IL-6 is a pleiotropic cytokine which is often released together with the proinflammatory cytokines TNF-{alpha} and IL-1ß, and is necessary for the induction of acute phase proteins and fever (28). However, recent investigations have shown that IL-6 and other members of the cytokine family using the gp130 transduction receptor exert rather antiinflammatory effects in the control of local inflammations, particularly in the lung (28–30). The antiinflammatory effects of both IL-10 and IL-6 are associated with the expression of members of the SOCS family, most importantly SOCS-3 (31, 32), which was found to be upregulated in alveolar macrophages following NO2 exposure. As implicated by their name, SOCS proteins are negative regulators of cytokine signaling that downregulate transcription and release of proinflammatory cytokines such as IL-1ß and TNF-{alpha} and other mediators that depend on the JAK/STAT pathway (33).

Taken together, our data suggest that in vivo exposure of rats to NO2 led to a priming of alveolar macrophages—e.g., detectable by increased SOCS-3 protein content—to respond to subsequent stimuli with an response characteristic for alternatively activated macrophages. This is even more surprising if one takes into account that in vitro stimulation of macrophages was performed with LPS, which is known as one of the most effective inducers of classical macrophage activation (34).

The mechanisms leading to the transition from the classically to the alternatively activated phenotype are still unclear. Resident alveolar macrophages may switch their phenotype in response to NO2, which may exert direct effects on macrophages resulting in alterations of the oxidative/antioxidative equilibrium. In addition, recognition and uptake of apoptotic cells, e.g., neutrophils, which are present at elevated numbers as a response to acute NO2 exposure, may also cause changes in macrophage activities (35–38). Furthermore, macrophages which already represent or gain the alternatively activated phenotype during epithelial passage may infiltrate the alveolar space whereby the proportion of classically versus alternatively activated macrophages is shifted to the latter.

Even though products of alternatively activated macrophages are involved in the downregulation of inflammatory processes and thus are helpful for the resolution of pulmonary inflammation, alternative activation of macrophages may also contribute to further progress and development of complex chronic diseases. As shown in the model of NO2 inhalation, alternatively primed macrophages exert an unusual response to bacterial products such as LPS. In vivo, an adequate response to a bacterial infection with the production of proinflammatory cytokines is required for the stimulation of macrophages and other cells to successfully fight pathogenic microorganisms. An alternative macrophage response with increased production of antiinflammatory cytokines such as IL-10 would favor the survival of invading bacteria (39). An increased susceptibility to bacterial and also to viral pulmonary infections has already been described following NO2 exposure in several experimental systems (40–42). Further investigations are in progress to address the question how the occurrence of alternatively activated macrophages in NO2-exposed animals modulate the response to other inflammatory stimuli, for example by stimulating different Toll-like receptor pathways under in vivo conditions.

It is widely accepted that human COPD is an inflammatory disease of the small airways and the lung parenchyma (19, 24). It is known that the inflammatory process is markedly different from asthma with respect to inflammatory cells, mediators (43), and the response to antiinflammatory treatment (44, 45). Smoking is the most important environmental risk factor for the occurrence of COPD (46). Because NO2 is one of the major and most reactive components of tobacco smoke, experimental inhalation models employing NO2 may provide a useful tool for the investigation of mechanisms contributing to the development of human COPD. In fact, the model of NO2 exposure of rats resembles some important features of the human disease, including histology, cellular infiltration, mucus production, and development of emphysema (47). The inflammatory reaction in human COPD is also characterized by an influx of neutrophil granulocytes, T cells, and macrophages into the alveolar compartment (24, 48). Macrophages are finally the most prominent cells and are found at markedly elevated levels in the BAL and sputum of patients with COPD (49, 50). Infiltrating leukocytes—mainly macrophages—are thought to contribute to the maintenance of the disease by production of proteases, oxygen, and nitrogen radicals, and release of inflammatory cytokines. The exact role of these cells and their products for the perpetuation of the inflammatory process is still poorly understood. Alternative activation of macrophages might explain some characteristic features of COPD. For example, repeated bacterial infections are assumed to be an important pathogenic factor for COPD. As already discussed for the NO2 model, occurrence of alternatively activated macrophages could decrease the resistance to pulmonary bacterial infections, thereby supporting the development of the chronic disease. Furthermore, although COPD is an inflammatory disease, corticosteroids have been found to be less useful in the treatment of affected patients (19, 44, 45), possibly because they have been shown to support the alternative activation of macrophages (1). Thus, the role of macrophages for the development of COPD has to be further investigated and perhaps newly defined, which could lead to alternative approaches for the treatment of this disease.


   Acknowledgments
 
The study was supported by the German Ministry for Education and Research Grant No. 01GC0901/5.


   Footnotes
 
* These authors contributed equally to the work presented in this article. Back

Received in original form April 29, 2002

Received in final form October 16, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Goerdt, S., and C. E. Orfanos. 1999. Other functions, other genes: alternative activation of antigen presenting cells. Immunity 10:137–142.[CrossRef][Medline]
  2. Hamilton, T. A., Y. Ohmori, J. M. Tebo, and R. Kishore. 1999. Regulation of macrophage gene expression by pro- and antiinflammatory cytokines. Pathobiology 67:241–244.[CrossRef][Medline]
  3. Goerdt, S., O. Politz, K. Schledzewski, R. Birk, A. Gratchev, P. Guillot, N. Hakij, C.-D. Klemke, E. Dippel, V. Kodelja, and C. E. Orfanos. 1999. Alternative versus classical activation of macrophages. Pathobiology 67:222–226.[CrossRef][Medline]
  4. Stein, M., S. Keshav, N. Harris, and S. Gordon. 1992. Interleukin 4 potently enhances murine macrophage mannose receptor activity: a marker of alternative immunologic macrophage activation. J. Exp. Med. 176:287–292.[Abstract/Free Full Text]
  5. Stumpo, R., M. Kauer, S. Martin, and H. Kolb. 1999. Alternative activation of macrophage by IL-10. Pathobiology 67:245–248.[CrossRef][Medline]
  6. Kodelja, V., C. Müller, O. Politz, N. Hakij, C. E. Orfanos, and S. Goerdt. 1998. Alternative macrophage activation-associated CC-chemokine-1, a novel structural homologue of macrophage inflammatory protein-1{alpha} with a Th2-associated expression pattern. J. Immunol. 160:1411–1418.[Abstract/Free Full Text]
  7. Buechler, C., M. Ritter, E. Orso, T. Langmann, J. Klucken, and G. Schmitz. 2000. Regulation of scavenger receptor CD163 expression in human monocytes and macrophages by pro- and antiinflammatory stimuli. J. Leukoc. Biol. 67:97–103.[Abstract]
  8. Ritter, M., C. Buechler, T. Langmann, E. Orso, J. Klucken, and G. Schmitz. 1999. The scavenger receptor CD163: regulation, promoter structure and genomic organization. Pathobiology 67:257–261.[CrossRef][Medline]
  9. Barth, P. J., S. Uhlarik, A. Bittinger, U. Wagner, and J. Ruschoff. 1994. Diffuse alveolar damage in the rat lung after short and long term exposure to nitrogen dioxide. Pathol. Res. Pract. 190:33–41.[Medline]
  10. Barth, P. J., B. Müller, U. Wagner, and A. Bittinger. 1995. Quantitative analysis of parenchymal and vascular alterations in NO2-induced lung injury in rats. Eur. Respir. J. 8:1115–1121.[Abstract]
  11. Chitano, P., V. Rado, A. Di Stefano, A. Papi, A. Boniotti, G. Zancuoghi, P. Boschetto, M. Romano, M. Salmona, A. Ciaccia, L. Fabbri, and C. E. Mapp. 1996. Effect of subchronic in vivo exposure to nitrogen dioxide on lung tissue inflammation, airway microvascular leakage, and in vitro bronchial muscle responsiveness in rats. Occup. Environ. Med. 53:379–386.[Abstract]
  12. Pagani, P., M. Romano, A. Erroi, M. Ferro, and M. Salmona. 1994. Biochemical effects of acute and subacute nitrogen dioxide exposure in rat lung and bronchoalveolar fluid. Arch. Environ. Contam. Toxicol. 27:426–430.[Medline]
  13. Kienast, K., M. Knorst, J. Müller-Quernheim, and R. Ferlinz. 1996. Modulation of IL-1 beta, IL-6, IL-8, TNF-alpha, and TGF-beta secretions by alveolar macrophages under NO2 exposure. Lung 174:57–67.[Medline]
  14. Drumm, K., R. Buhl, and K. Kienast. 1999. Additional NO2 exposure induces a decrease in cytokine spcific mRNA expression and cytokine release of particle and fiber exposed human alveolar macrophages. Eur. J. Med. Res. 25:59–66.
  15. Erroi, A., P. Pagani, M. Sironi, and M. Salmona. 1996. In vivo exposure to NO2 reduces TNF and IL-6 production by endotoxin-stimulated alveolar macrophages. Am. J. Physiol. 271:L132–L138.[Abstract/Free Full Text]
  16. Hubbard, A. K., K. M. Vetrano, J. B. Morris, and R. S. Thrall. 1994. Acute NO2 exposure alters inflammatory cell activation and particle clearance in silica-injected mice. J. Toxicol. Environ. Health 41:299–314.[Medline]
  17. Garn, H., A. Friedetzky, G. S. Davis, D. R. Hemenway, and D. Gemsa. 1997. T-lymphocyte activation in the enlarged thoracic lymph nodes of rats with silicosis. Am. J. Respir. Cell Mol. Biol. 16:309–316.[Abstract]
  18. Garn, H., A. Friedetzky, A. Kirchner, R. Jäger, and D. Gemsa. 2000. Experimental silicosis: a shift to a preferential IFN-{gamma}-based Th1 response in thoracic lymph nodes. Am. J. Physiol. Lung Cell. Mol. Physiol. 278:L1221–L1230.[Abstract/Free Full Text]
  19. Barnes, P. J. 2000. Chronic obstructive pulmonary disease. N. Engl. J. Med. 343:269–280.[Free Full Text]
  20. Nicod, L. P. 1999. Pulmonary defense mechanisms. Respiration 66:2–11.[CrossRef][Medline]
  21. Crapo, J. D., A. G. Harmsen, M. P. Sherman, and R. A. Musson. 2000. Pulmonary immunobiology and inflammation in pulmonary diseases. Am. J. Respir. Crit. Care Med. 162:1983–1986.[Free Full Text]
  22. Lohmann-Matthes, M. L., C. Steinmüller, and G. Franke-Ullmann. 1994. Pulmonary macrophages. Eur. Respir. J. 7:1678–1689.[Abstract]
  23. Chelen, C. J., Y. Fang, G. J. Freeman, H. Secrist, J. D. Marshall, P. T. Hwang, L. R. Frankel, R. H. DeKruyff, and D. T. Umetsu. 1995. Human alveolar macrophages present antigen ineffectively due to defective expression of B7 costimulatory cell surface molecules. J. Clin. Invest. 95:1415–1421.
  24. Cosio, M. G., and A. Guerassimov. 1999. Chronic obstructive pulmonary disease. Am. J. Respir. Crit. Care Med. 160:S21–S25.[Abstract/Free Full Text]
  25. Wang, Z., T. Zheng, Z. Zhu, R. J. Homer, R. J. Riese, H. A. Chapman, S. D. Shapiro, and J. A. Elias. 2000. Interferon {gamma} induction of pulmonary emphysema in the adult murine lung. J. Exp. Med. 192:1587–1599.[Abstract/Free Full Text]
  26. Bidani, A., C. Z. Wang, and T. A. Heming. 1996. Early effects of smoke inhalation on alveolar macrophage functions. Burns 22:101–106.[CrossRef][Medline]
  27. Salez, L., M. Singer, V. Balloy, C. Creminon, and M. Chignard. 2000. Lack of IL-10 synthesis by murine alveolar macrophages upon lipopolysaccharide exposure: comparison with peritoneal macrophages. J. Leukoc. Biol. 67:545–552.[Abstract]
  28. Xing, Z., J. Gauldie, G. Cox, H. Baumann, M. Jordana, X.-F. Lei, and M. K. Achong. 1998. IL-6 is an antiinflammatory cytokine required for controlling local or systemic acute inflammatory responses. J. Clin. Invest. 101:311–320.[Medline]
  29. Gadient, R. A., and P. H. Patterson. 1999. Leukemia inhibitory factor, interleukin 6, and other cytokines using the gp130 transducing receptor: roles in inflammation and injury. Stem Cells 17:127–137.[Abstract/Free Full Text]
  30. Ward, N. S., A. B. Waxmann, R. J. Homer, L. L. Mantell, O. Einarsson, Y. Du, and J. A. Elias. 2000. Interleukin-6-induced protection in hyperoxic acute lung injury. Am. J. Respir. Cell Mol. Biol. 22:535–542.[Abstract/Free Full Text]
  31. Donnelly, R. P., H. Dickensheets, and D. S. Finbloom. 1999. The interleukin-10 signal transduction pathway and regulation of gene expression in mononuclear phagocytes. J. Interferon Cytokine Res. 19:563–573.[CrossRef][Medline]
  32. Lebel, E., L. Vallieres, and S. Rivest. 2000. Selective involvement of interleukin-6 in the transcriptional activation of the suppressor of cytokine signaling-3 in the brain during systemic immune challenges. Endocrinology 141:3749–3763.[Abstract/Free Full Text]
  33. Chen, X. P., J. A. Losman, and P. Rothman. 2000. SOCS proteins, regulators of intracellular signaling. Immunity 13:287–290.[CrossRef][Medline]
  34. Adams, D. O. 1989. Molecular interactions in macrophage activation. Immunol. Today 10:33–35.[CrossRef][Medline]
  35. Haslett, C. 1999. Granulocyte apoptosis and its role in the resolution and control of lung inflammation. Am. J. Respir. Crit. Care Med. 160:S5–S11.[Abstract/Free Full Text]
  36. Fadok, V. A., D. L. Bratton, A. Konowal, P. W. Freed, J. Y. Westcott, and P. M. Henson. 1998. Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-ß, PGE2, and PAF. J. Clin. Invest. 101:890–898.[Medline]
  37. Byrne, A., and D. J. Reen. 2002. Lipopolysaccharide induces rapid production of IL-10 by monocytes in the presence of apoptotic neutrophils. J. Immunol. 168:1968–1977.[Abstract/Free Full Text]
  38. Kurosaka, K., N. Watanabe, and Y. Kobayashi. 2002. Potentiation by human serum of antiinflammatory cytokine production by human macrophages in response to apoptotic cells. J. Leukoc. Biol. 71:950–956.[Abstract/Free Full Text]
  39. Steinhauser, M. L., C. M. Hogaboam, S. L. Kunkel, N. W. Lukacs, R. M. Strieter, and T. J. Standiford. 1999. IL-10 is a major mediator of sepsis-induced impairment in lung antibacterial host defense. J. Immunol. 162:392–399.[Abstract/Free Full Text]
  40. Henry, M. C., J. Findlay, J. Spangler, and R. Ehrlich. 1970. Chronic toxicity of NO2 in squirrel monkeys. 3. Effect on resitance to bacterial and viral infection. Arch. Environ. Health 20:566–570.[Medline]
  41. Goldstein, E., M. C. Eagle, and P. D. Hoeprich. 1973. Effect of nitrogen dioxide on pulmonary bacterial defense mechanisms. Arch. Environ. Health 26:202–204.[Medline]
  42. Rose, R. M., J. M. Fuglestad, W. A. Skornik, S. M. Hammer, S. F. Wolfthal, B. D. Beck, and J. D. Brain. 1988. The pathophysiology of enhanced susceptibility to murine cytomegalovirus respiratory infection during short-term exposure to 5 ppm nitrogen dioxide. Am. Rev. Respir. Dis. 137:912–917.[Medline]
  43. Barnes, P. J. 1999. Molecular genetics of chronic obstructive pulmonary disease. Thorax 54:245–252.[Free Full Text]
  44. Keatings, V. M., A. Jatakanon, Y. M. Worsdell, and P. J. Barnes. 1997. Effects of inhaled and oral glucocorticoids on inflammatory indices in asthma and COPD. Am. J. Respir. Crit. Care Med. 155:542–548.[Abstract]
  45. Culpitt, S. V., W. Maziak, S. Loukidis, J. A. Nightingale, J. L. Matthews, and P. J. Barnes. 1999. Effect of high dose inhaled steroid on cells, cytokines, and proteases in induced sputum in chronic obstructive pulmonary disease. Am. J. Respir. Crit. Care Med. 160:1635–1639.[Abstract/Free Full Text]
  46. Markewitz, B. A., M. W. Owens, and D. K. Payne. 1999. The pathogenesis of chronic obstructive pulmonary disease. Am. J. Med. Sci. 318:74–78.[CrossRef][Medline]
  47. Hele, D. 2002. First Siena International Conference on animal models of chronic obstructive pulmonary disease, Certosa di Pontignano, University of Siena, Italy, September 30–October 2, 2001. Respir. Res. 3:12.
  48. O'Byrne, P. M. and D. S. Postma. 1999. The many faces of airway inflammation: asthma and chronic obstructive pulmonary disease. Am. J. Respir. Crit. Care Med. 159(Suppl.):S41–S66.
  49. Saetta, M., A. DiStefano, P. Maestrelli, A. Ferraresso, R. Drigo, A. Potena, A. Ciaccia, and L. Fabbri. 1993. Activated T-lymphocytes and macrophages in bronchial mucosa of subjects with chronic bronchitis. Am. Rev. Respir. Dis. 147:301–306.[Medline]
  50. Jeffery, P. K. 1998. The value of bronchial biopsies: assessment of inflammation in asthma and COPD. Eur. Respir. J. 8:1079–1085.



This article has been cited by other articles:


Home page
 J. Am. Soc. Nephrol.Home page
D. N. Grigoryev, M. Liu, H. T. Hassoun, C. Cheadle, K. C. Barnes, and H. Rabb
The Local and Systemic Inflammatory Transcriptome after Acute Kidney Injury
J. Am. Soc. Nephrol., March 1, 2008; 19(3): 547 - 558.
[Abstract] [Full Text] [PDF]

Home page
 ThoraxHome page
H. Fehrenbach, G. Zimmermann, E. Starke, V. A Bratu, D. Conrad, A. O Yildirim, and A. Fehrenbach
Nitrogen dioxide induces apoptosis and proliferation but not emphysema in rat lungs
Thorax, May 1, 2007; 62(5): 438 - 446.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Garn, H.
Right arrow Articles by Gemsa, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Garn, H.
Right arrow Articles by Gemsa, D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Crit. Care Med.
Copyright © 2003 American Thoracic Society.