Abstract
We previously described a reduction of silica–induced lung fibrosis in interleukin-10–deficient mice (IL-10−/−) (Huaux and colleagues; Am. J. Respir. Cell Mol. Biol. 1998;18:51–59). In the present study, we further dissect the exact functions of IL-10 in experimental silicosis. The reduced lung fibrotic response to silica in IL-10−/− mice was accompanied by a marked recruitment of TH1 CD4+ lymphocytes. However, treatment with anti-CD4 antibodies reduced silica–induced lung fibrosis in both IL-10−/− and IL-10+/+ mice, suggesting that this T cell population actually contributes to the extension of the fibrotic lesions in a manner that is independent of IL-10. In IL-10−/− mice, silica–induced lung production of the profibrotic mediator transforming growth factor (TGF)-β1 and the antifibrotic eicosanoid PGE2 were reduced and increased, respectively, relative to that in IL-10+/+ mice. In addition, in vitro experiments indicated that recombinant IL-10 upregulated TGF-β1 expression in alveolar macrophages while in contrast it downregulated PGE2 production and cyclooxygenase-2 expression in both lung fibroblasts and macrophages. Thus the net profibrotic activity of IL-10 in vivo appears to be mediated by its ability to stimulate the expression of the profibrotic cytokine TGF-β1 while suppressing the expression of cyclooxygenase-2 and thus production of the antifibrotic eicosanoid PGE2. These effects appear to be independent of the enhanced lung CD4+ T-lymphocytosis observed in IL-10–deficient mice.
The cellular mechanisms associated with fibrotic processes are currently thought to be the consequence of protracted injury and inflammation, leading to an excessive replication of mesenchymal cells and exuberant deposition of extracellular matrix proteins (1). In the lung, there is abundant evidence that alveolitis and fibrosis are orchestrated at least in part by proinflammatory cytokines such as tumor necrosis factor-α and interleukin (IL)-1. Indeed, by detecting, neutralizing, or increasing cytokine expression in different experimental models of lung fibrosis, it has been demonstrated that these proinflammatory cytokines are key mediators not only in the initiation and the extension of lung inflammation but also in the subsequent development of pulmonary fibrosis (2–5). Furthermore, mouse models targeting the specific overexpression of cytokines in the lung using the CC-10 promoter or adenoviral expression vectors have extended these findings to demonstrate the pro-fibrotic role of other cytokines including transforming growth factor (TGF)-β, interleukin (IL)-4, IL-10, IL-11, and IL-13 (6–11). Paradoxically, many of these cytokines also possess anti-inflammatory properties, especially on monocytes/macrophages (12–16), suggesting the possibility of alternative mechanisms of fibrosis other than chronic alveolitis and sustained expression of inflammatory factors. We previously reported that mice genetically deficient in IL-10 develop a reduced fibrotic lung response to silica particles that is associated with an exaggerated early inflammatory response relative to their wild-type counterparts (17). Although elevated IL-10 expression has been reported in human and experimental fibrotic lesions occurring in different organs, its exact role or roles in the pathogenesis of fibrosis in the lung or other organs has not been fully delineated and is still debated (18–22). The current study examined how the protracted expression of IL-10 could exacerbate silica-induced lung fibrosis.
Female C57BL/6J and IL-10–deficient mice on a C57BL/6J background were purchased from Jackson Laboratory (Bar Harbor, ME). Mice weighing between 18 and 22 g were used at 8 wk of age. The animals were housed in positive pressure air-conditioned units (25°C, 50% relative humidity) on a 12-h light/dark cycle.
A suspension of crystalline silica particles (DQ12; d50 = 2.2 μm, a gift from Dr. Armbruster, Essen, Germany) in sterile 0.9% saline was injected directly into lungs by intratracheal instillation at a dose of 2.5 mg/mouse. To allow sterilization and inactivation of any trace of endotoxin, particles were heated at 200°C for 2 h immediately before suspension and administration. All instillations (100 μl/mouse) were performed on anesthetized animals (sodium pentobarbital, 2 mg/mouse, intraperitoneally) after direct visualization of the trachea through a surgical incision in the neck. All data presented in this study are representative of two separate experiments.
Rat anti-mouse CD4 hybridoma (IgG2b, clone GK 1.5) in the form of ascite fluid was kindly provided by Dr. J. P. Coutelier (MEXP unit, ICP, Brussels, Belgium). This was used to block CD4+ T cell recruitment in silica-treated IL-10+/+ and IL-10−/− mice. One day before silica instillation, 500 μl of sterile ascite fluid or phosphate-buffered saline (PBS) were administered intraperitoneally. After silica treatment, injections were repeated every week until mice were killed (1 mo). CD4+ cell depletion after treatment with antibodies was confirmed by fluorescence-activated cell sorter analysis as follows. After hemolysis (3 min in 160 mM NH4Cl), spleen cells obtained from each group of treated mice were resuspended in Hanks' medium with 3% decomplemented fetal calf serum and 20 mM NaN3 with fluorescein isothiocyanate–conjugated rat anti-mouse CD4 (L3T4) monoclonal antibody (clone RM4–4; PharMingen, San Diego, CA). After immunostaining, the cells were fixed in paraformaldehyde (1.25%), and 104 cells were analyzed using a FACS-can apparatus (Becton-Dickinson, Franklin Lakes, NJ).
At selected time intervals after silica instillation, the animals were killed with sodium pentobarbital (20 mg/animal, intraperitoneally). The lungs were perfused and bronchoalveolar lavage (BAL) was performed by cannulating the trachea and lavaging the lungs 5× with 1 ml of NaCl 0.9%. The BAL fluid was centrifuged (1,000 rpm, 10 min, 4°C) and the cell-free supernatant used for biochemical measurements. Whole lungs were excised and placed into a Falcon tube (Becton-Dickinson) chilled on ice and 3 ml of cold NaCl 0.9% was added. The content of each tube was then homogenized using a Polytron PT1200 homogenizer (Kinematica AG; Littau, Lu, Switzerland) for 30 s. The tubes were centrifuged at 4°C, 5,000 rpm for 5 min and supernatants were kept frozen at –80°C until use.
Collagen deposition was estimated by measuring the hydroxyproline and soluble collagen contents in lung homogenates. For hydroxyproline assays, the lung was excised, homogenized, and hydrolyzed in 6N HCl overnight at 110°C. Hydroxyproline was assessed by high-performance liquid chromatography analysis (23) and data were expressed as micrograms of hydroxyproline per lung. Soluble collagen levels were estimated by Sircol collagen assay following the manufacturer's protocol (Biocolor, Newtownabbey, UK).
Active TGF-β1, interferon (IFN)-γ (R&D Systems, Minneapolis, MN), IgG1, IgG2a (BETHYL Laboratories, Montgomery, TX), and PGE2 (Amersham, Bucks, UK) concentrations in lung homogenates (see above) were measured using enzyme-linked immunosorbent assay (ELISA) kits following the respective manufacturer's protocols. The detection limits of these ELISAs were respectively 7 (pg/ml), 2 (pg/ml), 3.9 (ng/ml), 3.9 (ng/ml), and 16 (pg/ml).
Fibronectin and type I collagen contents were measured using standardized ELISAs as previously described (24).
The left lung was excised and fixed in Bouin's solution (Merck-Belgolabo, Overijse, Belgium). Paraffin-embedded sections were stained with hematoxylin and eosin, Masson's trichrome or toluidine blue for light microscopic examination. For immunohistochemistry, dewaxed and rehydrated tissue sections were subjected to endogenous peroxidase inactivation (0.5% H2O2 for 20 min) followed by three washes of 5 min in PGT buffer (PBS, 0.05% Tween 20, and 0.02% gelatine). The sections were then incubated in a humidified chamber with a monoclonal rat antimouse Lyt 1 antibody (CD4+ cells, diluted 25× in PBS, a gift from Dr. Van Ewijk, Rotterdam, The Netherlands). After three washes (5 min each) with PGT buffer, they were exposed for 1 h to the second antibody (polyclonal rabbit anti-rat immunoglobulin (Ig)G coupled with peroxidase; Dako, Copenhagen, Denmark) diluted 40-fold in PBS supplemented with 1% mouse serum. After washing three times in PGT buffer, peroxidase activity was visualized using 3,3′-diaminobenzidine tetrahydrochloride (Aldrich, Beerse, Belgium)-H2O2 as substrate. The staining was enhanced by incubation in a solution of 0.5% CuSO4 in saline for 15 min. Sections were counterstained with Harris hematoxylin, rinsed, dehydrated, and mounted in DPX (BDH, Poole, UK).
Mouse lung fibroblasts were isolated by an explant method (25). Using a sterile technique, the lungs of saline- or silica–treated mice were thoroughly perfused with sterile 0.9% saline via the right ventricle. The lungs were then excised, washed in PBS, and cut into small pieces. They were suspended in Dulbecco's MOD Eagle Medium (DMEM) (Life Technology-Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS), and 1% penicillin–streptomycin antibiotic (5000 U/ml) and incubated in a CO2 incubator at 37°C. Fibroblasts migrated from explants and allowed to grow to confluence, and then were used after the first cell passage. Confluent cell monolayers were treated for 24 h with the indicated doses of rIL-4, rIFN-γ, rTGF-β, rPDGF, or rIL-10 (R&D Systems) diluted in medium supplemented with 1% antibiotics (penicillin–streptomycin, 5000 U/ml), 1% glutamine (1 mM), 1% ascorbic acid (5 mg/ml) and 1% proline (0.2 mM). Fibroblast proliferation was assessed by [3H]thymidine incorporation (26). Type I collagen and α-smooth muscle actin (α-SMA) were measured by ELISA after sonication of the lung fibroblasts cultured in 24-well plates (24).
Alveolar macrophages (AM) recovered by BAL from saline and silica–treated mice were suspended in DMEM supplemented with 10% FBS, 1% antibiotics (penicillin–streptomycin 5000 U/ml) and 1% glutamate 1 mM, and plated onto culture dishes (1 × 106 cells/well). After 2 h of incubation at 37°C in a humidified incubator under 5% CO2 in air, nonadherent cells were removed by washing with PBS. Where indicated, AM were treated for 24 h with 10 ng/ml of mouse-recombinant IL-10 diluted in DMEM supplemented with 0.5% FBS.
Fibroblasts and AM from saline- and silica–treated mice were seeded in 6-well plates (1 × 106 cells/well) as described above. After washing, selected wells were replaced with fresh medium only or medium supplemented with IL-10 (10 ng/ml). After 15 min, the cells were lysed in 500 μl of Laemmli buffer (Bio-Rad, Hercules, CA) and boiled for 3 min before being loaded on precast Novex (Carlsbad, CA) SDS-polyacrylamide gels (8%) and transferred electrophoretically to nitrocellulose membranes (Hybond C; Amersham Biosciences, Piscataway, NJ). The membranes were then blocked in 5% nonfat dry milk, washed, and probed using antibodies specific for phosphorylated STAT3-Y705. Blots were reprobed with antiactin antibodies (Sigma, St. Louis, MO) as a control. A SuperSignal West Pico detection kit (Pierce, Rockford, IL) was used for detection.
Fibroblasts or AM from saline- and silica–treated mice were seeded in 6-well plates (106 cells/well) and total RNA was isolated with the Trizol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. For real-time reverse transcriptase polymerase chain reaction (RT-PCR), ∼ 700 ng of RNA was incubated with Dnase I (2 U/μl) (Ambion, Austin, TX) for 30 min at 37°C. The samples were then inactivated and reverse-transcribed with the Superscript II reverse transcriptase (Invitrogen) with random hexamers and poly(dT) oligos according to the manufacturer's protocol. An ABI PRISM 7,700 Sequence Detection System (Applied Biosystems, Foster City, CA) was used for real-time PCR analysis. For the current study, the comparative CT method recommended by the manufacturer (Applied Biosystems) was adopted with β-actin as endogenous reference housekeeping gene. For target genes, cyclooxygenase (COX)-2 and TGF-β1, PCR primers were designed using the Primers invitrogen program. The primer sequences were as follows: for COX-2, forward primer 5′-TTTGTTGAGTCATTCACCAGACAGAT-3′; reverse primer 5′-CAGTATTGAGGAGAACAGATGGGATT-3′; for TGF-β, forward primer 5′-CCCCACTGATACGCCTGAGT-3′; reverse primer 5′-AGCAGTGAGCGCTGAATCG-3′; for β-actin, forward primer 5′-AGAGGGAAATCGTGCGTGAC-3′; reverse primer 5′-CAATAGTGATGACCTGGCCGT-3′. In all cases, the thermal cycling condition was programmed according to the manufacturer's instructions, and PCR was performed at 95°C for 15 s followed by 60°C for 1 min using SYBR green buffer as fluorophore (Applied Biosystems). All samples were measured in triplicate. Six serial 1:10 dilutions of a positive control sample of cDNA were used as a standard curve in each reaction. The result was expressed as a ratio of product copies per sample to copies per sample of the housekeeping gene β-actin from the same RNA (respective cDNA) sample and PCR run.
Treatment-related differences were evaluated using t tests and one-way analysis of variance, followed by pairwise comparisons using the Student-Newman-Keuls test, as appropriate. Statistical significance was considered at P < 0.05.
In a previous study (17), we demonstrated that silica-treated IL-10−/− mice developed less lung fibrosis than the corresponding wild-type animals (IL-10+/+). This reduction in fibrosis was associated with a marked recruitment of lymphocytes into the lung. In this study, we sought to determine the lymphocyte subpopulation(s) that were recruited in silica-treated IL-10−/− animals and to assess their potential role in fibrosis. Immunohistochemical studies were performed on lung tissue sections to estimate the abundance of T (anti-CD4 or -CD8), B (B220), or natural killer (NK) (NK.1.1) lymphocytes. Figure 1
Figure 1. Immunohistochemical staining of CD4+ lymphocytes in IL-10+/+ (A) and IL-10−/− mice (B) one month after instillation of silica particles. The data shown are from a representative experiment of two independent experiments.
[More] [Minimize]To better delineate the role of recruited CD4+-lymphocytes, we investigated the effects of CD4+ T cell depletion on the fibrotic response induced by silica particles in IL-10+/+ and IL-10−/− mice. As observed in our previous study (17), deficiency of IL-10 resulted in decreased silica-induced lung fibrosis as confirmed by the lower levels of lung collagen in silica-treated IL-10−/− mice than those in the corresponding IL-10+/+ mice (Figures 2A and 2B)
Figure 2. Soluble collagen and type 1 collagen in lung homogenates of silica-treated IL-10−/− or IL-10+/+ mice with or without CD4+ T cell depletion. Deficiency in IL-10 resulted in reduced silica–induced lung fibrosis. In both types of mice, anti-CD4 treatment was associated with a decrease in lung collagen content. Bars represent means ± SEM of five to six animals. Significantly different from controls: *P < 0.05, **P < 0.01 (Student-Newman-Keuls multiple comparison test). The line drawn over the figure represents the mean value of the levels measured in saline-treated IL-10+/+ mice. No significant difference was noted between the levels in saline–treated IL-10+/+ and IL-10−/− mice. The data shown are from a representative experiment of two independent experiments. Open bars, IL-10+/+ silica; striped bars, IL-10+/+ silica + abCD4+; cross-hatched bars, IL-10−/− silica; filled bars, IL-10−/− silica + abCD4+.
[More] [Minimize]Both type-1 and type-2 immune responses have been implicated in the pathogenesis of fibrosis (27). We assessed whether lung responses in IL-10−/− and IL-10+/+ mice were associated with a preferential polarization by estimating IFN-γ and IgG2a levels (type-1 response) as well as IL-4 and IgG1 levels (type-2 response) in lung homogenates. IFN-γ and IgG2a levels were significantly higher in silica-treated IL-10−/− mice compared with corresponding IL-10+/+ animals (Figure 3)
Figure 3. IFN-γ and IgG2a levels in lung homogenates of IL-10−/− or IL-10+/+ mice 1 mo after instillation of silica particles with or without depletion of CD4+ T cells. IFN-γ and IgG2a levels were significantly higher in silica–treated IL-10−/− mice compared with corresponding IL-10+/+ animals, suggesting a preferential immune polarization toward a type-1 response in deficient animals. Bars represent means ± SEM of five to six animals. Significantly different from controls: *P < 0.05 (Student-Newman-Keuls multiple comparison test). The line drawn over the figure represents the mean value of the levels measured in saline–treated IL-10+/+ mice. No significant difference was noted between the levels in saline-treated IL-10+/+ and IL-10−/− mice. The data shown are from a representative experiment of two independent experiments. Open bars, IL-10+/+ silica; striped bars, IL-10+/+ silica + abCD4+; cross-hatched bars, IL-10−/− silica; filled bars, IL-10−/− silica + abCD4+.
[More] [Minimize]It has been proposed that fibrosis proceeds from an imbalance between pro- and antifibrotic factors, such as TGF-β and PGE2, respectively (28). To evaluate the role of an altered balance in the reduced fibrosis in IL-10–deficient mice, these two mediators were quantified in lung homogenates of IL-10–deficient and wild-type mice one month after silica treatment with or without depletion of CD4 T cells. Lung PGE2 levels were higher in silica–treated IL-10–deficient mice compared with those in wild-type mice, but the difference was not statistically significant. However, after anti-CD4 injection, PGE2 levels were significantly higher in IL-10−/− mice compared with similarly treated IL-10+/+ animals (Figure 4)
Figure 4. PGE2 and TGF-β levels in lung homogenates of IL-10−/− or IL-10+/+ mice 1 mo after instillation of silica particles with or without depletion of CD4+ T. After anti-CD4 injection, PGE2 levels were slightly increased in IL-10−/− mice compared with IL-10+/+ animals. TGF-β lung contents were significantly decreased in IL-10−/− mice compared with corresponding wild-type mice. Bars represent means ± SEM of five to six animals. Significantly different from controls: *P < 0.05 (Student-Newman-Keuls multiple comparison test). The line drawn over the figure represents the mean value of the levels measured in saline–treated IL-10+/+ mice. No significant difference was noted between the levels in saline–treated IL-10+/+ and IL-10−/− mice. The data shown are from a representative experiment of two independent experiments. Open bars, IL-10+/+ silica; striped bars, IL-10+/+ silica + abCD4+; cross-hatched bars, IL-10−/− silica; filled bars, IL-10−/− silica + abCD4+.
[More] [Minimize]To better understand the biological mechanism by which IL-10 can participate in the extension of silica-induced lung fibrosis, we studied its effects on pulmonary fibroblasts and AM, two cell types strongly implicated in the pathogenesis of silicosis. First, to demonstrate that these cells could actually respond to IL-10 directly, the effect of IL-10 treatment on signal transducer and activator of transcription (STAT)-3 phosphorylation was determined as an indicator of IL-10 receptor expression. Figure 5
Figure 5. IL-10–induced tyrosine phosphorylation of STAT 3 in fibroblasts from saline– and silica–treated mice and in macrophages, mainly from silica–treated mice. Fibroblasts and AM were treated without or with IL-10 (10 ng/ml) for 15 min. Total lysates were analyzed by Western blot with antibodies directed against phosphorylated STAT 3. The membrane was then reprobed with an anti-β-actin antibody. The data shown are from a representative experiment of three independent experiments.
[More] [Minimize]To evaluate the activity of IL-10 on pulmonary fibroblasts, we compared the in vitro effect of various concentrations of recombinant mouse IL-10 to that of PDGF (R&D Systems) and TGF-β (R&D Systems) on lung fibroblasts isolated from saline- and silica-treated mice. Although PDGF (10 ng/ml) significantly increased thymidine incorporation, rIL-10 (1–20 ng/ml) was unable to stimulate fibroblast proliferation (Figure 6A)
Figure 6. In vitro [3H] thymidine incorporation, type 1 collagen, and α-SMA expression in lung mouse fibroblasts from saline–treated C57BL/6 mice treated with IL-4, IFN-γ, IL-10, PDGF, or TGF-β. IL-10 had no significant effects on fibroblast proliferation or α-SMA and type 1 collagen expression. Bars represent means ± SEM of four to six wells. Significantly different from controls: **P < 0.01, ***P < 0.001 (Student-Newman-Keuls multiple comparison test). The data shown are from a representative experiment of four independent experiments.
[More] [Minimize]Because IL-10 deficiency in vivo was associated with increased lung PGE2 but decreased TGF-β content, we examined in vitro whether this could be due to a direct effect of IL-10 on COX-2 (a key enzyme in inducible PGE2 production) and TGF-β expression in pulmonary fibroblasts and AM purified from saline- or silica-treated mice. In comparison to unstimulated cells, IL-10 significantly downregulated COX-2 mRNA expression in both fibroblasts (Figure 7)
Figure 7. Expression of COX-2 and TGF-β mRNA in lung fibroblasts isolated from naive mice treated without or with IL-10 at the indicated doses. IL-10 downregulates COX-2 expression in lung fibroblasts. Bars represent means ± SEM of two different wells assayed in triplicate. Significantly different from controls: *P < 0.05 (Student-Newman-Keuls multiple comparison test). The data shown are from a representative experiment of two independent experiments. Open bars, CTL; cross-hatched bars, IL-10, 1 ng; horizontally striped bars, IL-10, 10 ng; vertically striped bars, IL-10, 20 ng.
[More] [Minimize]
Figure 8. Expression of COX-2 and TGF-β mRNA in AM isolated from naive mice treated without (open bars) or with (striped bars) IL-10. IL-10 downregulates COX-2 expression in AM, and in contrast upregulates TGF-β expression. Bars represent means ± SEM of two different wells assayed in triplicate. Significantly different from controls: *P < 0.05 (Student-Newman-Keuls multiple comparison test). The data shown are from a representative experiment of two independent experiments.
[More] [Minimize]Our in vitro and in vivo observations suggest a profibrotic role for IL-10, perhaps by its ability to upregulate TGF-β expression in AM and to downregulate PGE2 production in AM and fibroblasts. Paradoxically however, IL-10 deficiency caused a greater silica–induced expansion of lung CD4+ T cells relative to that in wild-type IL-10–sufficient mice. Since depletion of this T cell subpopulation reduced fibrosis, the significance of this increased T cell subpopulation in the IL-10–deficient mice is not immediately apparent, but does not appear to be the cause of the decreased fibrosis in this murine strain. However, IL-10 may play a role in terms of controlling the intensity of the fibrotic reaction in wild-type mice by limiting the T cell–dependent component of the fibrotic process.
Although several human and experimental studies have clearly demonstrated a relationship between the development of fibrosis and IL-10 expression, the exact role of this cytokine in the fibrotic reaction still remains controversial (17, 21, 29). First, several authors suggested a profibrotic function for IL-10 during the extension of lung fibrosis (17, 30, 31). Indeed, in a previous study, we have observed a less intense fibrosis in IL-10–deficient mice in response to silica particles compared with the wild-type mice (17). In the same IL-10–deficient mice, the injection of Schistosoma mansoni induced the formation of massive inflammatory granulomas in the liver without, however, being associated with more intense fibrotic lesions (30). Recently, Lee and colleagues have shown that transgenic mice overexpressing IL-10 (driven by CC10 promoter) exclusively in the lung spontaneously developed airway subepithelial fibrosis (31), demonstrating that in the lung, the TH2 cytokine IL-10 can have profibrotic functions. Remarkably, other anti-inflammatory TH2 cytokines such as IL-4, IL-13, IL-11, and TGF-β are also considered profibrotic cytokines (6–11). Indeed, by using constitutive or inducible overexpression systems, it has been demonstrated that IL-4, IL-11, or IL-13 can cause an enhanced accumulation of interstitial collagens in the airways (10, 32). It is also well established that TGF-β promotes extracellular matrix accumulation by upregulating collagen and fibronectin gene expression while inhibiting matrix degradation by decreasing the secretion of proteases and increasing secretion of protease inhibitors (33, 34).
Beside these studies supporting a profibrotic role of IL-10, some authors, in contrast, attributed antifibrotic properties to IL-10 (21, 22, 35). Indeed, IL-10–deficient mice showed significantly more severe CCl4-induced liver fibrosis than in the wild-type counterparts (21). In addition, administration of IL-10 adenoviral constructs inhibited bleomycin-induced lung fibrosis in mice (22). Recently, it has been suggested that the secretion of IL-10 induced by TGF-β1 may have an unexpected and paradoxical anti-fibrotic effect in a bleomycin-induced fibrosis model (35). These results suggest that IL-10 could control the development of the fibrotic reaction at least in certain experimental models and conditions.
These contradictory observations about IL-10 and its role in fibrosis could be interpreted as follows. During the inflammatory response, expression of anti-inflammatory cytokines such as IL-10 can limit both the recruitment of inflammatory cells and the activity of proinflammatory mediators such as tumor necrosis factor-α. In this capacity, IL-10 appears to play a beneficial role by limiting pulmonary inflammation that may promote subsequent collagen accumulation. At later more chronic stages, however, when the fibrotic reaction predominates and involves other cell components (mesenchymal cells and lymphocytes), the high amount of IL-10 produced could, in addition to its earlier anti-inflammatory action, act as a profibrotic mediator, conceivably by stimulating other pulmonary cells. According to this scenario then, the timing of IL-10 expression or its suppression may have diametrically opposite effects on fibrosis.
To further investigate the exact role of IL-10 and to better define its biological activities during silica-induced lung fibrosis, we analyzed the pulmonary responses to silica particles of IL-10–deficient mice. Our previous observations using these mice showed a marked recruitment of lymphocytes to the lung that was associated with a reduction of silica–induced fibrosis (17). In the current study, by immunohistochemical staining, the type of lymphocytes recruited was identified as CD4+ T lymphocytes (Figure 1). However the significance of such an increased recruitment of CD4+ T lymphocytes in the reduction of fibrosis observed in the absence of IL-10 is not immediately apparent. The importance of T lymphocytes in particle-induced pulmonary fibrosis remains controversial despite extensive studies. Hubbard reports that neither T cells nor the cells they influence affect the amount of collagen deposition in a silica model using mice genetically deficient in mature T cells (BALB/c nu/nu) and their T cell–sufficient counterparts (36). In contrast, Suzuki and colleagues found that collagen deposition was reduced in silica-treated athymic mice compared with their normal counterparts (37). In the present study, we explored the effects of CD4+ T lymphocyte depletion on silica–induced fibrosis in wild-type and IL-10–deficient strains. Because anti-CD4 antibodies reduced the amplitude of fibrosis in both strains, we conclude that CD4+ T cells play a detrimental role in experimental silicosis in an IL-10–independent manner. These results are in accordance with data showing a protective effect conferred by neutralization of CD4+ T cell recruitment in models of lung fibrosis induced by bleomycin (38) or radiation (39).
Recent studies show that T cells, especially the CD4+ subpopulation, purified from silicotic mice expressed IFN-γ but not IL-4, suggesting the importance of a Th-1 response in experimental silicosis (40). This possibility is further strengthened by a report showing that IFN−/− mice develop less fibrosis than their wild-type counterparts (41). The authors postulated that T lymphocytes producing IFN-γ may exacerbate macrophage activation and the production of cytokines that promote lung fibrosis. However, in addition to its potent ability to activate macrophages, IFN-γ possesses antifibrotic activity by its ability to directly inhibit several important fibroblast functions associated with fibrosis (42). Thus the net effect of this cytokine on fibrosis may depend on which target cells are present at a particular stage of the disease. Nevertheless, the studies with IFN-γ–deficient mice reveal that the profibrotic activity of this cytokine, as mediated by inflammatory/immune cells, appears to play a dominant role overall. Thus the loss of its antifibrotic activity on fibroblasts in IFN-γ–deficient mice could not compensate for the loss of its profibrotic activity on inflammatory/immune cells. In our model, the levels of IFN-γ and IgG2a were increased in lung homogenates of silica–treated IL-10−/− mice compared with wild-type mice, suggesting a type 1 immune polarization in these deficient animals. However in IL10−/− mice injected with anti-CD4 or not, IFN-γ production did not correlate with fibrosis. Thus, the reduced fibrosis in IL-10–deficient mice could not be due to the well-known antifibrotic properties of IFN-γ. Interestingly, anti-CD4 treatment inhibited IFN-γ production, confirming that in murine silicosis CD4+ T cells are the main producers of this cytokine (43). Our observations however do provide support for the view that in experimental silicosis TH1 CD4+ T cells possess profibrotic functions. The pronounced recruitment of these “profibrotic” cells in the absence of IL-10 may partially counter the loss of the profibrotic activity of IL-10, resulting in the incomplete inhibition of fibrosis in IL-10–deficient mice.
To better understand the biological mechanism by which IL-10 can participate in the extension of silica-induced lung fibrosis, we studied its effects on pulmonary fibroblasts and AM, two cells strongly implicated in the pathogenesis of silicosis. First, we verified the expression of a functional IL-10 receptor on these cells by documenting activation of STAT3 in response to IL-10 treatment (44). Our in vitro studies showed that IL-10 could not directly modulate fibrosis-associated functions of fibroblasts, such as proliferation and collagen as well as α-SMA expression, in contrast to other TH2 cytokines like IL-4 and IL-13 (27, 45). Consistent with our results, Liu and colleagues showed that IL-10 had no significant effect on human fetal lung fibroblasts (46). In contrast, results obtained with fibroblast cell lines show that IL-10 can downregulate constitutive and TGF-β–stimulated type I procollagen expression (22, 47). The basis for this apparent conflict is unknown at this time.
Although IL-10 cannot directly activate certain lung fibroblast functions, it is capable of regulating the production of several important regulators in lung fibrosis, namely TGF-β and PGE2. It is well accepted that TGF-β overexpressed during silicosis is one of the major fibrogenic factors participating in the extension of lung fibrosis (34). In lung homogenates of silica–treated animals, the levels of active TGF-β were decreased in the absence of IL-10 and thus could explain the reduction of fibrosis. These conclusions are supported by the data presented in IL-10 transgenic mice where expression of TGF-β is increased in the lung (6). A similar mode of action has been demonstrated for IL-4 (48) and IL-13 (11). In a similar fashion we showed by real-time PCR that IL-10 could directly stimulate TGF-β expression in AM from saline- or silica-treated mice. These observations support the concept of a profibrotic role for IL-10 via its stimulatory effect on macrophage TGF-β production in silica–induced fibrosis (34).
PGE2 is known to have antifibrotic activity by virtue of its ability to control several key fibroblast functions. First, PGE2 downregulates fibroblast proliferation and collagen synthesis in vitro (49–52). Second, enhanced bleomycin-induced pulmonary fibrosis in GM-CSF–deficient mice is associated with an impairment to elaborate PGE2 (53). Third, an increased fibroproliferative response is noted in the lungs of COX-2–deficient mice instilled with bleomycin or vanadium pentoxide (54, 55). Interestingly, PGE2 production is markedly upregulated in spleen cells of IL-10–deficient mice as compared with wild-type controls, consistent with the ability of IL-10 to regulate COX-2 expression (56). Thus PGE2 represents a good candidate to mediate the inhibition of lung fibrosis noted in IL-10−/− animals. Indeed we found that PGE2 levels measured in lung homogenates of the four groups of mice were inversely correlated with the intensity of fibrosis. Furthermore IL-10 inhibited COX-2 expression and PGE2 production in pulmonary fibroblasts and macrophages from saline– or silica–treated mice (Figures 7 and 8). These inhibitory effects of IL-10 are consistent with other reports concluding that IL-10 downregulates PGE2 expression by human synovial fibroblasts (57) or by rabbit AM (58). However, two other studies examining the activity of IL-10 on human fetal lung fibroblasts (46) or AM (59) were unable to observe similar effects. The basis for these discrepant results is not immediately apparent, but may be related to species and/or cell type differences.
In summary, the use of IL-10–deficient mice in this study has revealed that the roles of IL-10 in silica-induced lung fibrosis are complex, at least in part because of its pleiotropic effects on immune as well as nonimmune cells. The results show that IL-10 exacerbates TGF-β expression in lung macrophages while limiting PGE2 expression in both lung macrophages and fibroblasts, consistent with a net profibrotic effect. In contrast, IL-10 could also play a beneficial role in the extension of pulmonary fibrosis by limiting the expansion of detrimental CD4+ T cells, and thus could be considered under certain conditions to be an antifibrotic cytokine. However, the findings from IL-10–deficient mice suggest its profibrotic activity to be predominant in vivo, at least in this model.
This work was supported in part by the Belgian Federal Services for Scientific, Technical, and Cultural Affairs, the Actions de Recherche Concertées, Communauté Française de Belgique-Direction de la Recherche Scientifique, and by the National Institutes of Health. F.H. is research assistant with the Fonds National de la Recherche Scientifique (FNRS), Belgium.
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