Pulmonary overexpression of IL-10 augments lung fibrosis and Th2 responses induced by silica particles
Virginie Barbarin,1
Zhou Xing,2
Monique Delos,3
Dominique Lison,1 and
Francois Huaux1
1Industrial Toxicology and Occupational Medicine Unit, Faculty of Medicine; 3Laboratory of Pathology, University Hospital of Mont Godinne, Yvoir, Université catholique de Louvain, Brussels, Belgium; and 2Department of Pathology, McMaster University, Hamilton, Ontario, Canada
Submitted 1 September 2004
; accepted in final form 10 December 2004
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ABSTRACT
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Chronic inflammation and proinflammatory cytokines as well as T helper type 2 (Th2) cytokines have been involved in the pathogenesis of pulmonary injury and lung fibrosis. The actual role of IL-10 in lung fibrosis is still unclear because this cytokine has been identified as Th2 but possesses strong anti-inflammatory properties. To better dissect the potential role of IL-10 in silica-induced lung fibrosis, IL-10 was overexpressed in the lung of mice by adenoviral gene transfer during the inflammatory (administered at day 1) or the fibrotic (administered at day +30) stages of the disease. Pulmonary overexpression of IL-10 during both silica-induced lung inflammation and fibrosis exacerbated the fibrotic lesions as estimated by the measurement of hydroxyproline and other biochemical and histological markers. Increased expression of IL-10 significantly enhanced the number of lung lymphocytes and bronchoalveolar lavage fluid IgG1 but not IgG2a levels, indicating the induction of a Th2-like immune response. In addition, the production of the profibrotic Th2 cytokines IL-4 and IL-13 was also significantly increased upon IL-10 overexpression. No difference in transforming growth factor-
or PGE2 production was noted after adenoviral IL-10 treatment of silica-treated mice. Together, these data indicate that the increased expression of IL-10 significantly contributed to silica-induced lung fibrosis by exacerbating the Th2 response and the production of the profibrotic cytokines IL-4 and IL-13.
inflammation; T helper 2 cytokines; interleukin-10
CYTOKINES PROMOTE AND MAINTAIN the recruitment of leukocyte subpopulations, fibroblast activation, and proliferation, as well as collagen deposition during the development and the extension of lung fibrosis. T helper (Th) 2 cytokines such as IL-4 and IL-13 have been implicated in the development of pulmonary fibrosis (40, 54, 67), whereas Th1 cytokines such as IFN-
or IL-12 seem to possess opposite effects by controlling the fibrotic process (9, 20, 30, 54).
This paradigm was first suggested on the basis of in vitro studies in which Th2 cytokines (IL-4, IL-13) were found to upregulate fibroblast functions such as proliferation and collagen production (28), whereas a Th1 cytokine (IFN-
) had an opposite effect (53). These observations were confirmed in vivo since a preferential Th2 polarization was demonstrated in several animal (24, 57, 63) and human (62) studies of lung fibrosis. In mouse models developed to induce highly polarized Th1- or Th2-type inflammation in the lung, Th1-mediated inflammation was characterized by tissue damage, whereas Th2 directed wound healing and fibrosis (50). In addition, injection of IL-12, a prototypic Th1 cytokine, prevented the fibrotic reaction induced by Schistosoma infection in the liver (65) or in the lung (50) and attenuated bleomycin-induced lung fibrosis (30). IL-4 plays a profibrotic role since IL-4-deficient mice treated with bleomycin or developing tuberculosis developed significantly less pulmonary fibrosis compared with their wild-type counterparts (22, 25). Overexpression of Th2 cytokines in the lung, in transgenic mice or adenoviral constructs, has demonstrated that IL-4, IL-5, IL-9, and IL-13 participate in the development and extension of lung fibrosis (26, 37, 39, 59, 67). In addition, in idiopathic pulmonary fibrosis (IPF), IL-4 and IL-5 expression appeared to be increased with a concomitant reduction in the expression of IFN-
(62).
IL-10 has been identified as a Th2 cytokine (16). Thus IL-10 inhibits the production of IL-2, IL-3, lymphotoxin/TNF, IFN-
, and granulocyte-macrophage colony-stimulating factor by Th1 cells responding to antigens and antigen presenting cells, whereas Th2 cytokine synthesis is not significantly affected (17, 18). In addition to these Th2 properties, IL-10 has important immunosuppressive and anti-inflammatory activities. IL-10 downregulates the production of proinflammatory mediators by monocytes/macrophages (2, 7, 13, 17) and strongly regulates pulmonary inflammation in different animal models (58).
The actual role of IL-10 in lung fibrosis remains unsettled and is confused by its dual activities: IL-10 downregulates inflammation but is considered as a Th2 cytokine. Indeed, because of its anti-inflammatory activities, it has been first postulated and demonstrated that IL-10 may control chronic inflammation and thus the subsequent fibrotic process in a mouse model of liver fibrosis (38, 60). However, other authors have indicated that IL-10 may have a deleterious activity in lung fibrosis (27, 52). Interestingly, IL-10 overexpression was clearly shown in human lung fibrosis (43, 51).
To better dissect the activity of this cytokine, we overexpressed IL-10 in the lung in an experimental model of silicosis by adenoviral gene transfer during the alveolitis or the fibrotic stage of the disease and characterized the accompanying pulmonary responses.
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MATERIALS AND METHODS
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Animals.
Female C57BL/6J mice were obtained from our local breeding facility (Ludwig Institute, Brussels, Belgium). Mice weighing between 18 and 22 g were used and housed in positive-pressure air-conditioned units (25°C, 50% relative humidity) on a 12-h light-dark cycle. All procedures involving animals were approved by the animal use committee at Université Catholique de Louvain.
Instillation method.
A suspension of crystalline silica particles [Quartz DQ12; d50 (medium particle size) = 2.2 µm, 2.5 mg, a gift from Dr. L. Armbruster, Essen, Germany] in sterile 0.9% saline was injected directly into the lungs by transoral instillation at day 0 (26). To allow sterilization and inactivation of any trace of endotoxin, we heated particles at 200°C for 2 h immediately before suspension and administration. All instillations (50 µl/mouse) were performed on animals anesthetized with a mix of Ketalar (Warner-Lambert, Zaventem, Belgium) and Rompun (Bayer, Leverkusen, Germany) (1 and 0.2 mg /mouse intraperitoneally, respectively).
Adenovirus constructs.
IL-10 (AdIL-10) and control (AdCtl) adenoviral constructs were prepared as previously described (66). A dose of 108 x pfu of AdIL-10 or AdCtl vectors was diluted in 50 µl of PBS and delivered in mice by transoral instillation 1 day before or 30 days after silica treatment. The viral vector used in this study has a marked tropism for the epithelial cells of the lung and is highly efficient in transferring the IL-10 gene into these lining cells. In addition, it has been also demonstrated that IL-10 produced by this adenoviral gene transfer is biologically active because this approach can reconstitute the biological properties of IL-10 in IL-10-deficient mice (42).
Bronchoalveolar lavage and whole lung homogenates.
At selected time intervals after silica instillation, the animals were killed with pentobarbital sodium (20 mg/animal ip), and we performed a bronchoalveolar lavage (BAL) by cannulating the trachea and lavaging the lungs five times with a volume of 1 ml NaCl 0.9%. The BAL fluid (BALF) was centrifuged (1,000 rpm, 10 min, 4°C), and the cell-free supernatant was used for biochemical measurements. Aliquots of the cell suspensions were used to determine cell numbers and cell differentials (200 cells counted). This was performed on cytocentrifuge preparations fixed in methanol and stained with Diff Quick (Dade, Brussels, Belgium).
Separately, perfused whole lungs were excised and placed into a Falcon tube chilled on ice, and 3 ml of cold NaCl 0.9% were added. The content of each tube was then homogenized with a Ultra-Turrax T25 homogenizer (Janke and Kunkel, Brussels, Belgium) 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.
Biochemical analyses.
We assayed lactate dehydrogenase (LDH) activity in BALF spectrophotometrically by monitoring the reduction of nicotinamide adenine dinucleotide at 340 nm in the presence of lactate. Total proteins in BALF were determined by the pyrogallol red staining method (Technicon RA system; Bayer Diagnostics, Domont, France).
Silica measurement.
The amount of silica particles retained in the lung of mice instilled with AdIL-10 or AdCtl adenovirus was measured 2 mo after administration of 2.5 mg of silica. The concentration of silica was determined colorimetrically with the molybdenum blue method after digestion of the lungs in sodium hypochlorite (58a).
Collagen assays.
We estimated lung collagen deposition by measuring the hydroxyproline (OH-proline) and soluble collagen tissue contents. For OH-proline, the lung was excised, homogenized as described above, and hydrolyzed in 6 N HCl overnight at 110°C. OH-proline was assessed by high-performance liquid chromatography analysis (6), and data are expressed as micrograms of OH-proline per lung. Soluble collagen levels were estimated by the Sircol collagen assay following the manufacturers protocols (Biocolor, Newtownabbey, Northern Ireland) in the supernatant of centrifuged lung homogenates.
ELISA.
Fibronectin and type I collagen contents were measured in lung homogenates by standardized ELISA as detailed in a previous study (23, 25).
Mouse IL-10 (BD Biosciences, San Diego, CA), IL-4, IL-13, transforming growth factor (TGF-
; R&D Systems, Minneapolis, MN), IgG1, IgG2a (BETHYL laboratories, Montgomery, TX), and PGE2 (Amersham, Bucks, UK) concentrations were measured with ELISA kits following the manufacturers protocols. The detection limits of these ELISA are respectively 16 pg/ml, 2 pg/ml, 2 pg/ml, 7 pg/ml, 4 ng/ml, 4 ng/ml, and 16 pg/ml.
Statistics.
Treatment-related differences were evaluated by 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.
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RESULTS
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IL-10 expression after adenovirus administration.
To ascertain increased lung expression of IL-10 by adenoviral gene transfer, we first measured the level of this cytokine in the BAL of saline- or silica-treated mice administered with AdIL-10 or AdCtl. Figure 1 shows that administration of AdIL-10 dramatically increased the production of IL-10 as demonstrated by high levels both in saline- and silica-treated mice. The peak of IL-10 lung content was maximum at day 7 in saline mice and at day 3 in mice treated with silica. Low IL-10 concentrations were measured with the AdCtl construct.

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Fig. 1. Time course of IL-10 levels in bronchoalveolar lavage fluid (BALF) obtained from saline- or silica-treated mice administered with control (AdCtl) or IL-10 (AdIL-10) adenovirus [day 1 (d1)]. Bars represent means ± SE of 5 or 6 animals. IL-10 overexpression peaked at d7 in saline mice and at d3 in silica-treated mice.
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To determine whether overproduced IL-10 was biologically active, lung inflammation was assessed at day 3 after silica treatment. Protein concentration, number of neutrophils, and LDH activity in BAL were significantly decreased after injection of AdIL-10 compared with AdCtl [for example: polymorphonuclear neutrophils (103/ml), 76.8 ± 19.7 vs. 96.6 ± 11.4; proteins (g/l), 0.4 ± 0.04 vs. 0.6 ± 0.04; LDH (U/l), 193.4 ± 12.4 vs. 227.0 ± 44.4, respectively]. We concluded that the IL-10 adenoviral gene transfer approach allowed us to induce high lung expression of biologically active IL-10 in the lung.
Cellular parameters in mice treated with AdIL-10 and AdCtl.
Cellularity was analyzed in BALF 2 mo after saline or silica treatment when AdCtl or AdIL-10 were administered at day 1 and day +30. Overexpression of IL-10 in silica-treated mice was associated with a marked recruitment of lymphocytes as shown in Fig. 2A. Compared with AdCtl, the increase of lymphocytes was noted both after administration of AdIL-10 at day 1 and day +30. We also observed an increase of macrophages (Fig. 2C) and neutrophils (Fig. 2B) in AdIL-10- and silica-treated mice compared with AdCtl situations. These increases were, however, only significant for neutrophils at day +30 and for macrophages at day 1. We also observed a recruitment of macrophages and neutrophils in saline mice administered with AdIL-10 at day 1.

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Fig. 2. Alveolar lymphocyte (A), neutrophil (B), and macrophage (C) counts in BAL samples from saline- or silica-treated mice administered with AdCtl or AdIL-10. Bars represent means ± SE of 5 or 6 animals. Significantly different from AdCtl: *P < 0.05, **P < 0.01, ***P < 0.001 (Student-Newman-Keuls multiple-comparison test). Lymphocytes numbers were highly increased in mice treated with silica + AdIL-10 compared with AdCtl administration whatever the time of construct administration.
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Lung fibrosis in mice treated with AdIL-10 and AdCtl.
To assess the effect of lung IL-10 overexpression on the fibrotic process, OH-proline levels were measured at 2 mo in the lung homogenates of mice administered with AdIL-10 or AdCtl either at day 1 or at day +30 after silica treatment. After silica treatment, the levels of OH-proline were significantly increased in mice treated with AdIL-10 compared with mice treated with AdCtl, whatever the time of adenovirus administration (day 1 and day +30) (Fig. 3). We obtained similar results by measuring other markers of fibrosis such as type 1, soluble collagen, or fibronectin levels (data not shown). Collectively, these data suggested that overexpression of IL-10 both during the inflammatory or the fibrotic stages of experimental silicosis exacerbated lung fibrosis.

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Fig. 3. Hydroxyproline levels in lung homogenates obtained from saline- or silica-treated mice administered with AdCtl or AdIL-10. Bars represent means ± SE of 5 or 6 animals. Significantly different from AdCtl: *P < 0.05, ***P < 0.001 (Student-Newman-Keuls multiple-comparison test). Increased expression of IL-10 exacerbated silica-induced lung fibrosis as measured by hydroxyproline contents.
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Silica measurements.
To verify whether the increase of lung fibrosis in mice treated with AdIL-10 could be due to a reduced clearance of silica particles because of the anti-inflammatory properties of IL-10, the contents of silica particles remaining in the lung after 60 days were measured. We did not find any difference in terms of silica content among the groups considered (Fig. 4), demonstrating that modification in lung clearance cannot explain differences in the lung fibrosis responses (Fig. 3).

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Fig. 4. Silica contents in lung homogenates of saline and silica mice treated with AdCtl or AdIL-10. Bars represent means ± SE of 5 or 6 animals. No significant difference of silica content was observed between mice treated with AdCTL and AdIL-10.
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Levels of the anti- or profibrotic mediators PGE2 and TGF-
are not modified after AdIL-10 administration.
It has been demonstrated that IL-10 regulates the expression of PGE2 and TGF-
, two key mediators possessing important functions in the control or the extension of lung fibrosis, respectively (29, 44, 56). To investigate by which mechanism IL-10 can promote the fibrotic process in experimental silicosis, we assessed the levels of the anti- and profibrotic mediators PGE2 and TGF-
when lung IL-10 overexpression was maximum (i.e., 3 days after AdIL-10 administration in silica-treated mice). We found that neither PGE2 or TGF-
levels were modified by the overexpression of IL-10, denoting that the profibrotic functions of IL-10 were independent of PGE2 and TGF-
(Fig. 5).
Overproduction of IL-10 is associated with a preferential type 2 polarization and marked production of IL-4 and IL-13.
Silica-induced lung fibrosis in mice administered with AdIL-10 was accompanied by a marked accumulation of lymphocytes (Fig. 2A). To determine the phenotype of recruited lymphocytes and the immune polarization in these mice, BALF IgG2a and IgG1 levels (markers of the type 1 and type 2 immune response, respectively) were measured 2 mo after silica treatment. Compared with AdCtl, IgG1 levels were significantly higher in the BAL of mice treated with saline or silica and administered with AdIL-10 (day 1 or day +30) (Fig. 6). These results denoted a preferential type 2 immune response after overexpression of IL-10. In contrast, IgG2a levels were not changed after AdIL-10 or AdCtl administration in saline or silica mice. To support the preferential presence of a Th2 immune response induced by IL-10, we also measured the BALF levels of IL-4 and IL-13 3 days after AdIL-10 administration. AdIL-10 administration significantly increased the production of IL-4 and IL-13 in silica-treated mice compared with AdCtl (Fig. 7, A and B).

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Fig. 6. IgG1 and IgG2a levels in BALF of saline and silica mice treated with AdCTL or AdIL-10. Bars represent means ± SE of 5 or 6 animals. Significantly different from AdCtl: ***P < 0.001 (Student-Newman-Keuls multiple-comparison test). IgG1 levels were significantly higher in BAL of saline and silica mice treated with AdIL-10 compared with AdCtl, whatever the time of adenovirus administration.
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Fig. 7. IL-13 and IL-4 levels in BAL of saline and silica mice treated with control or IL-10 adenovirus at d3. Bars represent means ± SE of 5 or 6 animals. Significantly different from AdCtl: *P < 0.05 (Student-Newman-Keuls multiple-comparison test). IL-13 and IL-4 production was upregulated by IL-10 overexpression. ND, not detected.
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Altogether, these data suggest that the overexpression of IL-10 in the lung increased the polarization of the immune system toward a type 2 response and upregulated the expression of profibrotic cytokines such as IL-4 and IL-13.
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DISCUSSION
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In this study, we have shown that IL-10 overexpressed during either the inflammatory or the fibrotic stage of experimental silicosis may exert a profibrotic activity by upregulating the lung Th2 response induced by silica and by inducing IL-4 and IL-13 lung expression.
IL-10, a well-known immunoregulating factor, has been reported to reduce tissue inflammation and injury in several animal models and human lung disorders (17, 45). IL-10 downregulated the production of TNF-
and macrophage inflammatory protein-2 that occurs in response to systemic endotoxin or bacterial lipopolysaccharide exposure (55, 58). By downregulating neutrophil recruitment and the subsequent cytokine and oxidant generation, IL-10 also exerted an anti-inflammatory activity during acute lung inflammation and tissue damage associated with carrageenan-induced pleurisy (11). IL-10 also enhances the resolution of pulmonary inflammation by promoting neutrophil apoptosis (10) and regulates quartz-induced pulmonary inflammation in rats (14).
Because of its well-defined anti-inflammatory properties, it has been postulated that IL-10 may possess antifibrotic activities. Indeed, on the basis of the dogma that more proinflammatory mediators and inflammation lead to more fibrosis, several authors postulated that the expression of IL-10 may control not only the inflammation but also the subsequent fibrosis (34). This hypothesis was supported by some studies using acute models of fibrosis. Indeed, IL-10 controlled neutrophilic infiltration, hepatocyte proliferation, and liver fibrosis induced by carbon tetrachloride in mice (38, 60). The overexpression of IL-10 in mice also inhibited bleomycin-induced lung injury in vivo (1). It has also been suggested that the secretion of IL-10 induced by TGF-
1 may have an unexpected and paradoxical antifibrotic effect in a model of bleomycin-induced fibrosis (32).
Recently, a new scenario has been proposed postulating that a marked anti-inflammatory response established to control lung inflammation may also lead to fibrosis (27, 52). Numerous studies implicated Th2 cytokines (IL-4, TGF-
, IL-13) as essential mediators in the development of lung fibrosis (25, 29, 68). It is noteworthy that all these cytokines possess strong anti-inflammatory activities, especially on monocytes/macrophages. Importantly, some Th2 cytokines can directly upregulate fibroblast functions. Thus IL-4, IL-13, and TGF-
stimulate the proliferation of lung fibroblasts (25, 28) and induce collagen production (46, 48, 49, 53). These profibrotic cytokines are operative in vivo since 1) IL-4-deficient mice developed significantly less pulmonary fibrosis relative to wild-type mice after administration of bleomycin (25), 2) pulmonary selective expression of IL-13 caused subepithelial fibrosis (67), and 3) administration of TGF-
antibodies significantly reduced bleomycin-induced lung fibrosis (19). A preferential Th2 polarization has also been observed in human lung fibrotic diseases. Increased levels of IL-4, TGF-
, and IL-13 were detected in BAL cells recovered from patients with pulmonary fibrosis compared with healthy subjects (3, 21, 61, 62).
It is still not clear whether IL-10 may, as do the other Th2 cytokines, play a direct and essential role in the extension of fibrosis. Several recent human and experimental studies have clearly demonstrated an association between the development of lung fibrosis and IL-10 expression.
Bergeron and coauthors (5) observed that IL-10, in parallel with TGF-
, was quantitatively increased in lung biopsies of IPF patients compared with healthy subjects. Transgenic mice overexpressing IL-10 selectively in the lung via the 10-kDa Clara cell protein promoter spontaneously developed subepithelial fibrosis (36). In accordance with these data, we showed that the fibrotic response induced by silica particles was significantly less important in IL-10-deficient mice than in wild-type mice after silica treatment, suggesting that IL-10 plays a detrimental role during silica-induced fibrosis (4, 27).
To further dissect the role of IL-10, we examined the silica-induced lung fibrotic response in a model where IL-10 is overexpressed. To determine whether IL-10 by its anti-inflammatory activities may control inflammation and thus fibrosis or by its Th2 functions accentuate the fibrotic process itself, we administered IL-10 adenoviral constructs during the inflammatory stage (day 1) and during the development of fibrosis (day +30). Whatever the time of administration, an increase of lung fibrosis after AdIL-10 administration was observed. From these data, we first concluded that a reduced inflammatory response induced by IL-10 is not associated with a reduction of fibrosis. These data also indicate that IL-10 enhanced silica-induced lung fibrosis and can be considered as a profibrotic mediator. This evidence for a role of IL-10 in lung fibrosis establishment appears to contradict another study showing that overexpression of IL-10 by administrating hemagglutinating virus of Japan liposomes containing a human IL-10 expression vector suppressed bleomycin-induced lung fibrosis (1). The basis for this contradiction is not immediately evident but may be related to differences in overexpression model, the cell types infected, the intensity of IL-10 production, and the timing of analysis. Also, the pathogenic process in silica- and bleomycin-induced lung fibrosis probably implicates different immune cell populations (macrophages or eosinophils and T lymphocytes) and different mediators (41). It is thus possible that IL-10 possesses different activities depending on the cellular or the cytokine environment present in the lung.
The profibrotic effect of Th2 cytokines was often linked to an induction of TGF-
. This mode of action has been demonstrated for IL-4 (25) and IL-13 (37). In IL-10 transgenic mice, the expression of TGF-
was increased in the lung (36). In addition, in vitro, IL-10 stimulated TGF-
expression by alveolar macrophages (4). However, in this study, where IL-10 overexpression was induced by adenoviral gene transfer, we could not find a stimulation of TGF-
1 production. During silicosis, TGF-
is mainly expressed by macrophages (29). IL-10 overexpression in our model affected mainly lymphocyte recruitment and activation but not significantly macrophages. These observations may explain, in part, why we did not find any effect of IL-10 on the expression of TGF-
in this model.
Several experiments demonstrated that PGE2 downregulates important functions of fibroblasts such as proliferation and collagen synthesis (45) and can control the establishment of lung fibrosis (44, 8, 31). On the basis of in vitro studies, we suggested that IL-10 may downregulate PGE2 production by fibroblasts and macrophages (4). Consequently, a potential role of PGE2 in silica-lung fibrosis after AdIL-10 treatment was investigated. No effect of IL-10 overexpression on PGE2 levels was observed, suggesting that, in the model examined in this study, IL-10 exerts its profibrotic effect by another pathway than the inhibition of antifibrotic molecules, such as PGE2. Again, we can postulate that, in the model developed in this study, the effects of IL-10 overproduction affected mainly lymphocytes and fewer cells such as macrophages or fibroblasts, known as the main cellular sources of PGE2 (33, 47, 64).
We show in Fig. 2 a dramatic recruitment of lymphocytes in BAL of mice treated with AdIL-10. Consequently, we determined the phenotypes of these lymphocytes and demonstrated a preferential Th2 immune response. Both Th1 and Th2 polarization during experimental silicosis have already been reported. Although IL-4 expression was unchanged or decreased, Davis and colleagues (12) showed an increased IFN-
response in the lung of mice with silicosis, suggesting a Th1-like lymphocyte-mediated immune response. Opposite results showed that in wild-type animals, silica-induced fibrosis was correlated with markers of a Th2-like response such as upregulation of IL-4 levels in lung tissue and an increased immunoglobulin IgG1/IgG2a ratio in BAL (24). In the present study, BALF IgG2a levels (Th1) were not modified after silica or AdIL-10 administration. However, IgG1 levels significantly increased after silica and AdIL-10 administration, indicating a polarization toward a type 2 immune response. In addition, IL-13 and IL-4 levels were increased in silica-treated mice receiving AdIL-10 compared with controls whatever the time of administration. We can thus conclude that IL-10 overexpression stimulates the Th2 response in silica-induced fibrosis. Thus the marked presence of Th2 cytokines that possess direct profibrotic functions may in turn exacerbate lung fibrosis.
IL-10 exerts profibrotic activity both when it is overproduced during the inflammatory (AdIL-10, day 1) or the fibrotic reaction (AdIL-10, day +30). Because IL-10 already induced IL-13 and IL-4 3 days after administration of AdIL-10, we can speculate that the profibrotic effect of IL-10 is rapidly operative and could submerge its potential beneficial role exerted by regulating inflammation.
In summary, IL-10 overexpressed during either the early inflammation or the fibrotic stage exacerbated silica-induced lung fibrosis. IL-10 also stimulated the recruitment of Th2 lymphocytes as well as the production of profibrotic cytokines such as IL-4 and IL-13, which may in turn amplify the fibrotic process. On the basis of these observations, we conclude that IL-10 can be considered as a profibrotic cytokine in silica-induced lung fibrosis.
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GRANTS
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This work was supported in part by the FNRS Médicale and Actions de Recherche Concertées, Communauté française de Belgique, Direction de la Recherche Scientifique.
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ACKNOWLEDGMENTS
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We thank Johan Casters, Yousof Yakoub, and Francine Uwambayineme for excellent technical assistance. F. Huaux is a Postdoctoral Researcher with the Fonds de la Recherche Scientifique (FNRS), Belgium.
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FOOTNOTES
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Address for reprint requests and other correspondence: F. Huaux, Unit of Industrial Toxicology and Occupational Medicine, Université Catholique de Louvain, 30.54, Clos Chapelle-aux-Champs, 1200 Brussels, Belgium (E-mail: huaux{at}toxi.ucl.ac.be)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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