Abstract
We examined the effect of interleukin (IL)-9, a cytokine active on B and T lymphocytes and associated with bronchial asthma, on the development of lung fibrosis induced by crystalline silica particles. Therefore, we compared the response to silica (1 and 5 mg/animal, intratracheally) in transgenic mice that constitutively express high levels of IL-9 (Tg5) and their wild-type counterparts (FVB). At 2 and 4 mo after treatment with silica, histologic examination and measurement of lung hydroxyproline content showed that the severity of fibrosis was significantly less important in Tg5 mice than in their wild-type counterparts. Intraperitoneal injection of IL-9 in C57BL/6 mice also reduced the amplitude of silica-induced lung fibrosis. The reduction of lung fibrosis by IL-9 was associated with a significant expansion of the B-lymphocyte population, both in bronchoalveolar lavage (BAL) and in the pulmonary parenchyma. In wild-type animals, silica-induced fibrosis correlated with markers of a T helper 2–like response such as upregulation of IL-4 levels in lung tissue and an increased immunoglobulin (Ig) G1/IgG2a ratio in BAL. Immunohistochemical studies demonstrated that the upregulation of IL-4 associated with the development of fibrosis was mainly localized in inflammatory alveolar macrophages. In transgenic mice, the level of IL-4 in lung homogenates was not significantly affected by silica treatment, and a reduced IgG1/IgG2a ratio was observed upon treatment with silica. The levels of interferon- γ were significantly decreased after silica treatment in both strains. Together, these observations point to an antifibrotic effect of IL-9 in pulmonary fibrosis associated with a limitation of the type 2 polarization which accompanies lung fibrosis.
Chronic inhalation of crystalline silica may lead to the development of interstitial lung fibrosis, referred to as silicosis (1). Like other interstitial lung diseases, this pneumoconiosis is characterized by an exaggerated accumulation of mesenchymal cells and collagen production, and involves a complex interplay of diverse cell types and mediators, including cytokines (2, 3). Besides the well documented role of pulmonary macrophages, lymphocytes have occasionally been suggested to influence the pneumoconiotic process, but their potential role is not clearly understood and is still questioned (4-7). Increased numbers of lymphocytes in bronchoalveolar lavage (BAL), altered responsiveness of lung lymphocytes to lectins, and lung infiltration by CD4+ lymphocytes have been associated with experimental silicosis (8). The role of T lymphocytes was investigated by some authors and led to contradictory results. After silica instillation, a pulmonary fibrosis of equivalent severity was observed between mice genetically deficient in mature T cells (Balb/c nu/nu) and their T cell–sufficient counterparts, but more neutrophils and fewer macrophages were recovered from the lungs of T cell–deficient mice (6). In contrast, Suzuki and colleagues found that collagen deposition was reduced in silica-treated athymic mice compared with their normal counterparts (7). The possible contribution of B lymphocytes in silicosis has not been addressed. Conflicting results on the role of lymphocytes have also been reported in other experimental models of lung fibrosis (asbestos, bleomycin) (9-12). The recent suggestion that type 2 cytokines such as interleukin (IL)-4 might be associated with (13-16) and possibly directly involved (17) in lung fibrosis has stimulated a renewed interest in the potential role of lymphocytes in this pathogenic process. IL-9 is a T cell–derived cytokine active on both B and T lymphocytes (18). Genetic mapping analyses have led to the identification of this factor as a key mediator that determines susceptibility to asthma (19). This has been further supported by experimental data showing that increased IL-9 production in the lungs results in bronchial hyperresponsiveness and airway inflammation (20-22). In the present study, we found that the lung fibrotic response to silica was reduced by IL-9 and accompanied by an expansion of lymphocytes in the lung as well as a reduction of the T helper (Th)-2 response to silica.
Transgenic (Tg5) mice constitutively expressing high levels of IL-9 were described previously (23, 24). These mice express the IL-9 messenger RNA (mRNA) in all organs tested and have high serum IL-9 concentrations (above 1 μg/ml). IL-9–transgenics, their wild-type counterparts (FVB) and C57BL/6 mice were all female, 8 wk old, weighing 20 to 30 g, and obtained from our local breeding facility. The animals were housed in positive-pressure air-conditioned units (25°C, 50% relative humidity) and kept on a 12 h light/dark cycle.
To allow sterilization and inactivation of any trace of endotoxin, crystalline silica particles (DQ12; median aerodynamic diameter (d50), 2.2 μm) were heated at 200°C for 2 h immediately before suspension and administration. A suspension of DQ12 particles (1 or 5 mg) in sterile saline, or saline (controls), was injected directly into the lungs by intratracheal instillation. All instillations (100 μl/mouse) were performed on anesthetized animals (sodium pentobarbital, 2 mg/mouse, intraperitoneally) after surgical opening of the neck.
The mice were killed 2, 4, and 6 mo after instillation. A first group was used for biochemical and cellular analyses as well as immunoglobulin (Ig) G isotype determination in BAL fluid (BALF). After BAL, the lungs were excised and used for the measurement of cytokines and hydroxyproline content. Histopathology and immunohistology were performed on nonlavaged lungs excised from other groups of FVB and Tg5 mice 2 and 4 mo after instillation.
The animals were killed with sodium pentobarbital (20 mg/animal, intraperitoneally) and BAL was performed by cannulating the trachea and lavaging the lungs six times with a single volume of 1.5 ml of sterile NaCl 0.9%. This BALF was centrifuged (1,000 × g, 10 min, 4°C) and the cell-free supernatant used for biochemical measurements. BAL was carried on with three additional volumes (1.5 ml) of sterile 0.9% saline. After centrifugation, cell pellets from all the lavage fractions were pooled for each animal. Aliquots of the cell suspensions were used to determine cell numbers, and cell differentials (200 cells counted) were performed on cytocentrifuge preparations fixed in methanol and stained with Diff Quick (Dade, Brussels, Belgium). The remaining cells were used for fluorometric automated cell sorting (FACS) analysis.
BALF red blood cells were lysed by incubation for 5 min in 0.15 M NH4Cl. Fluorescent labeling of cells resuspended in Hanks' medium with 3% decomplemented fetal calf serum (FCS) and 10 mM NaN3 was performed with rat fluorescein isothiocyanate (FITC)–conjugated anti-CD8 (clone 53-6.7; ATCC, Manassas, VA) and biotinylated anti-CD4 (clone GK1.5; ATCC), followed by phycoerythrin (PE)-conjugated streptavidin (Becton-Dickinson, San Jose, CA). Double labelings were performed with biotinylated monoclonal antibodies (mAbs) against Mac-1 (clone M1/70; ATCC), followed by PE-conjugated streptavidin and FITC-conjugated anti-IgM (clone LOMM9; provided by H. Bazin, Catholic University of Louvain, Brussels, Belgium) or FITC-conjugated anti–Mac-1 (Cedarlane Labs, Ltd., Hornby, ON, Canada) plus PE-conjugated anti-CD5 (Pharmingen, San Diego, CA). After staining, cells were fixed in paraformaldehyde (1.25%), and fluorescence intensity was measured on 104 cells/sample on a FACScan apparatus (Becton-Dickinson). To exclude granulocytes, macrophages, and dead cells as well as silica particles, the lymphocyte population was gated according to side and forward scatters.
Whole lungs were excised and placed into a Falcon tube chilled on ice and 3 ml of cold NaCl 0.9% was added. The content of each tube was then homogenized for 30 s using a Polytron PT1200 homogenizer (Kinematica AG, Littau, Lu, Switzerland). The tubes were centrifuged at 4°C, 2,000 rpm, for 10 min and supernatants were kept frozen at −80°C until use. Mouse interferon (IFN)-γ and IL-4 concentrations were measured in homogenates by specific enzyme-linked immunosorbent assays (ELISA) obtained from Biosource International (Camarillo, CA) and R&D Systems (Minneapolis, MN), respectively. The detection limits of these ELISAs are 1 pg/ml for IFN-γ and 2 pg/ml for IL-4.
Lactate dehydrogenase (LDH) activity in BALF was assayed 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).
Collagen deposition was estimated by measuring the hydroxyproline content of the right lung. The lung was excised, homogenized, and hydrolyzed in 6 N HCl overnight at 110°C. Hydroxyproline was assessed by high-performance liquid chromatography analysis (25) and data are expressed as micrograms of hydroxyproline per lung.
IgG subclass levels were measured in BALF by ELISA. Briefly, polystyrene plates (Grenier, Nurtingen, Germany) were coated overnight with affinity-purified goat antibodies for rabbit IgG, followed by rabbit antibodies specific for these mouse IgG subclasses. After incubation for 2 h at 37°C with samples serially diluted in Tris-buffered saline (10 mM Tris, 10 mM merthiolate, and 130 mM NaCl, pH 7.4) supplemented with 5% FCS, biotinylated mAbs directed against IgG1 or IgG2a subclass were added for 2 h at 37°C. The fixation of labeled antibodies was measured with an avidin-peroxidase complex (26).
Left lungs were excised and fixed in Bouin solution (Merck- Belgabo, Leuven, Belgium). After dewaxing and rehydration, paraffin-embedded sections were stained with hematoxylin and eosin or Masson's trichrome staining for light microscopic examination. For immunohistochemistry stainings, endogenous peroxidase activity was quenched by exposure to hydrogen peroxide (0.5% in nanopure water) for 25 min followed by three washes of 5 min in PGT buffer (phosphate-buffered saline [PBS], 0.05% Tween 20, and 0.02% gelatine). An incubation was then performed for 1 h in a humidified room with 50 μL antimouse IL-4 mAb (hybridoma 11B11; ATCC; 10 μg/ml in PBS) or anti-B220 mAb (a gift from Dr. W. Van Ewijk, Erasmus University, Rotterdam, The Netherlands). After three washes (5 min each) with PGT buffer, tissue sections were exposed for 1 h to the second antibody (polyclonal rabbit against rat IgG coupled with peroxidase; Dako, Copenhagen, Denmark) diluted 40-fold in PBS supplemented with 1% mouse serum. Tissue sections were then rinsed and washed three times in PGT buffer. The peroxidase activity was revealed by 3-3′-diaminobenzidine tetrahydrochloride (Aldrich, Beerse, Belgium)– H2O2 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). Control stainings included omission, neutralization of the primary antibody with an excess of recombinant IL-4 (produced in our laboratory), or substitution of anti-IL-4 by an irrelevant rat mAb (LO-DNP-34, provided by H. Bazin).
A group of C57BL/6 mice was instilled intratracheally with 5 mg silica (DQ12) followed by different treatments with recombinant IL-9. One subgroup was injected intraperitoneally three times a week during the 2 mo after silica instillation with 0.5 μg of recombinant IL-9 produced and purified in our laboratory as described (27) and diluted in 200 μl of PBS containing 1% normal mouse serum. The second subgroup of animals treated with silica also received 0.5 μg IL-9 three times a week, but only during the second month after silica instillation. The third subgroup comprised mice instilled with silica but receiving only 200 μl of PBS containing 1% normal mouse serum three times a week during the 2 mo.
The amount of silica particles retained in the lung of Tg5 and FVB mice was measured 2 mo after administration of 5 mg DQ12. The concentration of silica was determined colorimetrically with the molybdenum blue method (28) after digestion in sodium hypochlorite.
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. For FACS analysis data, statistical analyses were performed by Mann–Whitney U statistical test for unpaired values using Instat software (GraphPad Software Inc., San Diego, CA). Statistical significance was considered at P < 0.05.
Wild-type mice and IL-9 transgenics received an intratracheal instillation with 1 or 5 mg of crystalline silica particles. Lung hydroxyproline content was measured to quantitatively monitor the fibrotic response. As shown in Figure 1, hydroxyproline levels increased with time and in a dose-dependent manner in both FVB and Tg5 animals. At all times studied (2, 4, and 6 mo), hydroxyproline levels were, however, lower in Tg5 mice than in wild-type animals with the same treatment. This difference between the two strains was statistically significant at 4 and 6 mo after the highest dose of silica (5 mg) and at 4 mo after treatment with 1 mg silica. Histologic analysis indicated a qualitatively different response to silica in Tg5 animals. In FVB mice, clear fibrotic areas were observed and were characterized, as in typical silicotic nodules, by an accumulation of collagen and mesenchymal cells (Figure 2a, panel A). The fibrotic response in the lung parenchyma of Tg5 mice was less marked and less organized than in wild-type animals. Tg5 animals treated with silica displayed lymphocytic nodules (Figure 2, upper panels, panel B) mostly consisting of B lymphocytes, as demonstrated by immunohistochemistry in the high- (B220+ cells; Figure 2, upper panels, panel C) and low-dose groups (not shown). Such lymphocytic nodules were absent in control and treated FVB mice. As expected, a reduced accumulation of collagen fibers was found in Tg5 animals treated with silica, which was consistent with the 20 to 30% reduction measured biochemically.
Fig. 1. Time course of lung hydroxyproline content (% of strain control) after silica instillation in FVB and Tg5 mice. Symbols represent means +/− standard error of the mean (SEM) of 8 to 12 animals. *Significantly different from FVB (P < 0.05, Student–Newman–Keuls multiple comparison test). Similar results were obtained in two independent experiments.
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Fig. 2. Representative lung sections 2 and 4 mo after silica instillation (DQ12, 5 mg). (Upper panels) Panel A: Silicotic nodule in a wild-type (FVB) mouse 4 mo after silica (original magnification: ×100). Masson trichrome staining. Panel B: Cellular nodules in a Tg5 4 mo after silica (original magnification: ×100). Masson trichrome staining. Panel C: Immunohistochemical staining of B lymphocytes (B220+) in a Tg5 mouse 4 mo after silica (original magnification: ×100). (Lower panels) Panel A: Immunohistochemical staining for IL-4 in the lung of a FVB mouse 2 mo after silica (original magnification: ×100). Panel B: Immunohistochemical staining for IL-4 in the lung of a Tg5 mouse 2 mo after silica (original magnification: ×100). Panel C: Immunohistochemical staining for IL-4 shows positive macrophages containing silica in a FVB mouse (polarized light; original magnification: ×400).
[More] [Minimize]The measurement of the amount of silica particles retained in the lungs of Tg5 and FVB mice 2 mo after administration of 5 mg DQ12 did not reveal a significant difference between the strains (1.73 ± 0.33 and 1.87 ± 0.38 mg, n = 7 and 11, respectively).
LDH activity and protein concentration were measured in BALF of silica-treated mice as markers of lung inflammation. At 2 and 4 mo after silica treatment (Table 1), LDH activity and protein levels in BALF increased dose-dependently in wild-type (FVB) and Tg5 mice. No significant difference was observed between the two strains except for a higher LDH activity in control Tg5 after 2 mo.
| LDH (IU/liter)* | Proteins (μg/liter)* | |||||||
|---|---|---|---|---|---|---|---|---|
| FVB | Tg5 | FVB | Tg5 | |||||
| 2 mo | ||||||||
| Saline | 27 ± 3 | 54 ± 7† | 24 ± 4 | 66 ± 7† | ||||
| 1 mg | 73 ± 6 | 95 ± 7 | 102 ± 13 | 114 ± 3 | ||||
| 5 mg | 156 ± 14 | 159 ± 8 | 219 ± 38 | 203 ± 14 | ||||
| 4 mo | ||||||||
| Saline | 45 ± 3 | 37 ± 10 | 69 ± 9 | 56 ± 10 | ||||
| 1 mg | 94 ± 10 | 134 ± 23 | 143 ± 22 | 175 ± 36 | ||||
| 5 mg | 177 ± 24 | 157 ± 15 | 328 ± 58 | 278 ± 46 | ||||
Cellularity of the BALF was also analyzed. At 2 mo after silica instillation, although a similar recruitment of macrophages, neutrophils, and CD4 and CD8 cells was observed, the number of B cells (IgM+ cells) present in BALF from silica-treated Tg5 mice was significantly higher than in FVB mice (Table 2). To determine in more detail the phenotype of the B-cell populations in BALF, FACS analysis was performed with anti-IgM, anti–Mac-1 and anti-CD5 antibodies. These three cell-surface antigens allow discrimination between the major B-lymphocyte subpopulations (29, 30). B-1 lymphocytes can be distinguished from conventional B cells (B-2 lymphocytes) by the expression of Mac-1 antigen. Although it was difficult to delineate two subpopulations of B lymphocytes on the basis of Mac-1 antigen expression, the expression of this antigen was clearly enhanced in B cells from IL-9 transgenics as compared with FVB mice, indicating that B-1 cells mainly contribute to this B cell population (Figure 3, panel A). Among B-1 cells, two subpopulations can further be distinguished on the basis of CD5 expression: B-1a cells are CD5+ and B-1b are CD5−. Staining of BAL cells (Figure 3, panel B) indicates that in IL-9 transgenics the accumulation of Mac-1+ cells was limited to CD5− cells (B-1b cells).
| Macrophages (× 103)* | Neutrophils (× 103)* | CD4 (× 103)* | CD8 (× 103)* | B Lymphocytes (× 103)* | ||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| FVB | Tg5 | FVB | Tg5 | FVB | Tg5 | FVB | Tg5 | FVB | Tg5 | |||||||||||
| 2 mo | ||||||||||||||||||||
| Saline | 49 ± 7 | 115 ± 12 | 0.2 ± 0.1 | 9 ± 5 | 0.2† | 8† | 0† | 1† | 0.1† | 11† | ||||||||||
| 1 mg | 348 ± 21 | 274 ± 32 | 125 ± 34 | 86 ± 13 | 20 ± 5 | 31 ± 5 | 10 ± 2 | 15 ± 4 | 39 ± 4 | 328 ± 57‡ | ||||||||||
| 5 mg | 758 ± 211 | 589 ± 164 | 534 ± 206 | 228 ± 48 | 85 ± 40 | 58 ± 13 | 39 ± 3 | 47 ± 9 | 90 ± 19 | 349 ± 53‡ | ||||||||||
| 4 mo | ||||||||||||||||||||
| Saline | 42 ± 10 | 124 ± 0.1 | 0.5 ± 0.3 | 2 ± 0.2 | 1† | 4† | 0† | 1† | 0† | 3† | ||||||||||
| 1 mg | 629 ± 92 | 290 ± 61 | 123 ± 33 | 185 ± 31 | 77 ± 14 | 62 ± 5 | 59 ± 22 | 28 ± 6 | 68 ± 18 | 258 ± 41‡ | ||||||||||
| 5 mg | 1,945 ± 363 | 1,090 ± 182§ | 1,068 ± 225 | 479 ± 125§ | 148 ± 12 | 118 ± 15 | 111 ± 21 | 55 ± 11 | 103 ± 24 | 245 ± 60‡ | ||||||||||
Fig. 3. Double-color FACS staining of BALF cells obtained from Tg5 and FVB mice treated 2 mo earlier with 5 mg of silica. The cells were labeled as described in Materials and Methods with anti–Mac-1–PE and anti–IgM–FITC (A) or anti–Mac-1– FITC and anti–CD5-PE (B). Results of analysis of lymphoid cells from FVB (left panels) and Tg5 mice (right panels) are shown for one representative animal, and indicated numbers correspond to the percentage of total cells for a pool of cells from five individuals. Similar results were obtained in two independent experiments.
[More] [Minimize]At 4 mo after silica instillation, B lymphocytes were also more abundant in BALF from silica-treated Tg5 than FVB mice. In addition, a reduced recruitment of macrophages and neutrophils was noticed in Tg5 mice as compared with FVB mice but the recruitment of CD4 and CD8 lymphocytes was not different.
The IL-9 transgenic mice used in this study showed elevated levels of this cytokine starting before birth. To assess whether the effect of IL-9 on lung fibrosis was immediate or required early and prolonged expression, we administered recombinant IL-9 to silica-treated C57BL/6 mice. As shown in Figure 4, animals that had received recombinant IL-9 during the 2 mo after administration of silica had significantly less lung hydroxyproline content than mice that did not receive IL-9 treatment. Hydroxyproline levels were also, though not significantly, reduced in mice treated only during the second month of the experiment. In a second experiment, the antifibrotic effect of recombinant IL-9 was reproduced (not shown) and BALF from B6 mice treated with silica and intraperitoneally injected with rIL-9 contained significantly more lymphocytes than did BALF from silica-treated animals that did not receive IL-9. FACS analysis revealed that the difference observed involved all classes of lymphocytes but, as in Tg5 transgenics, the difference was more marked for B lymphocytes (Figure 5).
Fig. 4. Hydroxyproline levels in lung tissue homogenates of C57BL/6 mice 2 mo after treatment with silica (DQ12). Recombinant IL-9 was administered by intraperitoneal injection (0.5 μg; three times/wk). Bars represent means +/− SEM of four to seven animals. *Significantly different (P < 0.05, Student–Newman– Keuls multiple comparison test).
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Fig. 5. BALF cell populations in C57BL/6 mice 2 mo after treatment with silica (DQ12). Recombinant IL-9 was administered by intraperitoneal injection (0.5 μg; three times/wk). BALF cells were labeled as described in Materials and Methods with anti-CD4, anti-CD8, or anti-IgM antibodies and analyzed by FACS. Bars represent means +/− SEM. *P < 0.05, ***P < 0.0001. (Mann–Whitney test).
[More] [Minimize]To examine the possible mechanisms involved in the reduction of lung fibrosis in Tg5 mice, we measured the lung content in IL-4 and IFN-γ, two cytokines that have been reported to have pro- and antifibrotic activity, respectively (17). In the absence of silica treatment, Tg5 mice had more IL-4 in lung tissue than did FVB mice (FVB and Tg5, respectively: 6.2 ± 0.6 and 13.5 ± 2 pg/ml at 2 mo; 9.2 ± 0.8 and 12.6 ± 3 pg/ml at 4 mo). In wild-type animals treated with silica, levels of IL-4 increased significantly and dose-dependently in lung tissue homogenates after 2 and 4 mo. Conversely, IFN-γ levels decreased in response to silica in FVB mice at both time points studied. By contrast, IL-9 transgenic mice showed no increase in IL-4 in response to silica treatment. The IFN-γ response to silica in Tg5 mice was not different from that observed in FVB animals (Figure 6).
Fig. 6. Levels of IL-4 and IFN-γ protein in lung homogenates of FVB and Tg5 mice after silica treatment. Bars represent means +/− SEM of four to six animals. Significantly different from controls: *P < 0.05, **P < 0.001 (Student–Newman–Keuls multiple comparison test).
[More] [Minimize]To determine which cell type was responsible for this production of IL-4, immunohistochemical staining for this cytokine was performed on lung tissue sections from FVB and Tg5 mice. In the absence of silica treatment, IL-4 was detected in association with perivascular and peribronchiolar lymphocytic infiltration in the lungs of Tg5 but not in FVB mice. In FVB mice treated with silica, fibrotic areas at 2 and 4 mo after treatment were characterized by intense immunoreactivity for IL-4 (Figure 2, lower panels, panel A). IL-4 was mainly localized in mononuclear cells (macrophages, mesenchymal cells, alveolar macrophages loaded with silica), as illustrated in Figure 2, lower panels, panel C. In Tg5 mice treated with silica, IL-4 positive cells were clearly less abundant (Figure 2, lower panels, panel B).
Because IgG2a and IgG1 are known to be associated with type 1 and type 2 immune responses, respectively, we measured their concentration in BALF to further determine the type 1/type 2 profile in the lungs. After two months, FVB and Tg5 mice (controls and treated) had similarly low concentrations of IgG1 and IgG2a in BALF (data not shown). At 4 mo after treatment, there was no significant difference in IgG isotypes between controls. In response to silica, increased IgG1 levels were observed in the two strains, however IgG1 levels in FVB mice treated with 5 mg silica were significantly higher than in corresponding Tg5 mice (Figure 7). In contrast, IgG2a levels were higher in silica-treated Tg5 mice than in their wild-type counterparts, indicating that IL-9 reduces the type 2 polarization associated with the response to silica.
Fig. 7. IgG1 and IgG2a concentrations in BALF of FVB and Tg5 mice 4 mo after silica treatment. Bars represent means +/− SEM of four to six animals. Significantly different: *P < 0.01; **P < 0.001 (Student–Newman–Keuls multiple comparison test). Similar results were obtained in two independent experiments.
[More] [Minimize]The main finding of this study is the reduced fibrotic response to silica in the lungs of IL-9 transgenics. The observation that injection of recombinant IL-9 was able to exert the same effect in C57BL/6 mice as constitutive IL-9 expression clearly demonstrates that the antifibrotic effect can be attributed to IL-9 and not to a genetic positioning effect in the transgenic construct. In addition, our data show that this activity of IL-9 is not restricted to a single genetic background because IL-9 transgenic mice were generated in the FVB/N background (H2q) and IL-9 administration experiments led to the same conclusions in C57BL/6 mice (H2b). However, it is not excluded that genetic factors could affect both the extent of silica-induced fibrosis as well as the IL-9–mediated protection. For instance, in preliminary experiments with DBA/2 mice which develop a marked lung fibrosis to silica associated with a mainly neutrophilic infiltration (31), this reaction was not substantially modified by IL-9 administration (data not shown), suggesting that distinct pathophysiologic mechanisms could underlie silica-induced fibrosis in different genetic backgrounds.
It has been reported that the production of mucus by bronchial cells is significantly increased in Tg5 mice (32), which might conceivably facilitate the clearance of silica particles from the lung. The absence of difference in the residual lung silica content between Tg5 and FVB mice 2 mo after instillation allows us to exclude the hypothesis that the reduction of the fibrosis in Tg5 mice is due to a better clearance of particles.
The effect of IL-9 on the lung fibrotic process induced by crystalline silica particles is associated with a recruitment of B lymphocytes in the lung parenchyma. A protective role of B lymphocytes in liver fibrosis has been suggested in a schistosomiasis model where mice deficient in B lymphocytes showed more fibrosis in the liver than did wild-type mice (33). The B-cell expansion induced by IL-9 is due mainly to Mac-1+ B cells, usually restricted to the peritoneal cavity and called B-1 lymphocytes. Previous studies have shown that Mac-1+ B cells express higher levels of the IL-9 receptor, and a specific expansion of B-1 cells in the peritoneal and pleurocardial cavities has been described in IL-9 transgenic mice (34). However, the function of this population remains ill-defined. The mechanisms by which B lymphocytes and in particular B-1 lymphocytes might contribute to reduce the lung fibrotic reaction to silica particles remain to be clarified. A plausible hypothesis could be that B lymphocytes produce cytokines or other factors that directly or indirectly limit fibroblast activation and/or their collagen production. However, the antifibrotic activity of IL-9 could also be completely independent from this recruitment of B-1 cells in the lungs.
Whereas little is known about the putative role of B lymphocytes in silicosis or other lung fibrosis models, several studies focused more on T lymphocytes. Velan and colleagues (35) suggested that in BALB/c mice, progression to lung fibrosis induced by silica was associated with an early influx of T lymphocytes in the lungs, whereas B lymphocytes were not detected. In an immunohistochemical study performed with the same strain of mice, an early influx of CD4-positive lymphocytes was also observed but a later accumulation of B lymphocytes was also described (36). Several investigators have tried to delineate the participation of T lymphocytes using mice deficient in mature T cells. On the one hand, Hubbard (6) reported that such mice showed less macrophage and lymphocyte inflammation than their T cell–sufficient counterparts, but equivalent degrees of pulmonary fibrosis. On the other hand, Suzuki and colleagues (7) and Callis and Lucas (37) observed increased lung inflammation but lower hydroxyproline content in the lungs from athymic mice treated with silica as compared with the wild-type animals, suggesting a possible profibrotic activity of T cells.
Contradictory reports on activities of T lymphocytes might reflect functional differences between subpopulations of T cells, such as type 1 and type 2 CD4+ T cells. Indeed, activation of the immune system is characterized by a functional dichotomy on the basis of the type of cytokines produced mainly by CD4+ T cells but also by other cells, such as macrophages. Indeed, cellular responses are generally associated with type 1 cytokines such as IL-2, IFN-γ, IL-12, and IL-18, whereas humoral immune responses usually involve type 2 cytokines such as IL-4, IL-5, IL-9, IL-10, and IL-13. Both cell types can contribute to B-cell activation but type 1 responses result, in the mouse, in the preferential production of IgG2a antibodies, whereas IgG1 is produced in the course of type 2 responses (38, 39). Several experimental and clinical studies have suggested that lung fibrotic processes are associated with a type 2 polarization (40-43), and substantially increased numbers of cells expressing IL-4 mRNA were observed in lung sections from patients with idiopathic pulmonary fibrosis (16). The possible contribution of type 2 cytokines to the fibrotic process is also strongly supported by in vitro studies demonstrating that primary cultures of fibroblasts proliferate and produce more collagen when incubated in the presence of IL-4, the prototypic type 2 cytokine (17). Other authors have found a Th-1–like response (upregulation of IFN-γ but not IL-4 transcripts) associated with the development of silicosis in C3H/HeN mice exposed by inhalation (44).
In an attempt to determine whether a particular immune profile was associated with lung fibrosis in our model, IgG subclasses (IgG1 and IgG2a) and cytokines (IL-4 and IFN-γ) were measured in BALF and lung homogenates, respectively. In wild-type mice, IL-4 levels in lung tissue were dose-dependently upregulated in response to silica, whereas IFN-γ levels were reduced in a similar manner. Immunohistochemistry confirmed the marked expression of IL-4 in the lungs of FVB mice, essentially by alveolar macrophages present both in the fibrotic areas and in alveolar spaces. Although IL-4 is usually considered a T-cell product, its production by lung alveolar macrophages has previously been reported in experimental radiation pneumonitis (13). The fact that macrophages are indeed responsible for IL-4 production in this model was confirmed by our (unpublished) observation that alveolar macrophages harvested from mice treated with silica particles produced higher in vitro amounts of IL-4 (about 100 pg/ 600,000 cells) than did cells from control animals (less than 10 pg/600,000 cells).
In Tg5 mice, the type 2 response induced by silica, based on the IgG1/IgG2a ratio and on IL-4 production, was significantly reduced, which is further consistent with an association between type 2 polarization and lung fibrosis in the present model. This observation contrasts with the promoting effect of IL-9 on allergic asthma development, because the latter disease is also known to be driven by type 2–mediated immune responses. The possible involvement of distinct cellular sources of IL-4 could, however, explain this apparent paradox. Allergic asthma results from allergen-specific activation of T cells leading to IL-4 production by Th-2 lymphocytes and downstream activation of allergic mediators. By contrast, in silica-treated mice, macrophages are the main producers of IL-4, which could both contribute to collagen production by fibroblasts and promote IgG1 production by B lymphocytes. This raises the possibility that IL-9 suppresses a type 2 response induced by macrophages but has the reverse activity where Th lymphocytes are concerned. Whether or not this effect on the immune profile observed in IL-9 transgenic mice plays a critical role in the antifibrotic activity of this cytokine needs to be addressed by further studies.
In conclusion, this study demonstrates the capacity of IL-9 to reduce the lung fibrotic process induced by crystalline silica particles in the mouse. This reduction of the fibrotic process is associated with a reduction of the type 2 immune response induced by silica and by an expansion of B lymphocytes in the lung parenchyma. These observations strongly support the hypothesis that lymphocytes and/or type 2 cytokines can play a regulating role in lung fibrotic processes.
This work was supported in part by the Belgian Federal Services for Scientific, Technical and Cultural Affairs and the Actions de Recherche Concertées, Communauté fran cşaise de Belgique—Direction de la Recherche scientifique. One author (J.-C.R.) is a research associate with the Fonds national de la Recherche scientifique, Belgium.
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Abbreviations: bronchoalveolar lavage, BAL; BAL fluid, BALF; enzyme-linked immunosorbent assay, ELISA; fluorometric automated cell sorting, FACS; fluorescein isothiocyanate, FITC; wild-type counterpart (mice), FVB (mice); interferon, IFN; immunoglobulin, Ig; interleukin, IL; lactate dehydrogenase, LDH; monoclonal antibody, mAb; phosphate-buffered saline, PBS; phycoerythrin, PE; standard error of the mean, SEM; transgenic (mice), Tg5 (mice); T helper, Th.
