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Published ahead of print on January 19, 2006, doi:10.1165/rcmb.2004-0383OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 34, pp. 573-580, 2006
© 2006 American Thoracic Society
DOI: 10.1165/rcmb.2004-0383OC

B Lymphocytes Are Critical for Lung Fibrosis Control and Prostaglandin E2 Regulation in IL-9 Transgenic Mice

Mohammed Arras, Jamila Louahed, Vincent Simoen, Virginie Barbarin, Pierre Misson, Sybille van den Brûle, Monique Delos, Laurent Knoops, Jean-Christophe Renauld, Dominique Lison and François Huaux

Unit of Industrial Toxicology and Occupational Medicine, Unit of Experimental Medicine and Ludwig Institute for Cancer Research, Faculty of Medicine, and Laboratory of Pathology, University Hospital of Mont-Godinne, Yvoir, Université catholique de Louvain, Brussels, Belgium

Correspondence and requests for reprints should be addressed to François Huaux, Ph.D., Unit of Industrial Toxicology and Occupational Medicine, Faculty of Medicine, UCL, Clos Chapelle-aux-Champs, 30.54, 1200 Brussels, Belgium. E-mail: huaux{at}toxi.ucl.ac.be


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
We previously showed that overexpression of IL-9 controls lung fibrosis induced by silica particles in mice (Arras and colleagues; Am J Respir Cell Mol Biol 2001;24:368–375). This protection was associated with an expansion of lung B lymphocytes. To explore the contribution of these cells in the protective effect of IL-9, we crossed IL-9 transgenic (IL-9+) and B-deficient (B–) mice. The antifibrotic effect of IL-9 was abolished in mice deficient in B lymphocytes (B–IL-9+) and restored by reconstituting these mice with B lymphocytes. The expression of the antifibrotic mediator prostaglandin (PG)E2 was markedly increased in the lung of IL-9+ mice at baseline, and similarly high levels were found in both wild-type and transgenic strains upon silica treatment. This PGE2 expression was completely abolished in B– mice, both at baseline and upon silica administration. In vitro, alveolar and peritoneal macrophages from IL-9+ mice had an increased capacity to produce PGE2 in response to LPS or silica. This capacity was markedly reduced in macrophages obtained from B– mice and restored by co-incubating macrophages with B lymphocytes from IL-9+ mice. The increased PGE2 response of IL-9+ macrophages was dependent on cyclooxygenase 2 expression, based on transcript analysis and inhibition by NS398. We conclude that B lymphocytes are essential for the protection against lung fibrosis and macrophage overexpression of PGE2 in IL-9 transgenic animals.

Key Words: B lymphocytes • cytokines • lipid mediators • lung • monocytes/macrophages


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Interleukin-9 (IL-9) was originally described in the mouse as a basic 32- to 39-kD single-chain, functional growth factor for a T cell clone (1), secreted from naive murine CD4+ T cells or several T cell lines (2). It has been reported that IL-9 is mainly produced by Th2 lymphocytes and exerts its effects on activated T and B lymphocytes, mast cells, epithelial cells, and hematopoietic systems (3). IL-9 was also recently shown to inhibit oxidative burst and TNF-{alpha} release by human monocytes through the production of TGF-beta and to block oxidative burst by human alveolar macrophages (4). An anti-inflammatory activity of exogenous IL-9 has been observed in a murine model of septic shock induced by Gram-negative bacteria (5). The biological activities of IL-9 have been further investigated in vivo in IL-9 transgenic mice (Tg5) that constitutively express multiple copies of this gene in all organs (6). In particular, IL-9 overexpression induces a B1 lymphocyte expansion in mice. These lymphocytes represent the main B cell population in the peritoneal and pleural cavities of these mice (7). It is now recognized that B1 lymphocytes play a role in the innate immunity. This B cell subset produces natural immunoglobulin (Ig)M and IgA antibodies, and thus provides a first line of defense against pathogens (8). However, B1-lymphocytes expanded in IL-9 transgenic mice fail to produce this type of antibody and might have distinct biological functions (9).

We have previously reported that IL-9 limits the severity of silica-induced lung fibrosis in mice (10). Consistent with the concept that type 2 immune responses promote fibrogenesis, the reduction of the fibrotic response by IL-9 was accompanied by a reduced Th2-type immune polarization (IL-4 and IgG1 lung levels) in silica-exposed Tg5 mice. IFN-{gamma} lung levels were decreased to a similar extent by silica treatment in Tg5 and wild-type mice. It remained, however, to explain how these antifibrotic effects could be induced by the overexpression of IL-9, a Th2 cytokine (11).

This protective effect of IL-9 was also accompanied by a remarkable expansion of B lymphocytes in the lungs of Tg5 mice treated with silica (10). We examine here whether these cells contribute to the antifibrotic effect of IL-9.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Mice
Tg5 mice and their wild-type counterparts (FVB) were described earlier (12), and B-deficient (B–) mice (muMT, C57BL/6 background strain) were provided by the Jackson Laboratory (Bar Harbor, ME) (13). muMT mice homozygous for the IgH-6tm1Cgn targeted mutation lack mature B cells, do not express membrane-bound IgM, and are used as model of B cell immunodeficiency. To obtain mice overexpressing IL-9 (heterozygous for the IL-9 transgene) but deficient in B lymphocytes, both strains were crossed. The F1 generation was backcrossed with muMT mice to obtain the following F2 phenotypes: mice wild-type for IL-9, competent (B+IL-9N) or deficient in B cells (B–IL-9N), and mice overexpressing IL-9 competent (B+IL-9+) or deficient in B cells (B–IL-9+). The different groups were identified on the basis of IL-9 serum levels measured by a bioassay (1) and the proficiency in B cells according to the presence of IgM in serum (by enzyme-linked immunosorbent assay). IL-9 transgenic mice express large amounts of IL-9 mRNA in all organs, including the lung, and high levels of bioactive IL-9 (0.1–2 µg/ml) are consistently detected in the serum of transgenic but never of wild-type mice (12). muMT mice have no detectable IgM in the serum (13). Female mice weighing between 20 and 25 g were used in all experiments. The animals were kept in a conventional animal facility and housed in positive-pressure air-conditioned units (25°C, 50% relative humidity) on a 12-h light/dark cycle.

Particles, Instillation, and Bronchoalveolar Lavage Methods
To allow sterilization and inactivation of any trace of endotoxin, crystalline silica particles (DQ12; median aerodynamic diameter [d50], 2.2 µm; a kind gift of Dr. Armbruster, Essen, Germany) were heated at 200°C for 2 h before suspension and administration. A 60-µl suspension of DQ12 particles (2.5 mg in sterile saline), or saline (NaCl 0.9%; controls), was injected intratracheally into the lungs. All instillations were performed on anesthetized animals (sodium pentobarbital, 2 mg/mouse, intraperitoneally) and after surgical opening of the neck. Bronchoalveolar lavage (BAL) and alveolar cell harvesting were performed as described previously (10).

Assessment of Fibrosis
Mice were killed 2 mo after instillation for the assessment of the fibrotic reaction. The right lung was removed and homogenized in PBS (3 ml/lung) with an Ultraturax (T25; Janke and Kunkel, Brussels, Belgium). Soluble collagen was measured in lung homogenates by the Sircol Collagen Assay Kit (Biocolor, Newtownabbey, Northern Ireland, UK) according to the manufacturer's instructions. Homogenates were hydrolyzed in 6N HCl overnight at 110°C and hydroxyproline content was assessed by high-performance liquid chromatography (14).

Left lungs were excised and fixed in Bouin solution (Merck-Belgolabo, Overijse, Belgium). After dewaxing and rehydration, paraffin-embedded sections were stained with hematoxylin and eosin or Masson's trichrome staining for light microscopic examination.

Passive Peritoneal Cell Transfer
Peritoneal cells from B+IL9+ F2 mice were passively transferred in B–IL-9+ F2 mice to examine the role of B cells in the fibrotic process. B+IL-9+ mice were killed by cervical dislocation, and a peritoneal lavage with saline was performed. Recovered cells (B1 cells, 30%; B2 cells, 3%; macrophages, 48%; eosinophils, 13%; T lymphocytes, 6%) were intravenously injected to B–IL-9+ mice (20.106 cells/mice). One day later, mice were intratracheally instilled with 2.5 mg of silica (DQ12).

Macrophage Cultures
For in vitro experiments, peritoneal macrophages were obtained from mice injected 3 d before with 1 ml of casein hydrolysate (6 g/100 ml saline; Sigma, St Louis, MO). After killing, the peritoneum was lavaged with 7 ml of sterile saline; the cells were washed and plated at a density of 1 x 106 macrophages/ml in DMEM supplemented with 10% fetal bovine serum (FBS) for 18 h at 37°C in a humidified incubator under 5% CO2 in air. Nonadherent cells were removed by washing twice with PBS and the cultures were stimulated with silica (50 µg/ml) or LPS (1µg/ml) for an additional 24 h. Alveolar macrophages recovered by BAL were plated at the same density during 2 h before silica or LPS stimulation. Co-cultures of macrophages with selected cell populations obtained by fluorescence-activated cell sorter (FACS) sorting (see below) were directly stimulated with silica or LPS during 24 h without preplating.

For cyclooxygenase (COX) inhibition experiments, adherent cells were pretreated with a COX-2–selective inhibitor, NS-398 (Alexis Biochemical, Carlsbad, CA) at concentrations of 0.5 or 5 µM, or indomethacin (1 µM) for 30 min before LPS stimulation.

FACS Analysis and Purification
BAL fluid red blood cells were lysed by incubation for 5 min in 0.15M 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 FAC-Scan apparatus (Becton-Dickinson, San Jose, CA). To exclude granulocytes, macrophages, and dead cells as well as silica particles, the lymphocyte population was gated according to side and forward scatters.

To purify B1 cells, peritoneal cells from mice treated with casein 3 d before were double-stained with biotinylated anti–Mac-1 (clone M1/70, ATCC) and FITC-coupled anti-B220 (clone RA3 3A1, ATCC). One hour later the cells were centrifuged at 1,200 rpm during 6 min and washed once with Hanks' medium for flow cytometry (HCF) containing 3% decomplemented FCS. Then, cells were incubated with streptavidin phycoerythrin (SAPE) (1/25; Becton-Dickinson, San Jose, CA) in HCF for 30 min at 4°C. Cells were finally washed and resuspended at a concentration of 107/ml. This preparation was filtered on a Nylon 50-µm filter and sorted by FACS (Becton-Dickinson). To purify B2 cells, the spleen was crushed in a Petri box with a syringe piston. Cells were filtered through a 70-µm cell strainer (Becton Dickinson, Franklin Lakes, NJ), double-stained as described above, and B220-positive cells isolated.

Prostaglandin E2 Immunoassay
Prostaglandin (PG)E2 levels in BAL fluid and culture supernatants were assessed by a specific enzymatic immunoassay (detection limit, 16 pg/ml; Amersham, Bucks, UK). The exact nature of the PG measured by immunoassay was confirmed by measuring a number of selected samples with high PGE2 content by gas chromatography (data not shown).

COX and PGES Expression
Peritoneal macrophages from the different strains were purified and plated as described above and adherent macrophages were stimulated with LPS (1 µg/ml). Twenty-four hours later, total RNA extraction was performed with Trizol according to the instructions of the manufacturer (Invitrogen, Carlsbad, CA). A quantity of 200 ng of RNA was reverse transcribed by Superscript Rnase H- Reverse Transcriptase (Invitrogen) with 350 pmol random hexamers (Eurogentec, Seraing, Belgium) in a final volume of 25 µl. The resulting cDNA was then diluted 20x and used as template in subsequent real-time PCR. Sequences of interest were amplified using the following forward primers: 5'-AGAGG GAAATCGTGCGTGAC-3' (mouse beta actin), 5'-AATGAGTACCG CAAACGCTTC-3' (mouse cyclooxygenase-2, COX-2), 5'-CAACGA CATGGAGACAATCTATCC-3' (mouse microsomal prostaglandin E synthetase, mPGES), 5'-CATCAAGGAGTCCCGAGAGAT-3' (mouse cyclooxygenase-1, COX-1) and reverse primers: 5'-CAATAGTGATGAC CTGGCCGT-3' (mouse beta actin), 5'-CAGCCATTTCCTTCTCTCCT GTA-3' (mouse COX-2), 5'-GGAAATGTATCCAGGCGATCA-3' (mouse mPGES), 5'-TAAGGCTTCAAGCCAAACCTC-3' (mouse COX-1).

PCR was primarily performed with Platinium Taq DNA polymerase (Invitrogen) according to manufacturer's instructions with the following temperature program: 2 min 94°C, (30 s 94°C, 30 s 55°C, 20 s 72°C) for 40 cycles, 5 min 72°C. DNA fragments were purified from a 1.5% agarose gel with Nucleospin Extract (Macherey-Nagel, Düren, Germany) and then serially diluted to serve as standards in real-time PCR. Reverse transcribed mRNAs were finally quantified by real-time PCR using SYBR Green technology on an ABI Prism 7,000 Sequence Detection System (Applied Biosystems, Foster City, CA) according to the following program: 2 min 50°C, 10 min 95°, (15 s 95°C, 1 min 60°C) for 40 cycles. Five microliters of diluted cDNA or standards were amplified with 300 nM described primers using SYBR Green PCR Master Mix (Applied Biosystems) in a total volume of 25 µl. PCR product specificity was verified by taking a dissociation curve and by agarose gel electrophoresis. Results were calculated as a ratio of COX-1, COX-2, or mPGES expression to the expression of the reference gene, beta actin.

Statistics
Treatment-related differences were evaluated using t tests and one-way ANOVA, followed by pairwise comparisons using the Student-Newman-Keuls test, as appropriate. Statistical significance was considered at P < 0.05.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
B Lymphocytes Modulate the Fibrotic Process in IL-9 Transgenic Mice
Because the antifibrotic effect of IL-9 went paired with an expansion of B cells in the lungs of Tg5 mice (10), we evaluated the role of these cells by comparing the response to silica in mice overexpressing IL-9 and genetically deficient in B lymphocytes (B–IL-9+) or not (B+IL-9+). Two months after instillation of silica particles, FACS analysis and cytospin preparations indicated a marked accumulation of B lymphocytes in the BAL of B+IL-9+ (mean ± SEM: 423 x 103 ± 83 x 103) and B+IL-9N (mean ± SEM: 221 x 103 ± 25 x 103) mice but not in B-deficient mice. No significant difference in BALF neutrophils, eosinophils, macrophages, or CD4+ and CD8+ lymphocyte numbers was observed between the B-competent and B-deficient wild-type or transgenic strains (data not shown).

Soluble collagen was measured in lung homogenates, and histologic analyses were performed 2 mo after silica instillation. In line with our previous report (10), B+IL-9+ mice developed less lung fibrosis than B+IL-9N mice. In contrast, the protective effect of IL-9 was not observed in B-deficient mice. Indeed, the fibrotic response in B–IL-9+ mice was significantly more severe than in B+IL-9+ mice (Figure 1). Interestingly, B–IL-9N mice were resistant to silica-induced lung fibrosis. Lung collagen deposition in this last strain was not increased by silica treatment compared with the corresponding B+IL-9N mice. To highlight the effect of silica on alveolar collagen deposition, the results are presented in percent of the values observed in saline-treated mice. Soluble collagen contents in saline-treated mice were as following: B+IL-9N, 79.45 ± 21.64 µg; B+IL-9+, 148 ± 29.88 µg; B–IL-9N,128 ± 35.47 µg; B–IL9+, 79.17 ± 18.64 µg/lung. Similar differences were observed by measuring lung hydroxyproline contents (data not shown).


Figure 1
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  		Figure 1. Soluble collagen content in the lung of F2 mice 2 mo after silica treatment (DQ12, 2.5 mg; shaded bars). Bars represent means ± SEM of 7–13 animals pooled from two independent experiments; bars with the same letter are not statistically different (Student-Newman-Keuls, P > 0.05). Control mice represent the animals treated with saline (open bars). IL-9: N, wild-type; +: overexpressing; B+: B-proficient; B–: B-deficient.

 
As illustrated in Figure 2, histologic studies showed that silicosis in B+IL-9N mice was characterized by the presence of numerous nodules affecting large areas of the parenchyma (Figure 2B). These silicotic lesions were characterized by well-organized foci of fibroblast with marked deposition of connective tissue matrix as well as inflammation featuring macrophages and lymphocytes (Figure 2C). In B+IL-9+ mice, fibrotic lesions were less developed and limited to smaller focal lymphocytic areas less well organized than in B+IL-9N mice (Figures 2E and 2F). Pulmonary lesions were very limited in B–IL-9N mice (Figures 2H and 2I) and were characterized by macrophage infiltration without apparent collagen accumulation. In B–IL-9+ mice, the fibrotic reaction was well established and of severity and composition similar to that in B+IL-9N animals (Figures 2K and 2L). No particular alveolar abnormality was noted between the saline-treated mice of the different strains studied (Figures 2A, 2D, 2G, and 2J). In accordance with a previous report (15), IL-9 overexpression in saline-instilled mice was associated with collagen deposition around airways and blood vessels (B+IL-9+ mice; Figure 2D, arrows).


Figure 2
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  		Figure 2. Pulmonary lesions of F2 mice 2 mo after silica treatment (2.5 mg). The panels present Masson's trichrome-stained lung sections from saline (A, D, G, and J)- or silica (B, C, E, F, H, I, K, and L)-treated mice. Representative lung sections of B+IL-9N mice are shown in A–C, B+IL-9+ mice in D–F, B–IL-9N mice in G–I, and B–IL-9+ in J–L. Arrows emphasize collagen deposition around airways and blood vessels in saline-treated B+IL-9+ animals. Original magnifications were x50 (A, D, G, and J), x100 (B, E, H, and K), or x400 (C, F, I, and L)

 
To assess the potential participation of B lymphocytes in the limitation of the lung fibrotic response in IL-9 transgenic mice, we used peritoneal cells from B+IL-9+ mice as a source of B lymphocytes. These cells comprised more than 30% of B1 lymphocytes and 3% of conventional B lymphocytes (B2). We transferred intravenously 20 x 106 of these cells into B–IL-9+ mice, 24 h before instillation of silica. To verify that the transferred B cells were functionally active, IgM levels were measured in serum. While IgM were not detected in the serum of B–IL-9+ mice treated with silica alone (< 5 ng/ml), significant IgM levels were measured after 2 mo in the serum of B–IL-9+ mice that had been injected with peritoneal cells and treated with silica (mean ± SEM, 93.2 ng/ml ± 43.4; n = 6). After 2 mo, we observed a reduced collagen deposition in the lungs of B–IL-9+ mice that had been transferred with peritoneal cells (Figure 3). Histologic studies confirmed the protective effects of B cell transfer on the fibrotic process (not shown). After 2 mo, B lymphocytes could not be detected in the lung, either by FACS analysis of BAL cells or by immunohistochemical detection on lung tissue sections, possibly reflecting the transient survival of B cells after transfer. A protective effect of peritoneal cell transfer was observed in three independent experiments with similar results.


Figure 3
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  		Figure 3. Effect of B cells transfer on soluble collagen content in the lung of F2 B–IL9+ mice 2 mo after silica treatment (20 x 106 cells intravenously followed by silica instillation 24 h later). Bars represent means ± SEM of 4–6 animals, bars with the same letter are not statistically different (Student-Newman-Keuls, P > 0.05). Open bar, B–IL9+ + saline; shaded bar, B–IL9+ + silica; black bar, B–IL9+ + silica + B cells.

 
B Lymphocytes Modulate the Production of PGE2 In Vivo
It is well accepted that lung fibrosis is related to an imbalance between the synthesis of profibrotic and antifibrotic mediators (16). We thus assessed whether the limitation of lung fibrosis in B+IL-9+ mice was associated with a concomitant decrease of the profibrotic cytokine TGF-beta1 expression. Silica particles increased BALF TGF-beta1 levels to the same extent in B+IL-9N and B+IL-9+ animals (2 mo, data not shown), ruling out a major contribution to explain the distinct response in the different strains. Since PGE2 appears as a key player in the limitation of the fibrosis process (17–19), we also examined its expression in the lungs of the different strains in response to silica. PGE2 levels in BAL fluid were markedly high in IL-9–overexpressing mice (473 ± 87 versus 7,786 ± 1,594 pg/ml at baseline and 12,797 ± 1,748 versus 17,881 ± 659 pg/ml upon silica treatment, in FVB and Tg5 mice, respectively). Similarly, in F2 B-competent mice, saline-instilled IL-9 transgenic mice showed higher PGE2 concentrations than nontransgenic animals. PGE2 concentrations were dramatically upregulated by silica in IL-9N mice, whereas silica had a nonsignificant additional effect on the constitutively high concentrations of PGE2 in IL-9+ mice (Figure 4). In mice that were deficient in B lymphocytes, low PGE2 concentrations were detected in the BAL fluid from both IL-9+ and IL-9N mice, irrespective of the administration of silica (Figure 4). We concluded that B lymphocytes are essential to upregulate PGE2 expression both in IL-9+ and IL-9N animals. Two months after silica administration, we could not detect increased PGE2 concentrations in the BAL fluid of B–IL-9+ mice that had been transferred with B cells (130 ± 64 versus 158 ± 44 pg/ml in naive and transferred mice, respectively).


Figure 4
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  		Figure 4. PGE2 levels in BAL fluid of F2 mice 2 mo after silica treatment. Bars represent means ± SEM of 4–6 animals; bars with the same letter are not statistically different (Student-Newman-Keuls, P > 0.05). IL-9: N, wild-type; +: overexpressing; B+: B-proficient; B–: B-deficient. Open bars, saline; shaded bars, silica.

 
To verify whether PGE2 overexpression in IL-9 transgenics may be implicated in the control of the fibrotic response, we treated FVB and Tg5 mice with COX inhibitors (indomethacin 2 mg/kg/d intraperitoneally or piroxicam 10 mg/kg/d intraperitoneally) starting 3 d before instillation of silica particles. After 2 mo of this regimen, the levels of PGE2 in BAL fluid were not significantly reduced and we could not demonstrate any significant effect on lung fibrosis parameters (soluble collagen, type I collagen, hydroxyproline, fibronectin), either in FVB or in Tg5 mice (data not shown). Altogether, these results indicated that IL-9 is a very potent stimulator of PGE2 expression in vivo.

B Lymphocytes Modulate the Production of PGE2 In Vitro
To further document the biological activity of IL-9 in PGE2 synthesis, we performed additional in vitro studies. Since the macrophage is an important cellular source of PGE2 and a key component of the lung response to silica particles (20, 21), we first stimulated alveolar macrophages from IL-9+ or IL-9N mice deficient or not in B cells with a conventional stimulus such as LPS, and measured their PGE2 production. LPS stimulation significantly increased PGE2 synthesis in macrophages purified from B-competent IL-9N animals in comparison with the untreated cells (Figure 5; 153 ± 51 pg/ml at baseline versus 730 ± 220 pg/ml upon LPS, P = 0.007). As shown in Figure 5, in macrophages purified from B-competent animals, a marked upregulation of PGE2 production was found in stimulated IL-9+ macrophages compared with their corresponding IL-9N cells, but not in cells from their B-deficient littermates. A similar increased capacity to produce PGE2 was found in LPS-stimulated alveolar (Figure 6A) and peritoneal macrophages (Figure 6B) from Tg5 compared with their FVB counterparts. Release of PGE2 was also significantly exacerbated in both types of macrophages exposed to silica particles (50 µg/ml) (Figures 6C and 6D). Similar effects were observed with two additional doses of silica: 25 and 75 µg/ml (data not shown). No difference in LPS- or silica-stimulated PGE2 production was, however, observed when resident instead of casein-primed peritoneal macrophages from both strains were compared (data not shown).


Figure 5
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  		Figure 5. PGE2 levels in culture supernatants of alveolar macrophages from F2 mice 24 h after LPS stimulation (1 µg/ml). Bars represent means ± SEM of three cultures; bars with the same letter are not statistically different (Student-Newman-Keuls, P > 0.05). IL-9: N, wild-type; +: overexpressing; B+: B-proficient; B–: B-deficient. Open bars, medium; filled bars, LPS.

 

Figure 6
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  		Figure 6. PGE2 levels in macrophages culture supernatants. Alveolar macrophages from FVB and Tg5 mice were stimulated 24 h with LPS (A, 1 µg/ml) or silica (C, 50 µg/ml). Peritoneal macrophages were recovered from FVB and Tg5 mice 3 d after intraperitoneal injection of casein (60 mg), plated overnight, and stimulated 24 h with LPS (B, 1 µg/ml) or silica (D, 50 µg/ml). Bars represent means ± SEM of three cultures; bars with the same letter are not statistically different (Student-Newman-Keuls, P > 0.05). A and B: open bars, medium; filled bars, LPS. C and D: open bars, medium; filled bars, silica.

 
To further study the effect of B cells on the production of PGE2 by macrophages, we examined the capacity of peritoneal macrophages to produce PGE2 when co-cultured in the presence or absence of B lymphocytes. When using macrophages obtained from B–IL-9+ mice, addition of B1 or B2 lymphocytes purified from B+IL-9+ mice restored a PGE2 production level equivalent to that measured in B-competent animals (Figure 7). B1 and B2 lymphocytes alone did not produce appreciable amounts of PGE2 (< 1 ng/106 cells).


Figure 7
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  		Figure 7. PGE2 levels in culture supernatants of peritoneal macrophages obtained from F2 B–IL-9+ co-cultured with isolated B-cells and stimulated with LPS (1 µg/ml). Bars represent means ± SEM of three to five cultures; bars with the same letter are not statistically different (Student-Newman-Keuls, P > 0.05). Open bars, medium; filled bars, LPS.

 
To explore the basis of the increased capacity to produce PGE2 in transgenics, we measured the expression of PGE2-related enzymes in macrophage cultures. At baseline, COX-1 was significantly more expressed in macrophages from Tg5 compared with FVB mice, and its expression was reduced upon LPS stimulation mainly in Tg5 cells (Figure 8A). COX2 expression was very low in unstimulated macrophages and upregulated by LPS. This increase was significantly higher in cells from Tg5 mice (Figure 8B). No significant difference in PGES expression was found between both strains (Figure 8C). Incubation of Tg5 macrophages with indomethacin (1 µM) or NS-398 (0.5–5 µM), a COX-2–selective inhibitor, completely abrogated PGE2 production in response to LPS, further confirming the role of COX-2 in the overproduction of PGE2 in this strain.


Figure 8
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  		Figure 8. COX-1 (A), COX-2 (B), and mPGES (C) expression in casein-elicited peritoneal macrophages from FVB and Tg5 mice after 6 h of LPS stimulation (1 µg/ml). Bars represent means ± SEM of three measurements; bars with the same letter are not statistically different (Student-Newman-Keuls, P > 0.05).

 
Collectively, these observations indicated that B-lymphocytes upregulate PGE2 production by IL-9 transgenic macrophages through a mechanism that is COX-2–dependent.


   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
In IL-9–overexpressing animals (Tg5), we previously observed an expansion of B lymphocytes in the blood, the peritoneum, and the pleuro-pericardial cavities (7), as well as in the lung during silicosis (10). In this article we show that B lymphocytes are involved in the protective effect of IL-9 against lung fibrosis induced by silica particles. Thus, in F2 animals obtained by crossing IL-9 transgenic (IL-9+) and B deficient (B–) mice, we reproduced the protective effect of IL-9 in B+IL-9+ but not in B–IL-9+ mice, and reconstitution with B cells restored the protection.

Exploring the mediators that might be involved in the control of the fibrotic response by IL-9 and/or B lymphocytes, we could previously exclude a role of IFN-{gamma} (10) and focused here our attention on a second antifibrotic mediator, PGE2. This eicosanoid is produced by various cell types and regulates a broad range of physiologic functions. In the immune system, PGE2 is mainly produced by macrophages and dendritic cells, and its effects are generally suppressive on Th1-related immune responses. PGE2 is produced in large quantities by macrophages in response to proinflammatory molecules such as IL-1 and LPS (21–23) and is therefore also considered as a proinflammatory mediator. In addition to its effects on inflammation, PGE2 suppresses fibroblast proliferation (24) and reduces collagen mRNA expression (25), thereby contributing to exert an antifibrotic activity. In vivo, consistent with an antifibrotic activity of PGE2, COX2 knockout mice were found more susceptible to bleomycin-induced lung fibrosis (17). We found that the alveolar macrophages of IL-9 transgenic animals produced more PGE2 than their wild-type counterparts, and it is likely that this cell type contributed significantly to the PGE2 levels measured in the BAL fluid of these animals. We cannot, however, exclude the possibility that other cell types (epithelial, fibroblasts) also contribute to this production. The very high PGE2 levels in the lung of B+IL-9+ mice provided a possible explanation for their protection against lung fibrosis. We were, however, not able to demonstrate that PGE2 mediates the reduction of the fibrotic response to silica in IL-9 transgenic mice. Why indomethacin treatment was ineffective to reduce PGE2 lung levels in the silicosis model is not immediately clear. Similar doses have been reported to inhibit the production of PGE2 in short-term treatment regimens (26, 27). We have recently shown in Tg5 mice that PGE2 levels at baseline or in response to a high dose of bleomycin was reduced by a similar short-term indomethacin treatment (28). It has, however, been suggested that after a long-term treatment with indomethacin, some resistance mechanisms take place (29). In B-deficient mice, the intensity of fibrosis induced by silica particles was not associated with modification in PGE2 levels. In this strain, the absence of B lymphocytes per se (30) and/or the contribution of other antifibrotic mediators (e.g., HGF [31]), deserves further investigations.

In peritoneal macrophages, casein-priming was necessary to reveal the increased expression of PGE2 in cells from IL-9+ animals. In contrast, upregulation of PGE2 production was observed in resident alveolar macrophages, and in BAL fluid of IL-9+ mice in the absence of experimental priming. This observation suggests that, in the lung, IL-9 modulates an early process involved in the physiologic differentiation of alveolar macrophages (induced, for example, by oxygen or surfactant components [32], or by airway antigens [33]).

The second main finding of this study is that the production of PGE2 by macrophages of IL-9 transgenics is modulated by B lymphocytes. Our in vitro studies indicate that B lymphocytes did not produce significant amounts of PGE2 but stimulated its production by macrophages. This effect was not specific to B1 lymphocytes, since it could also be obtained with B2 lymphocytes isolated from the spleen of IL-9 transgenics. While PGE2 is generally recognized to act in an inhibitory manner on immature and developing B lymphocytes (34), a modulating activity of B1 or B2 lymphocytes on PGE2 production is less well documented (35). It has been recently observed in vitro that B lymphocytes of patients with HIV stimulated monocytes to produce PGE2 (36). PGE2 is produced from arachidonic acid either through a COX-dependent pathway or via the nonenzymatic degradation of isoprostanes formed during a lipoperoxidation process (37). We show here that the overproduction of PGE2 induced by B cells in macrophages purified from IL-9 transgenic animals is mainly dependent on COX2 upregulation, which would be consistent with some proinflammatory signal acting as the intermediate mediator between B cells and macrophages. At this stage, we can only speculate about this mediator. B cells can produce a wide variety of proinflammatory (IL-1, TNF-{alpha}, TNF-beta) (38) and anti-inflammatory (IL-10) cytokines (39) and chemokines (MDC) (40) that are candidates for further investigation. Our in vitro observations also suggest that the beneficial effect of the reconstitution with B cells of silica-treated B–IL-9+ mice (Figure 3) is related to an indirect effect through, for instance, the activation of PGE2 expression by injected or resident macrophages.

Interestingly, we also found that, in B-deficient animals, IL-9 overexpression was associated with a profibrotic activity, which would be more consistent with its Th2 properties. There is indeed strong evidence that type 2 cytokines such as IL-4 and mainly IL-13 promote the development of pulmonary fibrosis (41, 42). Moreover, it has been shown that transgenic mice that selectively overexpress IL-9 in the airways showed an increased subepithelial deposition of collagen (15). A direct effect of IL-9 on fibroblasts seems very unlikely to explain this profibrotic activity because, in vitro, contrary to IL-4, IL-9 did not stimulate the proliferation of and/or collagen production by mouse lung fibroblasts (data not shown). However, it has been shown that IL-13 expression is upregulated in the lungs of IL-9 transgenic mice (43). Since this cytokine is a very potent mediator of fibrosis (42), it may mediate the effect of IL-9 on fibroblasts. This hypothesis is supported by the higher levels of IL-13 measured in the BAL fluid of silicotic B-deficient mice overexpressing IL-9 in comparison with the wild-type mice (11.3 ± 0.1 and 2.6 ± 0.5 pg/ml). Overall, the finding of this profibrotic activity of IL-9 helps to reconcile our previous observation with the Th2 properties of IL-9. The antagonistic activity of B-cells in IL-9+ provides an explanation for the apparent paradoxical activity of IL-9 in Tg5 mice (11).

In the absence of IL-9 overexpression—that is, when comparing the response in B+IL-9N and B–IL-9N mice (Figure 1)—B lymphocytes seemed to promote the fibrotic response to silica. There is very little information regarding the role of B cells in lung fibrosis and silicosis in particular. An increased number of conventional B lymphocytes (B2) has been reported in the lung of mice (44) or in lymph nodes of rats (45) after treatment with silica particles. The exact role of these B lymphocytes in the fibrotic process remains, however, unsettled and might be complicated by organ specificities. Indeed, in contrast to our finding in silicosis, others have reported that in a murine model of liver fibrosis induced by Schistosomia mansoni (46) B cells appeared to be involved in the limitation of fibrosis. In our model, it remains to understand why B cells appeared to have opposite activities in IL-9N and IL-9+ mice (Figure 1). A possible explanation is that the B cell populations involved are different or differentially activated in both strains. In particular, we can speculate that the subpopulation of B lymphocytes (i.e., B1 or B2) may possess opposite roles (i.e., protective or deleterious) during the development of lung fibrosis by expressing different sets of mediators. This hypothesis warrants further studies but, interestingly, it has been shown that B2 cells were identified as an important source of profibrotic cytokines such as IL-6 in skin fibrosis (47, 30). We have to explore in future investigations whether the resistance of B–IL-9N mice to silica could be related to the presence of a particular pathophysiologic mechanism controlling lung fibrosis extension.

Summing up, we have shown that B lymphocytes contribute to the protection against lung fibrosis induced by silica particles and the increased capacity of macrophages to produce PGE2 in mice overexpressing IL-9.


   Acknowledgments
 
The authors thank Yousof Yakoub, Johan Casters, and Monique Stevens for their excellent technical assistance.


   Footnotes
 
This work was supported in part by the Fonds de la Recherche scientifique médicale and the Actions de Recherche Concertées, Communauté française de Belgique-Direction de la Recherche scientifique. P.D.M. is Research Fellow and F.H. Postdoctoral Researcher with the Fonds National de la Recherche Scientifique (FNRS), Belgium.

Originally Published in Press as DOI: 10.1165/rcmb.2004-0383OC on January 19, 2006

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form December 2, 2004

Accepted in final form January 2, 2006


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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