AJP - Lung Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Lung Cell Mol Physiol 292: L107-L113, 2007. First published September 22, 2006; doi:10.1152/ajplung.00503.2005
1040-0605/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/1/L107    most recent
00503.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Misson, P.
Right arrow Articles by Huaux, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Misson, P.
Right arrow Articles by Huaux, F.

Type 2 immune response associated with silicosis is not instrumental in the development of the disease

Pierre Misson,1 Frank Brombacher,2 Monique Delos,3 Dominique Lison,1 and Francois Huaux1

1Unit of Industrial Toxicology and Occupational Medicine, Université catholique de Louvain, Belgium; 2Division of Immunology, Institute of Infectious Disease & Molecular Medicine, Faculty of Health Science, University of Cape Town, South Africa; and 3Laboratory of Pathology, University Hospital of Mont-Godinne, Yvoir, Belgium

Submitted 29 November 2005 ; accepted in final form 6 July 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
It has been proposed that the development of lung fibrosis is associated with a T helper type 2 response, mainly characterized by IL-4 and IL-13 production. We investigated the potential role of type 2 immune polarization in the silicotic process and examined the pulmonary response to silica particles in mice genetically deficient for IL-4. We found that IL-4–/– mice were not protected against the development of silicosis, suggesting that IL-4 is not essential for the development of this fibrotic disease. By evaluating the intensity of silica-induced lung fibrosis in mice deficient for IL-4 receptor {alpha} (IL-4R{alpha}), we showed that the establishment of pulmonary fibrosis was independent of both IL-4 and IL-13. Strong impairment of the type 2 immune response (IgG1) in the lungs of IL-4–/– and IL-4R{alpha}–/– mice did not affect the development of the disease. Measurement of IL-13{alpha}2 receptor expression and IgG2a, IL-12p70, and IFN-{gamma} levels in silica-treated IL-4–/– and IL-4R{alpha}–/– animals showed that the development of silicosis was not related to an IL-13 signaling pathway or a switch to a type 1 response in deficient animals. Our data clearly indicate that the type 2 immune response associated with silicosis in mice is not required for the development of this inflammatory and fibrotic disease.

inflammation; T helper type 2; lung fibrosis; cytokines

FIBROSIS IS A PROCESS CHARACTERIZED by an exaggerated accumulation of collagen and other extracellular matrix components, resulting in irreversible impairment of normal tissue architecture and function. Although the mechanisms leading to the development of fibrosis are still far from being fully understood, it is generally accepted that cytokines, and especially T helper type 2 (Th2) cytokines, are key mediators in the development of the fibrotic process. Several experimental and human studies have highlighted the profibrotic activities of Th2 cytokines, such as IL-4 and IL-13, as well as the association between the establishment of a fibrotic response and type 2 polarization of the immune response. This paradigm is supported by the findings of several reports on various organs, such as the lungs (37), skin (48), kidneys (50), and liver (53).

A number of experimental studies have implicated type 2 cytokines in the regulation of the fibrotic process in the lungs. Transgenic overexpression of IL-4 or IL-13 was shown to cause tissue fibrosis in mice (35, 38), suggesting strong profibrotic effects of these Th2 cytokines. Similar conclusions were drawn from in vitro experiments assessing cellular proliferation, collagen production, or myofibroblast differentiation in IL-4- or IL-13-treated pulmonary fibroblasts (9, 25, 27, 46, 55). In addition, targeting IL-4, IL-13, and/or their related receptors was accompanied by attenuation of bleomycin- and/or FITC-induced lung fibrosis in mice (16, 24, 29, 30, 36).

In experimental silicosis, both Th1 and Th2 polarization have been found to be associated with the development of the disease. We previously demonstrated that silica-induced fibrosis was correlated with markers of a Th2-like response. In mice, the development of silicosis was associated with upregulation of IL-4 levels in lung tissue and an increased IgG1/IgG2a ratio in bronchoalveolar lavage (BAL) fluid (1, 4, 23). IL-4 expression was also shown to be increased in the lungs of silica-treated rats (10). By contrast, overproduction of IFN-{gamma} by lung and thoracic lymph node T lymphocytes was reported in mice and rats developing silicosis (13, 14, 17, 18, 22). A predominance of Th1 immune response in silicosis was also evidenced by enhanced levels of serum IgG2a (51) but reduced serum IgG1 concentrations in silica-treated mice (8). However, the role of Th1 response in the fibrotic process has not been conclusively defined, since contradictory findings on the functions of IFN-{gamma} in experimental silicosis have been reported. Indeed, IFN-{gamma}–/– C57BL/6 mice exposed to silica developed lower (12) or equivalent (15) levels of lung collagen accumulation compared with wild-type mice. Furthermore, administration of recombinant IFN-{gamma} limited lung fibrosis through IL-4 and transforming growth factor-beta (TGF-beta) downregulation in a rat model (10). Together, these studies highlight the uncertainty that exists with regard to the respective roles of Th1 and Th2 immune responses in silicosis.

In this context, the present study was initiated to examine the role of type 2 immune polarization in the silicotic process by investigating the silica-induced pulmonary response in mice genetically deficient for IL-4, the prototypic Th2 cytokine.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. C57BL/6, IL-4-deficient C57BL/6 (43), and IL-4 receptor {alpha} (IL-4R{alpha})-deficient (41) mice, backcrossed to C57BL/6 for nine generations, were provided by Frank Brombacher (University of Cape Town, South Africa) and bred in our local animal facility. The mice were genotyped by PCR of tail biopsies as previously reported (31, 41). Eight- to 10-wk-old female mice were used for the study. The animals were housed in positive-pressure air-conditioned units (25°C, 50% relative humidity) on a 12-h light/dark cycles. The animal use committee at the Université Catholique de Louvain approved all experimental procedures involving mice.

Instillation method. For sterilization and inactivation of any traces of endotoxin, crystalline silica particles [DQ12, d50 (mean particle size) = 2.2 µm, kindly donated by Dr. L. Armbruster, Essen, Germany] were heated at 200°C for 2 h immediately before suspension and administration. The mice were anesthetized with a combination of Ketalar (N. V. Warner-Lambert, Zaventem, Belgium) and Rompun (Bayer, Leverkusen, Germany) (1 and 0.2 mg/mouse ip, respectively). A suspension of particles in sterile 0.9% saline was injected into the lungs via the trachea by transoral instillation (2.5 mg; 60 µl/mouse).

BAL and lung homogenates. We previously demonstrated that silica-induced inflammation in mice is acute in the first days following intratracheal instillation (3–5 days). Silica-induced lung fibrosis is progressive up to 120 days and detectable after 60 days (1, 23, 34, 39). On the basis of these data, we selected days 3 and 60 as the most relevant time points to assess lung inflammation and fibrosis, respectively. The animals were euthanized with pentobarbital sodium (20 mg/animal ip), and BAL was performed by cannulating the trachea and rising the lungs with 1 ml of sterile 0.9% NaCl. The BAL fluid was centrifuged (1,500 rpm, 10 min, 4°C), and the cell-free supernatant was used for biochemical measurements. The cell pellet was resuspended in 1 ml of sterile saline and used for total BAL cell number determination. Cell differentiation was performed on cytocentrifuge preparations fixed in methanol and stained with Diff-Quik (Baxter, Lessines, Belgium). After BAL, whole lungs were perfused with 5 ml of sterile 0.9% NaCl and then excised and transferred to 3 ml of cold PBS for hydroxyproline and soluble collagen measurements. The contents of each tube were subsequently homogenized for 30 s using an Ultra-Turax T25 homogenizer (Janke and Kunkel, Brussels, Belgium) and stored at –80°C.

Western blot analyses. Western blotting was used to detect IL-13R{alpha}2 protein expression. Twenty micrograms of protein from lung homogenates was loaded onto precast Novex (Carlsbad, CA) SDS-polyacrylamide gels (8%) and transferred electrophoretically to nitrocellulose membranes (Hybond C; Amersham Biosciences, Piscataway, NJ). The membranes were then blocked in 5% nonfat dry milk, washed, and probed with rat antibodies specific for mouse IL-13R{alpha} (R&D Systems, Oxon, UK). Blots were reprobed with anti-actin antibodies (Sigma, St. Louis, MO) as controls. A SuperSignal West Pico detection kit (Pierce, Rockford, IL) was used for detection.

Biochemical analyses. Lactate dehydrogenase (LDH) activity was assayed spectrophotometrically by monitoring the reduction in nicotinamide adenine nucleotide at 340 nm in the presence of lactate. Total proteins (TP) were determined by the pyrogallol red staining method (Technicon RA system; Bayer Diagnostics, Domont, France).

Collagen assays. Collagen deposition was evaluated by measuring the hydroxyproline and soluble collagen contents of lung homogenates. Hydroxyproline was assessed by HPLC analysis (6), and data were expressed as micrograms of hydroxyproline per lung. Soluble collagen levels were calculated by the Sircol collagen assay according to the manufacturer's protocol (Biocolor, Westbury, NY).

Histology. Animals were euthanized and perfused via the right ventricle with saline. Their lungs were inflated with 1 ml of 10% neutral buffered formalin and fixed overnight. After dehydration in 70% ethanol, the lungs were processed using standard procedures and embedded in paraffin. Sections were cut, mounted on slides, and stained with hematoxylin-eosin or Masson's trichrome.

Enzyme-linked immunosorbent assays. Mouse IgG1, IgG2a (Bethyl Laboratories, Montgomery, TX), IFN-{gamma}, and IL-12p70 (R&D Systems) concentrations in BAL fluid and lung homogenates were measured using ELISA kits following the manufacturer's instructions. The detection limits of these ELISAs were 3.9, 3.9, 5, and 8 pg/ml, respectively.

Statistics. Treatment-related differences were evaluated using t-tests or one-way analysis of variance, followed by pairwise comparisons with the Student-Newman-Keuls test, as appropriate. P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
IL-4 deficiency does not affect the intensity of silica-induced lung inflammation. Three days after intratracheal instillation, LDH activity, TP levels, and neutrophil number were assessed in the BAL fluid of IL-4+/+ and IL-4–/– animals to evaluate the influence of IL-4 deficiency on the extent of the silica-induced inflammatory reaction. Instillation of silica particles in IL-4+/+ mice led to marked pulmonary inflammation, characterized by a significant increase in LDH and protein levels (Fig. 1, A and B), as well as extensive recruitment of polymorphonuclear neutrophils (Fig. 1C), macrophages, and lymphocytes (data not shown) in the lungs. No statistically significant difference was observed for any of these inflammatory markers between IL-4+/+ and IL-4–/– mice. Similar observations were made during the chronic inflammatory stage of the disease (2 mo; data not shown). We concluded that IL-4 is not essential for silica-induced inflammation.


Figure 1
View larger version (5K):
[in this window]
[in a new window]

  		Fig. 1. IL-4 deficiency does not affect silica-induced lung inflammation. Three days after instillation, lactate dehydrogenase (LDH) (A), total proteins (B), and neutrophil numbers (C) were assessed in the bronchoalveolar lavage (BAL) fluid of saline- or silica-treated C57BL/6 IL-4+/+ and IL-4–/– mice. Bars represent means ± SE (n = 6). Significant differences (P < 0.001, not indicated in the graph) were noted between silica- and saline-treated mice, as assessed by the Student-Newman-Keuls multiple comparison test. No significant difference was observed for any of the markers between silica-treated IL-4+/+ and IL-4–/– mice. WT, wild-type.

 
IL-4 deficiency does not affect the intensity of silica-induced lung fibrosis. To determine whether IL-4 deficiency could affect the intensity of silica-induced lung fibrosis, we measured hydroxyproline and soluble collagen levels in the lung homogenates of IL-4+/+ and IL-4–/– mice 2 mo after instillation. As shown in GoGoFig. 4, both markers were significantly upregulated in silica-treated animals, clearly indicating the development of lung fibrosis at similar intensity in IL-4+/+ and IL-4–/– mice. The histological appearance of the lungs after silica treatment was also similar in both strains, and no difference was observed in the organization of the fibrotic tissue (parenchymal densification, micronodules including monocytes, polymorphonuclear neutrophils, or lymphocyte foci; Fig. 2). These results were supported by additional data obtained from Sv/129 IL-4+/+ and IL-4–/– mice (data not shown). We concluded that IL-4 is not a crucial mediator in the development of silica-induced fibrosis in mice.


Figure 2
View larger version (138K):
[in this window]
[in a new window]

  		Fig. 2. Histological examination reveals that silicosis is similar in both IL-4+/+ and IL-4–/– mice. Top: representative hematoxylin-eosin-stained lung sections from C57BL/6 IL-4+/+ (A) and IL-4–/– (C) mice 2 mo after silica administration. Original magnification x100. Bottom: representative green Masson's trichrome-stained lung sections from C57BL/6 IL-4+/+ (B) and IL-4–/– (D) mice showing silicotic nodules 2 mo after silica administration. Original magnification x400.

 

Figure 3
View larger version (7K):
[in this window]
[in a new window]

  		Fig. 3. IgG1 response is impaired in silica-treated IL-4–/– and IL-4 receptor {alpha}–/– (IL-4R{alpha}–/–) mice. Two months after instillation, IgG1 levels were assessed in the BAL fluid of saline- or silica-treated C57BL/6 IL-4+/+, IL-4–/–, and IL-4R{alpha}–/– mice. Bars represent means ± SE (n = 6). Significant differences (P < 0.001, not indicated in the graph) were noted between silica- and saline-treated IL-4+/+ mice, as assessed by the Student-Newman-Keuls multiple comparison test. ***P < 0.001 denotes a significant difference between silica-treated IL-4–/–/IL-4R{alpha}–/– and IL-4+/+ mice.

 

Figure 4
View larger version (7K):
[in this window]
[in a new window]

  		Fig. 4. IL-4R{alpha}–/– mice are not protected against the development of silicosis. Two months after instillation, hydroxyproline (A) and soluble collagen (B) were assessed in lung homogenates of saline- or silica-treated C57BL/6 IL-4+/+, IL-4–/–, and IL-4R{alpha}–/– mice. Bars represent means ± SE (n = 6). Significant differences (P < 0.001, not indicated in the graph) were noted between silica- and saline-treated mice, as assessed by the Student-Newman-Keuls multiple comparison test. No significant difference was observed for any of the markers between silica-treated IL-4+/+, IL-4–/–, or IL-4R{alpha}–/– mice.

 
IL-4R{alpha}–/– mice are not protected against the development of silicosis. IL-4 and IL-13 are both type 2 cytokines that exhibit many overlapping functions because they share a common receptor chain, IL-4R{alpha}, for signal transduction. To determine whether IL-13 could compensate for the absence of IL-4, we evaluated the development of silica-induced fibrosis in IL-4R{alpha}–/– mice. Wild-type and IL-4–/– mice were also treated concomitantly to allow adequate comparison and interpretation of the results.

To monitor the development of silica-induced type 2 immune response, we measured IgG1 concentrations in the BAL fluid of these animals 2 mo after instillation. As shown in Fig. 3, total IgG1 levels were significantly increased in silica-treated wild-type mice. This upregulation was drastically reduced in IL-4–/– animals, however, and even appeared to be completely absent in IL-4R{alpha}–/– mice, indicating considerable impairment of silica-induced type 2 immune response in both knockout strains.

We then measured hydroxyproline and soluble collagen levels in the lung homogenates of the same mice to evaluate the development of fibrosis in the absence of IL-4R{alpha}-mediated signal transduction. The results for both markers (Fig. 4) showed that, 2 mo after administration of silica particles, all three strains developed fibrosis of similar intensity. Histological analysis confirmed these observations (data not shown).

It has recently been recognized that IL-13R{alpha}2, formerly thought to function only as a decoy receptor, is essential for IL-13 signaling, TGF-beta production, and bleomycin-induced fibrosis (16). We therefore investigated whether IL-13R{alpha}2 expression was upregulated in the lungs of silica-treated mice and whether IL-13R{alpha}2 could compensate for the absence of IL-4R{alpha}. Whereas IL-13R{alpha}2 was strongly induced by silica in wild-type mice, IL-4R{alpha} deficiency was found to completely abrogate IL-13R{alpha}2 expression (Fig. 5). These data demonstrate that IL-13 did not compensate for IL-4 during the establishment of fibrosis in IL-4–/– mice.


Figure 5
View larger version (6K):
[in this window]
[in a new window]

  		Fig. 5. IL-13R{alpha}2 is not expressed in silica-treated IL-4R{alpha}–/– mice. Two months after saline or silica treatment, lungs of IL-4R{alpha}+/+ and IL-4R{alpha}–/– mice were analyzed for IL-13R{alpha}2 protein by Western blotting (see MATERIALS AND METHODS). Compared with saline, silica particles strongly induced pulmonary expression of IL-13R{alpha}2 in WT animals. By contrast, IL-13R{alpha}2 levels were not increased in silica-treated IL-4R{alpha}-deficient mice.

 
Silicosis in IL-4–/– and IL-4R{alpha}–/– mice is not associated with type 1 immune polarization. Although development of Th2 inflammation is the most widely evoked mechanism in the pathogenesis of silica-induced fibrosis, some authors have reported results disrupting this paradigm (12, 14).

To investigate whether establishment of silica-induced fibrosis in IL-4–/– and IL-4R{alpha}–/– mice may be associated with a type 1 immune response, we measured concentrations of IgG2a, IFN-{gamma}, and IL-12p70 in the BAL fluid and lung homogenates of wild-type, IL-4–/–, and IL-4R{alpha}–/– animals treated with silica. The results revealed that expression of these markers was not modified in any of the three strains examined in BAL fluid (Fig. 6) nor lung homogenates (data not shown). We concluded that silicosis development is not related to a switch to a type 1 response in the absence of IL-4- or IL-13-mediated signal transduction.


Figure 6
View larger version (8K):
[in this window]
[in a new window]

  		Fig. 6. Silicosis in IL-4–/– and IL-4R{alpha}–/– mice is not associated with type 1 immune polarization. Two months after instillation, IgG2a (A), IFN-{gamma} (B), and IL-12p70 (C) levels were assessed in the BAL fluid of saline- or silica-treated C57BL/6 IL-4+/+, IL-4–/–, and IL-4R{alpha}–/– mice. Bars represent means ± SE (n = 6). No significant difference was observed for any of the markers between either saline- or silica-treated IL-4+/+, IL-4–/–, or IL-4R{alpha}–/– mice.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study examined the role of type 2 immune polarization in the development of silica-induced lung fibrosis. The main finding of this report was that the Th2-like response associated with the development of silicosis is not essential for the establishment of the fibrotic process.

It is commonly proposed that the pathogenesis of fibroproliferative diseases is closely regulated by type 2 immune responses (37, 56). The importance of Th2 cytokines in the development of fibrotic lung diseases can be partially explained by in vitro studies, demonstrating a potent effect of IL-4 and IL-13 on the biology of key cell types involved in the pathogenesis of fibrosis. A number of studies have shown both IL-4 and IL-13 to stimulate proliferation of lung fibroblasts (24, 26) and induce synthesis of collagen (44, 46, 49) and growth factors, such as IGF-1 (55). Furthermore, recent in vitro data suggest that strong induction of FIZZ1 in alveolar type II epithelial cells by IL-4 and IL-13 constitutes another potential mechanism by which IL-4/IL-13 may play a role in the pathogenesis of lung fibrosis (36).

This hypothesis is supported by the results of several studies conducted in different animal models. Following bleomycin administration, Huaux et al. (24) reported that IL-4–/– mice developed significantly less lung fibrosis than IL-4+/+ animals. Similarly, the development of tuberculosis-associated fibrosis was reduced in infected IL-4–/– mice compared with IL-4+/+ controls (21). In line with these data, overexpression of IL-4 in the lungs of C57BL/6 mice was recently shown to be associated with fibrotic lesions, demonstrating that IL-4 exhibits strong profibrotic activity in the lungs (38).

However, conflicting observations have also been reported. Indeed, bleomycin-induced lung fibrosis was similar in both IL-4–/– and IL-4+/+ mice, and mice overexpressing IL-4 appeared to be protected from bleomycin-induced lung lesions (28). Moreover, neutralizing antibodies directed against IL-4 were unable to reduce bleomycin-induced lung fibrosis, unlike anti-IL-13 antibodies (5). Finally, FITC-induced lung fibrosis in BALB/c mice was not modified by IL-4 deficiency (30). These observations suggest that cellular and molecular events, as well as genetic factors, may contribute to the differential fibrotic effects of IL-4, as suggested by Ma et al. (38).

The role of IL-13 has also been extensively studied in experimental models of lung fibrosis. In murine models of FITC- or bleomycin-induced lung fibrosis, IL-13–/– and IL-13R{alpha}2–/– mice were shown to be significantly protected from fibrosis development (16, 30). Neutralizing antibodies against IL-13 or pulmonary administration of chimeric IL-13 immunotoxin molecule (IL-13-PE) had marked therapeutic effects on bleomycin-induced lung fibrosis (5, 29). Using a transgenic mouse model, Lee and colleagues (35) demonstrated that overexpression of IL-13 in the lungs led to the development of subepithelial and adventitial airway fibrosis, probably mediated by TGF-beta1 stimulation and activation. In murine schistosomiasis, egg-induced lung granuloma and fibrosis were found to be dependent on IL-4R{alpha} responsiveness, whereas IL-4/IL-13-stimulated alternative macrophage activation was not essential, as recently demonstrated in a macrophage/neutrophil-specific IL-4R{alpha}-deficient mouse model (20).

The Th2 paradigm is also supported by the results of several human studies, showing preferential Th2 polarization in fibrotic lung diseases. Increased levels of IL-4 and IL-13, but not IFN-{gamma}, were detected in BAL cells recovered from patients with pulmonary fibrosis, compared with healthy subjects (2, 19, 54). Flow cytometry analysis of intracellular cytokines of BAL cells also revealed a predominant Th2 profile in patients with idiopathic pulmonary fibrosis (IPF) (45). Finally, a pathogenic role has recently been suggested for IL-4 gene promoter polymorphisms in the etiology and pathogenesis of IPF (52).

Clearly, immune polarization status during the development of silica-induced lung fibrosis is still a matter for debate. Both Th1 (8, 13, 14, 17, 18, 22, 51) and Th2 responses (1, 4, 10, 22) have been found to be associated with the development of silicosis. Thus, in our study, we examined the potential role of type 2 immune polarization by investigating the functions of IL-4 and IL-13 in the fibrotic process in response to silica particles in C57BL/6 wild-type, IL-4–/–, and IL-4R{alpha}–/– mice. Upregulation of IgG1, but not IgG2a, in the BAL fluid of wild-type animals treated with silica indicated that the fibrotic process was associated with a Th2-like immune response. Two months after silica instillation, measurement of IgG1 levels in the BAL fluid of IL-4–/– and IL-4R{alpha}–/– mice showed this type 2 immune response to be strongly impaired in both strains of knockout mice. This is consistent with the results of several studies in IL-4–/– or IL-4R{alpha}–/– mice that have reported IL-4 to be the promoting factor of Th2 differentiation (31, 32, 41). IL-4-independent mechanisms have also been identified in in vivo Th2 development, however (7, 11, 40).

Evaluation of the different parameters of the silicotic response in IL-4–/– and IL-4R{alpha}–/– mice demonstrated that impairment of the silica-induced type 2 immune response did not influence the inflammatory response or intensity, or the histological characteristics of lung fibrosis in either mouse strain. Analysis of IgG2a, IL-12p70, and IFN-{gamma} levels in the BAL fluid of knockout animals proved that the development of pulmonary fibrosis in IL-4–/– and IL-4R{alpha}–/– mice could not be explained by a switch to a type 1 immune response in the absence of IL-4- or IL-13-mediated signal transduction. Other studies investigating lung fibrosis using Schistosoma mansoni eggs or gamma-herpesvirus showed that treated IL-4/IL-10- or IL-13/IL-10-deficient mice developed Th1-biased immune response (IFN-{gamma} overproduction), tissue damage, and limited fibrosis (47), whereas Th2-polarized mice (IL-12/IL-10- or IFN-{gamma}R-deficient mice) developed massive lung collagen deposition and upregulated Th2 cytokine expression (42, 47). These studies clearly demonstrate a shift between the two responses and their specific pathophysiological roles. However, these mutually exclusive responses do not occur in silica-induced lung fibrosis (Fig. 6). We propose that, whereas either a type 1 or type 2 immune response might well be induced during the different stages of silicosis, neither plays a critical role in the evolution of lung fibrosis.

During the last few years, a number of reports have been published on the involvement of CD4+ lymphocytes in the pathogenesis of silicosis (3, 12, 33). They demonstrated that treatment with anti-CD4 antibodies reduced the intensity of silica-induced lung fibrosis in mice, suggesting that this T cell population actually contributes to the establishment and/or evolution of fibrotic lesions. Our present data indicate that significant impairment of the Th2-like response does not influence the establishment of silica-induced fibrosis, which suggests that other CD4+ lymphocyte populations could be closely associated with the development of the disease. These observations are consistent with our previously published data on silicosis development, showing no clear association with alternative polarization of alveolar macrophages (M2) (39).

Our data thus demonstrate, contrary to other models of lung fibrosis, that the Th2-like immune response associated with the development of silicosis is not required for the establishment of the fibrotic process.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
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. Misson is a Research Fellow and F. Huaux is a Research Associate with the Fonds de la Recherche Scientifique, Belgium.


   ACKNOWLEDGMENTS
 
We thank Francine Uwambayinema, Yousof Yakoub, and Johan Casters for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: F. Huaux, 53.02, Ave. E. Mounier, 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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Arras M, Huaux F, Vink A, Delos M, Coutelier JP, Many MC, Barbarin V, Renauld JC, Lison D. Interleukin-9 reduces lung fibrosis and type 2 immune polarization induced by silica particles in a murine model. Am J Respir Cell Mol Biol 24: 368–375, 2001.[Abstract/Free Full Text]
  2. Atamas SP, Yurovsky VV, Wise R, Wigley FM, Robinson CJG, Henry P, Alms WJ, White B. Production of type 2 cytokines by CD8+ lung cells is associated with greater decline in pulmonary function in patients with systemic sclerosis. Arthritis Rheum 42: 1168–1178, 1999.[CrossRef][ISI][Medline]
  3. Barbarin V, Arras M, Misson P, Delos M, McGarry B, Phan SH, Lison D, Huaux F. Characterization of the effect of interleukin-10 on silica-induced lung fibrosis in mice. Am J Respir Cell Mol Biol 31: 78–85, 2004.[Abstract/Free Full Text]
  4. Barbarin V, Xing Z, Delos M, Lison D, Huaux F. Pulmonary overexpression of IL-10 augments lung fibrosis and Th2 responses induced by silica particles. Am J Physiol Lung Cell Mol Physiol 288: L841–L848, 2005.[Abstract/Free Full Text]
  5. Belperio JA, Dy M, Burdick MD, Xue YY, Li K, Elias JA, Keane MP. Interaction of IL-13 and C10 in the pathogenesis of bleomycin-induced pulmonary fibrosis. Am J Respir Cell Mol Biol 27: 419–427, 2002.[Abstract/Free Full Text]
  6. Biondi PA, Chiesa LM, Storelli MR, Renon P. A new procedure for the specific high-performance liquid chromatographic determination of hydroxyproline. J Chromatogr Sci 35: 509–512, 1997.[ISI][Medline]
  7. Brewer JM, Conacher M, Hunter CA, Mohrs M, Brombacher F, Alexander J. Aluminium hydroxide adjuvant initiates strong antigen-specific Th2 responses in the absence of IL-4- or IL-13-mediated signaling. J Immunol 163: 6448–6454, 1999.[Abstract/Free Full Text]
  8. Brown JM, Pfau JC, Holian A. Immunoglobulin and lymphocyte responses following silica exposure in New Zealand mixed mice. Inhal Toxicol 16: 133–139, 2004.[CrossRef][ISI][Medline]
  9. Buttner C, Skupin A, Rieber EP. Transcriptional activation of the type I collagen genes COL1A1 and COL1A2 in fibroblasts by interleukin-4: analysis of the functional collagen promoter sequences. J Cell Physiol 198: 248–258, 2004.[CrossRef][ISI][Medline]
  10. Chen Y, Chen J, Dong J, Liu WY. Antifibrotic effect of interferon gamma in silicosis model of rat. Toxicol Lett 155: 353–360, 2005.[CrossRef][ISI][Medline]
  11. Cunningham AF, Serre K, Toellner KM, Khan M, Alexander J, Brombacher F, MacLennan ICM. Pinpointing IL-4-independent acquisition and IL-4-influenced maintenance of Th2 activity by CD4 T cells. Eur J Immunol 34: 686–694, 2004.[CrossRef][ISI][Medline]
  12. Davis GS, Holmes CE, Pfeiffer LM, Hemenway DR. Lymphocytes, lymphokines, and silicosis. J Environ Pathol Toxicol Oncol 20, Suppl 1: 53–65, 2001.[Medline]
  13. Davis GS, Pfeiffer LM, Hemenway DR. Expansion of interferon-gamma-producing lung lymphocytes in mouse silicosis. Am J Respir Cell Mol Biol 20: 813–824, 1999.[Abstract/Free Full Text]
  14. Davis GS, Pfeiffer LM, Hemenway DR. Interferon-gamma production by specific lung lymphocyte phenotypes in silicosis in mice. Am J Respir Cell Mol Biol 22: 491–501, 2000.[Abstract/Free Full Text]
  15. Desaki M, Sugawara I, Iwakura Y, Yamamoto K, Takizawa H. Role of interferon-gamma in the development of murine bronchus-associated lymphoid tissues induced by silica in vivo. Toxicol Appl Pharmacol 185: 1–7, 2002.[CrossRef][ISI][Medline]
  16. Fichtner-Feigl S, Strober W, Kawakami K, Puri RK, Kitani A. IL-13 signaling through the IL-13 alpha 2 receptor is involved in induction of TGF-beta 1 production and fibrosis. Nat Med 12: 99–106, 2006.[CrossRef][ISI][Medline]
  17. Garn H, Friedetzky A, Davis GS, Hemenway DR, Gemsa D. T-lymphocyte activation in the enlarged thoracic lymph nodes of rats with silicosis. Am J Respir Cell Mol Biol 16: 309–316, 1997.[Abstract]
  18. Garn H, Friedetzky A, Kirchner A, Jager R, Gemsa D. Experimental silicosis: a shift to a preferential IFN-gamma-based Th1 response in thoracic lymph nodes. Am J Physiol Lung Cell Mol Physiol 278: L1221–L1230, 2000.[Abstract/Free Full Text]
  19. Hancock A, Armstrong L, Gama R, Millar A. Production of interleukin 13 by alveolar macrophages from normal and fibrotic lung. Am J Respir Cell Mol Biol 18: 60–65, 1998.[Abstract/Free Full Text]
  20. Herbert DR, Holscher C, Mohrs M, Arendse B, Schwegmann A, Radwanska M, Leeto M, Kirsch R, Hall P, Mossmann H, Claussen B, Forster I, Brombacher F. Alternative macrophage activation is essential for survival during schistosomiasis and downmodulates T helper 1 responses and immunopathology. Immunity 20: 623–635, 2004.[CrossRef][ISI][Medline]
  21. Hernandez-Pando R, Aguilar D, Hernandez ML, Orozco H, Rook G. Pulmonary tuberculosis in BALB/c mice with non-functional IL-4 genes: changes in the inflammatory effects of TNF-alpha and in the regulation of fibrosis. Eur J Immunol 34: 174–183, 2004.[CrossRef][ISI][Medline]
  22. Huaux F, Arras M, Tomasi D, Barbarin V, Delos M, Coutelier JP, Vink A, Phan SH, Renauld JC, Lison D. A profibrotic function of IL-12p40 in experimental pulmonary fibrosis. J Immunol 169: 2653–2661, 2002.[Abstract/Free Full Text]
  23. Huaux F, Lardot C, Arras M, Delos M, Many MC, Coutelier JP, Buchet JP, Renauld JC, Lison D. Lung fibrosis induced by silica particles in NMRI mice is associated with an upregulation of the p40 subunit of interleukin-12 and Th-2 manifestations. Am J Respir Crit Care Med 20: 561–572, 1999.
  24. Huaux F, Liu T, McGarry B, Ullenbruch M, Phan SH. Dual roles of IL-4 in lung injury and fibrosis. J Immunol 170: 2083–2092, 2003.[Abstract/Free Full Text]
  25. Huaux F, Liu TJ, McGarry B, Ullenbruch M, Xing Z, Phan SH. Eosinophils and T lymphocytes possess distinct roles in bleomycin-induced lung injury and fibrosis. J Immunol 171: 5470–5481, 2003.[Abstract/Free Full Text]
  26. Ingram JL, Rice A, Geisenhoffer K, Madtes DK, Bonner JC. Interleukin-13 stimulates the proliferation of lung myofibroblasts via a signal transducer and activator of transcription-6-dependent mechanism–a possible mechanism for the development of airway fibrosis in asthma. Chest 123: 422S–424S, 2003.
  27. Ingram JL, Rice AB, Geisenhoffer K, Madtes DK, Bonner JC. IL-13 and IL-1 beta promote lung fibroblast growth through coordinated up-regulation of PDGF-AA and PDGF-R alpha. FASEB J 18: 2004.
  28. Izbicki G, Or R, Christensen TG, Segel MJ, Fine A, Goldstein RH, Breuer R. Bleomycin-induced lung fibrosis in IL-4-overexpressing and knockout mice. Am J Physiol Lung Cell Mol Physiol 283: L1110–L1116, 2002.[Abstract/Free Full Text]
  29. Jakubzick C, Choi ES, Joshi BH, Keane MP, Kunkel SL, Puri RK, Hogaboam CM. Therapeutic attenuation of pulmonary fibrosis via targeting of IL-4- and IL-13-responsive cells. J Immunol 171: 2684–2693, 2003.[Abstract/Free Full Text]
  30. Kolodsick JE, Toews GB, Jakubzick C, Hogaboam C, Moore TA, McKenzie A, Wilke CA, Chrisman CJ, Moore BB. Protection from fluorescein isothiocyanate-induced fibrosis in IL-13-deficient, but not IL-4-deficient, mice results from impaired collagen synthesis by fibroblasts. J Immunol 172: 4068–4076, 2004.[Abstract/Free Full Text]
  31. Kopf M, Le Gros G, Bachmann M, Lamers MC, Bluethmann H, Kohler G. Disruption of the murine IL-4 gene blocks Th2 cytokine responses. Nature 362: 245–248, 1993.[CrossRef][Medline]
  32. Kuhn R, Rajewsky K, Muller W. Generation and analysis of interleukin-4 deficient mice. Science 254: 707–710, 1991.[Abstract/Free Full Text]
  33. Kumar RK. Quantitative immunohistologic assessment of lymphocyte populations in the pulmonary inflammatory response to intratracheal silica. Am J Pathol 135: 605–614, 1989.[Abstract]
  34. Lardot C, Huaux FA, Broeckaert FR, Declerck PJ, Delos M, Fubini B, Lison DF. Role of urokinase in the fibrogenic response of the lung to mineral particles. Am J Respir Crit Care Med 157: 617–628, 1998.
  35. Lee CG, Homer RJ, Zhu Z, Lanone S, Wang X, Koteliansky V, Shipley JM, Gotwals P, Noble P, Chen Q, Senior RM, Elias JA. Interleukin-13 induces tissue fibrosis by selectively stimulating and activating transforming growth factor beta(1). J Exp Med 194: 809–821, 2001.[Abstract/Free Full Text]
  36. Liu TJ, Jin H, Ullenbruch M, Hu B, Hashimoto N, Moore B, McKenzie A, Lukacs NW, Phan SH. Regulation of found in inflammatory zone 1 expression in bleomycin-induced lung fibrosis: role of IL-4/IL-13 and mediation via STAT-6. J Immunol 173: 3425–3431, 2004.[Abstract/Free Full Text]
  37. Lukacs NW, Hogaboam C, Chensue SW, Blease K, Kunkel SL. Type 1/type 2 cytokine paradigm and the progression of pulmonary fibrosis. Chest 120: 5S–8S, 2001.
  38. Ma B, Blackburn MR, Lee CG, Homer RJ, Liu W, Flavell RA, Boyden L, Lifton RP, Sun CX, Young HW, Elias JA. Adenosine metabolism and murine strain-specific IL-4-induced inflammation, emphysema, and fibrosis. J Clin Invest 116: 1274–1283, 2006.[CrossRef][ISI][Medline]
  39. Misson P, van den Brule S, Barbarin V, Lison D, Huaux F. Markers of macrophage differentiation in experimental silicosis. J Leukoc Biol 76: 926–932, 2004.[Abstract/Free Full Text]
  40. Mohrs M, Holscher C, Brombacher F. Interleukin-4 receptor alpha-deficient BALB/c mice show an unimpaired T helper 2 polarization in response to Leishmania major infection. Infect Immun 68: 1773–1780, 2000.[Abstract/Free Full Text]
  41. Mohrs M, Ledermann B, Kohler G, Dorfmuller A, Gessner A, Brombacher F. Differences between IL-4- and IL-4 receptor alpha-deficient mice in chronic leishmaniasis reveal a protective role in IL-13 receptor signaling. J Immunol 162: 7302–7308, 1999.[Abstract/Free Full Text]
  42. Mora AL, Woods CR, Garcia A, Xu JG, Rojas M, Speck SH, Roman J, Brigham KL, Stecenko AA. Lung infection with gamma-herpesvirus induces progressive pulmonary fibrosis in Th2-biased mice. Am J Physiol Lung Cell Mol Physiol 289: L711–L721, 2005.[Abstract/Free Full Text]
  43. Noben-Trauth N, Sacks D, Paul WE. IL-4 and IL-4 receptor deficient mice reveal parasite strain differences in the ability to control Leishmania major infection (Abstract). FASEB J 12: A1084, 1998.
  44. Postlethwaite AE, Holness MA, Katai H, Raghow R. Human fibroblasts synthesize elevated levels of extracellular matrix proteins in response to interleukin 4. J Clin Invest 90: 1479–1485, 1992.[ISI][Medline]
  45. Rottoli P, Magi B, Perari MG, Liberatori S, Nikiforakis N, Bargagli E, Cianti R, Bini L, Pallini V. Cytokine profile and proteome analysis in bronchoalveolar lavage of patients with sarcoidosis, pulmonary fibrosis associated with systemic sclerosis and idiopathic pulmonary fibrosis. Proteomics 5: 1423–1430, 2005.[CrossRef][ISI][Medline]
  46. Saito A, Okazaki H, Sugawara I, Yamamoto K, Takizawa H. Potential action of IL-4 and IL-13 as fibrogenic factors on lung fibroblasts in vitro. Int Arch Allergy Immunol 132: 168–176, 2003.[CrossRef][ISI][Medline]
  47. Sandler NG, Mentink-Kane MM, Cheever AW, Wynn TA. Global gene expression profiles during acute pathogen-induced pulmonary inflammation reveal divergent roles for Th1 and Th2 responses in tissue repair. J Immunol 171: 3655–3667, 2003.[Abstract/Free Full Text]
  48. Scaletti C, Vultaggio A, Maggi E, Romagnani S, Piccinni MP. Microchimerism and systemic sclerosis. Int Arch Allergy Immunol 125: 196–202, 2001.[CrossRef][ISI][Medline]
  49. Sempowski GD, Derdak S, Phipps RP. Interleukin-4 and interferon-gamma discordantly regulate collagen biosynthesis by functionally distinct lung fibroblast subsets. J Cell Physiol 167: 290–296, 1996.[CrossRef][ISI][Medline]
  50. Tipping PG, Holdsworth SR. T cells in glomerulonephritis. Springer Semin Immunopathol 24: 377–393, 2003.[CrossRef][ISI][Medline]
  51. van Zijverden M, van der Pijl A, Bol M, van Pinxteren FA, de Haar C, Penninks AH, van Loveren H, Pieters R. Diesel exhaust, carbon black, and silica particles display distinct Th1/Th2 modulating activity. Toxicol Appl Pharmacol 168: 131–139, 2000.[CrossRef][ISI][Medline]
  52. Vasakova M, Striz I, Slavcev A, Jandova S, Kolesar L, Sulc J. Th1/Th2 cytokine gene polymorphisms in patients with idiopathic pulmonary fibrosis. Tissue Antigens 67: 229–232, 2006.[CrossRef][ISI][Medline]
  53. Wahl SM, FrazierJessen M, Jin WW, Kopp JB, Sher A, Cheever AW. Cytokine regulation of schistosome-induced granuloma and fibrosis. Kidney Int 51: 1370–1375, 1997.[ISI][Medline]
  54. Wallace WA, Ramage EA, Lamb D, Howie SE. A type 2 (Th2-like) pattern of immune response predominates in the pulmonary interstitium of patients with cryptogenic fibrosing alveolitis (CFA). Clin Exp Immunol 101: 436–441, 1995.[ISI][Medline]
  55. Wynes MW, Frankel SK, Riches DWH. IL-4-induced macrophage-derived IGF-I protects myofibroblasts from apoptosis following growth factor withdrawal. J Leukoc Biol 76: 1019–1027, 2004.[Abstract/Free Full Text]
  56. Wynn TA. Fibrotic disease and the T(H)1/T(H)2 paradigm. Nat Rev Immunol 4: 583–594, 2004.[CrossRef][ISI][Medline]



This article has been cited by other articles:


Home page
 Am. J. Respir. Crit. Care Med.Home page
T. R. McMillan, B. B. Moore, J. B. Weinberg, K. M. Vannella, W. B. Fields, P. J. Christensen, L. F. van Dyk, and G. B. Toews
Exacerbation of Established Pulmonary Fibrosis in a Murine Model by Gammaherpesvirus
Am. J. Respir. Crit. Care Med., April 1, 2008; 177(7): 771 - 780.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
C. T. Migliaccio, M. C. Buford, F. Jessop, and A. Holian
The IL-4R{alpha} pathway in macrophages and its potential role in silica-induced pulmonary fibrosis
J. Leukoc. Biol., March 1, 2008; 83(3): 630 - 639.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
B. B. Moore and C. M. Hogaboam
Murine models of pulmonary fibrosis
Am J Physiol Lung Cell Mol Physiol, February 1, 2008; 294(2): L152 - L160.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/1/L107    most recent
00503.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Misson, P.
Right arrow Articles by Huaux, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Misson, P.
Right arrow Articles by Huaux, F.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.