This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Belperio, J. A. Articles by Keane, M. P. Search for Related Content PubMed PubMed Citation Articles by Belperio, J. A. Articles by Keane, M. P.

Interaction of IL-13 and C10 in the Pathogenesis of Bleomycin-Induced Pulmonary Fibrosis

Department of Medicine, Division of Pulmonary and Critical Care Medicine, UCLA School of Medicine, Los Angeles, California; and Division of Pulmonary and Critical Care Medicine, Yale University, New Haven, Connecticut

Address correspondence to: Dr. Michael P. Keane, UCLA, Department of Medicine, Division of Pulmonary and Critical Care Medicine, 900 Veteran Ave, 14-154 Warren Hall, Los Angeles, CA 90095-1922. E-mail: mpkeane{at}mednet.ucla.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The initial stimulus for inflammatory cell recruitment and the mechanisms responsible for the perpetuation and evolution of chronic inflammation, granulation tissue formation, and fibrosis have not been fully elucidated. Although interleukin (IL)-13, a Th2 cytokine, has been shown to have direct effects on fibroblasts that support fibroproliferation, it is also a potent inducer of a novel CC chemokine, C10, which is chemotactic for mononuclear phagocytes. The macrophage/mononuclear phagocyte has been shown to have a role in the pathogenesis of pulmonary fibrosis, serving as an important source of growth factors that regulate extracellular matrix synthesis. In this study we demonstrate that IL-13 and C10 are elevated in the pathogenesis of bleomycin-induced pulmonary fibrosis. Neutralization of IL-13, but not IL-4, attenuated bleomycin-induced pulmonary fibrosis and levels of C10, suggesting that IL-13 has an important role in the development of pulmonary fibrosis. IL-13 is a potent inducer of C10 in vivo, and neutralization of C10 attenuated bleomycin-induced pulmonary fibrosis and intrapulmonary macrophage numbers. This suggests that IL-13 has a role in the development of pulmonary fibrosis that is independent of its direct effect on fibroblasts and is evidence for an interaction between Th2 cytokines and specific CC chemokines.

Abbreviations: bronchoalveolar lavage, BAL • BAL fluid, BALF • enzyme-linked immunosorbent assay, ELISA • interferon-, IFN- • interleukin, IL • idiopathic pulmonary fibrosis, IPF • macrophage inflammatory protein, MIP • matrix metalloproteinase, MMP • phosphate-buffered saline, PBS • polymerase chain reaction, PCR • transforming growth factor, TGF • tissue inhibitor of metalloproteinase, TIMP • tumor necrosis factor, TNF

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Idiopathic pulmonary fibrosis (IPF) is a devastating disease with a median survival of 5 yr and objective response rates of less than 30% to conventional treatment. Despite these dismal response rates, the standard of care remains corticosteroids either with or without a second immunosuppressive agent (1). This treatment strategy reflects our limited understanding of the immune mechanisms and mediators involved in the pathogenesis of pulmonary fibrosis, as well as the lack of truly efficacious agents to delay the progression of the disease. Inflammatory cell recruitment is associated with initiation of the pathogenesis of interstitial lung disease (2). Similarly, bleomycin-induced pulmonary injury evokes a route and dose-dependent pulmonary inflammatory response characterized by increases in mononuclear cells, granulocytes, fibroblast proliferation, and collagen synthesis (3, 4). Although the proinflammatory cytokines, tumor necrosis factor (TNF), interleukin (IL)-6, and IL-1, have been shown to have important roles in the development of bleomycin-induced pulmonary fibrosis, only a few specific chemotactic mediators have been implicated (5–10). Animal models, such as bleomycin-induced pulmonary fibrosis, have demonstrated the presence and contribution of CC chemokines in the pathogenesis of fibrosis. Time-dependent expression of MCP-1 has been reported in response to bleomycin challenge in rodents (11, 12). Macrophage inflammatory protein (MIP)-l protein and mRNA expression in lung tissue homogenates has also been found to be elevated after bleomycin challenge (13, 14). However, depletion of MIP-1 did not completely abrogate either the inflammatory or fibrotic response to bleomycin. This suggests the existence of other mediators with similar or overlapping activities.

IL-13 is a Th2 cytokine that has been shown to have an important role in the pathogenesis of fibroproliferative disorders, asthma, and airway remodeling (15–17). C10 is a CC chemokine that has been cloned in the mouse (18–20). This novel chemokine shares significant homology with the human CC chemokine, MIP-1 (21). Both of these chemokines are chemotactic for monocytes and T cells (22). C10 is expressed in hematopoietic cells and fibroblasts and is stimulated by exposure to IL-3, IL-4, IL-13, and GM-CSF (18, 20). Furthermore, C10 is differentially regulated by Th1 and Th2 cytokines (23). C10 has been shown to be present in a variety of chronic inflammatory disorders (24–26). These findings demonstrate an important role for C10 in chronic inflammation associated with a Th2 cytokine phenotype and macrophage recruitment.

In this study we demonstrate that IL-13 and C10 are elevated in the pathogenesis of bleomycin-induced pulmonary fibrosis. Both IL-4 and IL-13 were elevated during the pathogenesis of bleomycin-induced pulmonary fibrosis. Neutralization of IL-13, but not IL-4, attenuated bleomycin-induced pulmonary fibrosis and levels of C10, suggesting that IL-13 has an important role in the development of pulmonary fibrosis. IL-13 is a potent inducer of C10 in vivo, and neutralization of C10 attenuated bleomycin-induced pulmonary fibrosis and intrapulmonary macrophage numbers. This suggests that IL-13 has a role in the development of pulmonary fibrosis that is independent of its direct effect on fibroblasts, and is evidence for an interaction between Th2 cytokines and specific CC chemokines.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents
Polyclonal neutralizing anti-C10 antibodies were purchased from R&D Systems (Minneapolis, MN). Polyclonal anti-murine IL-4 and IL-13 antibodies were produced by the immunization of rabbits with murine recombinant IL-4 and IL-13 (R&D Systems) in multiple intradermal sites with complete Freund's adjuvant. The specificity of the antibodies was assessed by Western blot analysis and enzyme-linked immunosorbent assay (ELISA) against a panel of other recombinant cytokines. The antibodies were specific in our sandwich ELISA without cross-reactivity to a panel of cytokines, including IRAP, IL-1, IL-2, IL-6, TNF-, IP-10, MIG, and other members of the CXC and CC chemokine families. The C10 antibody is a neutralizing antibody as determined by its ability to block C10 activity in a monocyte chemotaxis assay. The neutralizing capacity of the IL-4 antibody was confirmed using an in vitro assay of a CTLL-2 cell line (27). The neutralizing capacity of the IL-13 antibody was assessed using a proliferation assay with a premyeloid TF1 cell line (28). The "antiprotease" buffer for tissue homogenization consisted of 1x phosphate-buffered saline (PBS) with one Complete tablet (Boehringer Mannheim Corporation, Indianapolis, IN) per 50 ml.

Animal Model of Pulmonary Fibrosis
Female CBA/J mice (6–8 wk) were purchased from The Jackson Laboratory (Bar Harbor, ME). For the purpose of using IL-4^-/- mice an additional time course of IL-4 and C10 expression was performed in C57B6 mice (female, 6–8 wk), which were also obtained from The Jackson Laboratory. We used these strains of mice because they are both well-characterized inbred strains that are susceptible to bleomycin-induced pulmonary fibrosis. Mice were maintained in specific pathogen–free conditions and provided with food and water ad libitum. To induce pulmonary fibrosis, mice were treated with intratracheal bleomycin (Blenoxane; a gift from Bristol Myers Co., Evansville, IN) (1 U/kg) on Day 0 as previously described (29, 30). Control animals received only sterile saline as previously described (29, 30). Briefly, mice were anesthetized with 250 µl of 12.5 µg/ml ketamine injected intraperitoneally, followed by intratracheal instillation of 1 U/kg bleomycin in 25 µl sterile isotonic saline. At various time points after instillation, animals were killed, bronchoalveolar lavage (BAL) was performed, and both lungs were removed for homogenization as described below. In separate experiments, bleomycin-treated mice were passively immunized by intraperitoneal injection of anti-C10, anti–IL-13, anti–IL-4, or appropriate control antibodies on Days 0, 2, 4, and 6, before maximal fibrosis. All studies were approved by the UCLA institutional animal care and use committee.

BAL and Lung Tissue Preparation
BAL was performed as previously described (31). The trachea was exposed and intubated using a 1.7-mm OD polyethylene catheter. BAL was performed by instilling PBS containing 5 mM EDTA in 1-ml aliquots. A total of 2 ml/mouse was lavaged, retrieved, and centrifuged at 900 x g for 15 min. The cell-free supernatants were assayed by specific ELISAs. Bleomycin- or saline (control)-treated lungs were homogenized and sonicated in "anti-protease" buffer using a method as previously described (29, 30). Specimens were centrifuged at 900 x g for 15 min, filtered through 1.2-µm Sterile Acrodiscs (Gelman Sciences, Ann Arbor, MI), and frozen at -70°C until thawed for assay by specific ELISA. Additionally, some lungs were fixed in 4% paraformaldehyde and embedded in paraffin for immunohistochemical analysis.

Cytokine ELISA
Cytokines were quantitated using an ELISA as previously described (32, 33). Briefly, flat-bottomed 96-well microtiter plates (Nunc, Rosskilde, Denmark) were coated with 50 µl/well of the appropriate polyclonal antibody (1 ng/µl in 0.6 M NaCl, 0.26 M H₃BO₄, and 0.08 N NaOH, pH 9.6) for 24 h at 4°C, then washed with PBS and 0.05% Tween-20 (wash buffer). Nonspecific binding sites were blocked with 2% bovine serum albumin. Plates were rinsed and samples were added (50 µl/well), followed by incubation for 1 h at 37°C. Plates were then washed and 50 µl/well of the appropriate biotinylated polyclonal antibody (3.5 ng/µl in wash buffer, and 2% FCS) added for 45 min at 37°C. Plates were washed three times, streptavidin-peroxidase conjugate (Bio-Rad Laboratories, Richmond, CA) added, and the plates incubated for 30 min at 37°C. Chromogen substrate (Dako, Carpinteria, CA) was then added, and the plates were incubated at room temperature to the desired extinction. Plates were read at 490 nm in an automated microplate reader (Bio-Tek Instruments, Inc., Winooski, VT). Standards were 1/2 log dilutions of recombinant cytokine (50 µl/well). Sensitivity of the assay was > 50 pg/ml for each cytokine.

Western Blot Analysis
Western blot analysis was performed as previously described (29, 34). Total protein extracts were made by homogenizing lungs in TNE lysis buffer (20 mM Tris-HCl pH 8, 150 mM NaCl, 1% Nonidet P-40, 2.5 mM EDTA) supplemented with 2 ng/ml aprotinin and 35 ng/ml phenylmethyl sulfonyl fluorane. Cell extracts were incubated on ice for 30 min, followed by centrifugation at 4°C for 30 min. Supernatants were then removed and assayed for total protein content using BCA Protein assay reagents (Pierce, Rockford, IL) and comparison to known amounts of bovine serum albumin. 1 µg of total protein was loaded in each well of a 12% polyacrylamide gel, and extracts were subjected to SDS-PAGE. The separated proteins were transferred to polyvinylidene fluoride membrane (Pierce) by electrophoretic transfer overnight in Tris-glycine buffer (20 mM Tris, 150 mM glycine pH 8.0, methanol added to a final concentration of 20% [vol/vol]). Blots were blocked in 5% skim milk in TBST buffer (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.05% Tween-20) for 2 h at room temperature, followed by incubation with polyclonal goat primary antibody against IL-13 (R&D Systems) diluted 1:1,000 in blocking solution for 2 h at room temperature. Blots were washed for three 10-min washes in TBST and were incubated for 1 h at room temperature in rabbit-anti-goat HRP-conjugated secondary antibody (BioRad, Hercules, CA) at a 1:10,000 dilution. Blots were again washed for four 10-min washes in TBST, and proteins were visualized following incubation of the blots in SuperSignal chemiluminescent substrate solution according to the manufacturer's protocol (Pierce) and exposure to XAR-5 film (Kodak, Rochester NY).

Real Time Quantitative Polymerase Chain Reaction for Cytokine Gene Expression
Total lung RNA was extracted from mouse lungs using Trizol reagent (Gibco-BRL, Grand Island, NY) according to manufacturer's instructions. One microgram of total RNA was reversed transcribed into cDNA and amplified using TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, CA). To exclude amplification of genomic DNA, samples were also run in the absence of RT. Real-time quantitative polymerase chain reaction (PCR) was performed using the ABI Prism 7700 Sequence Detector and SDS analysis software (Applied Biosystems). For both IL-13 and C10 and 18S, TaqMan Pre Developed Assay Reagents (PDAR) (Applied Biosystems) were used for amplification. Reactions were assembled in 96-well reaction plates using TaqMan Universal PCR Master Mix (2x); the appropriate PDAR; and template (cDNA). PCR was performed under the following conditions: 50°C for 2 min; 95°C for 10 min; 40 cycles at 95°C for 15 s; and 60°C for 1 min. Negative controls (no template cDNA) were performed on each PCR plate. Quantitative analysis of gene expression was done using the comparative C_T (C_T) methods, in which C_T is the threshold cycle number (the minimum number of cycles needed before the product can be detected) (35). The arithmetic formula for the C_T method is described as the difference in threshold cycles for a target (i.e.; IL-13 or C10) and an endogenous reference (i.e., our housekeeping gene 18 s). The amount of target normalized to an endogenous reference (i.e., C10 or IL-13 in bleomycin-treated mice) and relative to a calibration normalized to an endogenous reference (i.e., C10 or IL-13 in saline-treated mice) is given by 2^–CT (35). The calculation of 2^–CT then gives a relative value when comparing the target to the calibrator, which we designate in this context as fold increase in bleomycin-treated mice as compared with saline-treated controls.

Immunolocalization of C10 and Macrophages
Paraffin-embedded tissue from control and bleomycin-treated lung was processed for immunohistochemical localization of C10 or macrophages (Mac-3), using a method previously described (32). Briefly, tissue sections were dewaxed with xylene and rehydrated through graded concentrations of ethanol. Tissue nonspecific binding sites were blocked using normal goat serum (BioGenex, San Ramon, CA). Tissue sections were overlaid with 1:500 dilution of either control (rabbit) or polyclonal rabbit anti-C10 antibodies (R&D Systems) or control (rat) or polyclonal rat anti Mac-3 (PharMingen, San Diego, CA) antibodies. The tissue sections were washed with TRIS-buffered saline and then incubated for 60 min with secondary goat anti-rabbit biotinylated antibodies (BioGenex) or goat anti rat biotinylated antibodies. The tissue sections were then washed in TRIS-buffered saline and incubated with alkaline phosphatase conjugated to streptavidin (BioGenex). Tissue sections were then incubated with Vectastain ABC reagent (Vector Laboratories, Burlingame, CA) followed by the peroxidase substrate, DAB reagent (Vector Laboratories). After optimal color development, tissue sections were immersed in sterile water, counterstained with Lerners hematoxylin, and cover slipped using an aqueous mounting solution.

Hydroxyproline Assay
Total lung collagen was determined by analysis of hydroxyproline as previously described (30, 36). Briefly, lungs were harvested at Day 16 (time of maximal fibrosis) after bleomycin administration and homogenized in 2 ml PBS (pH 7.4) with a Tissue Tearor (BioSpecs Products, Inc., Bartlesville, OK). One-half milliliter of each sample (both lungs) was then digested in 1 ml 6N HCL for 8 h at 120°C. Five microliters of citrate/acetate buffer (5% citric acid, 7.24% sodium acetate, 3.4% sodium hydroxide, 1.2% glacial acetic acid, pH 6.0) and 100 µl chloramine T solution (282 mg chloramine T, 2 ml n-propanol, 2 ml H2O, 16 ml citrate/acetate buffer) were added to 5 µl of sample, and the samples were left at room temperature for 20 min. Next, 100 µl Ehrlich's solution (2.5 g 4-[dimethylamino] benzaldehyde [4-DMAB]; Aldrich, Milwaukee, WI), 9.3 ml n-propanol, 3.9 ml 70% perchloric acid (Eastman Kodak, Rochester, NY) was added to each sample and the samples were incubated for 15 min at 65°C. Samples were cooled for 10 min and read at 550 nm on a Beckman DU 640 spectrophotometer (Fullerton, CA). Hydroxyproline (Sigma Immunochemicals, St. Louis, MO) concentrations from 0–10 µg/ml were used to construct a standard curve.

Assessment of Pulmonary Inflammatory Cells
Lung single-cell suspension preparations were made using a method previously described (29, 30). Briefly, lungs were harvested at Day 8 from bleomycin-treated animals that had been treated with either anti-C10 or control antibodies. Lungs were minced with scissors to a fine slurry in 15 ml digestion buffer (RPMI, 5% FCS, 1 mg/ml collagenase [Boehringer Mannheim Corporation, Indianapolis, IN], 30 µg/ml DNase [Sigma]). Lung slurry was enzymatically digested for 45 min at 37°C. Any undigested fragments were further dispersed by drawing the solution up and down through the bore of a 10-ml syringe. The total lung cell suspension was pelleted, resuspended, and spun through a 20% Percoll gradient. Cell counts and viability were determined using trypan blue exclusion on a hemocytometer. Cytospins were prepared and stained with Diff Quick (Sigma). Differential cell counts were performed on a light microscope.

Statistical Analysis
Data were analyzed on a Dell computer using the Statview 5.0 statistical package (Abacus Concepts, Inc., Berkeley, CA). Comparisons were made using the unpaired t test. Data were considered statistically significant if P values were 0.05 or less.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Lung Tissue from Mice Treated with Bleomycin Have Increased Levels of IL-13
IL-13 has been implicated in the pathogenesis of both fibroproliferative disorders and diseases such as asthma in which Th2 type cytokines predominate (15, 16, 37). Therefore, we measured IL-13 in the bleomycin-induced pulmonary fibrosis model. Total RNA was isolated from lungs of bleomycin-treated mice or saline-treated controls. Using quantitative PCR, we found that IL-13 was significantly elevated at Days 2, 4, 8, and 12 as compared with saline-treated controls (Figure 1). Furthermore, lung tissue from bleomycin-treated mice demonstrated greater levels of IL-13 protein as assessed by Western blot analysis (Figure 1).

View larger version (34K): [in this window] [in a new window]   Figure 1. (A) Time course of IL-13 mRNA as measured by real-time quantitative PCR from lung tissue of mice exposed to either intratracheal bleomycin or saline control at Day 0. Data is presented as fold increase in bleomycin-treated lung tissue as compared with saline-treated controls (n = 6 in each group). (B) Time course of C10 mRNA as measured by real-time quantitative PCR from lung tissue of mice exposed to either intratracheal bleomycin or saline control at Day 0. Data is presented as fold increase in bleomycin-treated lung tissue as compared with saline-treated controls (n = 6 in each group). (C) Time course of C10 production from BALF specimens of mice exposed to either intratracheal bleomycin (squares) or saline control (triangles) at Day 0, as measured by specific ELISA (n = 6 in each group). (D) Time course of IL-4 production in lung tissue of mice exposed to either intratracheal bleomycin (squares) or saline control (triangles) at Day 0. IL-4 was measured by specific ELISA from lung tissue and normalized to ng per lung (n = 6 in each group). (E) Western blot analysis of lung tissue IL-13 at Day 12 from mice exposed to either intratracheal bleomycin or saline control at Day 0. A quantity of 50 ng of IL-13 was loaded as a control (n = 3 in each group). *P < 0.05.

BAL Fluid and Lung Tissue from Mice Treated with Bleomycin Express Greater Levels of C10
Because both IL-13 and IL-4 are potent inducers of C10, we next assessed whether C10 was elevated during the pathogenesis of bleomycin-induced pulmonary fibrosis. We obtained lung tissue from bleomycin- and saline control–treated mice at each time point, and measured C10 by specific ELISA and real-time quantitative PCR. Lung tissue from bleomycin-treated animals demonstrated greater levels of C10 as compared with saline-treated controls at Days 2, 4, 8, 12, and 16 (P < 0.05), as measured by real-time quantitative PCR (Figure 1). Similarly, BAL fluid (BALF) from bleomycin-treated animals demonstrated greater levels of C10 as compared with saline-treated controls at Days 2, 4, 8, 12, and 16 (P < 0.05) (Figure 1). Immunolocalization demonstrated that the macrophage was the predominant cellular source of C10 (Figure 2). Furthermore, the greater levels of C10 paralleled the time of maximal pulmonary fibrosis (Day 16) as determined by total lung hydroxyproline as previously described (29). These results suggest a temporal relationship between elevated levels of IL-13 and C10 and the development of pulmonary fibrosis.

View larger version (175K): [in this window] [in a new window]   Figure 2. Representative photomicrograph of the immunolocalization of C10 and Mac-3 in lung tissue at Day 16 after intratracheal bleomycin administration (magnification: x312). (A) Saline-treated lung tissue demonstrating the absence of staining for C10. (B) Bleomycin-treated lung tissue immunostained with control Abs demonstrating the lack of nonspecific staining. (C, D) Same lung specimen immunostained for C10 and demonstrating localization to macrophages. (E, F ) Same lung specimen immunostained with control Abs and the macrophage-specific marker Mac-3, respectively.

View larger version (22K): [in this window] [in a new window]   Figure 3. (A) Lung hydroxyproline levels at Day 16 from mice administered intratracheal bleomycin (Day 0) and treated with either anti–IL-13 antibodies or control antibodies. Antibodies were administered by intraperitoneal injection at Days 0, 2, 4, and 6. Saline-treated mice received no antibody (n = 6 lungs in each group). (B) C10 levels from BALF at Day 6 from mice administered intratracheal bleomycin (Day 0) and treated with either anti–IL-13 antibodies or control antibodies. Antibodies were administered by intraperitoneal injection at Days 0, 2, 4, and 6. Saline-treated mice received no Ab (n = 6 lungs in each group). *P < 0.05.

Passive Immunization with Neutralizing C10 Antibodies Reduces Bleomycin-Induced Pulmonary Fibrosis
Based on our findings that IL-13 was playing an important role in the pathogenesis of pulmonary fibrosis and that IL-13 is a potent inducer of C10, we next assessed whether neutralization of C10 in the in vivo model of bleomycin-induced pulmonary fibrosis would attenuate the fibrotic response. Bleomycin-treated mice were passively immunized with neutralizing C10 antibodies or control antibodies on Days 0, 2, 4, and 6. Neutralization of C10 led to a reduction in pulmonary fibrosis, as compared with control antibodies as assessed by hydroxyproline levels (Figure 4). Furthermore, neutralization of C10 had no effect on IL-13 mRNA expression as assessed by quantitative PCR (Figure 4) .This supports the notion that C10 is an important chemokine in the pathogenesis of pulmonary fibrosis and supports the notion of a serial pathway, IL-13 C10 pulmonary fibrosis.

View larger version (19K): [in this window] [in a new window]   Figure 4. (A) Lung hydroxyproline levels at Day 16 from mice administered intratracheal bleomycin (Day 0) and treated with either anti-C10 antibodies or control antibodies. Antibodies were administered by intraperitoneal injection at Days 0, 2, 4, and 6. Saline-treated mice received no Ab (n = 6 lungs in each group). (B) IL-13 mRNA as measured by real-time quantitative PCR from lung tissue at Day 12 from mice administered intratracheal bleomycin (Day 0) and treated with either anti-C10 antibodies or control antibodies. Antibodies were administered by intraperitoneal injection at Days 0, 2, 4, and 6. Data is presented as fold increase in bleomycin-treated lung tissue as compared with saline-treated controls. Saline-treated mice received no Ab (n = 6 lungs in each group).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The realization that Th1 and Th2 cytokines are expressed by a variety of cells, and that the function of these cytokines are different, suggests that an imbalance in the expression of Th1 and Th2 cytokines may be important in dictating different immunopathologic responses (43, 44). Although animal models of pulmonary fibrosis have provided insight into a role for Th2 cytokines in the mediation of pulmonary fibrosis, recent studies have confirmed this profile in IPF. Although there is a pattern for the existence of both Th1 (characterized by the expression of interferon [IFN]-) and Th2 (characterized by the expression of IL-4 and IL-5) cytokines in IPF lung tissue, the presence of Th2 cytokines predominated over the expression of IFN- (45). This pattern of cytokine expression may be related to the potential role for the humoral response in the pathogenesis of IPF, or be related to the inability of IFN- to tilt the balance away from an IL-4/IL-13–dependent profibrotic environment. In further support of an imbalance of the presence of Th2 cytokines as compared with IFN- is the finding that IFN- levels are inversely related to the levels of type III pro-collagen in the BALF of IPF patients (46). These findings have been further substantiated with the recent suggestion that treatment of IPF patients who had failed to respond to glucocorticoids with IFN- may be beneficial, and suggest that the persistent imbalance in the expression of Th1 and Th2 cytokines in the lung may be a mechanism for the progression of pulmonary fibrosis (47). Similarly, both CBA/J and C57B6 mice have been shown to develop a Th2 cytokine profile in response to bleomycin (30, 34, 48, 49).

In the present study we investigated the role of the Th2 cytokines, IL-13 and IL-4, and the CC chemokine, C10, in the pathogenesis of pulmonary fibrosis. We found IL-13 to be elevated during the pathogenesis of pulmonary fibrosis, and the development of fibrosis was attenuated by the neutralization of IL-13. This is consistent with previous observations that IL-13 is a profibrotic cytokine. IL-13 induces the expression of fibroblast-derived type I and III pro-collagens in a similar magnitude as IL-4 and transforming growth factor (TGF)-ß (50). IL-13 inhibits IL-1–induced matrix metalloproteinase (MMP)-1 and MMP-3 production, and enhances tissue inhibitor of metalloproteinase (TIMP)-1 generation from fibroblasts (50). Recently, IL-13 was selectively expressed in the lungs using a Clara cell promoter (17). The phenotype of the transgenic mice expressing IL-13 demonstrated airway epithelial cell hypertrophy, mucus cell metaplasia, the hyperproduction of neutral and acidic mucus, and subepithelial airway fibrosis (17). Furthermore, IL-13 has been shown to activate TGF-ß (51). These data demonstrate both the profibrotic effects of IL-13 on collagen homeostasis and the potential differential regulation of collagen homeostasis in fibroblast subtypes by IL-13.

Because IL-13 is also a potent inducer of the CC chemokine C10, we investigated the role of C10 in our model. C10 is differentially regulated by Th1 and Th2 cytokines (23). Bone marrow–derived macrophages produce C10 in response to IL-4, IL-10, and IL-13 in a dose-dependent manner (23). In contrast, IFN- inhibits IL-3– and GM-CSF–induced expression of C10 (23). We found that C10 was significantly elevated during the pathogenesis of bleomycin-induced pulmonary fibrosis, and that neutralization of C10 led to a reduction in fibrosis as assessed by hydroxyproline levels. Neutralization of IL-13 lead to a reduction in levels of C10 in the BALF. Furthermore, transgenic mice that express IL-13 on the Clara cell promoter demonstrate elevated levels of C10 in BALF consistent with the induction of C10 by IL-13 (Jack A. Elias, unpublished observation), and elevated levels of mRNA in lung tissue (52). These observations are further support for the induction of C10 by IL-13 in vivo, and suggest that the fibrotic effects of IL-13 may be mediated, at least in part, through C10. Although the fibrotic response in the transgenic mice differs from that following bleomycin injury, this can be explained by the fact that the inflammatory response to bleomycin takes place primarily in the distal airspace/parenchyma of the lung and not the airway. Therefore, the Clara cell–driven IL-13 transgenic mouse model is more of a primary airway model, with secondary effects on the distal airspaces, whereas the bleomycin model is more of a distal airspace/parenchymal model. C10 has been shown to be present in a variety of chronic inflammatory disorders (24–26). High expression of C10 has been found in the cerebellum and spinal cord of mice with experimental autoimmune encephalitis (26). In a model of chronic peritoneal inflammation there was delayed (24 h) but sustained elevation in C10 levels in peritoneal fluid (24). These findings demonstrate an important role for C10 in chronic inflammation associated with a Th2 cytokine phenotype.

We found that IL-4 was elevated from lung tissue of bleomycin-treated mice. This is consistent with a recent study that demonstrated elevated levels of IL-4 during the pathogenesis of bleomycin-induced pulmonary fibrosis (49). Although IL-4 is also a potent inducer of C10, our findings would suggest that IL-4 is not playing an important role in either the induction of C10 or the development of fibrosis in the bleomycin model. Neutralization of IL-4 had no effect on bleomycin-induced pulmonary fibrosis. These findings were confirmed using IL-4^-/- mice. This is consistent with previous studies in which pulmonary expression of IL-4 in transgenic mice leads to little or no fibrosis, suggesting a disparity between the in vitro and in vivo effects (53). Similarly, IL-4 depletion studies and studies with IL-4^-/- mice fail to demonstrate an indispensable role for IL-4 in models of Th2-mediated inflammation and fibrosis (54–56).

Neutralization of C10 led to a reduction in intrapulmonary macrophages, suggesting that C10 may be promoting fibrosis through the recruitment of macrophages. This is consistent with the finding that intracerebroventricular injection of C10 protein promoted the recruitment of large numbers of macrophages (26). Furthermore, we found that the macrophage was the predominant cellular source of C10, suggesting the presence of an autocrine pathway that amplifies and perpetuates the inflammatory response. The macrophage/mononuclear phagocyte has been shown to have a central role in the pathogenesis of pulmonary fibrosis, serving as an important source of growth factors that regulate extracellular matrix synthesis (57). In addition to secreting profibrotic cytokines such as platelet-derived growth factor and TGF-ß, macrophages are important sources of metalloproteinases which play an important role in repair and tissue remodeling (58). The macrophage has been shown to play a key role in the transition from inflammation to repair in cutaneous wound healing (59). Diseases such as IPF are characterized by the accumulation of collagen in the interstitium and alveolar space. Studies have demonstrated that in patients with IPF there is an impairment of collagen turnover with decreased collagenolytic activity (60, 61).

In summary, we have shown that both IL-13 and C10 are important cytokines in the pathogenesis of bleomycin-induced pulmonary fibrosis. C10 appears to be promoting fibrosis through the recruitment of macrophages, which are a further source of C10, thereby amplifying the inflammatory response. Furthermore, our findings suggest that IL-13 has profibrotic actions that are independent of the known direct effects on fibroblasts and demonstrates an important co-operative interaction between Th2 cytokines and CC chemokines in the development of pulmonary fibrosis.

	Acknowledgments

 
This work was supported, in part, by National Institutes of Health grants P01HL67665, HL03906 (M.P.K), HL04493 (J.A.B.), HL61004, HL64242, HL66571, and P50HL56389 (J.A.E). M.P.K is the holder of a Dalsemer Scholar Award from the American Lung Association. J.A.B. holds a Research Award from the American Lung Association and the American Lung Association of California.

Received in original form January 22, 2002

Received in final form May 10, 2002

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. American Thoracic Society. 2000. Idiopathic pulmonary fibrosis: diagnosis and treatment. International consensus statement. Am. J. Respir. Crit. Care Med. 161:646–664.[Free Full Text]
2. Crystal, R. G., P. B. Bitterman, S. I. Rennard, A. J. Hance, and B. A. Keogh. 1984. Interstitial lung diseases of unknown cause: disorders characterized by chronic inflammation of the lower respiratory tract (first of two parts). N. Engl. J. Med. 310:154–166.[Medline]
3. Chandler, D. B. 1990. Possible mechanisms of bleomycin-induced fibrosis. Clin. Chest Med. 11:21–30.[Medline]
4. Bowden, D. H. 1984. Unraveling pulmonary fibrosis: the bleomycin model. Lab. Invest. 50:487–488.[Medline]
5. Jones, K. P., S. P. Reynolds, S. J. Capper, S. Kalinka, J. H. Edwards, and B. H. Davies. 1991. Measurement of interleukin-6 in bronchoalveolar lavage fluid by radioimmunoassay: differences between patients with interstitial lung disease and control subjects. Clin. Exp. Immunol. 83:30–34.[Medline]
6. Jordana, M., C. Richards, L. B. Irving, and J. Gauldie. 1988. Spontaneous in vitro release of alveolar-macrophage cytokines after the intratracheal instillation of bleomycin in rats: characterization and kinetic studies. Am. Rev. Respir. Dis. 137:1135–1140.[Medline]
7. Phan, S. H., and S. L. Kunkel. 1992. Lung cytokine production in bleomycin-induced pulmonary fibrosis. Exp. Lung Res. 18:29–43.[Medline]
8. Piguet, P. F., M. A. Collart, G. E. Grau, Y. Kapanci, and P. Vassalli. 1989. Tumor necrosis factor/cachectin plays a key role in bleomycin-induced pneumopathy and fibrosis. J. Exp. Med. 170:655–663.[Abstract/Free Full Text]
9. Piguet, P., C. Vesin, G. Grau, and R. C. Thompson. 1993. Interleukin 1 receptor anatgonist (IL-1ra) prevents or cures pulmonary fibrosis elicited in mice by bleomycin or silica. Cytokine 5:57–61.[Medline]
10. Suwabe, A., K. Takahashi, S. Yasui, S. Arai, and F. Sendo. 1988. Bleomycin-stimulated hamster alveolar macrophages release interleukin-1. Am. J. Pathol. 132:512–520.[Abstract]
11. Zhang, K., M. Gharaee-Kermani, M. L. Jones, J. S. Warren, and S. H. Phan. 1994. Lung monocyte chemoattractant protein-1 gene expression in bleomycin-induced pulmonary fibrosis. f. J. Immunol. 153:4733–4741.[Abstract]
12. Brieland, J. K., M. L. Jones, C. M. Flory, G. R. Miller, J. S. Warren, S. R. Phan, and J. C. Fantone. 1993. Expression of monocyte chemoattractant protein-1 (MCP- 1) by rat alveolar macrophages during chronic lung injury. Am. J. Respir. Cell Mol. Biol. 9:300–305.
13. Smith, R. E., R. M. Strieter, S. H. Phan, and S. L. Kunkel. 1996. CC chemokines: novel mediators of the profibrotic inflammatory response to bleomycin challenge. Am. J. Respir. Cell Mol. Biol. 15:693–702.[Medline]
14. Smith, R. E., R. M. Strieter, S. H. Phan, N. W. Lukacs, G. B. Huffnagle, C. A. Wilke, M. D. Burdick, P. Lincoln, H. Evanoff, and S. L. Kunkel. 1994. Production and function of murine macrophage inflammatory protein-1 alpha in bleomycin-induced lung injury. J. Immunol. 153:4704–4712.[Abstract]
15. Wills-Karp, M., J. Luyimbazi, X. Xu, B. Schofield, T. Y. Neben, C. L. Karp, and D. D. Donaldson. 1998. Interleukin-13: central mediator of allergic asthma [see comments]. Science 282:2258–2261.[Abstract/Free Full Text]
16. Grunig, G., M. Warnock, A. E. Wakil, R. Venkayya, F. Brombacher, D. M. Rennick, D. Sheppard, M. Mohrs, D. D. Donaldson, R. M. Locksley, and D. B. Corry. 1998. Requirement for IL-13 independently of IL-4 in experimental asthma [see comments]. Science 282:2261–2263.[Abstract/Free Full Text]
17. Zhu, Z., R. J. Homer, Z. Wang, Q. Chen, G. P. Geba, J. Wang, Y. Zhang, and J. A. Elias. 1999. Pulmonary expression of interleukin-13 causes inflammation, mucus hypersecretion, subepithelial fibrosis, physiologic abnormalities, and eotaxin production. J. Clin. Invest. 103:779–788.[Medline]
18. Orlofsky, A., M. S. Berger, and M. B. Prystowsky. 1991. Novel expression pattern of a new member of the MIP-1 family of cytokine-like genes. Cell Regul. 2:403–412.[Medline]
19. Berger, M. S., C. A. Kozak, A. Gabriel, and M. B. Prystowsky. 1993. The gene for C10, a member of the beta-chemokine family, is located on mouse chromosome 11 and contains a novel second exon not found in other chemokines. DNA Cell Biol. 12:839–847.[Medline]
20. Orlofsky, A., E. Y. Lin, and M. B. Prystowsky. 1994. Selective induction of the beta chemokine C10 by IL-4 in mouse macrophages. J. Immunol. 152: 5084–5091.[Abstract]
21. Wang, W., K. B. Bacon, E. R. Oldham, and T. J. Schall. 1998. Molecular cloning and functional characterization of human MIP-1 delta, a new C–C chemokine related to mouse CCF-18 and C10. J. Clin. Immunol. 18:214–222.[Medline]
22. Berger, M. S., D. D. Taub, A. Orlofsky, T. R. Kleyman, B. Coupaye-Gerard, D. Eisner, and S. A. Cohen. 1996. The chemokine C10: immunological and functional analysis of the sequence encoded by the novel second exon. Cytokine 8:439–447.[Medline]
23. Orlofsky, A., Y. Wu, and M. B. Prystowsky. 2000. Divergent regulation of the murine CC chemokine C10 by Th(1) and Th(2) cytokines. Cytokine 12:220–228.[Medline]
24. Wu, Y., M. B. Prystowsky, and A. Orlofsky. 1999. Sustained high-level production of murine chemokine C10 during chronic inflammation. Cytokine 11:523–530.[Medline]
25. Hogaboam, C. M., C. S. Gallinat, D. D. Taub, R. M. Strieter, S. L. Kunkel, and N. W. Lukacs. 1999. Immunomodulatory role of C10 chemokine in a murine model of allergic bronchopulmonary aspergillosis. J. Immunol. 162:6071–6079.[Abstract/Free Full Text]
26. Asensio, V. C., S. Lassmann, A. Pagenstecher, S. C. Steffensen, S. J. Henriksen, and I. L. Campbell. 1999. C10 is a novel chemokine expressed in experimental inflammatory demyelinating disorders that promotes recruitment of macrophages to the central nervous system. Am. J. Pathol. 154:1181–1191.[Abstract/Free Full Text]
27. Hu-Li, J., J. Ohara, C. Watson, W. Tsang, and W. E. Paul. 1989. Derivation of a T cell line that is highly responsive to IL-4 and IL-2 (CT.4R) and of an IL-2 hyporesponsive mutant of that line (CT.4S). J. Immunol. 142:800–807.[Abstract]
28. McKenzie, A. N., J. A. Culpepper, R. de Waal Malefyt, F. Briere, J. Punnonen, G. Aversa, A. Sato, W. Dang, B. G. Cocks, S. Menon, et al. 1993. Interleukin 13, a T-cell-derived cytokine that regulates human monocyte and B-cell function. Proc. Natl. Acad. Sci. USA 90:3735–3739.[Abstract/Free Full Text]
29. Keane, M. P., J. A. Belperio, T. A. Moore, B. B. Moore, D. A. Arenberg, R. E. Smith, M. D. Burdick, S. L. Kunkel, and R. M. Strieter. 1999. Neutralization of the CXC chemokine, macrophage inflammatory protein-2, attenuates bleomycin-induced pulmonary fibrosis. J. Immunol. 162:5511–5518.[Abstract/Free Full Text]
30. Keane, M. P., J. A. Belperio, D. A. Arenberg, M. D. Burdick, Z. J. Xu, Y. Y. Xue, and R. M. Strieter. 1999. IFN-gamma-inducible protein-10 attenuates bleomycin-induced pulmonary fibrosis via inhibition of angiogenesis. J. Immunol. 163:5686–5692.[Abstract/Free Full Text]
31. Laichalk, L. L., S. L. Kunkel, R. M. Strieter, J. M. Danforth, M. B. Bailie, and T. J. Standiford. 1996. Tumor necrosis factor mediates lung antibacterial host defense in murine Klebsiella pneumonia. Infect. Immun. 64:5211–5218.[Abstract]
32. Arenberg, D. A., S. L. Kunkel, P. J. Polverini, M. Glass, M. D. Burdick, and R. M. Strieter. 1996. Inhibition of interleukin-8 reduces tumorigenesis of human non-small cell lung cancer in SCID mice. J. Clin. Invest. 97:2792–2802.[Medline]
33. Arenberg, D. A., S. L. Kunkel, P. J. Polverini, S. B. Morris, M. D. Burdick, M. Glass, D. T. Taub, M. D. Iannetoni, R. I. Whyte, and R. M. Strieter. 1996. Interferon--inducible protein 10 (IP-10) is an angiostatic factor that inhibits human non-small cell lung cancer (NSCLC) tumorigenesis and spontaneous metastases. J. Exp. Med. 184:981–992.[Abstract/Free Full Text]
34. Keane, M. P., J. A. Belperio, M. D. Burdick, and R. M. Strieter. 2001. IL-12 attenuates bleomycin-induced pulmonary fibrosis. Am. J. Physiol. Lung Cell. Mol. Physiol. 281:L92–L97.[Abstract/Free Full Text]
35. Xiang, S. D., E. M. Benson, and I. S. Dunn. 2001. Tracking membrane and secretory immunoglobulin alpha heavy chain mRNA variation during B-cell differentiation by real-time quantitative polymerase chain reaction. Immunol. Cell Biol. 79:472–481.[Medline]
36. Smith, R. E., R. M. Strieter, S. H. Phan, N. W. Lukacs, G. B. Huffnagle, C. A. Wilke, M. D. Burdick, P. Lincoln, H. Evanoff, and S. L. Kunkel. 1994. Production and function of murine macrophage inflammatory protein-1a in bleomycin-induced lung injury. J. Immunol. 153:4704–4712.
37. Vogel, G. 1998. Interleukin-13's key role in asthma shown [news; comment]. Science 282:2168.
38. Vaillant, P., O. Menard, J.-M. Vignaud, N. Martinet, and Y. Martinet. 1996. The role of cytokines in human lung fibrosis. Monaldi Arch. Chest Dis. 51:145–152.[Medline]
39. Phan, S. H. 1995. New strategies for treatment of pulmonary fibrosis. Thorax 50:415–421.[Medline]
40. Panos, R. J., and T. E. King. 1991. Idiopathic pulmonary fibrosis. In I. J. P. Lynch and R. A. DeRemee, editors. Immunologically mediated pulmonary diseases. J. B. Lippincott Co., Philadelphia, PA. 1–39.
41. Gauldie, J., M. Jordana, and G. Cox. 1993. Cytokines and pulmonary fibrosis. Thorax 48:931–935.[Abstract]
42. Crystal, R. G., P. B. Bitterman, S. I. Rennard, A. J. Hance, and B. A. Keogh. 1984. Interstitial lung diseases of unknown cause: disorders characterized by chronic inflammation of the lower respiratory tract. N. Engl. J. Med. 310:154–166, 235–244.
43. Romagnani, S. 1992. Induction of Th1 and Th2 responses: a key role for the natural immune response? Immunol. Today 13:379–381.[Medline]
44. Romagnani, S. 1999. Th1/Th2 cells. Inflamm. Bowel Dis. 5:285–294.[Medline]
45. Wallace, W. A. H., E. A. Ramage, D. Lamb, and E. M. Howie. 1995. A type 2 (Th2-like) pattern of immune response predominates in the pulmonary interstitium of patients with crytogenic fibrosingalveolitis (CFA). Clin. Exp. Immunol. 101:436–441.[Medline]
46. Kuroki, S., A. Ohta, N. Sueoka, O. Katoh, H. Yamada, and M. Yamaguchi. 1995. Determination of various cytokines and type III procollagen aminopeptide levels in bronchoalveolar lavage fluid of the patients with pulmonary fibrosis: inverse correlation between type III procollagen aminopeptide and interferon- in progressive fibrosis. Br. J. Rheumatol. 34:31–36.[Abstract/Free Full Text]
47. Ziesche, R., E. Hofbauer, K. Wittmann, V. Petkov, and L. H. Block. 1999. A preliminary study of long-term treatment with interferon gamma-1b and low-dose prednisolone in patients with idiopathic pulmonary fibrosis (see comments). N. Engl. J. Med. 341:1264–1269.[Abstract/Free Full Text]
48. Moore, B. B., R. Paine III, P. J. Christensen, T. A. Moore, S. Sitterding, R. Ngan, C. A. Wilke, W. A. Kuziel, and G. B. Toews. 2001. Protection from pulmonary fibrosis in the absence of ccr2 signaling. J. Immunol. 167:4368–4377.[Abstract/Free Full Text]
49. Gharaee-Kermani, M., Y. Nozaki, K. Hatano, and S. H. Phan. 2001. Lung interleukin-4 gene expression in a murine model of bleomycin-induced pulmonary fibrosis. Cytokine 15:138–147.[Medline]
50. Oriente, A., N. S. Fedarko, S. E. Pacocha, S. K. Huang, L. M. Lichtenstein, and D. M. Essayan. 2000. Interleukin-13 modulates collagen homeostasis in human skin and keloid fibroblasts. J. Pharmacol. Exp. Ther. 292:988–994.[Abstract/Free Full Text]
51. Lee, C. G., R. J. Homer, Z. Zhu, S. Lanone, X. Wang, V. Koteliansky, J. M. Shipley, P. Gotwals, P. Noble, Q. Chen, R. M. Senior, and J. A. Elias. 2001. Interleukin-13 induces tissue fibrosis by selectively stimulating and activating transforming growth factor beta(1). J. Exp. Med. 194:809–821.[Abstract/Free Full Text]
52. Zhu, Z., B. Ma, T. Zheng, R. J. Homer, C. G. Lee, I. F. Charo, P. Noble, and J. A. Elias. 2002. IL-13-induced chemokine responses in the lung: role of CCR2 in the pathogenesis of IL-13-induced inflammation and remodeling. J. Immunol. 168:2953–2962.[Abstract/Free Full Text]
53. Rankin, J. A., D. E. Picarella, G. P. Geba, U. A. Temann, B. Prasad, B. DiCosmo, A. Tarallo, B. Stripp, J. Whitsett, and R. A. Flavell. 1996. Phenotypic and physiologic characterization of transgenic mice expressing interleukin 4 in the lung: lymphocytic and eosinophilic inflammation without airway hyperreactivity. Proc. Natl. Acad. Sci. USA 93:7821–7825.[Abstract/Free Full Text]
54. Chensue, S. W., K. Warmington, J. H. Ruth, N. Lukacs, and S. L. Kunkel. 1997. Mycobacterial and schistosomal antigen-elicited granuloma formation in IFN-gamma and IL-4 knockout mice: analysis of local and regional cytokine and chemokine networks. J. Immunol. 159:3565–3573. [Published erratum appears in J. Immunol. 162:3106][Abstract]
55. Metwali, A., D. Elliott, A. M. Blum, J. Li, M. Sandor, R. Lynch, N. Noben-Trauth, and J. V. Weinstock. 1996. The granulomatous response in murine Schistosomiasis mansoni does not switch to Th1 in IL-4-deficient C57BL/6 mice. J. Immunol. 157:4546–4553.[Abstract]
56. Pearce, E. J., A. Cheever, S. Leonard, M. Covalesky, R. Fernandez-Botran, G. Kohler, and M. Kopf. 1996. Schistosoma mansoni in IL-4-deficient mice. Int. Immunol. 8:435–444.[Abstract/Free Full Text]
57. Keane, M. P., P. M. Henson, and R. M. Strieter. 2000. Inflammation, injury, and repair. In J. F. Murray, J. A. Nadel, R. J. Mason, and H. A. Boushey, editors. Textbook of Respiratory Medicine, 3rd ed. W.B. Saunders, Philadelphia. 495–538.
58. Riches, D. W. H. 2000. Monocytes, macrophages, and dendritic cells of the lung. In J. F. Murray, J. A. Nadel, R. J. Mason, and H. A. Boushey, editors. Textbook of Respiratory Medicine, 3rd ed. WB Saunders, Philadelphia. 385–412.
59. Singer, A. J., and R. A. Clark. 1999. Cutaneous wound healing. N. Engl. J. Med. 341:738–746.[Free Full Text]
60. Selman, M., M. Montano, C. Ramos, and R. Chapela. 1986. Concentration, biosynthesis and degradation of collagen in idiopathic pulmonary fibrosis. Thorax 41:355–359.[Abstract]
61. Pardo, A., M. Selman, R. Ramirez, C. Ramos, M. Montano, G. Stricklin, and G. Raghu. 1992. Production of collagenase and tissue inhibitor of metalloproteinases by fibroblasts derived from normal and fibrotic human lungs. Chest 102:1085–1089.[Abstract/Free Full Text]

This article has been cited by other articles:

				T J Williams and J W Wilson Challenges in pulmonary fibrosis: 7 {middle dot} Novel therapies and lung transplantation Thorax, March 1, 2008; 63(3): 277 - 284. [Abstract] [Full Text] [PDF]

				T. Zheng, W. Liu, S.-Y. Oh, Z. Zhu, B. Hu, R. J. Homer, L. Cohn, M. J. Grusby, and J. A. Elias IL-13 Receptor {alpha}2 Selectively Inhibits IL-13-Induced Responses in the Murine Lung J. Immunol., January 1, 2008; 180(1): 522 - 529. [Abstract] [Full Text] [PDF]

				S. P. Chapoval, A. Al-Garawi, J. M. Lora, I. Strickland, B. Ma, P. J. Lee, R. J. Homer, S. Ghosh, A. J. Coyle, and J. A. Elias Inhibition of NF-{kappa}B Activation Reduces the Tissue Effects of Transgenic IL-13 J. Immunol., November 15, 2007; 179(10): 7030 - 7041. [Abstract] [Full Text] [PDF]

				A. L. Coelho, M. A. Schaller, C. F. Benjamim, A. Z. Orlofsky, C. M. Hogaboam, and S. L. Kunkel The Chemokine CCL6 Promotes Innate Immunity via Immune Cell Activation and Recruitment J. Immunol., October 15, 2007; 179(8): 5474 - 5482. [Abstract] [Full Text] [PDF]

				E. M. Pierce, K. Carpenter, C. Jakubzick, S. L. Kunkel, K. R. Flaherty, F. J. Martinez, and C. M. Hogaboam Therapeutic Targeting of CC Ligand 21 or CC Chemokine Receptor 7 Abrogates Pulmonary Fibrosis Induced by the Adoptive Transfer of Human Pulmonary Fibroblasts to Immunodeficient Mice Am. J. Pathol., April 1, 2007; 170(4): 1152 - 1164. [Abstract] [Full Text] [PDF]

				M. P. Keane, B. N. Gomperts, S. Weigt, Y. Y. Xue, M. D. Burdick, H. Nakamura, D. A. Zisman, A. Ardehali, R. Saggar, J. P. Lynch III, et al. IL-13 Is Pivotal in the Fibro-Obliterative Process of Bronchiolitis Obliterans Syndrome J. Immunol., January 1, 2007; 178(1): 511 - 519. [Abstract] [Full Text] [PDF]

				P. Misson, F. Brombacher, M. Delos, D. Lison, and F. Huaux Type 2 immune response associated with silicosis is not instrumental in the development of the disease Am J Physiol Lung Cell Mol Physiol, January 1, 2007; 292(1): L107 - L113. [Abstract] [Full Text] [PDF]

				Y. M. Shim, Z. Zhu, T. Zheng, C. G. Lee, R. J. Homer, B. Ma, and J. A. Elias Role of 5-Lipoxygenase in IL-13-Induced Pulmonary Inflammation and Remodeling J. Immunol., August 1, 2006; 177(3): 1918 - 1924. [Abstract] [Full Text] [PDF]

				C. G. Lee, H.-R. Kang, R. J. Homer, G. Chupp, and J. A. Elias Transgenic Modeling of Transforming Growth Factor-{beta}1: Role of Apoptosis in Fibrosis and Alveolar Remodeling. Proceedings of the ATS, July 1, 2006; 3(5): 418 - 423. [Abstract] [Full Text] [PDF]

				V. N. Lama, H. Harada, L. N. Badri, A. Flint, C. M. Hogaboam, A. McKenzie, F. J. Martinez, G. B. Toews, B. B. Moore, and D. J. Pinsky Obligatory Role for Interleukin-13 in Obstructive Lesion Development in Airway Allografts Am. J. Pathol., July 1, 2006; 169(1): 47 - 60. [Abstract] [Full Text] [PDF]

				B. Ma, W. Liu, R. J. Homer, P. J. Lee, A. J. Coyle, J. M. Lora, C. G. Lee, and J. A. Elias Role of CCR5 in the Pathogenesis of IL-13-Induced Inflammation and Remodeling. J. Immunol., April 15, 2006; 176(8): 4968 - 4978. [Abstract] [Full Text] [PDF]

				S. J. Cho, M. J. Kang, R. J. Homer, H. R. Kang, X. Zhang, P. J. Lee, J. A. Elias, and C. G. Lee Role of Early Growth Response-1 (Egr-1) in Interleukin-13-induced Inflammation and Remodeling J. Biol. Chem., March 24, 2006; 281(12): 8161 - 8168. [Abstract] [Full Text] [PDF]

				S. C. Wesselkamper, S. A. McDowell, M. Medvedovic, T. P. Dalton, H. S. Deshmukh, M. A. Sartor, L. M. Case, L. N. Henning, M. T. Borchers, C. R. Tomlinson, et al. The Role of Metallothionein in the Pathogenesis of Acute Lung Injury Am. J. Respir. Cell Mol. Biol., January 1, 2006; 34(1): 73 - 82. [Abstract] [Full Text] [PDF]

				J. H. Kim, H. Y. Kim, S. Kim, J.-H. Chung, W. S. Park, and D. H. Chung Natural Killer T (NKT) Cells Attenuate Bleomycin-Induced Pulmonary Fibrosis by Producing Interferon-{gamma} Am. J. Pathol., November 1, 2005; 167(5): 1231 - 1241. [Abstract] [Full Text] [PDF]

				J. Mestas, M. D. Burdick, K. Reckamp, A. Pantuck, R. A. Figlin, and R. M. Strieter The Role of CXCR2/CXCR2 Ligand Biological Axis in Renal Cell Carcinoma J. Immunol., October 15, 2005; 175(8): 5351 - 5357. [Abstract] [Full Text] [PDF]