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IL-13-Induced Chemokine Responses in the Lung: Role of CCR2 in the Pathogenesis of IL-13-Induced Inflammation and Remodeling¹

Zhou Zhu^*, Bing Ma^*, Tao Zheng^*, Robert J. Homer^,, Chun Geun Lee^*, Israel F. Charo, Paul Noble^* and Jack A. Elias^2,*

^* Section of Pulmonary and Critical Care Medicine, Departments of Internal Medicine and Pathology, Yale University School of Medicine, New Haven, CT 06520; Pathology and Laboratory Medicine Service, Veterans Affairs-Connecticut Health Care System, West Haven, CT 06516; and San Francisco General Hospital, Gladstone Institution of Cardiovascular Division, University of California, San Francisco, CA 94143

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
IL-13 stimulates inflammatory and remodeling responses and contributes to the pathogenesis of human airways disorders. To further understand the cellular and molecular events that mediate these responses, we characterized the effects of IL-13 on monocyte chemotactic proteins (MCPs) and compared the tissue effects of transgenic IL-13 in mice with wild-type (+/+) and null (-/-) CCR2 loci. Transgenic IL-13 was a potent stimulator of MCP-1, -2, -3, and -5. This stimulation was not specific for MCPs because macrophage-inflammatory protein (MIP)-1, MIP-1, MIP-2, MIP-3, thymus- and activation-regulated chemokine, thymus-expressed chemokine, eotaxin, eotaxin 2, macrophage-derived chemokines, and C10 were also induced. The ability of IL-13 to increase lung size, alveolar size, and lung compliance, to stimulate pulmonary inflammation, hyaluronic acid accumulation, and tissue fibrosis, and to cause respiratory failure and death were markedly decreased, whereas mucus metaplasia was not altered in CCR2^-/- mice. CCR2 deficiency did not decrease the basal or IL-13-stimulated expression of target matrix metalloproteinases or cathepsins but did increase the levels of mRNA encoding 1-antitrypsin, tissue inhibitor of metalloproteinase-1, -2, and -4, and secretory leukocyte proteinase inhibitor. In addition, the levels of bioactive and total TGF-₁ were decreased in lavage fluids from IL-13 transgenic mice with -/- CCR2 loci. These studies demonstrate that IL-13 is a potent stimulator of MCPs and other CC chemokines and document the importance of MCP-CCR2 signaling in the pathogenesis of the IL-13-induced pulmonary phenotype.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Inflammatory and remodeling responses are prominent features of disorders of the airway and parenchyma of the lung. These responses are readily apparent in chronic obstructive pulmonary disease (COPD),³ which is characterized by inflammation with increased numbers of CD8⁺ lymphocytes, macrophages, eosinophils, and granulocytes (1, 2, 3, 4), and a remodeling response that causes the alveolar septal destruction and changes in compliance that are characteristic of pulmonary emphysema (1). Asthma is characterized by chronic tissue inflammation with increased numbers of eosinophils, macrophages, and lymphocytes, which are believed to cause the subepithelial fibrosis, mucus metaplasia, and myocyte hyperplasia that are prominent features of the remodeled asthmatic airway (5, 6). Surprisingly, the mechanisms that are responsible for the generation of each of these abnormalities have not been adequately defined, and the relationship(s) between the inflammatory and remodeling responses in these tissues have not been elucidated.

IL-13 is a pleiotropic 12-kDa product of a gene at chromosome 5 q31 that is produced in large quantities by Th2 cells. A large number of studies have demonstrated that IL-13 is overproduced in asthma and have implicated IL-13 in the pathogenesis of the Th2 inflammation and airway remodeling that are characteristic of this disorder (7, 8, 9). The asthma-relevant effector functions of IL-13 can be appreciated in studies from our laboratory that demonstrated the lung-specific constitutive overexpression of IL-13 produced eosinophil-, lymphocyte-, and macrophage-rich inflammation, airway remodeling with subepithelial fibrosis, mucus metaplasia, and airways hyperresponsiveness on methacholine challenge (10). It has long been speculated that asthma and COPD may not be absolutely distinct entities and that similar mechanisms may contribute to the pathogenesis of both diseases (11). Support for this hypothesis and for the contention that IL-13 plays an important role in the pathogenesis of COPD comes from studies that described the emphysema-like decrease in lung elastic recoil in asthma (12) and from our studies that demonstrated that the inducible overexpression of IL-13 in the adult murine lung generated COPD-like inflammation, alveolar enlargement, lung enlargement, enhanced compliance, and mucus metaplasia (13). IL-13 also plays an important role in the pathogenesis of a variety of other disorders including respiratory syncytial virus infection (14), hepatic fibrosis (15), and fungal pneumonitis (16). Surprisingly, the mechanisms that are responsible for IL-13-induced inflammatory, proteolytic, and fibrotic tissue responses are poorly understood. In particular, the cellular and molecular events that allow IL-13 to recruit and activate leukocytes and the relationship(s) between these inflammatory cells and subsequent tissue remodeling have not been defined.

A coordinated network of chemokines and chemokine receptors plays a key role in the generation of the complex pathology and physiology of asthma and other inflammatory pulmonary disorders (16, 17, 18, 19, 20, 21). Monocyte chemotactic protein (MCP) family chemokines are believed to play particularly important roles in these disorders. This is based on the demonstration that MCP-1 is a potent stimulator of mast cell mediator release, T cell chemotaxis, basophil chemotaxis, and tissue fibrosis, which also enhances naive T cell acquisition of a Th2 cell phenotype (19, 22). It is also based on the demonstration that MCP-1, acting via its major receptor, CCR2, is the major recruiter of macrophages at sites of allergic tissue inflammation (23) and that MCP-1, MCP-3, and MCP-4 are expressed in a exaggerated fashion in tissues from patients with asthma (20, 21, 24, 25). Previous studies from our laboratory demonstrated that IL-13 is a potent stimulator of eotaxin production in vivo (10). MCP-3, macrophage-derived chemokine (MDC), and C10 have also been demonstrated to be induced in an IL-13-dependent fashion in vivo, and IL-13 stimulates MCP-1, MDC, and eotaxin production in vitro (14, 26, 27). Surprisingly, little else is known about the chemokine responses in IL-13-stimulated tissues, and virtually nothing is known about the roles that individual chemokines and their receptors play in the pathogenesis of the inflammatory and remodeling responses induced by IL-13.

We hypothesized that IL-13 is a potent stimulator of CC chemokines in vivo and that the signaling of specific populations of these chemokines plays a crucial role in the induction of selected aspects of the IL-13 phenotype. To test this hypothesis, we characterized the pulmonary MCP and CC chemokine response in transgenic mice in which IL-13 was overexpressed in a lung-specific fashion. We also selectively assessed the role of CCR2 signaling in IL-13 effector pathways by comparing the phenotypes induced by transgenic IL-13 in mice with wild type (+/+) and null (-/-) CCR2 loci. These studies demonstrate that IL-13 is a potent inducer of MCP-1, -2, -3, and -5 in vivo. They also demonstrate that this stimulation is not MCP-specific because IL-13 also stimulates a large number of other chemokines in the lung. Furthermore, these studies demonstrate that IL-13-induced alveolar enlargement, lung enlargement, compliance alterations, inflammation, hyaluronic acid (HA) accumulation, pulmonary fibrosis, and respiratory failure and death are mediated by mechanisms that are, at least partially, CCR2-dependent, whereas IL-13-induced mucus metaplasia is mediated by a CCR2-independent pathway(s). Mechanistic insights are also provided because these studies demonstrated that IL-13 induces increased levels of lung antiproteases and decreased levels of total and bioactive TGF-₁ in the absence of CCR2.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Transgenic mice

Two types of overexpression transgenic mice were generated in our laboratories and used in these studies. Both are on a C57BL/6 background and use the Clara cell 10-kDa protein (CC10) promoter to target transgene expression to the lung. In the CC10-IL-13 mice, the CC10 promoter drives the expression of murine IL-13 in a constitutive fashion. The methods that were used to generate and characterize these mice were described previously (10). To allow IL-13 to be expressed in a temporally regulated fashion, CC10-reverse tetracycline transactivator (rtTA)-IL-13 mice were used. These are dual transgenic mice that use the rtTA and doxycycline (dox) to activate transgene expression. The IL-13 transgene in these mice is activated by putting dox in the animal’s drinking water. In the absence of dox, low-level or no IL-13 is produced. The constructs that were used and the methods that were used to generate and characterize these mice have been previously described (13). The phenotypes of the CC10-IL-13 and the CC10-rtTA-IL-13 mice were virtually identical when their respective transgenes were activated for appropriate intervals. In both modeling systems, IL-13 caused a mononuclear cell- and eosinophil-rich tissue inflammatory response, alveolar enlargement, subepithelial and parenchymal fibrosis, mucus metaplasia, and crystal deposition, as previously described (10, 13). In keeping with the chronic nature of the IL-13 production in the CC10-IL-13 mice, the phenotype of these animals progressed most rapidly. These animals died prematurely from a terminal inflammatory-fibrodestructive lung disorder.

CCR2 null mice (CCR2^-/-) were generated on a 129Sv and then bred to a C57BL/6 genetic background as previously reported (28). CC10-IL-13 and CC10-rtTA-IL-13 mice with wild type (+/+) and null (-/-) CCR2 loci were generated by breeding the IL-13 overexpressing mice with the CCR2^-/- animals. Genotyping was accomplished as previously described (10, 13, 28). For all the experiments, littermate wild-type mice with CCR2^+/+ or CCR2^-/- loci were used as controls.

Dox water administration

In experiments performed with CC10-rtTA-IL-13 transgene⁺ animals and their littermate controls, all animals were maintained on normal water until they were 1 mo of age. They were then randomized to receive either normal water or water with dox for the duration of the experiment. Dox was administered at 500 mg/L in 4% sucrose and was kept in dark brown bottles to prevent light-induced degradation.

Bronchoalveolar lavage

Lung inflammation was assessed by bronchoalveolar lavage (BAL) as previously described (13, 29). The BAL samples from each animal were then pooled and centrifuged. The number and types of cells in the cell pellet were determined as previously described (13, 29). The supernatants were stored at -20°C until used.

Lung volume and compliance assessments

Lung volume and compliance were assessed as previously described (13). Animals were anesthetized, the trachea was cannulated, and the lungs were removed and inflated with PBS at 25 cm. The size of the lung was evaluated via volume displacement.

Histologic evaluation

H&E and periodic acid Schiff with diastase (D-PAS) stains were performed after pressure fixation with Streck solution (Streck Laboratories, St. La Vista, NE) in the Research Histology Laboratory of the Department of Pathology at Yale University School of Medicine (New Haven, CT) as previously described (13).

Morphometric analysis

Alveolar size was estimated from the mean chord length of the airspace as previously described by our laboratory (13). This measurement is similar to the mean linear intercept, a standard measure of air space size, but has the advantage that it is independent of alveolar septal thickness. When CC10-IL-13 mice were being evaluated, at least four animals were studied at each time point. When CC10-rtTA-IL-13 mice were being evaluated, at least four animals that had received dox water were studied at each time point. Chord length increases with alveolar enlargement.

Calculation of HMI

The histologic mucus index (HMI) provides a measurement of the percentage of epithelial cells that are D-PAS⁺ per unit of airway basement membrane. It was calculated from D-PAS-stained sections as previously described by our laboratories (13).

mRNA analysis

mRNA levels were evaluated by RT-PCR analysis as previously described (13). The primers that were used have been described (13). New primers are in Table I. -Actin was used as an internal standard. Amplified PCR products were detected using ethidium bromide gel electrophoresis, quantitated electronically, and confirmed by nucleotide sequencing.

BAL IL-13 and chemokine levels were quantitated using commercial ELISA kits (R&D Systems, Minneapolis, MN) per the manufacturer’s instructions.

Immunohistochemistry

Immunostains for MCP-1 were performed as previously described (13). The primary Ab, a polyclonal goat anti-mouse MCP-1 Ab (Santa Cruz Biotechnology, Santa Cruz, CA) was applied at a 1/100 dilution. To verify the specificity of the reactions, the primary Ab was incubated with MCP-1-specific peptide (Santa Cruz Biotechnology) at 1:1 ratio for 2 h before being applied to the tissues, which were then counterstained with hematoxylin.

In situ hybridization

Lung tissues were fixed in formaldehyde and processed into paraffin. Five-micron sections were cut, deparaffinized, and treated with proteinase K (20 µg/ml, 37°C, 20 min). Tissues were then treated with 0.1 M triethylnolamine/0.25% acetic anhydride (pH 8) for 10 min at room temperature and rinsed in PBS. The MCP-1 probe was generated by cloning a fragment of mouse MCP-1 cDNA into pBS II KS that contains T3 and T7 primer sequences flanking the multiple cloning sites (Stratagene, La Jolla, CA). The primers, 5'-CCT CTA GAC AGC ACC AGC CAA C-3' and 5'-ATC TCG AGC ATC ACA GTC CGA GTC-3' with XbaI and XhoI restriction enzyme sites incorporated, were used to amplify a 515-bp fragment from total lung RNA of an IL-13 transgene-positive mouse. The RT-PCR product was digested with XbaI and XhoI and cloned into the vector pBS II KS. Sense and antisense RNA probes were generated, labeled with a digoxigenin RNA labeling kit (Roche, Indianapolis, IN), denatured at 65°C, and added to commercially available hybridization buffer (Ambion, Austin, TX) at 6 ng/µl, and the hybridization mixture was incubated with tissue overnight at 52°C. The tissues were then washed twice with 4x SSC for 5 min at room temperature, twice with 2x SSC for 10 min at 37°C, and incubated with RNase A (10 µg/ml) for 45 min at 37 °C. This was followed by two 10-min washes in 2x SSC at room temperature and three 20-min washes in 0.2x SSC at 50°C. Probe was detected by overnight incubation with sheep Abs to digoxigenin labeled with alkaline phosphatase (Roche) followed by 4-nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indoyl-phosphate, as described by the manufacturer.

Quantification of lung collagen

Lungs were obtained from CC10-IL-13 transgene⁺ mice and transgene littermate controls. Total lung collagen content was quantitated using the hydroxyproline method, as described (30).

Quantification of HA

The levels of BAL HA were measured using a competitive enzyme-linked immunosorbent-like assay using biotinylated HA binding protein as described previously (31, 32). Microtiter plates are coated with HA by combining rooster comb HA, carbodiimide HCl, and HCl. Samples are incubated with biotinylated HA binding protein for 1 h and then added to the wells. The plate is then agitated, washed, and developed with HRP streptavidin and exposed to peroxidase substrate for 30 min. OD₄₀₅ is evaluated. Samples are compared with a simultaneously performed standard curve.

TGF- bioassay

To measure the bioactivity of TGF- in BAL fluids, we used mink lung epithelial cells permanently transfected with a construct containing the TGF--responsive human plasminogen activator inhibitor-1 promoter fused to a luciferase reporter gene (TMLC; a gift from J. Munger, New York University Medical Center, New York, NY). These cells were seeded into 12-well tissue culture plates (10⁵ cells/well/ml) in DMEM supplemented with 10% FCS and allowed to attach for 5 h. They were then washed and incubated in triplicate in mixtures containing 200 µl of BAL fluid and 800 µl of assay medium (DMEM with 2.5% FCS). These incubations were performed in the presence and absence of saturating quantities of neutralizing Abs specific for TGF-₁, TGF-₂, or TGF-₃ (R&D Systems). The luciferase activities in these cells were measured 16 h later using the Luciferase Assay System (Promega, Madison, WI) according to the manufacturer’s instructions. The bioactivity attributed to TGF-₁ (TGF-₁ bioactivity) was defined as the difference in the luciferase activities of identical cells incubated in the absence and presence of anti-TGF-₁.

Statistics

Normally distributed data are expressed as means ± SEM and assessed for significance by Student’s t test or ANOVA as appropriate. Data that were not normally distributed were assessed for significance using the Wilcoxon rank sum test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effect of IL-13 on CC chemokines in the lung

To further define the mechanisms that underlie IL-13-induced tissue alterations, we assessed the levels of mRNA encoding CC chemokines that might be expected to contribute to these responses. As shown in Fig. 1A, IL-13 was a potent stimulator of MCP moieties, including MCP-1, MCP-2, MCP-3, and MCP-5 (Fig. 1). These chemokine-inductive effects of IL-13 were not specific for MCP-cytokine moieties because prominent induction of eotaxin, eotaxin-2, C10, MDC, macrophage-inflammatory protein (MIP)-1, MIP-1, MIP-2, MIP-3, thymus- and activation-regulated chemokine (TARC), and thymus-expressed chemokine (TECK) were also noted (Fig. 1A). These inductive responses were seen in lungs from both CC10-rtTA-IL-13 and CC10-IL-13 mice. In the former, mRNA increases could be seen after as little as 1–4 wk of dox administration depending on the moiety being assessed (see below). Prominent chemokine mRNA induction was seen in all cases after 1 mo of dox administration (Fig. 1A), and induction was still present after 3 mo of dox administration (data not shown). In the latter, chemokine induction was seen at all time points that were assessed (1–3 mo) (Fig. 1A and data not shown). However, IL-13 was not a nonspecific stimulator of CC chemokines because the levels of mRNA encoding RANTES were not significantly altered at any time point in either of our transgenic modeling systems (Fig. 1 and data not shown).

View larger version (47K): [in this window] [in a new window]   FIGURE 1. Effect of IL-13 on chemokine gene expression and protein production. A, Whole lung RNA was obtained from 2-mo-old transgene+ CC10-IL-13 mice and transgene- littermate controls (left panel) and from transgene+ CC10-rtTA-IL-13 mice and transgene- littermate that had been started on normal water or dox water at 1 mo of age and maintained on this regimen for 4 wk (right panel). RT-PCR was used to evaluate the levels of mRNA encoding the noted chemokines. B, BAL fluids were obtained from CC10-rtTA-IL-13 transgene+ mice that were placed on normal water or dox water at 1 mo of age and maintained on these regimens for the noted periods of time. BAL chemokine levels were evaluated via ELISA. The noted values represent the means ± SEM of a minimum of four animals at each time point (*, p < 0.05).

Localization of MCP-1 in lungs from IL-13 transgenic mice

Because MCP-1 was induced in a potent and rapid fashion, immunohistochemistry (IHC) and in situ hybridization (ISH) were used to define the sites of MCP-1 accumulation and production in lungs from transgene^- and transgene⁺ animals. With these methodologies, MCP-1 protein and mRNA were not appreciated in lungs from transgene^- animals. In contrast, MCP-1 protein and mRNA were readily apparent in macrophages from lungs from IL-13 transgene⁺ animals (Fig. 2). In these immunohistochemical evaluations, preincubation of our primary Ab with MCP-1 peptide effectively abrogated the detection of MCP-1 in these tissues (Fig. 2A). In the ISH evaluations, significant staining with the sense probe was not detected (Fig. 2B). This demonstrates the specificity of these approaches. These studies demonstrate that the macrophage is the major site of production and storage of MCP-1 in lungs from IL-13 transgene⁺ animals.

View larger version (89K): [in this window] [in a new window]   FIGURE 2. IHC and ISH localization of MCP-1 protein and mRNA. A, IHC was used to localize MCP-1 in tissues from transgene+ and transgene- CC10-IL-13 mice using Ab that was incubated with (Peptide +) or without (Peptide -) MCP-1-specific peptide. B, ISH was performed with tissues from transgene+ CC10-IL-13 mice using antisense and sense probes. Arrows point to macrophages.

To determine whether MCP signaling via CCR2 played an important role in the pathogenesis of IL-13-induced alterations in lung size, lung volume, alveolar size, and lung compliance, we compared these parameters in CC10-rtTA-IL-13 transgene⁺ mice with +/+ and -/- CCR2 loci. Lung size, lung volume, alveolar size, and lung compliance were similar in lungs from CCR2^-/- mice and wild-type littermate controls (Fig. 3 and data not shown). In accord with previous observations (13), dox induction of IL-13 caused an impressive increase in all of these parameters in mice with +/+ CCR2 loci. In contrast, IL-13 did not have the same effect in mice that were deficient in CCR2. After 1 or 3 mo of dox administration, the size and volume of lungs from CC10-rtTA-IL-13⁺CCR2^-/- mice were significantly smaller than the lungs from CC10-rtTA-IL-13⁺CCR2^+/+ animals (Fig. 3, A and B, and data not shown). Alveolar size and lung compliance were also significantly decreased in the CC10-rtTA-IL-13⁺CCR2^-/- animals (Fig. 3C and data not shown). Similar decreases in lung size, lung volume, and morphologic and histologic parameters of alveolar size were seen in comparisons of CC10-IL-13⁺CCR2^+/+ and CC10-IL-13⁺CCR2^-/- animals. These differences were seen in animals as young as 1 mo of age and were still readily apparent in 3-mo-old animals (data not shown).

View larger version (55K): [in this window] [in a new window]   FIGURE 3. Effect of CCR2 deficiency on IL-13-induced alterations in lung size, lung volume, and alveolar size. CC10-rtTA-IL-13 transgene- and transgene+ mice with +/+ and -/- CCR2 loci were generated and placed on dox water at 1 mo of age. At 2 mo of age their lungs were then removed and lung size (A), lung volume, and chord length (B), and histology (H&E, at x4 original magnification) (C) were assessed. The noted values represent the means ± SEM of a minimum of four animals in each group (*, p < 0.001).

To determine whether CCR2 played a key role in IL-13-induced inflammation, we compared the cellular characteristics of BAL fluids from transgene⁺ and transgene^- CC10-IL-13 mice with +/+ or -/- CCR2 loci. The number of cells that were recovered and their differentials were similar in BAL of transgene^- littermate control and CCR2^-/- animals (Fig. 4). IL-13 increased BAL cell recovery dramatically and significantly increased the percentages of these cells that were lymphocytes and eosinophils (Fig. 4 and data not shown). A deficiency in CCR2 decreased the total number of cells that were recovered in BAL fluids from CC10-IL-13 transgene⁺ mice (Fig. 4). CCR2 deficiency, however, did not alter the differential of the cells that were recovered (data not shown). A deficiency in CCR2 also decreased total BAL cell yield without altering BAL cell differential in CC10-rtTA-IL-13 transgene⁺ mice on dox for 1–3 mo (data not shown). These studies demonstrate that CCR2 plays a crucial role in determining the intensity but not the nature of IL-13-induced pulmonary inflammation.

View larger version (31K): [in this window] [in a new window]   FIGURE 4. Effect of CCR2 deficiency on BAL cellularity. BAL was performed in 3-mo-old CC10-IL-13 transgene+ mice and transgene- littermate controls that were either +/+ or -/- at their CCR2 loci and total cell recovery was assessed. The noted values represent the means ± SEM of a minimum of four animals (*, p < 0.01).

D-PAS tissue stains and HMI calculations were used to determine whether CCR2 was involved in the pathogenesis of IL-13-induced mucus metaplasia. As previously described (10), D-PAS staining cells were not appreciated in airways from transgene^- mice, and prominent mucus metaplasia was seen in CC10-IL-13 transgene⁺ animals. A deficiency of CCR2 did not alter these IL-13-induced responses because similar HMI values and D-PAS staining patterns were seen in CC10-IL-13⁺ mice with -/- and +/+ CCR2 loci (data not shown).

Role of CCR2 in IL-13-induced fibrosis and HA accumulation

Qualitative histologic techniques (trichrome stains) and quantitative biochemical approaches were used to determine whether CCR2 played a significant role in IL-13-induced pulmonary fibrosis and HA accumulation. In these studies, we compared these collagen and HA parameters in CC10-IL-13 transgene⁺ mice with +/+ and -/- CCR2 loci. Similar amounts of collagen and BAL HA were noted in the lungs from wild-type littermate control mice and CCR2^-/- animals. IL-13 caused an impressive increase in lung collagen and BAL HA content that could be easily appreciated with the histochemical and biochemical measurement techniques (Fig. 5 and data not shown). In contrast, these increases in lung collagen and BAL HA content were significantly reduced in CC10-IL-13 mice with null CCR2 loci (Fig. 5). Thus, CCR2 plays a critical role in IL-13-induced tissue fibrosis and HA accumulation.

View larger version (25K): [in this window] [in a new window]   FIGURE 5. Effect of CCR2 deficiency on IL-13-induced lung fibrosis and BAL HA accumulation. A, Collagen content of CC10-IL-13 transgene+ and transgene- animals with wild-type and null CCR2 loci are compared. B, Levels of BAL HA are compared. The noted values represent the means ± SEM of a minimum of four animals at each time point (*, p < 0.01).

In the CC10-IL-13 mice, progressive lung pathology is noted. At 1 mo of age, these animals manifest peribronchial inflammation, mucus metaplasia, alveolar enlargement, and subepithelial fibrosis (10). With time, the IL-13-induced pathologies progress, causing these animals to die prematurely. To determine whether CCR2-dependent pathways play a role in this respiratory failure, we compared the survival of CC10-IL-13 mice with +/+ and -/- CCR2 loci. The CC10-IL-13⁺CCR2^+/+ mice start to die when they are 90–110 days old, and 100% were dead by the time they were 128 days old. As can be seen in Fig. 6, a deficiency of CCR2 significantly improved the survival of these animals. Overall, CC10-IL-13⁺CCR2^-/- mice had a mean survival of 220 days. This demonstrates that CCR2 plays a critical role in the pathogenesis of IL-13-induced pathologies that lead to the death of these animals.

View larger version (14K): [in this window] [in a new window]   FIGURE 6. Effect of CCR2 deficiency on survival of IL-13 transgenic mice. Comparisons were made of the survival of CC10-IL-13+CCR2+/+ mice (•), CC10-IL-13+CCR2-/- mice (), and wild-type mice (). Each point represents the survival of a minimum of eight animals (*, p < 0.01, comparing transgene+ animals with +/+ and -/- CCR2 loci).

A deficiency of CCR2 can modify IL-13-induced phenotypes by altering IL-13 production or by modifying IL-13 effector functions. To determine whether CCR2 regulated CC10 promoter-driven IL-13 elaboration, we compared the levels of BAL IL-13 in CC10-IL-13 transgene⁺ and transgene^- mice with +/+ and -/- CCR2 loci. In all cases, virtually identical levels of IL-13 were noted (data not shown). In accord with this observation, similar levels of MCP-1 were noted in BAL fluids from these animals (data not shown). This demonstrates that CCR2 alters IL-13-induced phenotypes by modifying IL-13 effector function.

Effect of CCR2 deficiency on lung proteases

We reasoned that a deficiency of CCR2 could modulate the IL-13 alveolar phenotype by decreasing the production of respiratory proteases. To test this hypothesis, we initially compared the levels of mRNA encoding lung-relevant matrix metalloproteinases (MMPs) and cathepsins in wild-type and CCR2^-/- mice. Comparable levels of mRNA encoding MMP-2, MMP-9, MMP-12, and cathepsins S, L, K, and B were noted in lungs from wild-type and CCR2^-/- animals (Fig. 7A and data not shown).

View larger version (33K): [in this window] [in a new window]   FIGURE 7. Effect of CCR2 deficiency on protease and antiprotease mRNA. Whole lung mRNA was obtained from 2-mo-old CC10-IL-13 transgenic- and transgene+ mice with +/+ and -/- CCR2 loci. The levels of mRNA encoding the noted proteases (A) and antiproteases (B) were evaluated via RT-PCR. -Actin mRNA was used as internal control for even loading.

Effect of CCR2 deficiency on lung antiproteases

To determine whether the alterations in IL-13 effector function in the absence of CCR2 were due to alterations in antiproteases, the levels of mRNA encoding 1-AT, TIMP-1, TIMP-2, TIMP-4, SLPI, and cystatin-C were evaluated in lungs from IL-13 transgene^- and transgene⁺ mice with +/+ or -/- CCR2 loci. Similar levels of expression of 1-antitrypsin (1-AT), tissue inhibitor of metalloproteinase (TIMP-2), secretory leukocyte proteinase inhibitor (SLPI), and cystatin-C were seen in wild-type and CCR2^-/- mice (Fig. 7B). Interestingly, the levels of expression of TIMP-1 and TIMP-4 were increased in CCR2^-/- animals (Fig. 7B). As previously reported (13), IL-13 was a potent inducer of TIMP-1 and had lesser stimulatory effects on TIMP-2 and TIMP-4 while inhibiting 1-AT in wild-type animals (Fig. 7B). A deficiency of CCR2, however, caused an impressive increase in the levels of 1-AT and modest increases in the levels of mRNA encoding TIMP-1, TIMP-2, TIMP-4, and SLPI in IL-13-producing transgenic animals (Fig. 7B). When viewed in combination, these studies demonstrate that the protective effects of CCR2 deficiency in our transgenic system are associated with the enhanced expression of 1-AT, TIMP-1, TIMP-2, TIMP-4, and SLPI.

Effect of CCR2 deficiency on IL-13-induced TGF-₁

Members of the MCP cytokine family can stimulate TGF-₁ production, and TGF-₁ is a potent stimulator of HA production and tissue fibrosis (18, 33, 34, 35). Studies were thus undertaken to determine whether the decreased tissue fibrosis and HA production seen in IL-13-producing transgenic mice with -/- CCR2 loci were due to a decrease in IL-13-induced TGF-₁ production. In these studies, TGF-₁ was assessed using mink lung epithelial cells transfected with a promoter-reporter gene construct that is known to be TGF-₁ responsive. In these assays, we quantitated the levels of spontaneously active and total TGF-₁ by measuring the TGF-₁ bioactivity in BAL fluids from IL-13-producing mice with +/+ and -/- CCR2 loci before and after acidification, respectively. Spontaneously activated TGF-₁ was not present in BAL fluids from wild-type mice or CCR2^-/- animals (Fig. 8A). As previously reported by our laboratory (36), the BAL fluids from IL-13-producing mice had significant levels of TGF-₁ bioactivity in the absence of acid activation and even greater levels of TGF-₁ bioactivity after acidification (Fig. 8B). Interestingly, the levels of spontaneously bioactive and total TGF-₁ in BAL fluids from CC10-IL-13⁺CCR2^-/- mice were significantly lower than those in BAL fluids from CC10-IL-13⁺CCR2^+/+ animals (Fig. 8). These studies demonstrate that CCR2 plays a critical role in IL-13 induction and activation of TGF-₁ in the lung.

View larger version (26K): [in this window] [in a new window]   FIGURE 8. Effect of CCR2 deficiency on TGF-1 bioactivity. BAL fluids were obtained from CC10-IL-13 transgene+ and transgene- littermate controls with +/+ or -/- CCR2 loci. The levels of TGF-1 bioactivity in these fluids were assessed before (spontaneous bioactivity) (A) or after (total activity) (B) acid activation using mink lung cells permanently transfected with constructs containing plasminogen activator inhibitor promoter driving a luciferase reporter gene, as described in Materials and Methods. The noted values represent the means ± SEM of a minimum of four animals in each category (*, p < 0.05, comparing transgene+ animals with +/+ and -/- CCR2 loci).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Chemokines are small, 8- to 10-kDa cytokines (reviewed in Refs. 18, 37 , and 41) that have been subdivided into four supergene families (CXC, CC, C, and CXXXC) based on the position of either one of the two cysteine residues located near the N terminus of each protein. The CC and CXC chemokine groups are large and contain over 50 identified ligands. Although in vitro characterizations would suggest that there is impressive redundancy in this system, examinations of a limited number of ligands in vivo have demonstrated that their production is organized in a coordinated manner and that their effector functions can be restricted to different stages of disease development and/or pathology (17, 18, 39, 44). Thus, in vivo, a deficiency of an individual ligand or its receptor can cause striking alterations in tissue phenotype (16, 19). An impressive finding in our study is the potent and rapid induction of MCP-1 and other MCP moieties by IL-13 in the murine lung. Because MCP-1 signals predominantly via CCR2 and MCP-2, -3, and -5 are also key CCR2 ligands (16, 19), the roles of MCP signaling and CCR2 were evaluated by characterizing the IL-13 responses in mice with +/+ and -/- CCR2 loci. Because prior studies demonstrated that MCP-1 has different effects when exogenously administered during the sensitization vs the elicitation/effector phases of type I and type II granulomatous immune responses (45), care was taken to use a system that allowed the effector mechanisms of IL-13 to be selectively analyzed (see below). These studies highlight a previously unappreciated relationship between IL-13 and CCR2 by demonstrating that IL-13 increases alveolar and lung size, induces tissue inflammation, alters pulmonary compliance, increases the levels of BAL HA, induces tissue fibrosis, and causes respiratory failure and death via CCR2-dependent pathways, while inducing mucus metaplasia via CCR2-independent pathways. All in all, these studies demonstrate that CCR2 signaling plays a critical role in the pathogenesis of IL-13-induced inflammatory and remodeling responses in the lung.

COPD is a generic term that encompasses chronic bronchitis, small airways disease, and emphysema. It affects approximately 18 million people in the U.S. and millions more around the world. A macrophage-rich inflammatory response and alveolar remodeling are prominent features of the lung pathology that is seen in these patients. Premature death is also a dreaded consequence of COPD. To date, however, the mechanisms of these responses have not been defined. We previously demonstrated that the transgenic overexpression of IL-13 causes a COPD-like phenotype in the murine lung characterized by alveolar enlargement, lung enlargement, compliance alterations, and eventually respiratory failure and death. In these studies, we demonstrate that these inflammatory and emphysematous parameters are markedly decreased in the absence of CCR2. We also demonstrate that the IL-13 effector pathway alterations that are responsible for the different phenotypes seen in the presence and absence of CCR2 cannot be attributed to a decreased ability of IL-13 to induce MMPs or cathepsins but are associated with increased levels of mRNA encoding the antiproteases 1-AT, TIMP-1, TIMP-2, TIMP-4, and SLPI. These are the first studies to provide insights into the mechanisms that may mediate the macrophage-rich inflammation and the first to demonstrate a critical role for any chemokine signaling pathway in COPD or models of these disorders. They are also the first to demonstrate this novel relationship between CCR2 signaling and antiprotease gene expression. When viewed in combination, these studies allow for the tempting speculation that interventions that regulate CCR2 activation may be useful in the treatment of patients with COPD and that the benefits of these interventions may be mediated, in part, by the ability of CCR2 signaling to regulate the levels of these important antiproteases.

MCP-1 overproduction has been noted in a large number of inflammatory, granulomatous, and fibrotic disorders. Immunoneutralization studies have demonstrated that MCP-1 plays an important proinflammatory role in these responses. This is nicely illustrated in murine asthma models where treatment with neutralizing anti-MCP-1 Abs diminished the inflammatory and physiologic abnormalities noted after allergen challenge (17, 46). In accord with these findings, type II granulomatous responses have also been shown to be decreased in CCR2^-/- animals (47). Surprisingly, more recent studies by Kim et al. (19) and Blease et al. (48) demonstrated that CCR2^-/- mice have a propensity to mount exaggerated Th2 responses when sensitized and challenged with aeroallergen or infected with Aspergillus fumigatus, respectively. Because IL-13 is a major Th2 effector cytokine, our findings are in accord with the findings in the immunoneutralization studies and support the contention that MCP signaling via CCR2 plays an important role in mediating Th2 effector pathways. A superficial analysis of our data, however, might lead one to believe that our findings disagree with those of Blease and Kim and their coworkers. There are, however, crucial differences in the methodologies that were used in these studies that we feel can explain the different results and provide new important insights into the role of CCR2 in IL-13-mediated and Th2-dominated inflammatory disorders. Specifically, in the studies by Kim et al. (19) and Blease et al. (48), Ag sensitization, Th2 cell generation, Th2 cytokine production, and cytokine effector pathway activation were all undertaken in CCR2^-/- mice. Thus, in these systems, it is virtually impossible to determine whether a change in the final tissue phenotype is the result of an alteration at the level of sensitization, Th2 cell number, Th2 cytokine production, or cytokine effector pathway activation. Importantly, in the studies by Blease et al. (48) and Kim et al. (19), impressive increases in the levels of IL-13 and MCP-1 were seen. This demonstrates that, in these experiments, CCR2 deficiency enhanced the activity of at least one of the three pre-effector events (sensitization, Th2 cell generation, and Th2 cytokine elaboration) involved in the generation of the Th2 response. In contrast, our studies were performed with a system that bypasses these stages and only assesses the role of CCR2 in the effector pathways of IL-13. As a result, comparable levels of IL-13 and MCP-1 were seen in transgenic mice with +/+ and -/- CCR2 loci. When our data and the studies of Blease et al. (48) and Kim et al. (19) are combined, it is tempting to propose that CCR2 signaling has different effects in different phases of tissue inflammation. In particular, CCR2 appears to inhibit one or all of the pre-effector phases of Th2 inflammation, thereby decreasing Th2 cytokine elaboration. In contrast, CCR2 also plays a critical role in mediating the effector functions of the IL-13 (and possibly other Th2 cytokines) that has been elaborated. This hypothesis can account for our findings and the findings in the literature. Support for this concept can also be seen in studies that demonstrate that MCP-1 is a potent stimulator of IL-13 production and that T cells sensitized in the presence of MCP-1 manifest enhanced Th2 responses on subsequent Ag challenge (22, 45). Additional investigation will be required to test this hypothesis and define the pathways CCR2 uses to transmit its inhibitory and stimulatory signals. If this hypothesis is correct, however, it would have important implications for therapeutic interventions based on CCR2. Specifically, IL-13-induced pathologies would be appropriately antagonized by agents that block or interfere with CCR2 signaling only if they are given during effector phases of a disorder (after immune sensitization and IL-13 elaboration have occurred). In contrast, agents with CCR2 agonist properties might be useful in attempts to control IL-13 elaboration if they are administered during the pre-effector phases of response generation.

Structural alterations, collectively referred to as airway remodeling, are well documented in asthmatic airways and are believed to contribute to the natural history of this disorder (5, 6). Subepithelial fibrosis and elevated levels of HA are prominent features of this remodeling response, and IL-13 is believed to be an important contributor to the pathogenesis of these alterations (5, 6). Our studies provide insights into mechanisms that likely contribute to these responses by demonstrating that IL-13-induced tissue fibrosis and HA accumulation are decreased in mice with null CCR2 loci. Previous studies from our laboratory demonstrated that the fibrotic effects of IL-13 are mediated by its ability to induce and activate TGF-₁ in the lung. These studies also demonstrated that macrophages are the major source of TGF-₁ in lungs from IL-13-overexppressing transgenic mice (36). In accord with these observations, studies that demonstrate that MCP-1 can stimulate TGF-₁ production (18, 34, 35) and studies that demonstrate that TGF-₁ is a potent stimulator of HA production and tissue fibrosis (33, 49), our studies demonstrate that the diminished fibrogenic and HA stimulating effects of IL-13 in the absence of CCR2 are associated with the diminished production and activation of TGF-₁. These are the first studies to demonstrate that CCR2 signaling is a critical event in cytokine induction and activation of TGF-₁ in vivo and to define this relationship between CCR2 and the regulation of HA production. Because CCR2 is the major CCR involved in the recruitment and activation of mononuclear cells and mononuclear cells are the major TGF-₁-producing cells in our transgenic mice, it is tempting to speculate that the diminished remodeling response seen in IL-13-producing CCR2^-/- mice is a direct result of the diminished recruitment and/or activation of mononuclear cells in the CCR2-deficient animals.

IL-13 was originally described as an IL-4-like cytokine and noted to have effector properties relevant to Th2 inflammation. More recent studies from our laboratory and others demonstrated that IL-13 is a powerful regulator of tissue remodeling and fibrotic responses in vivo and in vitro (13, 15). IL-13 has also been implicated in the pathogenesis of the inflammation and remodeling responses in a variety of disorders including asthma, COPD, pulmonary fibrosis, scleroderma, hepatic fibrosis, and nodular sclerosing Hodgkin’s disease (15, 50, 51, 52). In the present study, we demonstrate that IL-13 is a broad-spectrum stimulator of CC chemokines with the capacity to increase the production of MCPs and a variety of other chemokine moieties. We also demonstrate that MCP-mediated signaling via CCR2 plays a key role in the pathogenesis of IL-13-induced inflammatory, fibrotic, and proteolytic effector responses in vivo. As a result of these observations, it is reasonable to believe that CCR2 plays a similarly important role in the pathogenesis of the inflammatory and remodeling responses in these important diseases. This establishes the MCP-CCR2 ligand pathway as a worthwhile site for further investigation designed to identify therapeutic agents that can be used to treat these and other IL-13-mediated disorders.

	Acknowledgments

 
We thank the scientific institutions and investigators that provided the reagents that were used and Susan Ardito and Kathleen Bertier for their excellent secretarial and administrative assistance.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants HL-56389, HL-61904, and HL-64242 (to J.A.E.) and HL-60539 (to P.N.).

² Address correspondence and reprint requests to Dr. Jack A. Elias, Section of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Yale University School of Medicine, 333 Cedar Street, 105 LCI, P. O. Box 205087, New Haven, CT 06520-8057. E-mail address: jack.elias{at}yale.edu

³ Abbreviations used in this paper: COPD, chronic obstructive pulmonary disease; MCP, monocyte chemotactic protein; MDC, macrophage-derived chemokine; HA, hyaluronic acid; rtTA, reverse tetracycline transactivator; dox, doxycycline; BAL, bronchoalveolar lavage; D-PAS, periodic acid Schiff with diastase; HMI, histologic mucus index; IHC, immunohistochemistry; ISH, in situ hybridization; MIP, macrophage-inflammatory protein; TARC, thymus- and activation-regulated chemokine; MMP, matrix metalloproteinase; 1-AT, 1-antitrypsin; TIMP, tissue inhibitor of metalloproteinase; SLPI, secretory leukocyte proteinase inhibitor.

Received for publication October 17, 2001. Accepted for publication January 4, 2002.

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