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The Journal of Immunology, 2006, 176: 4968-4978.
Copyright © 2006 by The American Association of Immunologists

Role of CCR5 in the Pathogenesis of IL-13-Induced Inflammation and Remodeling1

Bing Ma*, Wei Liu*, Robert J. Homer{dagger}, Patty J. Lee*, Anthony J. Coyle{ddagger}, Jose M. Lora{ddagger}, Chun Geun Lee* and Jack A. Elias2,*

* Section of Pulmonary and Critical Care Medicine, Yale University School of Medicine, New Haven, CT 06520; {dagger} Department of Pathology, Yale University School of Medicine, New Haven, CT 06519, and Pathology and Laboratory Medicine Service, Veterans Affairs-Connecticut Health Care System, West Haven, CT 06516; and {ddagger} Department of Biology, Inflammation Division, Millennium Pharmaceuticals, Cambridge, MA 02139


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
IL-13 is a major effector at sites of Th2 inflammation and tissue remodeling. In these locations, it frequently coexists with the CCR5 chemokine receptor and its ligands MIP-1{alpha}/CCL3 and MIP-1beta/CCL4. We hypothesized that CCR5 induction and activation play important roles in the pathogenesis of IL-13-induced tissue responses. To test this hypothesis, we evaluated the effects of IL-13 on the expression of CCR5 in the murine lung. We also compared the effects of lung-targeted transgenic IL-13 in mice treated with anti-CCR5 or an Ab control and mice with wild-type or null CCR5 loci. These studies demonstrate that IL-13 is a potent stimulator of epithelial cell CCR5 expression. They also demonstrate that CCR5 neutralization or a deficiency of CCR5 significantly decreases IL-13-induced inflammation, alveolar remodeling, structural and inflammatory cell apoptosis, and respiratory failure and death. Lastly, these studies provide mechanistic insights by demonstrating that CCR5 is required for optimal IL-13 stimulation of select chemokines (MIP-1{alpha}/CCL3, MIP-1beta/CCL4, MCP-1/CCL-2), matrix metalloproteinase-9 and cell death regulators (Fas, TNF, TNFR1, TNFR2, Bid), optimal IL-13 inhibition of {alpha}1-antitrypsin, and IL-13-induction of and activation of caspases-3, -8, and-9. Collectively, these studies demonstrate that CCR5 plays a critical role in the pathogenesis of IL-13-induced inflammation and tissue remodeling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Interleukin-13 is a pleiotropic cytokine product of a gene on chromosome 5 at Q31 that is one of the major effector molecules at sites of Th2 inflammation and remodeling (1, 2, 3). Studies from our laboratory and others have used overexpression transgenic modeling and other approaches to define the effector properties of this cytokine. These studies demonstrated that IL-13 is a potent stimulator of eosinophil-, lymphocyte-, and macrophage-rich inflammation, mucus metaplasia, tissue fibrosis, and parenchymal remodeling (2, 3, 4, 5, 6, 7). They also demonstrated that, in the lung, IL-13 induces asthma-like airway hyperresponsiveness on methacholine challenge (3, 4, 6, 8). In accord with these findings, IL-13 dysregulation has been documented and is felt to play an important role in the pathogenesis of a variety of diseases including asthma, idiopathic pulmonary fibrosis, hepatic fibrosis, fungal pneumonitis, viral pneumonia, nodular-sclerosing Hodgkin’s disease, and chronic obstructive pulmonary disease (1, 2, 9, 10, 11, 12, 13, 14, 15, 16).

In accord with its importance in inflammation and disease pathogenesis, numerous investigators have sought to define the mechanisms that IL-13 uses to induce tissue alterations. These studies demonstrated that the effects of IL-13 are mediated, in great extent, by its ability to regulate a number of downstream genes and effector pathways including responses involving TGF-beta, chitinases, and matrix metalloproteinases (MMPs)3 (7, 17, 18, 19). Previous studies from our laboratory also demonstrated that IL-13 stimulation of macrophage and epithelial cell chemokine production and signaling via CCR-1 and CCR-2 are also key events in the generation of tissue inflammation and remodeling (7, 20). The contributions of other CC chemokine receptors, however, have not been investigated.

CCR-5 is a G protein-coupled chemokine receptor that binds MIP-1{alpha}/CCL3, MIP-1beta/CCL4, and RANTES/CCL5, serves as a coreceptor for HIV, and is expressed on a variety of cells including dendritic cells, macrophages, CD8 cells, memory CD4 cells, stromal cells, and at high levels on Th1 lymphocytes (21, 22, 23, 24, 25). CCR-5 plays a critical role in Th1 inflammation and immunity where it is required for the successful control of a variety of infectious agents including tuberculosis, cryptococcus, and toxoplasma (21, 22, 26, 27, 28), and is expressed in exaggerated quantities in T cell (Tc)-1-dominated responses including those in tuberculosis, sarcoidosis, Wegener’s granulomatosus, rheumatoid arthritis, periodontitis, and acute and chronic transplant rejection (22, 28, 29, 30, 31). The association between CCR5 and Th1 responses is so striking that CCR5 is frequently used as a marker of Th1 cells (30, 32, 33, 34, 35, 36). However, CCR5 can also be found on Th2 cells (37, 38). In addition, interventions that abrogate CCR5 or interfere with its ligand binding have been shown to alter Th2-induced inflammatory responses (39, 40), and hypofunctional polymorphisms of CCR5 (CCR5 {delta} 32) have been reported to be associated with a decreased risk of asthma in some populations (41, 42). Surprisingly, the role of CCR5 in the pathogenesis of IL-13-induced effector responses at sites of Th2 inflammation has not been investigated.

We hypothesized that CCR5 signaling plays an important role in the pathogenesis of IL-13-induced tissue alterations in vivo. To test this hypothesis, we characterized the expression of CCR5 in transgenic mice in which IL-13 was overexpressed in a lung-specific fashion and defined the effects of CCR5 neutralization or a null mutation of CCR5 on IL-13-induced inflammation and remodeling in these animals. These studies demonstrate that IL-13 is a potent stimulator of epithelial cell and to a lesser extent macrophage CCR5 gene expression. They also demonstrate that CCR5 neutralization or a deficiency of CCR5 significantly decreases IL-13-induced inflammation, alveolar remodeling, structural and inflammatory cell apoptosis, and respiratory failure and death. Lastly, they demonstrate that IL-13 stimulates the production of select chemokines, MMPs, and cell death regulators and activates pulmonary caspases via CCR5-dependent mechanisms.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Transgenic mice

Two types of overexpression transgenic mice were generated in our laboratories and used in these studies. Both 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 (6). To allow IL-13 to be expressed in a temporally regulated fashion, CC10-rtTA-IL-13 mice were used. These are dual transgenic mice that use the reverse tetracycline transactivator (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 (5). 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 (5, 6).

CCR5null mutant (–/–) mice, generated by Kuziel et al. (43), were obtained from The Jackson Laboratory after breeding for >10 generations onto a C57BL/6 background. CC10-IL-13 mice with wild-type (+/+) or null (–/–) CCR5 loci were generated by breeding C57BL/6 background IL-13 overexpressing mice with the CCR5–/– animals. PCR was used to define the transgenic status of all offspring, using primers that detected rtTA and/or the junction region of the murine IL-13-human growth hormone construct. The CCR5 loci were evaluated by PCR using primers: upper, 5'-ATTCTCCACACCCTGTTTCG-3' and lower, 5'-GTTCTCCTGTGGATCGGGTA-3' which detects a 388-bp reaction product.

Dox water administration

In experiments performed with CC10-rtTA-IL-13 transgene (+) (Tg+) 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 kept in dark brown bottles to prevent light-induced degradation.

Treatment with anti-CCR5 Abs

To define the role(s) of CCR5 in the IL-13 phenotype, we compared the phenotypes of the CC10-rtTA-IL-13 transgene (–) (Tg–) and Tg+ mice that had been randomized to receive rat monoclonal anti-CCR5 (Research Diagnostics) or an isotype control (rat IgG2c) Ig (control Ig) (500 µg i.p. every other day). Two days later, they were randomized to normal or dox water and maintained on this regimen for 2 wk. At the end of this interval, the animals were sacrificed and pulmonary phenotype was assessed as described below. The specificity and neutralizing capacity of this antiserum have been previously defined by our laboratories (44).

Bronchoalveolar lavage (BAL)

Lung inflammation was assessed by BAL as previously described (5, 6). 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 (5, 6). The supernatants were stored at –20°C until used.

Lung volume and compliance assessments

Lung volume and compliance were assessed as previously described (5). In brief, 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 stains were performed after pressure fixation with Streck solution (Streck Laboratories) in the Research Histology Laboratory of the Department of Pathology at Yale University School of Medicine as previously described (5).

Morphometric analysis

Alveolar size was estimated from the mean chord length of the airspace as previously described by our laboratory (5). At least four animals were studied at each time point. Chord length increases with alveolar enlargement.

mRNA analysis

mRNA levels were evaluated by RT-PCR and real-time RT-PCR analysis using whole lung RNA as previously described (5, 6). The primers that were used for the matrix MMP and cathepsin evaluations have been described (5, 6, 44). In the RT-PCRassays, amplified PCR products were detected using ethidium bromide gel electrophoresis, quantitated electronically, and confirmed by nucleotide sequencing.

Quantification of IL-13, chemokines, and IFN-{gamma}

BAL IL-13, chemokine, and IFN-{gamma} levels were quantitated using commercial ELISA kits (R&D Systems) as per the manufacturer’s instructions.

In situ hybridization

In situ hybridization was undertaken as previously described (6, 18). Formaldehyde fixation and a cDNA encoding the portion of CCR5 between nucleotides 10 and 921 were used. The CCR5 probe was generated by cloning the cDNA into pBS II KS that contains T3 and T7 primer sequences flanking a multiple cloning site (Stratagene). Sense and antisense RNA probes were generated and labeled with a digoxigenin RNA labeling kit (Roche).

CCR5 immunohistochemistry

CCR5 immunohistochemistry was performed as previously described by our laboratory (44).

TUNEL evaluations

End labeling of exposed 3'-OH ends of DNA fragments in paraffin-embedded tissue was undertaken with the TUNEL in situ cell death detection kit AP (Roche Diagnostics) using the instructions provided by the manufacturer. Staining specificity was assessed by comparing the signal that was seen when terminal transferase was included and excluded from the reaction. After staining, a minimum of 20 fields of alveoli were randomly chosen and 2000 nuclei were counted per lung. The labeled cells were expressed as a percentage of total nuclei.

Caspase evaluations

Whole lung lysates were prepared and Western evaluations were undertaken as previously described by our laboratory (44). Abs that selectively reacted to caspase-3, caspase-9, poly(ADP)ribose polymerase (PARP), and beta-tubulin were purchased from Santa Cruz Biotechnology. The bioactivities of caspases-3, -8, and -9 were measured with colorimetric assays using the CaspACE assay system (Promega) and the Caspase-8 and Caspase-9 Colorimetric Activity assay kits (Chemicon International), respectively, as previously described by our laboratory (44, 45).

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
 Disclosures
 References
 
Effect of IL-13 on CCR5

Previous studies from our laboratory demonstrated that transgenic IL-13 is a potent stimulator or MIP-1{alpha}/CCL-3 and MIP-1beta/CCL-4 in the murine lung (7). Studies were thus undertaken to define the effects of IL-13 on their receptor, CCR5. In lungs from transgene (–) (Tg–) mice, the levels of CCR5 mRNA and protein were near or below the limits of detection of our assays (Fig. 1, A–C). In contrast, the levels of CCR5 mRNA and protein were markedly increased in lungs from CC10-IL-13 Tg+ animals (Fig. 1, A–C). To localize this receptor, immunohistochemistry was undertaken. These studies demonstrated that CCR5 protein is a prominent finding in epithelial cells and was also apparent in occasional macrophages in lungs from Tg+ mice (Fig. 1D and data not shown). This staining was CCR5-specific because it was not noted in transgenic mice with null CCR5 loci and was not seen in the absence of the primary Ab (data not shown). In addition, the Ab that was used detected an appropriately sized molecule on Western blot evaluations of proteins from lungs from Tg+ animals (data not shown). This protein appeared to be produced in these cells because in situ hybridization revealed similar induction of CCR5 mRNA in epithelial cells and to a lesser degree in macrophages in lungs from IL-13 Tg+ animals (Fig. 1E and data not shown). A similar pattern of induction and localization of CCR5 was seen in lungs from CC10-rtTA-IL-13 mice in which transgene activation was induced with dox water (data not shown). In all cases, CCR5 activation did not appear to be mediated by IFN-{gamma} because similar levels of IFN-{gamma} mRNA and protein were seen in lungs from Tg+ and Tg– mice on normal or dox water (data not shown). These studies demonstrate that IL-13 stimulates CCR5 in epithelial cells and macrophages in the murine lung.


Figure 1
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  		FIGURE 1. IL-13 regulation of pulmonary CCR5. Tg– and Tg+ mice were given normal or dox water for 1 mo. The levels of mRNA encoding CCR5 were evaluated by RT-PCR (A) and real-time RT-PCR (B) and CCR5 protein was evaluated with Western analysis (C). CCR5 protein and mRNA were also localized undertaken using immunohistochemistry (D, x100) and in situ hybridization (E, x20), respectively. The arrows highlight positively staining epithelial cells. Each evaluation in A and C–E is representative of a minimum of four similar experiments. The values in B represent the mean ± SEM of evaluations in a minimum of four animals (*, p < 0.05).

 
Role of CCR5 in IL-13-induced inflammation

To understand the roles of CCR5 and its ligands in the generation of IL-13-induced tissue alterations, we compared the inflammation in CC10-rtTA-IL-13 Tg+ mice that had been randomized to normal water or dox water at 1 mo of age and treated with antiserum against CCR5 or control antiserum. We also bred CC10-IL-13 Tg+ mice with CCR5null (–/–) mice and compared the effects of transgenic IL-13 in mice with (+/+) and null (–/–) CCR5 loci. As previously reported (6, 7, 20), transgenic IL-13 caused significant increases in BAL total cell, eosinophil, macrophage, and lymphocyte recovery and an impressive eosinophil- and mononuclear cell-rich tissue inflammatory response (Fig. 2, A–C). Treatment with anti-CCR5 did not alter the number or differential of the cells that were recovered in BAL fluids or tissues from lungs from Tg– mice on normal or dox water (Fig. 2, A–C). In contrast, this intervention significantly decreased BAL total cell, eosinophil, macrophage and lymphocyte recovery, and the tissue inflammatory response in lungs from dox-treated Tg+ animals (Fig. 2, A–C). Similar alterations were seen in comparisons of BAL and tissues from CC10-IL-13 Tg+ mice with (+/+) or (–/–) CCR5 loci (Fig. 2, D–F). Thus, CCR5 is a critical regulator of the intensity and nature of IL-13-induced pulmonary inflammation.


Figure 2
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  		FIGURE 2. Effect of CCR5 neutralization/ablation on IL-13-induced inflammation. In A–C, Tg– and Tg+ mice were placed on dox water and treated with anti-CCR5 or control Ig. In D–F, Tg– and Tg+ mice with (+/+) and (–/–) CCR5 loci were evaluated at 3 mo of age. BAL total cell recovery (A and D), differential cell recovery (B and E), and tissue inflammation (C and F, x40) were evaluated. The values represent the mean ± SEM of evaluations in a minimum of four mice in each group (*, p ≤ 0.05) (Eo, eosinophil; neu, neutrophil; lym, lymphocyte; mac, macrophage). C and E are representative of a minimum of four similar evaluations.

 
Role of CCR5 in IL-13-induced alveolar remodeling

To define the role of CCR5 in the pathogenesis of IL-13-induced alveolar remodeling, we compared the alterations in lung size and volume, alveolar size, and lung compliance in Tg+ mice on dox water that had been treated with anti-CCR5 or control Ig and Tg+ mice with (+/+) and null CCR5 loci. In accord with previous observations (5), dox induction of IL-13 caused an impressive increase in all of these parameters (Fig. 3, A–D, and data not shown). Treatment with anti-CCR5 did not significantly alter these parameters in lungs from wild-type mice on normal or dox water (Fig. 3, A–D). In contrast, lungs from dox-treated Tg+ treated with anti-CCR5 were significantly smaller (Fig. 3A), had smaller lung volumes (Fig. 3B), and were less compliant (Fig. 3B) than lungs from Tg+ mice treated with control Ig. Alveolar size was similarly decreased when assessed with light microscopic (Fig. 3C) or morphometric (Fig. 3D) approaches. Similar decreases in alveolar remodeling were noted in comparisons of lungs from Tg+ mice with (+/+) and (–/–) CCR5 loci (Fig. 3, E–G, and data not shown). Importantly, treatment with anti-CCR5 and genetic ablation of CCR5 did not alter IL-13-induced mucus metaplasia (data not shown), illustrating the specificity of CCR5 in IL-13-induced tissue remodeling responses. These studies demonstrate that CCR5 plays a critical and specific role in the pathogenesis of IL-13-induced alveolar remodeling in the murine lung.


Figure 3
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  		FIGURE 3. Role of CCR5 in IL-13-induced remodeling. Tg– and Tg+ mice were placed on dox water for 1 mo. In A, they were fixed to pressure and photographed. In B–D, the mice were treated with anti-CCR5 or control Ig for 2 wk. In E–G, Tg– and Tg+ mice with (+/+) and (–/–) CCR5 loci were evaluated at 3 mo of age. Lung size (A), lung volume (B and E), histology (C and F; x20) and chord length (D and G) were evaluated. A, C, and F are representative of a minimum of five similar evaluations. The values in the rest of the panels represent the mean ± SEM of evaluations in a minimum of five mice in each group (*, p < 0.05).

 
Role of CCR5 in IL-13 elaboration

A deficiency of CCR5 could modify transgene-induced tissue response by altering the production of IL-13 or modulating IL-13 effector responses. To determine whether alterations in CCR5 regulated the production of IL-13, we compared the levels of BAL IL-13 in Tg+ and Tg– mice treated with anti-CCR5 or control Ig and Tg+ with (+/+) and null CCR5 loci. IL-13 was not readily apparent in BAL fluids from Tg– on normal or dox water that had been treated with anti-CCR5 or control Ig (Fig. 4). In contrast, significant levels of BAL IL-13 were appreciated in Tg+ mice on dox water. These levels were similar in mice treated with anti-CCR5 or the control Ig (Fig. 4A). Comparisons of BAL fluids from Tg+ mice with (+/+) and (–/–) CCR5 loci also did not reveal differences in IL-13 content (Fig. 4B). Thus, treatment with anti-CCR5 and null mutations of CCR5 altered IL-13-induced tissue responses by modifying IL-13 effector pathway activation.


Figure 4
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  		FIGURE 4. Role of CCR5 in transgenic IL-13 production. In A, Tg– and Tg+ mice were placed on dox water and treated with anti-CCR5 or control Ig. In B, Tg– and Tg+ mice with (+/+) and (–/–) CCR5 loci were evaluated at 3 mo of age. BAL IL-13 was evaluated by ELISA. The values represent the mean ± SEM of evaluations in a minimum of five mice in each group.

 
Role of CCR5 in IL-13-induced chemokine elaboration

To investigate the mechanism(s) by which a deficiency of CCR5 decreased IL-13-induced inflammation, we compared the expression of selected chemokines in Tg+ mice treated with anti-CCR5 or control Ig. In accord with previous observations from our laboratory (7), the levels of MIP-1{alpha}/CCL-3, MIP-1beta/CCL-4, MCP-1/CCL-2, MCP-2/CCL-8, MCP-3/CCL-7, MCP-5/CCL-12, MIP-2/CXCL-2/3, MDC/CCL-22, eotaxin/CCL-11, eotaxin 2/CCL24, thymus and activation-regulated chemokine (TARC)/CCL-17, thymus-expressed chemokine (TECK)/CCL-25, and C10/CCL-6 were near or below the limits of detection of our assays in Tg– mice and were significantly increased by transgenic IL-13 (Fig. 5, A–D, and data not shown). Anti-CCR5 significantly diminished the ability of IL-13 to stimulate MIP-1{alpha}/CCL-3, MIP-1beta/CCL-4, and MCP-1/CCL-2 mRNA accumulation (Fig. 5, A–D). In all cases, comparable decreases in the levels of BAL chemokines were also noted (Fig. 5, B–D, and data no shown). In contrast, IL-13 did not stimulate RANTES/CCL-5 and anti-CCR5 did not alter the ability of IL-13 to stimulate the expression of MCP-2/CCL-8, MCP-3/CCL-7, MCP-5/CCL-12, MIP-2/CXCL-2/3, MDC/CCL-22, Eotaxin/CCL-11, eotaxin 2/CCL24, TARC/CCL-17, TECK/CCL-25, C10/CCL-6, and RANTES/CCL-5 (Fig. 5A and data not shown). Similar alterations in the expression and production of these chemokines were noted in comparisons of Tg + mice with (+/+) and (–/–) CCR5 loci (Fig. 5, E–I, and data not shown). These studies demonstrate that IL-13 stimulates pulmonary chemokines via CCR5-dependent and -independent pathways with the CCR5 ligands MIP-1{alpha}/CCL-3 and MIP-1beta/CCL-4 and MCP-1/CCL-2 being induced by the former and MCP-2/CCL-8, MCP-3/CCL-7, MCP-5/CCL-12, MIP-2/CXCL-2/3, MDC/CCL-22, eotaxin/CCL-11, eotaxin 2/CCL-24, TARC/CCL-17, TECK/CCL-25, and C10/CCL-6 being stimulated by the latter.


Figure 5
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  		FIGURE 5. Role of CCR5 in IL-13-induced chemokine responses. In A–D, Tg– and Tg+ mice were placed on dox water and treated with anti-CCR5 or control Ig. In E–I, Tg– and Tg+ mice with (+/+) and (–/–) CCR5 loci were evaluated at 3 mo of age. Chemokine mRNA was evaluated by RT-PCR (A and E) and real-time RT-PCR (F). BAL chemokines (B–D and G–I) were evaluated by ELISA. Each evaluation in A and E is representative of a minimum of four similar experiments. The values in the remaining panels represent the mean ± SEM of evaluations in a minimum of four animals (*, p ≤ 0.05).

 
Role of CCR5 in IL-13-induced protease and antiprotease alterations

Because a deficiency of CCR5 could modulate the IL-13-induced inflammatory and alveolar phenotypes by regulating the production of proteases and or antiproteases, studies were undertaken to test this hypothesis. This was done by comparing the levels of mRNA encoding lung-relevant MMPs, cathepsins, and antiproteases in Tg– and Tg+ mice treated with anti-CCR5 or control Ig and Tg+ mice with wild-type and null CCR5 loci. Comparable levels of mRNA encoding MMP-2, MMP-9, MMP-12, MMP-14, cathepsin B, cathepsin H, cathepsin K, cathepsin L, cathepsin S, {alpha}1-antitrypsin, ({alpha}1-AT), tissue inhibitor of metalloproteinases (TIMPs) 1–4, and secretory leukocyte protease inhibitor (SLPI) were noted in lungs from Tg– mice treated with anti-CCR5 or control Ig and Tg– with wild-type and null CCR5 loci (Fig. 6, A and B). In these mice, the levels of mRNA encoding a number of these moieties were near or below the limits of detection of our assays. In accord with previous studies from our laboratory (5), transgenic IL-13 increased expression of these MMPs, cathepsins, and SLPI while inhibiting the expression of {alpha}1-AT (Fig. 6, A and B). Interestingly, the neutralization or ablation of CCR5 decreased the ability of IL-13 to stimulate the accumulation of MMP-9 mRNA (Fig. 6, A–C) and protein (Fig. 6D) and inhibit the expression of {alpha}1-AT (Fig. 6, A and B). It did not alter the effects of IL-13 on the other MMPs, cathepsins, and antiproteases (Fig. 6, A and B). Thus, IL-13 selectively stimulates MMP-9 and inhibits {alpha}1-AT via CCR5-dependent activation pathways.


Figure 6
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  		FIGURE 6. Role of CCR5 in IL-13-induced protease and antiprotease responses. In A, Tg– and Tg+ mice were placed on dox water and treated with anti-CCR5 or control Ig (anti-CCR5–). In B–D, Tg– and Tg+ mice with (+/+) and (–/–) CCR5 loci were evaluated at 3 mo of age. Protease and antiprotease mRNA was evaluated by RT-PCR (A and B) and MMP-9 mRNA and protein were evaluated by real-time RT-PCR (C) and Western blot (D), respectively. The values in C represent the mean ± SEM of evaluations in a minimum of four animals. Each of the other panels is representative of a minimum of four similar experiments.

 
Role of CCR5 in IL-13-induced DNA injury and cell death

In keeping with the proposed role of apoptosis in the pathogenesis of the alveolar remodeling in emphysema (46, 47, 48, 49), TUNEL evaluations were used to compare the DNA injury and cell death in lungs from Tg– and Tg+ mice that had been treated with anti-CCR5 or control Ig and Tg+ mice with (+/+) and (–/–) CCR5 loci. In Tg– mice on normal or dox water, less than or equal to 4% of cells were TUNEL+ (Fig. 7, A and B). In contrast, dox induction of IL-13 caused a significant increase in TUNEL staining in lungs from Tg+ mice (Fig. 7, A and B). After 2 wk of dox, ~22% of the cells were TUNEL+ with staining being readily appreciated in alveolar epithelial cells and, to a lesser degree, in macrophages, lymphocytes, and eosinophils (data not shown). Treatment with anti-CCR5 did not alter these responses in lung from Tg– mice on normal or dox water (Fig. 7, A and B, and data not shown). Treatment with anti-CCR5 did, however, cause a significant decrease in DNA injury and cell death in dox-treated Tg+ animals (Fig. 7, A and B). Similar decreases in DNA injury and cell death were seen with TUNEL evaluations of lungs from Tg+ mice with (+/+) and (–/–) CCR5 loci (Fig. 7, C and D). These studies demonstrate that IL-13 is a potent inducer of DNA injury and apoptosis in the murine lung. They also demonstrate that these responses are mediated by pathways that are, at least partially, CCR5 dependent.


Figure 7
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  		FIGURE 7. Role of CCR5 in IL-13-induced DNA injury and cell death. In A and B, Tg– and Tg+ mice were placed on dox water and treated with anti-CCR5 or control Ig. In C and D, Tg– and Tg+ mice with (+/+) and (–/–) CCR5 loci were evaluated at 3 mo of age. TUNEL evaluations (A and C, x40) and TUNEL quantification (B and D) were undertaken. Each evaluation in A and C is representative of a minimum of four similar experiments. The values in the remaining panels represent the mean ± SEM of evaluations in a minimum of four animals (*, p ≤ 0.05).

 
Role of CCR5 in IL-13-induced cell death

To further understand the mechanism(s) by which CCR5 regulated DNA injury and cell death, the expression and activities of caspases and cell death regulators were evaluated in mice that were treated with anti-CCR5 or control Ig and Tg+ mice with (+/+) and (–/–) CCR5 loci. In Tg– mice, the levels of mRNA encoding FasL, Fas, TNF, TNFR-1, TNFR-2, caspase-3, caspase-8, caspase-9, and Bid were near or below the limits of detection in our assays and were not significantly altered by CCR5 neutralization (Fig. 8, A–C). In contrast, IL-13 was a potent stimulator of the levels of mRNA encoding each of these moieties (Fig. 8, A–C). Western blot and bioactivity evaluations also demonstrated that IL-13 activated caspases-3, -8, and -9, and, as a consequence, increased the accumulation of truncated (t) Bid and the cleavage of the caspase target PARP (Fig. 8, D and E). These events were CCR5 dependent because anti-CCR5 treatment or a null mutation of CCR5 decreased the levels of mRNA encoding these moieties, the levels of caspase-3, -8, and -9 bioactivity and activation, the levels of tBid, and the levels of cleaved PARP (Fig. 8). When viewed in combination, these studies demonstrate that IL-13 is a potent activator of the extrinsic/death receptor and intrinsic/mitochondrial apoptosis pathways and that these activation events are, at least in part, CCR5 dependent.


Figure 8
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  		FIGURE 8. Role of CCR5 in IL-13-regulation of cell death pathways. In A and D, Tg– and Tg+ mice were placed on dox water and treated with anti-CCR5 or control Ig. In B, C, and E, Tg– and Tg+ mice with (+/+) and (–/–) CCR5 loci were evaluated at 3 mo of age. mRNA was evaluated by RT-PCR (A and B) and real-time RT-PCR (C). In D, caspases-3 and -9, Bid, tBid, and PARP are evaluated by Western analysis. In E, caspases-3, -8, and -9 bioactivities were evaluated. Each evaluation in A, B, and D is representative of a minimum of four similar experiments. The values in the remaining panels represent the mean ± SEM of evaluations in a minimum of five animals (*, p ≤ 0.05).

 
Role of CCR5 in IL-13-induced respiratory death

In the CC10-IL-13 mice, progressive lung pathology is noted. As previously reported (7), these mice die prematurely due to a fibrodestructive lung disorder. To determine whether CCR5-dependent pathways play a role in this respiratory failure, we compared the survival of CC10-IL-13 mice with (+/+) and (–/–) CCR5 loci. The Tg+/CCR5 +/+ mice started to die when they were ~100 days old and 100% were dead by the time they were 150 days old. As can be seen in Fig. 9, a deficiency of CCR5 significantly improved the survival of these animals. Overall, Tg+/CCR5–/– mice had a mean survival of 191 days (p < 0.002). Interestingly, Tg+/CCR5+/– mice had an intermediate phenotype (Fig. 9). These studies demonstrate that CCR5 plays a critical role in the pathogenesis of the IL-13-induced pathologies that lead to the death of these animals.


Figure 9
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  		FIGURE 9. Effect of CCR5 deficiency on the survival of IL-13-transgenic mice. Comparisons were made of the survival of IL-13 Tg+ mice with wild-type ({blacksquare}), heterozygous (•), and null ({blacktriangleup}) CCR5 loci. Each point represents the survival of a minimum of six animals.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
To further understand the mechanisms that are used by IL-13 to generate tissue inflammatory and remodeling responses, we used transgenic systems developed in our laboratory to characterize the role(s) of CCR5 in the pathogenesis of IL-13-induced alterations in the lung. These studies demonstrate that IL-13 is a potent stimulator of epithelial cell CCR5 mRNA and its ligands MIP-1{alpha}/CCL3 and MIP-1beta/CCL4. They also demonstrate that CCR5-ligand interactions play an important role in the pathogenesis of IL-13-induced tissue responses because IL-13-induced inflammation and alveolar remodeling were decreased and the survival of IL-13-transgenic mice was significantly increased by immunologic and genetic interventions that blocked or abrogated CCR5. Insights into the mechanisms of these responses were also provided by demonstrating that CCR5 is an integral component of the chemokine, protease, antiprotease, and cell death cascades that IL-13 uses to stimulate MIP-1{alpha}/CCL3, MIP-1beta/CCL4, MCP-1/CCL2, and MMP-9, inhibit {alpha}1-AT, and activate initiator and effector caspases.

CCR5 has been strongly associated with Th1/Tc1 responses (30, 33, 34, 35, 36). CCR5 signaling and/or CCR5-mediated cell recruitment have also been shown to play important roles in the pathogenesis of the type I immune responses in a variety of diseases and disease models including tuberculosis, toxoplasmosis, cryptococcosis, myocardial infarctions, arterial graft rejection, and arthritis (21, 22, 26, 27, 28, 50, 51, 52). In accord with this concept, studies from our laboratory recently demonstrated that the prototypic Th1/Tc1 cytokine, IFN-{gamma} induces CCR5 on pulmonary interstitial cells where it plays a critical role in the pathogenesis of IFN-{gamma}-induced inflammation and remodeling and cigarette smoke-induced pulmonary emphysema (44). In contrast, the present studies demonstrate that CCR5 also plays an essential role in the pathogenesis of the inflammatory and remodeling responses induced by the prominent Th2 effector, IL-13. On superficial analysis, these observations would appear to be mutually contradictory. On deeper evaluation, however, they may instead be complimentary with the role that CCR5 plays in the pathogenesis of IFN-{gamma}-induced and IL-13-induced responses representing different aspects of the true in vivo CCR5 effector repertoire. In accord with this broader view, in addition to Th1 cells, CCR5 is expressed on Th2 cells (37, 38) and stromal cells (25). In addition, the observation that CCR5 signaling plays a key role in IL-13-induced tissue responses is in accord with and provides a potential explanation for previous reports demonstrating that CCR5-based interventions abrogate fungus and aeroallergen-induced Th2 inflammatory responses (39, 40, 53) and the observation that the hypofunctional {delta}32 CCR5 polymorphism is associated with a decreased risk of asthma in select study populations (41, 42).

Chemokines are small, 8- to 10-kDa cytokines that have been subdivided into four supergene families (CXC, CC, C, and CXXC) based on sequence and structural similarities. Although in vitro characterization would suggest that there is impressive redundancy in this system, examinations of an ever-increasing number of ligands in vivo have demonstrated that a deficiency of an individual ligand or its receptor can cause striking alterations in tissue phenotype. This is due, in part, to the organized nature of the chemokine response, which allows the effector functions of specific moieties to be restricted to a specific stage of disease development and/or site of pathology. It has been proposed that the inflammatory and physiologic responses characteristic of Th2 pulmonary inflammation are the result of the coordinated interactions of a variety of CC chemokines including eotaxin/CCL11, MCP-1/CCL2, and MDC/CCL22 (7, 20, 54, 55). Our studies add to our knowledge of this response by demonstrating, for the first time, that IL-13, a major effector at sites of Th2 inflammation, induces MIP-1{alpha}/CCL3 and MIP-1beta/CCL4 while simultaneously enhancing the expression of their receptor CCR5 on epithelial cells and macrophages. They also demonstrate, for the first time, that IL-13 stimulates MCP-1/CCL2 via a CCR5-dependent mechanism. Previously, studies from our laboratory demonstrated that MCP-1/CCL2 and CCR2 play critical roles in the pathogenesis of Th2 and IL-13-induced tissue responses (7, 54). They also demonstrated that IL-13 stimulates MIP-1{alpha}/CCL3 via a mechanism that is, at least in part, CCR1 dependent (20). When viewed in combination, it is tempting to speculate that the ameliorative effects of CCR5 neutralization and/or ablation on IL-13-induced tissue responses are due, at least in part, to the decreased ability of IL-13 to stimulate MCP-1 in this setting. It is also tempting to speculate that optimal IL-13-inducted tissue responses require the coordinated interaction of CCR1, CCR2, and CCR5 activated signaling pathways.

The ability of IL-13 to induce alveolar enlargement, lung enlargement, and compliance alterations was decreased in mice treated with anti-CCR5 and mice with null mutations of CCR5. These findings were due to alterations in effector pathway activation because there were similar levels of IL-13 in BAL fluids from Tg+ mice treated with anti-CCR5 or the control Ab and Tg+ mice with wild-type or null mutant CCR5 loci. Previous studies from our laboratory demonstrated that IL-13 causes lung and alveolar enlargement and compliance alterations via a mechanism(s) that involves MMPs and {alpha}1-AT (5, 7, 17, 20). In accord with these findings, the present studies demonstrate that IL-13 stimulation of MMP-9 and IL-13 inhibition of {alpha}1-AT are mediated via CCR5-dependent mechanisms. Thus, it is tempting to speculate that the decrease in IL-13-induced alveolar remodeling that was seen after CCR5 neutralization and/or abrogation is mediated, at least in part, by these CCR5-dependent protease and antiprotease alterations.

Cell death can be induced by a complex series of triggers and pathways. However, the majority of the signals engage the cell death machinery at the level of the cell membrane or at the level of the mitochondria. The membrane (extrinsic) pathway triggers "death receptors" which subsequently activate caspase-8. In the intrinsic mitochondrial response, BH3 domain-only family members such as Bid are activated to tBid and interact with Bax-type proteins to form or interact with mitochondrial pores releasing cytochrome c, activate caspase-9, and induce cell death (56). Our studies demonstrate that IL-13 is a potent stimulator of a TUNEL+ cell death response and that this induction is partially dependent on CCR5. To further understand the mechanism(s) by which CCR5 regulates this IL-13-induced cell death response, we characterized the intrinsic and extrinsic pathways in mice treated with anti-CCR5 or control Ig and mice with wild-type and a null CCR5 loci. These studies demonstrate that IL-13 activates both the intrinsic and extrinsic cell death pathways. They also demonstrate that CCR5 contributes to this response in a variety of ways including regulating the levels of mRNA encoding Fas, TNF, TNFR1, TNFR2, the activation of Bid and the levels of expression, activation, and bioactivity of caspases-3, -8, and -9. These are the first studies to demonstrate that IL-13 is a potent stimulator of apoptosis in the murine lung and the first to demonstrate that CCR5 plays a key role in IL-13 activation of both the death receptor and mitochondrial cell death pathways. These observations, however, are not without precedent because CCR5 has been shown to be a coreceptor for HIV where it activates Fas and caspase-8 and induces CD4 cell death (57) and IL-13 is known to stimulate epithelial cell apoptosis at sites of gastrointestinal pathology (58, 59). It is clear from these studies that CCR5 plays a critical role in and is a multifunctional regulator of IL-13-induced death responses in the lung.

Because IL-13 stimulated MIP-1{alpha}/CCL3 and MIP-1beta/CCL4 while inducing CCR5-dependent tissue remodeling, it is reasonable to speculate that the binding of these ligands to CCR5 plays a critical role(s) in the pathogenesis of these CCR5-mediated tissue responses. In accord with this speculation, MIP-1{alpha}/CCL3 and MIP-1beta/CCL4 can stimulate monocyte-like cell and lymphocyte MMP-9 production (60, 61) and MIP-1{alpha}/CCL3 plays a critical role in neuronal apoptosis in models of CNS neurodegeneration (62). Our studies add to our understanding of the biology of these chemokines by highlighting their unique roles in IL-13-induced alveolar remodeling and alveolar epithelial apoptosis. Additional investigation will be required, however, to define the relative contributions of each chemokine moiety in these complex responses.

In summary, our studies demonstrate that IL-13 is a potent stimulator of CCR5 and its ligands and that signaling via CCR5 is an important event in the pathogenesis of IL-13-induced inflammation, tissue remodeling, cell death, and survival. They also demonstrate the CCR5 plays an important role in IL-13 induction of chemokines and proteases, IL-13 inhibition of {alpha}1-AT and IL-13 activation of death receptor and mitochondrial cell death pathways. Exaggerated IL-13 production has been implicated in the pathogenesis of a variety of disorders including asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, scleroderma, hepatic fibrosis, ulcerative colitis, and nodular-sclerosing Hodgkin’s disease. The present studies suggest that the effects of IL-13 in these disorders may be beneficially controlled via interventions that control the expression and/or signaling of CCR5. This establishes the CCR5 pathway as a worthy site for future investigations to identify therapeutic agents that can be used to treat these and other IL-13-mediated disorders.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the National Institutes of Health Grants HL 56389, HL 064242, HL 66571, and HL 078744 (to J.A.E.). Back

2 Address correspondence and reprint requests to Dr. Jack A. Elias, Section of Pulmonary and Critical Care Medicine, Yale University School of Medicine, 300 Cedar Street (S441 TAC), P.O. Box 208057, New Haven, CT 06520-8057. E-mail address: jack.elias{at}yale.edu Back

3 Abbreviations used in this paper: MMP, matrix metalloproteinase; Tc, T cell; rtTA, reverse tetracycline transactivator; dox, doxycycline; tBid, truncated Bid; BAL, bronchoalveolar lavage; PARP, poly(ADP)ribose polymerase; MDC, macrophage-derived chemokine; TARC, thymus and activation-regulated chemokine; TECK, thymus-expressed chemokine; {alpha}1-AT, {alpha}1-antitrypsin; TIMP, tissue inhibitor of metalloproteinase; SLPI, secretory leukocyte protease inhibitor 1. Back

Received for publication December 9, 2005. Accepted for publication January 30, 2006.


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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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