The Role of the Th2 CC Ligand CCL17 in Pulmonary Fibrosis John A. Belperio, Maria Dy, Lynne Murray, Marie D. Burdick, Ying Y. Xue, Robert M. Strieter and Michael P. This information is current as Keane of October 2, 2021. J Immunol 2004; 173:4692-4698; ; doi: 10.4049/jimmunol.173.7.4692 http://www.jimmunol.org/content/173/7/4692 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The Role of the Th2 CC Chemokine Ligand CCL17 in Pulmonary Fibrosis1

John A. Belperio, Maria Dy, Lynne Murray, Marie D. Burdick, Ying Y. Xue, Robert M. Strieter, and Michael P. Keane2

Increasing evidence suggests that the development of pulmonary fibrosis is a Th2-mediated process. We hypothesized that the CC that are associated with a Th2 profile (CCL17 and CCL22) have an important role in the development of pulmonary fibrosis. We measured CCL17 and CCL22 during the pathogenesis of bleomycin-induced pulmonary fibrosis. We found that both CCL17 and CCL22 were significantly elevated through day 20 as compared with control mice. Peak expression of CCL22 preceded the peak levels of CCL17, as measured by real-time quantitative PCR. CCR4 is the receptor for CCL17 and CCL22 therefore, to further characterize the role of CCL17 and CCL22, we measured CCR4 mRNA in lung tissue of bleomycin-treated mice by real-time quantitative PCR. CCR4 was significantly elevated in bleomycin-treated mice Downloaded from as compared with control mice. Immunolocalization demonstrated that CCR4 was expressed predominantly on . Neutralization of CCL17, but not CCL22, led to a reduction in pulmonary fibrosis. Immunolocalization of bleomycin-treated lung tissue and human idiopathic pulmonary fibrosis tissue specimens showed that epithelial cells expressed CCL17. These findings demonstrate a central role for Th2 chemokines and the in the pathogenesis of pulmonary fibrosis and are further support for the role of a Th2 phenotype in the pathogenesis of pulmonary fibrosis. The Journal of Immunology, 2004, 173: 4692Ð4698. http://www.jimmunol.org/

diopathic pulmonary fibrosis (IPF)3 is a devastating dis- There is evidence suggesting that the profile of the ease with a median survival of 3Ð5 years and objective natural immune/inflammatory response determines the disease I response rates of Ͻ30% to conventional treatment. De- phenotype responsible for either resolution or progression to end- spite these dismal response rates the standard of care remains stage fibrosis. Supporting evidence is derived from studies dem- corticosteroids either with or without a second immunosuppres- onstrating that IFNs, especially IFN-␥, have profound suppressive sive agent (1). This treatment strategy reflects our limited un- effects on the production of extracellular matrix , such as derstanding of the immune mechanisms and mediators involved collagen and fibronectin (10, 11). The administration of IFN-␥ in in the pathogenesis of pulmonary fibrosis, as well as the lack of vivo can cause a reduction of extracellular matrix in animal models by guest on October 2, 2021 truly efficacious agents to delay the progression of the disease. of fibrosis (10Ð12). Furthermore we have shown that IL-12 atten- Inflammatory cell recruitment is associated with initiation of uates bleomycin-induced pulmonary fibrosis in an IFN-␥-depen- the pathogenesis of interstitial lung disease (2). In an attempt to dent manner (13). In contrast the Th2 , IL-4 and IL-13, understand the pathogenesis of pulmonary fibrosis, animal mod- induce the expression of fibroblast-derived type I and III procol- els have been used. Bleomycin-induced pulmonary injury lagens in a similar magnitude as TGF-␤ (14). Similarly IL-13 has evokes a route and dose-dependent pulmonary inflammatory re- been shown to induce fibrosis by selectively stimulating produc- sponse characterized by increases in mononuclear cells, gran- tion and activation of TGF-␤ (15). We have shown that IL-13 ulocytes, fibroblast proliferation, and collagen synthesis (3, 4). promotes bleomycin-induced fibrosis, in part, through the elabo- Studies using the bleomycin model of pulmonary fibrosis have ration of CCL6 (9). Neutralization of IL-13 lead to a reduction in demonstrated the presence and contribution of CC chemokines fibrosis and macrophage numbers (9). Similar use of an IL-13 im- in the pathogenesis of fibrosis (5Ð9). munotoxin chimeric molecule that antagonizes the effect of IL-13 lead to a reduction in bleomycin-induced pulmonary fibrosis (16). Chemokines and their receptors are essential components of Th1- and Th2-mediated responses (17). CXCR3 and CCR5 are Division of Pulmonary and Critical Care Medicine, David Geffen School of Medicine, University of California, Los Angeles, CA 90095 mainly expressed on Th1 cells whereas CCR3, CCR4, and CCR8 Received for publication December 22, 2003. Accepted for publication August are more characteristic of Th2 cells (17, 18). These findings dem- 2, 2004. onstrate that chemokines are important in the amplification and The costs of publication of this article were defrayed in part by the payment of page polarization of the immune response. There is increasing evidence charges. This article must therefore be hereby marked advertisement in accordance that pulmonary fibrosis is predominantly a Th2-mediated process. with 18 U.S.C. Section 1734 solely to indicate this fact. Therefore we hypothesized that CCL17 and CCL22 acting via 1 This work was supported in part by National Institutes of Health Grants P50HL67665 and HL03906 (to M.P.K.), HL04493 (to J.A.B.), and P50HL67665, CCR4 would have an important role in the pathogenesis of pul- HL66027, and CA87879 (to R.M.S.). monary fibrosis. In this study we demonstrate that CCL17 has an 2 Address correspondence and reprint requests to Dr. Michael P. Keane, Department important role in the development of pulmonary fibrosis via the of Medicine, Division of Pulmonary and Critical Care Medicine, University of Cal- recruitment of lymphocytes and macrophages. The predominant ifornia, 900 Veteran Avenue, 14-154 Warren Hall, Los Angeles, CA 90095-1922. E-mail address: [email protected] cell expressing CCR4 was the macrophage and neutralization of 3 Abbreviations used in this paper: IPF, idiopathic pulmonary fibrosis; BAL, bron- CCL17 led to reduced numbers of intrapulmonary macrophages as choalveolar lavage; PDGF, platelet-derived . well as lymphocytes.

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 The Journal of Immunology 4693

Materials and Methods and amplified using TaqMan Reverse Transcription Reagents (PE Applied Reagents Biosystems, Foster City, CA). To exclude amplification of genomic DNA, samples were also run in the absence of reverse transcription. Real-time Polyclonal neutralizing anti-CCL22 and anti-CCL17 Abs and polyclonal quantitative PCR was performed using the ABI Prism 7700 Sequence De- goat IgG were purchased from R&D Systems (Minneapolis, MN). Neu- tector and SDS analysis software (PE Applied Biosystems). For CCL17, tralizing capacity was tested by the ability to inhibit chemotaxis of CEM- CCL22 and CCR4 and 18S TaqMan Pre-Developed Assay Reagents (PE NKR cells to CCL17 or CCL22. Anti-CCR4 Ab was purchased from R&D Applied Biosystems) were used for amplification. Reactions were assem- Systems. Anti-Mac-3 Ab was purchased from BD Pharmingen (San Diego, bled in 96-well reaction plates using TaqMan Universal PCR Master Mix CA). The “antiprotease” buffer for tissue homogenization consisted of 1ϫ (2ϫ); the appropriate Pre-Developed Assay Reagent; and template PBS with one Complete tablet (Boehringer Mannheim, Indianapolis, IN) (cDNA). PCR was performed under the following conditions: 50¡C for 2 per 50 ml. 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. Human tissue specimens Quantitative analysis of expression was done using the comparative C (⌬C ) methods, in which C is the threshold cycle number (the mini- Human tissue specimens were obtained from consenting individuals in ac- T T T mum number of cycles needed before the product can be detected) (23). cordance with institutional review board approval. All had clinical and The arithmetic formula for the ⌬C method is described as the difference radiographic findings consistent with the diagnosis of IPF, and all had T in threshold cycles for a target (i.e., CCL17 or CCL22) and an endogenous pathologic confirmation of the diagnosis of usual interstitial pneumonia reference (i.e., our housekeeping gene 18S). The amount of target normal- made by open lung biopsy. None of the patients had been previously ized to an endogenous reference (i.e., CCL22, CCL17 or CCR4 in bleo- treated or were currently being treated with corticosteroids or other immu- mycin-treated mice) and relative to a calibration normalized to an endog- nosuppressive agents. enous reference (i.e.; CCL17, CCL22, or CCR4 in saline-treated mice) is Ϫ⌬⌬ Ϫ⌬⌬ Animal model of pulmonary fibrosis given by 2 CT (23). The calculation of 2 CT then gives a relative value when comparing the target to the calibrator, which we designate in Female CBA/J mice (6Ð8 wk) were purchased from The Jackson Labo- this context as fold increase in bleomycin-treated mice as compared with Downloaded from ratory (Bar Harbor, ME). We used these strains of mice as they are a saline-treated controls (9). well-characterized inbred strain of mice that are susceptible to bleomycin- induced pulmonary fibrosis. Mice were maintained in specific pathogen- Immunohistochemistry free conditions and provided with food and water ad libitum. To induce Paraffin-embedded tissue from control and bleomycin-treated lung was pulmonary fibrosis, mice were treated with intratracheal bleomycin processed for immunohistochemical localization of CCR4, CCL17, or (Blenoxane, a gift from Bristol Myers, Evansville, IN) (1 U/kg) on day 0 macrophages (Mac-3), using a method as previously described (21). Sim-

as previously described (9, 19, 20). Control animals received only sterile http://www.jimmunol.org/ ilarly, paraffin-embedded tissue from human IPF or control lung was pro- saline as previously described (9, 19, 20). Briefly, mice were anesthetized cessed for immunohistochemical localization of CCL17 as previously de- with ketamine injected i.p. followed by intratracheal instillation of 1 U/kg scribed (24, 25). Briefly, tissue sections were dewaxed with xylene, and bleomycin in 25 ␮l sterile isotonic saline. At various time points postin- rehydrated through graded concentrations of ethanol. Tissue nonspecific stillation, animals were euthanized, and both lungs were removed for ho- binding sites were blocked using normal goat serum (BioGenex Labora- mogenization as described below. In separate experiments bleomycin- tories, San Ramon, CA). Tissue sections were overlaid with 1/500 dilution treated mice were passively immunized by i.p. injection with 4 ␮gof of either control (rabbit) or polyclonal (rabbit) anti-CCL17, CCL22, or anti-CCL17, anti-CCL22, or 4 ␮g of polyclonal goat IgG as a control, daily CCR4 Abs (R&D Systems) or control (rat) or polyclonal (rat) anti-Mac-3 until day 8 before maximal fibrosis. All studies were approved by the (BD Pharmingen) Abs. The tissue sections were washed with Tris-buffered University of California, Los Angeles, CA, institutional animal care and saline and then incubated for 60 min with secondary goat anti-rabbit bio- use committee. tinylated Abs (BioGenex Laboratories) or goat anti-rat biotinylated Abs. by guest on October 2, 2021 Lung tissue preparation The tissue sections were then washed in Tris-buffered saline and incubated with alkaline phosphatase conjugated to streptavidin (BioGenex Labora- Bleomycin- or saline-treated (control) lungs were homogenized and soni- tories). Tissue sections were then incubated with Vectastain ABC reagent cated in antiprotease buffer using a method as previously described (9, 19, (Vector laboratories, Burlingame, CA) followed by the peroxidase sub- 20). Specimens were centrifuged at 900 ϫ g for 15 min, filtered through 1.2 strate, DAB reagent (Vector Laboratories, Burlingame, CA). After optimal ␮m Sterile Acrodiscs (Gelman Sciences, Ann Arbor, MI), and frozen at color development, tissue sections were immersed in sterile water, coun- Ϫ70¡C until thawed for assay by specific ELISA or collagen assay. Ad- terstained with Lerners hematoxylin, and cover slipped using an aqueous ditionally, some lungs were fixed in 4% paraformaldehyde and embedded mounting solution. in paraffin for immunohistochemical analysis. Collagen assay Chemokine ELISA Total lung collagen was measured using the Sircol Collagen assay (Bio- CCL17 and CCL22 were quantitated using an ELISA as previously de- color, Belfast, Northern Ireland) according to manufacturers instructions scribed (21, 22). Briefly, flat-bottom 96-well microtiter plates (Nunc, Rosk- and as previously described (26Ð28). Briefly, lungs were harvested and ilde, Denmark) were coated with 50 ␮l/well of the appropriate polyclonal homogenized as described earlier. Sircol dye (1000 ␮l) was added to 100 ␮ Ab (1 ng/ l in 0.6 M NaCl, 0.26 M H3B04, and 0.08 N NaOH, pH 9.6) for ␮l of collagen standard or test samples (lung homogenate) and placed on 24hat4¡C and then washed with PBS, and 0.05% Tween 20 (wash buffer). a mechanical rocker for 30 min. Samples were then centrifuged at 5000 ϫ Nonspecific binding sites were blocked with 2% BSA. Plates were rinsed g for 5 min to pack the collagen dye complex at the bottom of the tube. and samples were added (50 ␮l/well), followed by incubation for1hat Unbound dye solution was then removed and 1 ml of a 0.5 M NaOH 37¡C. Plates were then washed and 50 ␮l/well of the appropriate biotin- solution was added to the remaining pellet of collagen-bound dye. Follow- ylated polyclonal Ab (3.5 ng/␮l in wash buffer, and 2% FCS) added for 45 ing solubilization, samples were transferred to a 96-well plate and read at min at 37¡C. Plates were washed three times, streptavidin-peroxidase con- 540 nm on a Beckman DU-640 spectrophotometer (Fullerton, CA) jugate (Bio-Rad Laboratories, Richmond, CA) added, and the plates incu- bated for 30 min at 37¡C. Chromogen substrate (DAKO, Carpinteria, CA) Assessment of infiltrating pulmonary leukocytes was then added, and the plates were incubated at room temperature to the Bronchoalveolar lavage (BAL) was performed to obtain cells for FACS desired extinction. Plates were read at 490 nm in an automated microplate analysis of leukocyte subsets. The trachea was exposed and intubated with reader (Bio-Tek Instruments, Winooski, VT). Standards were log(1/2) di- a 1.7-mm outer diameter polyethylene catheter. BAL was performed by lutions of recombinant chemokine (50 ␮l/well). Sensitivity of the assay Ͼ instilling PBS containing 5 mM EDTA in 1 ml aliquots. Three milliliters was 50 pg/ml for each chemokine. Our ELISA was highly specific with- of PBS were instilled per mouse. Cell counts and viability were determined out cross-reactivity to a panel of cytokines, including IL-1Ra, IL-1, IL-2, ␣ ␥ using trypan blue exclusion on a hemocytometer. Single cell suspensions IL-4, IL-6, TNF- , IFN- , CXC chemokine ligand 9, and other members were stained with anti-CD45 Tri-Color (Caltag Laboratories, South San of the CXC and CC chemokine families. Francisco, CA) to allow gating on CD45-positive cells for analysis of leu- Real-time quantitative PCR for cytokine gene expression kocytes. For analysis of lung leukocyte cell populations the following Abs directly conjugated to FITC or PE (BD Pharmingen) were used, CD3, CD4, Total lung RNA was extracted from mouse lungs using TRIzol reagent CD8, NK1.1, Gr-1, and Moma-1. Cells were analyzed on a FACScan flow (Invitrogen Life Technologies, Grand Island, NY) according to manufac- cytometer (BD Biosciences, San Jose, CA) using CellQuest software (BD turer’s instructions. Total RNA (1 ␮g) was reversed transcribed into cDNA Biosciences). 4694 CCL17 AND PULMONARY FIBROSIS

Fibroblast Proliferation bleomycin-treated mice or saline-treated controls. Using quantita- Murine lung fibroblasts were cultured as previously described (19, 24). tive PCR we found that CCL17 was significantly elevated at days Briefly, murine pulmonary fibroblasts were grown to 80% confluence and 2, 8, 12, and 20 as compared with saline-treated controls (Fig. 1A). passaged. At the time of the fourth passage, pulmonary fibroblast purity Furthermore lung tissue from bleomycin-treated mice demon- Ͼ was 99% as determined by the absence of nonspecific esterase, Factor strated greater levels of CCL17 as assessed by ELISA (Fig. VIII-related Ag, or cytokeratin immunostaining. The cells were Ͼ90% positive for vimentin, laminin, and fibronectin and were Ͼ90% negative 1B). Similarly using quantitative PCR we found that CCL22 was for ␣-smooth muscle actin and desmin. This technique allowed the estab- significantly elevated at days 2, 4, 8, 12, and 20 (Fig. 1C). Lung lishment of murine pulmonary fibroblast cell lines. On the day of use, tissue from bleomycin-treated mice also demonstrated greater lev- pulmonary fibroblasts were plated out in 96-well plates at a concentration els of CCL22 protein as assessed by ELISA (Fig. 1D). Interest- of 5000 cells/well and allowed to adhere overnight. Fibroblasts were then washed free of serum and cultured for 24 h under serum-free conditions. At ingly, peak levels of CCL17 as compared with CCL22 were 40- 24 h 5% FCS was again added along with varying concentrations of fold greater as measured by ELISA (Fig. 1, B and D) CCL22, CCL17, or platelet-derived growth factor (PDGF), and fibroblasts were cultured for a further 72 h. [3H]Thymidine was added 12 h before harvesting using a cell harvester (Brandel, Gaithersburg, MD). [3H]Thy- midine incorporation was assessed using a Beckman LS 1801 scintillation CCR4 is elevated in bleomycin-induced pulmonary fibrosis counter (Beckman Instruments, Schaumburg, IL) and expressed as cpm. Because CCL17 and CCL22 share a common receptor, CCR4, we Statistical analysis next assessed lung tissue from bleomycin-treated or saline-treated control mice at each time point for the presence of CCR4 by real- Data were analyzed on a Dell computer using the Statview 5.0 statistical time PCR. Lung tissue from bleomycin-treated animals demon- package (Abacus Concepts, Berkeley, CA). Comparisons were made using Downloaded from the unpaired t test. Data were considered statistically significant for values strated greater levels of CCR4, as compared with saline-treated p Յ 0.05. controls at days 2, 4, 8, 12, and 20 (Fig. 2). These findings sug- gested that CCR4 might be playing a role in the pathogenesis and Results the development of pulmonary fibrosis. To further characterize the Lung tissue from mice treated with bleomycin has increased role of CCR4, we performed immunolocalization for CCR4 in levels of CCL22 and CCL17 bleomycin-treated lung tissue. As shown in Fig. 3, CCR4 is pre- We measured CCL17 and CCL22 in the bleomycin-induced pul- dominantly expressed on macrophages. This localization to mac- http://www.jimmunol.org/ monary fibrosis model. Total RNA was isolated from lungs of rophages was confirmed with staining for Mac-3 (Fig. 3). by guest on October 2, 2021

FIGURE 1. CCL17 and CCL22 are significantly elevated in bleomycin-treated as compared with saline-treated controls. A, Time course of CCL17 expression as measured by real-time quantitative PCR from lung tissue of mice exposed to either intratracheal bleomycin or saline control at day 0. Data are presented as fold increase in bleomycin-treated lung tissue as compared with saline-treated controls (n ϭ 6 in each group). B, Time course shown of CCL17 production in lung tissue of mice exposed to either intratracheal bleomycin or saline control at day 0. CCL17 was measured by specific ELISA from lung tissue and normalized to nanograms per lung (n ϭ 6 in each group). C, Time course of CCL22 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 are presented as fold increase in bleomycin-treated lung tissue as compared with saline-treated controls (n ϭ 6 in each group). D, Time course of CCL22 production in lung tissue of mice exposed to either intratracheal bleomycin or saline control at day 0. CCL22 was measured by specific ELISA from lung tissue and normalized to ng per lung (n ϭ 6 in each group). The Journal of Immunology 4695

FIGURE 2. Time course of CCR4 expression as measured by real-time quantitative PCR from lung tissue of mice exposed to either intratracheal bleomycin or saline control at day 0. Data are presented as fold increase in bleomycin-treated lung tissue as compared with saline-treated controls (n ϭ 6 in each group).

FIGURE 4. Lung collagen levels at day 16 from mice administered in- Neutralization of CCL17 but not CCL22 attenuates tratracheal bleomycin (day 0) and treated with either anti-CCL17, anti- CCL22 Abs, or control Abs. Abs were administered by daily i.p. injection bleomycin-induced pulmonary fibrosis until day 8. Saline-treated mice received no Ab (n ϭ 6 lungs in each Downloaded from To further investigate the role that CCL17 and CCL22 were group). playing in the pathogenesis of bleomycin-induced pulmonary fibrosis we next assessed whether neutralization of CCL17 or CCL22 by passive immunization would attenuate pulmonary nization with neutralizing anti-CCL17 Abs led to a significant reduc- fibrosis. Passive immunization with specific neutralizing Abs to tion in CD3, CD4, and CD8 lymphocytes, NK cells, macrophages, CCL17 every 24 h for 8 days led to a significant reduction in and neutrophils, as compared with controls, at day 8. (Fig. 5) pulmonary fibrosis, as compared with control Ab-treated mice http://www.jimmunol.org/ as assessed by collagen levels (Fig. 4). Interestingly, using a CCL17 does not stimulate fibroblast proliferation similar strategy of neutralization of CCL22 had no effect on Having shown that anti-CCL17 reduces fibrosis we were next in- pulmonary fibrosis (Fig. 4). Neither anti-CCL17 nor anti- terested to see whether CCL17 had any direct effect on pulmonary CCL22 have any effect on collagen content in the lungs of un- fibroblasts. Pulmonary fibroblasts were isolated from CBA/J challenged mice (data not shown). mouse lungs and stimulated with various concentrations of CCL17, whereas PDGF was used as a positive control. Prolifera- Passive immunization with neutralizing CCL17 Abs is associated tion at 72 h was measured using incorporation of tritiated thymi- with reduced BAL leukocyte and macrophage numbers dine. As shown in Fig. 6, CCL17 had no effect on fibroblast pro- by guest on October 2, 2021 Having shown that neutralization of CCL17 attenuates bleomycin- liferation. Furthermore we found no evidence of CCR4 mRNA induced fibrosis, we subsequently set out to determine the potential expression from isolated fibroblasts as assessed by real-time PCR. mechanisms for this effect. Single cell suspensions were prepared These findings are further evidence for a role for CCL17 in the from BAL fluid of bleomycin-treated mice who had received either pathogenesis of bleomycin-induced pulmonary fibrosis other than anti-CCL17 Abs or control Abs as previously described. Cellular through an effect on fibroblast proliferation, specifically leukocyte populations were studied by FACS analysis at day 8. Passive immu- and macrophage recruitment.

FIGURE 3. Representative photomicrograph of the immunolocalization of CCR4 and macro- phages in lung tissue at day 16 postintratracheal bleomycin administration (magnification, ϫ312). A, Bleomycin-treated lung tissue immunostained for CCR4 demonstrating localization to macro- phages is shown. B, Same lung specimen immuno- stained with control Abs demonstrating the lack of nonspecific staining. C) Same lung specimen im- munostained with the macrophage specific marker Mac-3. D, Same lung specimen immunostained with control Abs demonstrating the lack of nonspe- cific staining. 4696 CCL17 AND PULMONARY FIBROSIS

FIGURE 6. Murine pulmonary fibroblast proliferation at 72 h in re- sponse to media alone (control), platelet-derived growth factor (PDGF, 200 FIGURE 5. FACS analysis of BAL leukocyte populations at day 8 in ng/ml), CCL17 (1, 10, 50 and 100 ng/ml). [3H]Thymidine was added to the saline-treated mice or bleomycin-treated mice that received either anti- cultures 12 h before fibroblast harvest. [3H]Thymidine incorporation was CCL17 or control Abs. Bleomycin or saline was administered at day 0 by expressed as cpm. Data represent mean Ϯ SEM, n ϭ minimum of six in intratracheal injection. Abs were administered by daily i.p. injection until each group. day 8. Saline-treated mice received no Ab (n ϭ 6 lungs in each group). Downloaded from pathogenesis of IPF, or may be related to the inability of IFN-␥ to Immunolocalization of CCL17 is predominantly associated with tilt the balance away from Th2-dependent profibrotic environment. epithelial cells in bleomycin-induced fibrosis and human IPF In further support of an imbalance of the presence of Th2 cyto- lung tissue kines, as compared with IFN-␥,isthefinding that IFN-␥ levels are Having demonstrated an important role for CCL17 in bleomycin- inversely related to the levels of type III procollagen in the BAL

induced pulmonary fibrosis we were next interested to know what fluid of IPF patients (32). http://www.jimmunol.org/ the predominant cellular source of CCL17 was in the lungs of mice Chemokines and their receptors are essential components of treated with bleomycin. Furthermore we were interested to know Th1- and Th2-mediated responses (17). CXCR3 and CCR5 are whether CCL17 was expressed in human IPF lung tissue and if so, mainly expressed on Th1 cells whereas CCR3, CCR4, and CCR8 what was the predominant cellular source. Using immunohisto- are more characteristic of Th2 cells (17, 18). We found elevated chemistry, we found that the predominant cells in bleomycin- levels of the Th2 chemokines, CCL17 and CCL22, and their treated lungs and IPF lung tissue that expressed CCL17 were ep- shared receptor, CCR4, in the bleomycin model of pulmonary fi- ithelial cells (Figs. 7 and 8). brosis. This supports a role for these CC chemokines in the de- velopment of fibrosis. Previous researchers have shown that Discussion CCL17 is elevated in BAL fluid of patients with eosinophilic pneu- by guest on October 2, 2021 The natural history and sequence of events that dictate the patho- monia that correlated with the presence of IL-4, IL-5, and IL-13 in genesis of pulmonary fibrosis is not well characterized. Numerous BAL fluid (33). Similarly Katoh et al. (34) have shown that factors that regulate immune and inflammatory responses have CCR4ϩCD4ϩ T cells in BAL fluid from patients with eosinophilic been implicated in the pathogenesis of pulmonary fibrosis. Regard- pneumonia correlated with BAL fluid levels of CCL17, CCL22, less of the initial inciting agent, the hallmark of pulmonary fibrosis and IL-5. This suggests an important role for CCL17 in the re- is chronic inflammation and deposition of extracellular matrix. The cruitment of Th2 cells to the lung. Further support for this notion phenotype of this chronic inflammation appears to be highly as- is seen in the report of increased expression of CCR4 on lympho- sociated with a Th2 phenotype of cytokine expression (29). cytes in the airway mucosa of atopic asthmatics (35). This was Th1 and Th2 cytokines are expressed by a variety of cells and associated with the expression of CCL17 and CCL22 in airway the function of these cytokines are different, suggesting that an epithelial cells upon allergen challenge (35). Furthermore, CCL22 imbalance in the expression of Th1 and Th2 cytokines may be has been shown to be inducible in bronchial epithelial cells (36). important in dictating different immunopathological responses (30, Similarly we found that CCL17 localized to the epithelium both in 31). Although there is a pattern for the existence of both Th1 the bleomycin model and IPF tissue specimens. Interestingly it has (characterized by the expression of IFN-␥) and Th2 cytokines in been shown that CCR4-expressing lymphocytes were lacking in IPF lung tissue, the presence of Th2 cytokines predominated over the airways of patients with sarcoidosis (35). Sarcoidosis is con- the expression of IFN-␥ (29). This pattern of cytokine expression sidered to be a predominantly Th1-mediated disease, and T cells may be related to the potential role for the humoral response in the infiltrating the airways of patients with sarcoidosis were found to

FIGURE 7. Photomicrograph showing im- munolocalization of CCL17 from bleomycin- treated lung tissue specimens. A, Bleomycin- treated lung section (magnification, ϫ312) stained with anti-CCL17 Abs demonstrating immunolocalization to epithelial cells. B, Bleomycin-treated lung section (magnifica- tion, ϫ312) stained with control Abs demon- strating the lack of nonspecific staining. The Journal of Immunology 4697

FIGURE 8. Photomicrograph showing immunolocalization of CCL17 from IPF lung tissue specimens. A, IPF lung section (magnification, ϫ312) stained with anti-CCL17 Abs demonstrating immunolocalization to epithelial cells. B, IPF lung section (magnification, ϫ312) stained with control Abs demon- strating the lack of nonspecific staining.

express IFN-␥ and CXCR3 (35). In a model of chronic fungal in T cell-deficient nude mice as compared with euthymic mice, Downloaded from asthma, the absence of CCR4 was associated with increased clear- although these findings were in contrast to an earlier study show- ance of Aspergillus and the attenuation of many of the Th2 cyto- ing similar fibrosis in nude and euthymic mice (44). Resistant kine-associated features of this particular disease with decreased BALB/c mice that were pretreated with cyclophosphamide were airway responsiveness at day 30 (37). There was however no dif- more susceptible to bleomycin than control mice, and this could be ference in the airway remodeling that is seen with chronic As- overcome with the administration of T cells from control mice pergillus infection (37). (45). Depletion of CD4 or CD8 lymphocytes has been shown to http://www.jimmunol.org/ Neutralization of CCL17 reduced pulmonary fibrosis. Interest- attenuate bleomycin-induced pulmonary fibrosis (46). Interest- ingly neutralization of CCL22 had no effect on fibrosis. This might ingly, combined depletion of CD4 and CD8 lymphocytes has a seem surprising given that both CCL22 and CCL17 bind to CCR4. greater than additive benefit over depletion of either subset alone However peak gene expression and protein levels of CCL17 were (46). One of the reasons for the apparent disparities among these 10- and 40-fold higher, respectively, than CCL22. This would sug- different studies may be the role of T regulatory cells. There is gest that in this model of pulmonary fibrosis CCL17 is playing a increasing evidence for the presence of regulatory T cells in both more important role than CCL22. Another possibility is that the CD4 and CD8 populations and selective depletion of one or CCL17 is acting independently of CCR4. Currently there is no more subsets may have differing effects (47). by guest on October 2, 2021 evidence of an alternative receptor for CCL17. Neutralization of In summary, we have shown that in the context of bleomycin- CCL17 was associated with a decrease in inflammatory cells in- induced pulmonary fibrosis there is increased expression of cluding macrophages. Furthermore macrophages were the predom- CCL17, CCL22, and CCR4. CCR4 is highly expressed on mac- inant cells expressing CCR4. This supports the notion that the rophages. Depletion of CCL17 attenuated fibrosis and pulmonary macrophage is a key inflammatory cell in the development of fi- inflammatory cell numbers. Furthermore, we have shown that brosis. In human IPF lung tissue it has been shown that insulin like CCL17 is localized to the epithelium in both the bleomycin model growth factor-1 immunolocalizes to alveolar macrophages and in- and human IPF lung tissue suggesting an important role for the terstitial macrophages (38). The degree of clinical impairment and epithelium in the pathogenesis of pulmonary fibrosis. It has re- collagen deposition in the interstitium correlated with the degree of cently been suggested that epithelial injury may have an important staining of CD68-positive interstitial macrophages (38). These role in the pathogenesis of IPF (48, 49). Our findings support an findings support a role for interstitial macrophages in the patho- integrated role for the epithelium, inflammatory cells, and Th2 genesis of IPF. Furthermore, there is increasing evidence that mac- type chemokines in the pathogenesis of pulmonary fibrosis. At- rophages can polarize along Th1 or Th2 pathways (39, 40). Our tempts at switching the fibrotic phenotype toward a Th1 response results showing that CCR4 is significantly expressed on macro- may be more effective than targeting individual ligands or phages would suggest that this might be true in pulmonary fibrosis. receptors. The expression of CCL17 in epithelial cells in bleomycin-treated lungs and IPF suggests that the epithelium has an important role in the recruitment of macrophages in IPF. Further indirect support for References 1. American Thoracic Society. 2000. Idiopathic pulmonary fibrosis: diagnosis and the role of CCL17 in macrophage recruitment during the patho- treatment. International consensus statement. Am. J. Respir. Crit. Care Med. genesis of bleomycin-induced pulmonary fibrosis is the lack of a 161:646. direct effect of CCL17 on fibroblast proliferation. 2. Crystal, R. G., P. B. Bitterman, S. I. Rennard, A. J. Hance, and B. A. Keogh. 1984. 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