IL-33 Induces IL-13−Dependent Cutaneous Fibrosis Andrew L. Rankin, John B. Mumm, Erin Murphy, Scott Turner, Ni Yu, Terrill K. McClanahan, Patricia A. Bourne, This information is current as Robert H. Pierce, Rob Kastelein and Stefan Pflanz of September 25, 2021. J Immunol 2010; 184:1526-1535; Prepublished online 30 December 2009; doi: 10.4049/jimmunol.0903306

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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 © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

IL-33 Induces IL-13–Dependent Cutaneous Fibrosis

Andrew L. Rankin,* John B. Mumm,† Erin Murphy,‡ Scott Turner,‡ Ni Yu,‡ Terrill K. McClanahan,‡ Patricia A. Bourne,‡ Robert H. Pierce,‡ Rob Kastelein,* and Stefan Pflanz*

IL-33 is constitutively expressed in epithelial barrier tissues, such as skin. Although increased expression of IL-33/IL-33R has been correlated with fibrotic disorders, such as scleroderma and progressive systemic sclerosis, the direct consequences of IL-33 release in skin has not been reported. To determine the effects of dysregulated IL-33 signaling in skin, we administered IL-33 s.c. and mon- itored its effects at the injection site. Administration of IL-33 resulted in IL-33R–dependent accumulation of , CD3+ lymphocytes, F4/80+ mononuclear cells, increased expression of IL-13 mRNA, and the development of cutaneous fibrosis. Con- sistent with extensive cutaneous tissue remodeling, IL-33 resulted in significant modulation of a number of extracellular matrix- associated , including collagen VI, collagen III, and tissue inhibitor of metalloproteases-1. We establish that IL-33–induced Downloaded from fibrosis requires IL-13 using IL-13 knockout mice and eosinophils using DdblGATA mice. We show that bone marrow-derived eosinophils secrete IL-13 in response to IL-33 stimulation, suggesting that -derived IL-13 may promote IL-33–induced cutaneous fibrosis. Collectively, our results identify IL-33 as a previously unrecognized profibrotic mediator in skin and highlight the cellular and molecular pathways by which this pathology develops. The Journal of Immunology, 2010, 184: 1526–1535.

ibrosis is characterized by the excess accumulation of Recent studies have revealed an association between IL-33/ST2 http://www.jimmunol.org/ extracellular matrix (ECM) components, including colla- and the development of fibrotic disorders, such as scleroderma and F gen (1, 2). The progressive replacement of parenchymal progressive systemic sclerosis (20, 21). However, the consequences tissues with ECM is observed in fibrotic diseases, such as systemic of dysregulated IL-33 activity in skin have not been reported. To sclerosis, idiopathic pulmonary fibrosis, and liver cirrhosis, lead- model chronic IL-33 release caused by sustained tissue damage, we ing to impaired organ function. Fibrosis is estimated to contribute repeatedly administered IL-33 s.c. and show that it induces ST2- to nearly 45% of deaths in the developed world. Although fibrosis dependent cutaneous fibrosis and inflammation. IL-33–induced fi- is postulated to result from chronic irritation or damage to the brosis is associated with altered expression of ECM-modifying affected organs, the cellular and molecular factors that sustain the genes. Importantly, we demonstrate that IL-13 is a critical down- fibrotic cascade remain poorly understood (1, 3–5). stream mediator of IL-33–induced cutaneous fibrosis. In addition, by guest on September 25, 2021 IL-33(IL-1F11,NF-HEV)isthemostrecentlydiscoveredmember we show that complete development of IL-33–induced fibrosis re- of the IL-1 family (6). IL-33 is constitutively expressed in quires eosinophils and RAG-dependent lymphocytes. For the first barrier tissues, such as skin, where it is found preferentially localized time, our data reveal profibrotic properties of IL-33 and collectively to the nucleus of epithelial and endothelial cells (7–10). The receptor suggest that IL-33 may function as an endogenous DAMP involved for IL-33 is composed of two subunits, IL-1RAcP (IL-1R3) and ST2 in the initiation of fibrotic disease. (IL-1R4) (6, 11). IL-1RAcP is widely expressed, whereas ST2 ex- pression is restricted to cell types that include Th2 cells, eosinophils, Materials and Methods , invariant NKT cells, and mast cells (6, 12–16). Consistent Animals with the expression of ST2 by Th2-associated cell types, in vivo B6/129.IL-1RAcP2/2, B6.IL-1R12/2, B6.RAG2/2, and 129S6.IL-132/2 systemic administration of recombinant IL-33 induces Th2 cytokine mice were housed in specific -free conditions at Schering-Plough production, , and mucous hypersecretion in the lung and Biopharma according to Institutional Animal Care and Use Committee gut (6, 14). IL-33 is released by cells undergoing necrotic cell death guidelines (22–25). B6.ST22/2 mice were a kind gift from the Neurath and in this respect IL-33 is thought to function as damage-associated laboratory, University of Mainz, Mainz, Germany (26). BALB/c. Δ w/v 2/2 molecular pattern (DAMP) (17–19). dblGATA, WB/B6F1.cKit , B6.IL-4 , and appropriate control mice were obtained from The Jackson Laboratory (Bar Harbor, ME) (27, 28). B6/129 mice were obtained from The Jackson Laboratory. C57BL/6 and *Department of Immunology, †Department of Oncology, and ‡Department of Exper- 129S6 mice were obtained from Taconic Farms (Germantown, NY). Age- imental Pathology and Pharmacology, Schering-Plough Biopharma, Palo Alto, CA and sex-matched mice, 8–16 wk of age, were used for the experiments 94304 described. Received for publication October 9, 2009. Accepted for publication November 20, Mice were injected s.c. daily for 7 d with 5 mg mouse serum albumin 2009. (MSA) or recombinant mIL-33. Recombinant IL-33 (aa 112–270) was produced in Escherichia coli by Aragen Biosciences (Morgan Hill, CA) and Address correspondence and reprint requests to Dr. Stefan Pflanz, Schering-Plough , Biopharma, 901 California Avenue, Palo Alto, CA 94304. E-mail address: stefan. contained 0.5 U/ml endotoxin. Twenty-four hours after the final injection, pfl[email protected] the injection site was harvested for analysis. Experimental protocols were approved by the Schering-Plough Biopharma Institutional Animal Care and The online version of this article contains supplemental material. Use Committee. Abbreviations used in this paper: bmEos, bone marrow-derived eosinophils; Ct, cycle threshold; DAMP, damage-associated molecular pattern; ECM, extracellular matrix; Quantitative real-time PCR KO, knockout; MMP, matrix metalloprotease; MSA, mouse serum albumin; Mut, mutant; TIMP, tissue inhibitor of metalloproteases; WT, wild type. RNA isolation was performed by standard techniques and expression was calculated using the D-DCt method (using the mean cycle threshold Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 [Ct] value for ubiquitin and the gene of interest for each sample). Primers www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903306 The Journal of Immunology 1527 were obtained commercially from Applied Biosystems (Foster City, CA) oxide for 10 min. Slides were heat retrieved with citrate buffer at pH 6.1 or designed using Primer Express (PE Biosystems, Foster City, CA). The (Catalog S1699, DakoCytomation) for 4 min at 123˚C using a Biocare equation 1.8e (Ct ubiquitin 2 Ct gene of interest) 3 104 was used to obtain Decloaker chamber, then cooled for 15 min, followed by a running tap the normalized values. water rinse. Slides were mounted on a Dako Autostainer and covered with fresh TBS to prevent drying of sections. Sections were then incubated with Histology anti-CD3 or anti-F4/80 Abs at room temperature for 60 min and rinsed with TBS. CD3-stained sections were incubated for 30 min in Rabbit Skin samples from the injection site were harvested and formalin fixed for Envision-Plus (Catalog K4011, DakoCytomation). F4/80 stained sections 24 h. Samples were then paraffin embedded and 5 mm sections cut. Sections were incubated for 30 min in Rat Probe and then Rat Polymer-HRP were stained with H&E, modified Masson’s trichrome, or astra blue and (Catalog RT517L, Biocare Medical, Concord, CA). Slides were rinsed violet red according to the manufacturer’s directions (American Master- with TBS and developed with DAB-Plus (DakoCytomation), counter- Tech, Lodi, CA). To quantify and eosinophil skin infiltration, astra stained in modified Mayer’s hematoxylin and blued in 0.3% ammonia blue and violet red-stained sections were scanned using a Mirax Midi slide water, followed by a tap water rinse. CD3+ and F4/80+ cells were counted scanner (Zeiss, Jena, Germany). Mast cells were identified based on the as described previously. characteristic astra blue staining of mast cell granules and the presence of a singular nucleus, whereas eosinophils were identified by violet red Collagen quantification cytoplasmic staining colocalized with a bilobed nucleus. A minimum cross- sectional area of 0.5 mm2 skin per mouse was randomly selected and sur- Total soluble collagen levels in the skin were quantified using the sircol veyed to obtain manual cell counts. Images were obtained using a bright- assay according to the manufacturer’s directions (Biocolor, Carrickfergus, field microscope (Olympus Model BX51, Olympus, Center Valley, PA) U.K.). Briefly, a 6 mm dermal punch biopsy was isolated from the injection with attached digital camera (QImaging Model Retiga 2000R, QImaging, site and s.c. fat removed. Samples were homogenized in 3 ml 0.5 M acetic Surrey, British Columbia, Canada) and were white balanced using Adobe acid, supplemented with complete protease inhibitors (Roche, Indian- Photoshop elements (v.2.0, Adobe Systems, San Jose, CA). apolis, IN). After overnight extraction at 4˚C, the samples were spun down and 0.2 ml extract was assayed to determine collagen content. Results are Downloaded from Immunohistochemistry reported as total micrograms of collagen per punch biopsy. Immunohistochemical studies were performed on formalin-fixed, paraffin- Bone marrow-derived eosinophils embedded tissue sections using a cross-reactive rabbit polyclonal Ab against anti-human CD3 (A0452; DakoCytomation, Carpenteria, CA, di- Bone marrow-derived eosinophils (bmEos) were generated as described (29). lution 1:200) and a rat mAb against anti-mouse F4/80 (BM8; eBioscience, Briefly, bone marrow was isolated from C57BL/6 and ST22/2 mice and San Diego, CA, dilution 1:400). Paraffin-embedded tissues were sectioned single-cell suspensions prepared. Cells were plated at 106/ml in RPMI 1640 at 5 mm thickness, deparaffinized, and quenched with 3% hydrogen per- containing 20% FBS (Irvine Scientific, Santa Ana, CA), 2 mM glutamine, http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 1. Injection of recombinant IL-33 s.c. induces fibrosis and inflammation. A, Photomicrographs show representative skin sections obtained from MSA- injected (control, left column) or IL-33–injected (right column) mice stained with H&E (top row) or Masson’s trichrome (bottom row); A, indicates s.c. adipose tissue; F, indicates s.c. fibrotic tissue. Scale bars indicate 200 mm (original magnification 310). B, Graph shows collagen content in dermal punches isolated from MSA- (control) or IL-33–injected mice. Symbols indicate values obtained from individual mice and lines indicate the average values throughout the figure. pppp = 0.001; Student t test. C, Photomicrographs show representative skin sections obtained from MSA- (control) or IL-33–injected mice stained with astra blue and violet red for identification of mast cells and eosinophils, respectively. Scale bars indicate 50 mm (original magnification 360). D, Graph shows the number of eosinophils in skin sections obtained from MSA- (open circles) or IL-33–injected (closed circles) mice. pppp = 0.001; Student t test. E, Photomicrographs show representative immunohistochemical staining for CD3 on skin sections obtained from MSA- (control, left panel)orIL-33–injected(right panel) mice. Scale bars indicate 50 mm (original magnification 340). F, Graphs show the number of CD3+ cells in skin sections obtained from MSA- (control) or IL-33–injected (closed circles) mice. Experiments were repeated independently at least two times with comparable results. 1528 IL-33 MEDIATES SUBCUTICULAR FIBROSIS Downloaded from

FIGURE 2. IL-33-induced inflammation and fibrosis are IL-1RAcP and ST2 dependent. Photomicrographs show representative H&E-stained skin sections obtained from WT (top row)and(A)IL-1RAcP2/2,(B)ST22/2,and (C)IL-1R12/2 (middle row) mice injected with IL-33. Masson’s trichrome staining is shown in the bottom row for (A)IL-1RAcP2/2,(B)ST22/2,and(C) http://www.jimmunol.org/ IL-1R12/2 mice. Scale bars indicate 200 mm (original magnification 310). Experiments were performed with at least five mice per group and were re- peated independently at least two times with comparable results.

25 mM HEPES, 13 MEM nonessential amino acids, 1 mM sodium pyruvate (Life Technologies BRL, Rockville, MD), 50 mM b-mercaptoethanol (Sigma-Aldrich, St. Louis, MO), 100 ng/ml , and 100 ng/ml FLT3-L (Peprotech). After 4 d, cells were replated in RPMI 1640 containing 20% FBS, 2 mM glutamine, 25 mM HEPES, 13 MEM nonessential amino acids, 1 mM sodium pyruvate, 50 mM b-mercaptoethanol, and 10 ng/ml by guest on September 25, 2021 rmIL-5. Cells were harvested 12 d after the start of the culture. For in vitro stimulations, 5 3 105 cells/ml were either left untreated or stimulated with 10 ng/ml rmIL-33. Supernatants were harvested after 24 h. IL-13 (Pepro- Tech, Rocky Hill, NJ) ELISAs were performed according to the manu- facturer’s directions. Flow cytometric analysis A total of 1 3 105 bmEos were Fc-blocked with 5 mg/ml anti-CD16/32 Ab (2.4G2; BD Biosciences, San Jose, CA) for 15 min. Cells were subsequently washed and coincubated with anti–mSiglec-F-PE (E50-2440; BD Bio- sciences) and anti–mST2-FITC (DJ8; MD Biosciences, Zurich, Switzerland) or control Abs (BD Biosciences) for 30 min in PBS containing 2% BSA (Sigma-Aldrich). Samples were washed and data collected on a BD LSRII flow cytometer (BD Biosciences). Data were analyzed using FlowJo software FIGURE 3. Increased expression of Th2 and ECM-associated genes in version 7.2.4 (Tree Star, Ashland, OR). IL-33– injected skin. A, TAQman analysis of IL-4, IL-5, and IL-13. B,ECM- associated genes. C,TGF-b mRNA expression in skin samples obtained from Statistical analysis MSA- (open circles) or IL-33–injected (closed circles) mice. Dots represent Statistical analyses to compare collagen content in skin and cell numbers values obtained from individual mice; lines indicate the average result. Ex- between experimental and control groups were performed using Student t tests, periments were repeated independently at least two times with comparable with p values # 0.05 considered significant. Studies using TAQman analysis results. pppp =0.008;ppp =0.016;pp = 0.032; Mann-Whitney U test. to compare levels between control and experimental groups were analyzed using the Mann-Whitney U test. p values # 0.05 were con- sidered significant. Statistical analyses were performed using GraphPad Prism site and development of subcuticular fibrosis as evidenced by version 4.02 for Windows (GraphPad Software, San Diego, CA). Masson’s trichrome staining (Fig. 1A). Soluble collagen levels were increased 4-fold in skin punch biopsies obtained from IL- Results 33–injected mice (Fig. 1B). The majority of infiltrating leukocytes IL-33 s.c. administration induces fibrosis and inflammation consisted of granulocytic leukocytes with dense clusters of these To assess the consequences of dysregulated IL-33 release in skin, cells occasionally observed in s.c. adipose tissue (Fig. 1A,1C). IL-33 or control (MSA) were injected s.c. everyday for 7 d. Histochemical staining revealed that the majority of infiltrating leu- IL-33– and MSA-injected mice did not exhibit any discernible kocytes were eosinophils and that s.c. injection of IL-33 caused sig- changes in behavior over the course of the injection series (e.g., nificant accumulation of these cells compared with MSA-injected pruritus) nor was any overt skin pathology observed. Nevertheless, skin (Fig. 1C,1D). Expression levels of eosinophil major basic histological analysis of skin from IL-33– but not MSA-injected protein in skin from IL-33– and control protein-treated mice yielded mice revealed prominent inflammation and edema at the injection a similar pattern of results (data not shown). CD3+ mononuclear The Journal of Immunology 1529 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 4. IL-33–induced fibrosis is IL-13 dependent. A, Photomicrographs show representative H&E-stained (top row) and Masson’s trichrome-stained (bottom row) skin sections obtained from WT (left column) and IL-132/2 mice (right column) treated with IL-33. Scale bars indicate 200 mm (original magnification 310). B, Graph shows collagen content in dermal punches obtained from MSA- (control) and IL-33–treated WT and IL-132/2 knockout (KO) mice. Symbols represent values obtained from individual mice and lines indicate the average values throughout the figure. pppp , 0.0001; Student t test. C, TAQman analysis of collagen VIa, collagen IIIa, (D) MMP12, MMP13, and TIMP1 in skin samples obtained from individual IL-33–treated WT or IL-132/2 KO mice. pppp = 0.008; pp = 0.016 Mann-Whitney U test. E, TAQman analysis of fibronectin in individual skin samples obtained from MSA- (control) or IL-33–treated WT and IL-132/2 KO mice. F, Graph shows the number of eosinophils in skin sections obtained from IL-33–injected or IL-132/2 KO mice. G, TAQman analysis of IL-5 and RANTES mRNA expression in skin samples obtained from MSA- (control) or IL-33–treated WT or IL-132/2 KO mice. Experiments were repeated independently at least two times with comparable results. cells were also significantly increased in skin obtained from IL- velopment of IL-33–induced cutaneous fibrosis and inflammation we 33–injected mice, whereas mast cells and B220+ cells were present treated IL-1R12/2 mice s.c. with IL-33. IL-1R12/2 mice injected with at similar numbers in skin sections obtained from mice injected with IL-33 developed inflammation and subcuticular fibrosis in a manner IL-33 or MSA (Fig. 1E,1F and data not shown). No pathological that was indistinguishable from control mice indicating that IL-1a changes were observed in skin sections adjacent to the injection site and -1b signaling is not required for IL-33–induced skin pathology (data not shown). (Fig. 2C). Collectively, these results demonstrate that repeated s.c. administration of IL-33 induces an ST2/IL-1RAcP–dependent skin IL-33–induced skin pathology is ST2 and IL-1RAcP dependent inflammation and fibrosis. To determine whether IL-33–induced inflammation is dependent on signaling through the IL-33Rs, ST2, and IL-1RAcP, we next injected Modulation of ECM-associated gene expression in IL-33– ST22/2 and IL-1RAcP2/2 mice s.c. with IL-33. Neither IL-1RAcP2/2 induced fibrotic lesions nor ST22/2 mice exhibited histological signs of inflammation or We next examined gene expression in skin obtained from IL- subcuticular fibrosis after receiving IL-33 injections (Fig. 2A,2B), and 33–injected mice. Consistent with the ability of IL-33 to induce skin sections from IL-33– and MSA control-injected IL-1RAcP2/2 expression of Th2 , skin samples obtained from IL-33– or ST22/2 mice were indistinguishable by histology (data not shown). injected mice had significantly elevated expression of IL-4, IL-5, IL-1RAcP is a shared subunit of the IL-33R that also pairs with IL-1R1 and IL-13, whereas expression of the Th1- and Th17-associated to form the heterodimeric receptor for IL-1a and -1b (22). To de- cytokines, IFN-g and IL-17, respectively, remained unchanged termine whether signaling through IL-1R1 is required for the de- (Fig. 3A and data not shown). As repeated injection of IL-33– 1530 IL-33 MEDIATES SUBCUTICULAR FIBROSIS induced the development of subcuticular fibrosis locally, we also inadvertently impact expression of IL-4 by disrupting regulation of examined expression of a panel of ECM-associated genes (Sup- IL-4 transcription (32). As it has also been reported that systemic IL-4 plemental Table I). IL-33–injected skin exhibited substantially in- overexpression via transgene can promote collagen accumulation in creased expression of various components of the ECM, such as skin, we assessed the requirement for IL-4 in the development of IL- collagen VIa, collagen IIIa, and fibronectin 1 (Fig. 3B, Supple- 33–induced skin pathology (33). H&E-stained skin sections pre- mental Table I). In addition, expression of ECM-modifying com- pared from IL-42/2 mice injected with IL-33, exhibited in- ponents, such as tissue inhibitor of metalloprotease (TIMP)-1, flammation that was grossly indistinguishable from WT mice matrix metalloprotease (MMP)-12, and MMP-13 were also sig- injected with IL-33 (Fig. 5C). In addition, the collagen content in nificantly elevated (Fig. 3B). Interestingly, IL-33 treatment also skin samples obtained from IL-42/2 and WT mice injected with IL- resulted in significantly reduced expression of several collagen 33 was similar, indicating that IL-33–induced inflammation and isoforms, including collagens II, IV, XI, XIII, XVI, and XVIII fibrosis are not dependent on IL-4 (Fig. 5D). Collectively, these (Supplemental Table I). Thus, these results highlight the novel results indicate that IL-33–induced fibrosis develops via an IL-13– observation that IL-33 can modulate expression of ECM-associated dependent and IL-4–independent mechanism. genes in the skin. TGF-b and IL-13 are two cytokines known to play a critical role in promoting fibrotic disease in multiple animal IL-33–induced skin fibrosis is dependent on eosinophils models (1). Skin isolated from IL-33–injected mice expressed The accumulation of eosinophils at the site of IL-33 injection levels of TGF-b that were similar to those observed in MSA- suggested that this cell type may promote IL-33–induced fibrosis. To injected skin samples (Fig. 3C). Because expression of IL-13, but determine whether IL-33–induced fibrosis requires eosinophils, we not TGF-b, was significantly increased in IL-33–injected skin Downloaded from samples these results suggested that IL-33–induced fibrosis may develop in an IL-13–dependent manner. IL-33–induced fibrosis is IL-13 dependent To determine whether IL-33–induced skin pathology requires IL-13, we injected IL-132/2 mice with IL-33 s.c. for 7 d and then examined skin samples histologically and quantified collagen content. Skin http://www.jimmunol.org/ sections prepared from IL-132/2 mice injected with IL-33 revealed only minor trichrome staining in the subcutis (Fig. 4A) and the col- lagen content in skin obtained from IL-33–injected IL-132/2 mice was found to be significantly reduced compared with wild-type (WT) animals treated with IL-33 (Fig. 4B). Strikingly, the soluble collagen content in skin obtained from IL-33–injected IL-132/2 mice was similar to levels observed in control protein-treated animals sug-

gesting that IL-13 acts as a critical mediator of IL-33–induced skin by guest on September 25, 2021 fibrosis. Expression levels of collagen VIa, collagen IIIa, MMP-12, MMP-13, and TIMP-1 were reduced in skin obtained from IL-33– injected IL-132/2 mice compared with WT mice (Fig. 4C,4D), whereas expression of fibronectin was similarly induced by IL-33 in WT and IL-132/2 mice indicating that IL-33–dependent expression of fibronectin occurs via an IL-13–independent mechanism (Fig. 4E). Although local collagen deposition was reduced in skin from IL-33– injected IL-132/2 mice, inflammatory infiltrates were still clearly present (Fig 4A). Comparing IL-33–injected WTand IL-132/2 mice, a nonsignificant trend toward reduced eosinophil infiltrates was ob- served in the IL-132/2 mice (Fig. 4F). IL-5 is known as a critical cytokine promoting eosinophil maturation and survival, and the RANTES (CCL5) is known as a key factor for re- cruitment of eosinophils via CCR3 signaling (30, 31). We therefore investigated mRNA levels of IL-5 and RANTES in skin of IL-33– injected WT and IL-132/2 mice: no differential expression for IL-5 and RANTES was detected (Fig. 4G), suggesting that neither IL-5 nor RANTES expression was IL-13 dependent. In contrast, expression of other CCR3-dependent , such as CCL7 (MCP-3), CCL11 (eotaxin), and CCL24 (eotaxin-2), were reduced in IL-33–injected IL-132/2 mice compared with WT mice (Supplemental Fig. 1). These data suggest that expression of CCL7, CCL11, and CCL24, but not CCL5 and IL-5 are regulated via IL-13–dependent pathways in this FIGURE 5. IL-33–induced fibrosis and inflammation is IL-4 independent. model. A, TAQman analysis of IL-13 and (B) IL-4 expression in skin samples ob- 2 2 IL-4 is not required for IL-33–induced skin fibrosis tained from MSA- (control) or IL-33–injected WT or IL-13 / (KO) mice. 2/2 C, Photomicrographs show representative H&E-stained skin sections ob- As expected, IL-13 expression in skin samples from IL-13 mice tained from IL-33–injected WT (left image) and IL-42/2 (right image) mice. was undetectable, but we also noted that IL-4 expression was greatly Scale bars indicate 200 mm (original magnification 310). D, Graph shows reduced (Fig. 5A,5B). The coding sequences for IL-4 and IL-13 are collagen content in dermal punches isolated from individual MSA- (control) tightly linked in an 11 kb region of mouse 11 and or IL-33–injected WT or IL-42/2 (KO) mice. Experiments were repeated previous studies have indicated that targeted deletion of IL-13 may independently at least two times with comparable results. The Journal of Immunology 1531 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 6. IL-33–induced fibrosis requires eosinophils. A, Graph shows collagen content in dermal punches obtained from MSA- (control) and IL-33–treated WT and ΔdblGATA mice (mutant [Mut]). Symbols represent values obtained from individual mice and lines indicate the average values throughout the figure. pp =0.01; ppp = 0.0021, Student t test. B, Photomicrographs show representative H&E-stained skin sections obtained from WT (left image)andΔdblGATA mice (right image). Scale bars indicate 200 mm (original magnification 310). C, Photomicrographs show representative F4/80 immunohistochemical staining on skin sections prepared from an MSA- (control) or IL-33–injected ΔdblGATA mice. Scale bar indicates 50 mm (original magnification 340). D, Graph shows the number of F4/80+ cells in skin obtained from MSA- (control) or IL-33–treated ΔdblGATA mice. pppp , 0.0001, Student t test. E, Histograms show levels of Siglec-F on bmEos from WT (top left panel)andST22/2 (bottom left panel)miceandthelevelsofST2(right panel) on Siglec-F+ bmEos from WT (black line), ST22/2 (gray line) mice, or WT cells stained with control Ab (dashed line). F, Graph indicates the concentration of IL-13 in supernatants obtained from IL-33–stimulated WT and ST22/2 (KO)-derived bone marrow eosinophils. Error bar indicates the SEM from duplicate measurements. G, TAQman analysis of IL-13 in skin samples obtained from IL-33–injected WT and ΔdblGATA (Mut) mice. Experiments were repeated independently at least two times with comparable results. injected ΔdblGATA mice with IL-33 (27). The eosinophil- deficient reduced compared with WT mice (Fig. 6A). Interestingly, the colla- status of ΔdblGATA mice was confirmed after completing the IL-33 gen content in skin from IL-33–treated ΔdblGATA mice was also treatment regimen by measuring blood eosinophils using a hemato- significantly increased compared with skin samples obtained from logic analyzer (data not shown). Notably, the collagen content in skin control (MSA)-injected mice (Fig. 6A). Thus, these results indicate obtained from ΔdblGATA mice injected with IL-33 was significantly that IL-33–induced fibrosis requires eosinophils for complete 1532 IL-33 MEDIATES SUBCUTICULAR FIBROSIS development and that additional cell types also contribute. H&E- control protein-treated mice and IL-33–treated WT mice, in- stained skin sections prepared from ΔdblGATA and WT mice in- dicating that RAG-dependent lymphocytes contribute to the de- jected with IL-33 revealed that both strains had developed in- velopment of IL-33–induced fibrosis (Fig. 7A). Consistent with the flammation in subcuticular tissue (Fig. 6B). The predominant cell reduced fibrosis exhibited by IL-33–treated RAG-deficient mice, type present in skin obtained from IL-33–injected ΔdblGATA mice IL-13 expression levels were also decreased in IL-33 treated were mononuclear cells morphologically resembling monocytes. RAG2/2 mice compared with WT mice treated with IL-33 (Fig. In fact, many of the infiltrating mononuclear cells in skin samples 7C). Notably, eosinophil infiltrates were found in H&E-stained skin obtained from IL-33–treated ΔdblGATA mice expressed F4/80 (Fig. specimens from IL-33–injected RAG2/2 mice lending further 6C). F4/80+ mononuclear cells were present in significantly increased support to the suggestion that eosinophils may serve as a source of numbers in skin samples obtained from IL-33–treated ΔdblGATA IL-33–induced IL-13 in vivo (Fig. 7B). mice compared with MSA-treated mice (Fig. 6D). We also observed some F4/80+ monocyte-like cells in WT skin injected with IL-33; IL-33–mediated cutaneous fibrosis is mast cell independent however, we were unable to quantify these cells because of abundant Several reports have suggested the potential for IL-33 to stimulate eosinophil infiltrates that also express F4/80 [data not shown and mast cells (6, 35–39). To investigate the possible contributions of (34)]. Nevertheless, these data indicate that a monocyte-like cell type skin resident mast cells to IL-33–mediated subcuticular fibrosis, can accumulate in IL-33–induced cutaneous lesions. we injected mast cell-deficient cKitw/v mice with IL-33 (40). No histological differences with regard to infiltrates were noted be- IL-33 stimulates production of IL-13 by bmEos tween H&E-stained skin sections prepared from cKitw/v and WT To further dissect the mechanism of IL-13–dependent cutaneous fi- control mice injected with IL-33, indicating that mast cells or mast Downloaded from brosis in this model, we next sought to determinewhether eosinophils cell activation are not required for IL-33–induced skin pathology could be a source of IL-13 after stimulation with IL-33. To these ends, (Supplemental Fig. 2A). Similarly, no overt difference in collagen 2/2 we prepared bmEos from WTand ST2 mice (29). bmEos cultures deposits was revealed by Masson’s trichrome staining (Supple- 2/2 prepared from WTand ST2 mice had comparable growth kinetics mental Fig. 2A). This result is consistent with the observation that + and viability and yielded .94% Siglec-F cells in resultant cultures s.c. IL-33 injection of WT mice does not result in a local increase

(Fig. 6E and data not shown). ST2 expression was consistently low in mast cell numbers (Supplemental Fig. 2B). http://www.jimmunol.org/ but detectable on the surface of Siglec-F+ bmEos from WT mice and as expected ST2 expression was undetectable on the surface of bmEos prepared from ST22/2 mice (Fig. 6E). Notably, WT bmEos Discussion secreted abundant IL-13 in response to stimulation with IL-33, Fibrosis is thought to be a consequence of chronic tissue irritation or whereas IL-13 production by IL-33–stimulated ST22/2 bmEos was damage (1, 3–5). However, the cellular and molecular factors that undetectable (Fig. 6F). Thus, bmEos produce IL-13 in response to promote the accumulation of ECM in fibrotic disease remain poorly stimulation with IL-33 in an ST2-dependent fashion. Consistent with defined. IL-33 is a recently described IL-1 family member that is the reduced fibrosis exhibited by IL-33–treated eosinophil-deficient constitutively expressed in barrier tissues, such as skin. IL-33 is mice, IL-13 expression levels were also decreased in IL-33–treated released on necrotic cell death and therefore has been suggested to by guest on September 25, 2021 ΔdblGATA mice compared with WT mice treated with IL-33 (Fig. function as DAMP (17–19). As IL-33 has been associated with fi- 6G). These results suggest that IL-13 production by IL-33–stimulated brotic disease, we examined the consequences of dysregulated IL- eosinophils may promote IL-33–induced cutaneous fibrosis. 33 signaling in the skin by repeated s.c. administration of IL-33 (7– 9, 20, 41, 42). We show that IL-33 induces cutaneous fibrosis and RAG-dependent lymphocytes contribute to IL-33–induced intense inflammation that is associated with large numbers of in- fibrosis filtrating eosinophils, CD3+ cells and F4/80+ myeloid cells. IL-33– CD3+ lymphocytes also accumulated at the site of IL-33 injection. induced fibrosis develops through an IL-13–dependent mechanism To determine whether lymphocytes are required for the de- that requires both eosinophils and RAG-dependent lymphocytes. velopment of fibrosis, we injected IL-33 into RAG2/2 mice, which We also show that IL-33– stimulated bmEos produce IL-13, thus lack IL-33–responsive Th2 and iNKT cell populations as well as B highlighting a novel cellular source of IL-33–induced IL-13 pro- cells and gd T cells (24). Skin samples obtained from IL-33–treated duction. Collectively, these studies identify IL-33 as a novel factor RAG2/2 mice contained collagen levels intermediate between sufficient to induce cutaneous fibrosis.

FIGURE 7. RAG-dependent lymphocytes contribute to IL-33–induced fibrosis. A, Graph shows collagen content in dermal punches obtained from MSA- (control) and IL-33–treated WTand RAG2/2 (KO) mice. Symbols represent values obtained from individual mice and lines indicate the average values. ppp = 0.009; pppp = 0.0001, Student t test. B, Photomicrographs show representative H&E-stained skin sections obtained from WT (left panel) and RAG2/2 (right panel) mice injected with IL-33. Scale bars indicate 200 mm (original magnification 310). C, TAQman analysis of IL-13 in skin samples obtained from IL-33– injected WT and RAG2/2 (KO) mice. Experiments were repeated independently at least two times with comparable results. The Journal of Immunology 1533

Injection of IL-33 s.c. resulted in the development of an ST2-de- phils respond to IL-33 in vivo remains to be examined, nevertheless pendent fibrotic lesion, which was evidenced by trichrome staining in these results suggest that eosinophils may promote the development subcuticulartissueandincreasedcollagencontent indermalpunches. of IL-33–induced fibrosis by producing IL-13. Transgenic over- ThepathologicchangesinducedbyIL-33wereassociatedwithaltered expression of IL-13 by keratinocytes is sufficient to induce the ac- expression of ECM genes in the collagen, MMP, TIMP, and bone cumulation of eosinophils in skin (46), suggesting that IL-33– morphogenetic protein families.Inparticular,theexpression levelsof induced production of IL-13 by eosinophils may contribute to collagen VIa and collagen IIIa were significantly increased in skin a positive feedback loop for eosinophil recruitment to sites of IL-33 samples obtained from IL-33–injected mice suggesting that these release through IL-13–dependent expression of CCL7, CCL11, and collagen subtypes may contribute to the increased collagen de- CCL24 (Supplemental Fig. 1). position in IL-33–treated skin. TIMP-1 and TIMP-2 expression were In addition to eosinophils, RAG-dependent lymphocytes con- also increased by IL-33 treatment. TIMP family members can inhibit tribute to driving IL-33–induced fibrosis. Th2 cells and iNKT cells MMP-mediated collagenase activity, which may promote IL-33– are both CD3+ populations that express ST2 and produce IL-13 in induced collagen accumulation by decreasing collagen turnover response to stimulation with IL-33 (6, 12, 16). Staining for CD3 in (43). Our findings indicate that IL-33 regulates the expression of fibrotic lesions revealed significant accumulation of these cells in these genes through an IL-13–dependent pathway. IL-13 has been IL-33–treated skin. By contrast, B220+ B cells, which are another shown to directly modulate expression of collagen VIa, collagen III, RAG-dependent cell type, were observed infrequently in IL-33– and TIMP-1 in previous studies (44, 45). Interestingly, fibronectin-1 expression was increased independently of IL-13 in IL-33–injected induced fibrotic lesions and their numbers were not modulated in skin samples, suggesting that expression of some ECM components response to injection with IL-33. We also have not detected sur- Downloaded from may be regulated directly through IL-33– or other IL-13–independent face expression of ST2 on B cells by flow cytometry (data not downstream mediators. Overall, our data highlight IL-33–mediated shown). gd T cells are a CD3+ RAG-dependent cell type that alterations in the expression levels of various collagen subtypes as comprises a small percentage of total lymphocytes. Vg3Vd1 well as enzymes known to affect ECM remodeling. It should be noted T cells are a specialized subset of gd T cells, termed “dendritic that our studies use a recombinant truncated form of IL-33 (aa 112– epidermal T cells,” that are skin-resident and have been shown to

270) and we cannot formally exclude the possibility that full length contribute to wound repair (63, 64). Expression of ST2 by gd T http://www.jimmunol.org/ IL-33 (aa 1–270) may have differential effects invivo. However, after cell subsets has not been reported, thus we cannot formally ex- production of both IL-33 forms via in vitro transcription and trans- clude a potential contribution of these cells to IL-33–induced fi- lation, we have been unable to detect differences in the invitro assays brosis. These data suggest that Th2, iNKT, or possibly gd T cells (unpublished observations). may also contribute to the development of IL-33–induced fibrosis. IL-13 is a profibrotic cytokine that is sufficient for the induction of + fibrosis in skin and lung (46, 47). In addition, IL-13 is required for F4/80 monocyte-like cells also infiltrated IL-33–induced fibrotic the development of fibrosis in some animal models (48–51). The lesions. Macrophages have been reported to express ST2 and to factors that regulate IL-13 expression in fibrotic disease remain respond to IL-33 in vitro (65, 66). In addition, monocytes can be poorly understood. In this study, we show that IL-33–induced fi- differentiated in the presence of IL-4/IL-13 in vitro to become al- by guest on September 25, 2021 brosis requires IL-13 but not IL-4. IL-4 can signal through the type I ternatively activated M2 macrophages, which contribute to the tis- IL-4 receptor, whereas both IL-4 and IL-13 can signal through the sue remodeling. In vivo, macrophage infiltration is observed in type II IL-4 receptor (52). Our results suggest that the type I IL-4 different fibrotic disease models (67–72). Future work will char- receptor is not required for IL-33–induced fibrosis. IL-13 can signal acterize the F4/80+ infiltrates in IL-33–induced fibrotic lesions to through the type II IL-4 receptor and IL-13Ra2, which both can determine whether these cells are directly responsive to IL-33. contribute to the development of fibrosis (53–55). Future studies Clinical studies have identified a positive correlation with IL-33 will examine the requirement for specific IL-13 receptors in the and/or IL-33R and several fibrotic diseases. Sera obtained from development of IL-33–induced fibrosis. systemic sclerosis patients and idiopathic pulmonary fibrosis patients In addition to IL-13, TGF-b is another cytokine with profibrotic exhibiting acute exacerbation contained elevated levels of sST2, the properties in several organs, including skin (56, 57). Although our endogenous soluble receptor antagonist of IL-33 (20, 41). Moreover, data show that mRNA expression of TGF-b is not modulated in IL- abundant staining for ST2 is observed in sclerotic lesions, whereas 33–injected skin (Fig. 3C), our results do not exclude a role for its IL-33 protein exhibits marked downregulation in early sclerotic skin involvement in IL-33–induced cutaneous fibrosis as TGF-b activity lesions (21). In addition, both IL-33 and ST2 were expressed at is regulated at multiple posttranscriptional levels and our assays significantly elevated levels in hepatic fibrotic lesions obtained from have not exhaustively examined TGF-b activity (58, 59). In- patients (42). Collectively, the data implicate the IL-33/IL-33R terestingly, in mice that overexpress IL-13 in lung tissue, blocking pathway as a potential mediator of pathogenesis in fibrotic diseases. TGF-b activity partially inhibited the development of fibrosis in- Future studies will examine the impact of neutralizing endogenous dicating that TGF-b expression can be induced downstream of IL- IL-33 on the development of fibrosis. 13 (60). Because we observe no evidence of fibrosis in IL-13–de- ficient mice injected with IL-33, our results argue that if TGF-b contributes to IL-33–induced cutaneous fibrosis then its activity is Acknowledgments likely restricted to effects downstream of IL-13. We thank S. Jungers and F. Shen for technical assistance with flow cytom- The full development of IL-33–induced cutaneous fibrosis re- etry and hematologic analysis. The many helpful discussions with E. Bow- quires both eosinophils and RAG-dependent lymphocytes. Eosin- man, D. Cua, C. Tato and M. Kleinschek were much appreciated throughout ophils have been shown to contribute to the development of fibrosis these studies. in transgenic mice that overexpress IL-13 in the lung (61). Impor- tantly, human eosinophils secrete IL-13 in response to stimulation Disclosures with IL-5 and produce IL-8 after stimulation with IL-33 in vitro (15, Schering-Plough Biopharma (formerly DNAX) is fully funded by the 62). We demonstrate that murine bmEos express ST2 and secrete IL- Schering-Plough Corporation. The authors have no further conflicting 13 in response to stimulation with IL-33. Whether murine eosino- financial interests. 1534 IL-33 MEDIATES SUBCUTICULAR FIBROSIS

References 27. Yu, C. N., A. B. Cantor, H. Yang, C. Browne, R. A. Wells, Y. Fujiwara, and S. H. Orkin. 2002. Targeted deletion of a high-affinity GATA-binding site in the 1. Wynn, T. A. 2008. Cellular and molecular mechanisms of fibrosis. J. Pathol. 214: GATA-1 promoter leads to selective loss of the eosinophil lineage in vivo. J. Exp. 199–210. Med. 195: 1387–1395. 2. Sivakumar, P., and A. M. Das. 2008. Fibrosis, chronic inflammation and new 28. Metwali, A., D. Elliott, A. M. Blum, J. Li, M. Sandor, R. Lynch, N. Noben- pathways for drug discovery. Inflamm. Res. 57: 410–418. Trauth, and J. V. Weinstock. 1996. The granulomatous response in murine 3. Friedman, S. L. 2008. Mechanisms of hepatic fibrogenesis. Gastroenterology 134: 1655–1669. Schistosomiasis mansoni does not switch to Th1 in IL-4-deficient C57BL/6 4. Trojanowska, M., and J. Varga. 2007. Molecular pathways as novel therapeutic mice. J. Immunol. 157: 4546–4553. targets in systemic sclerosis. Curr. Opin. Rheumatol. 19: 568–573. 29. Dyer, K. D., J. M. Moser, M. Czapiga, S. J. Siegel, C. M. Percopo, and 5. Selman, M., and A. Pardo. 2006. Role of epithelial cells in idiopathic pulmonary H. F. Rosenberg. 2008. Functionally competent eosinophils differentiated ex fibrosis: from innocent targets to serial killers. Proc. Am. Thorac. Soc. 3: 364–372. vivo in high purity from normal mouse bone marrow. J. Immunol. 181: 4004– 6. Schmitz, J., A. Owyang, E. Oldham, Y. L. Song, E. Murphy, T. K. McClanahan, 4009. G. Zurawski, M. Moshrefi, J. Z. Qin, X. X. Li, et al. 2005. IL-33, an - 30. Johnson, Z., C. A. Power, C. Weiss, F. Rintelen, H. Ji, T. Ruckle, M. Camps, 1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T. N. Wells, M. K. Schwarz, A. E. Proudfoot, and C. Rommel. 2004. Chemokine T helper type 2-associated cytokines. Immunity 23: 479–490. inhibition—why, when, where, which and how? Biochem. Soc. Trans. 32: 366– 7. Baekkevold, E. S., M. Roussigne´, T. Yamanaka, F. E. Johansen, F. L. Jahnsen, 377. F. Amalric, P. Brandtzaeg, M. Erard, G. Haraldsen, and J. P. Girard. 2003. 31. Hogan, S. P., H. F. Rosenberg, R. Moqbel, S. Phipps, P. S. Foster, P. Lacy, Molecular characterization of NF-HEV, a nuclear factor preferentially expressed A. B. Kay, and M. E. Rothenberg. 2008. Eosinophils: biological properties and in human high endothelial venules. Am. J. Pathol. 163: 69–79. role in health and disease. Clin. Exp. Allergy 38: 709–750. 8. Carriere, V., L. Roussel, N. Ortega, D. A. Lacorre, L. Americh, L. Aguilar, 32. Guo, L. Y., J. Hu-Li, J. F. Zhu, C. Pannetier, C. Watson, G. J. McKenzie, A. N. G. Bouche, and J. P. Girard. 2007. IL-33, the IL-1-like cytokine ligand for ST2 J. McKenzie, and W. E. Paul. 2001. Disrupting Il13 impairs production of IL-4 receptor, is a chromatin-associated nuclear factor in vivo. Proc. Natl. Acad. Sci. specified by the linked allele. Nat. Immunol. 2: 461–466. USA 104: 282–287. 33. Elbe-Bu¨rger, A., A. Egyed, S. Olt, R. Klubal, U. Mann, K. Rappersberger, 9. Ku¨chler, A. M., J. Pollheimer, J. Balogh, J. Sponheim, L. Manley, A. Rot, and G. Stingl. 2002. Overexpression of IL-4 alters the homeostasis in the D. R. Sorensen, P. M. De Angelis, H. Scott, and G. Haraldsen. 2008. Nuclear skin. J. Invest. Dermatol. 118: 767–778. Downloaded from interleukin-33 is generally expressed in resting endothelium but rapidly lost 34. McGarry, M. P., and C. C. Stewart. 1991. Murine eosinophil granulocytes bind upon angiogenic or proinflammatory activation. Am. J. Pathol. 173: 1229–1242. the murine macrophage-monocyte specific monoclonal antibody F4/80. J. Leu- 10. Moussion, C., N. Ortega, and J. P. Girard. 2008. The IL-1-like cytokine IL-33 is koc. Biol. 50: 471–478. constitutively expressed in the nucleus of endothelial cells and epithelial cells 35. Allakhverdi, Z., D. E. Smith, M. R. Comeau, and G. Delespesse. 2007. Cutting in vivo: a novel ‘alarmin’? PLoS One 3: e3331. edge: The ST2 ligand IL-33 potently activates and drives maturation of human 11. Chackerian, A. A., E. R. Oldham, E. E. Murphy, J. Schmitz, S. Pflanz, and mast cells. J. Immunol. 179: 2051–2054. R. A. Kastelein. 2007. IL-1 receptor accessory protein and ST2 comprise the IL- 36. Moulin, D., O. Donze´, D. Talabot-Ayer, F. Me´zin, G. Palmer, and C. Gabay.

33 receptor complex. J. Immunol. 179: 2551–2555. 2007. Interleukin (IL)-33 induces the release of pro-inflammatory mediators by http://www.jimmunol.org/ 12. Lo¨hning, M., A. Stroehmann, A. J. Coyle, J. L. Grogan, S. Lin, J. C. Gutierrez- mast cells. Cytokine 40: 216–225. Ramos, D. Levinson, A. Radbruch, and T. Kamradt. 1998. T1/ST2 is preferen- 37. Iikura, M., H. Suto, N. Kajiwara, K. Oboki, T. Ohno, Y. Okayama, H. Saito, tially expressed on murine Th2 cells, independent of , interleukin 5, S. J. Galli, and S. Nakae. 2007. IL-33 can promote survival, adhesion and cy- and , and important for Th2 effector function. Proc. Natl. Acad. tokine production in human mast cells. Lab. Invest. 87: 971–978. Sci. USA 95: 6930–6935. 38. Ho, L. H., T. Ohno, K. Oboki, N. Kajiwara, H. Suto, M. Iikura, Y. Okayama, 13. Suzukawa, M., R. Koketsu, M. Iikura, S. Nakae, K. Matsumoto, H. Nagase, S. Akira, H. Saito, S. J. Galli, and S. Nakae. 2007. IL-33 induces IL-13 pro- H. Saito, K. Matsushima, K. Ohta, K. Yamamoto, and M. Yamaguchi. 2008. duction by mouse mast cells independently of IgE-FcepsilonRI signals. J. Leu- Interleukin-33 enhances adhesion, CD11b expression and survival in human koc. Biol. 82: 1481–1490. eosinophils. Lab. Invest. 88: 1245–1253. 39. Ali, S., M. Huber, C. Kollewe, S. C. Bischoff, W. Falk, and M. U. Martin. 2007. 14. Kondo, Y., T. Yoshimoto, K. Yasuda, S. Futatsugi-Yumikura, M. Morimoto, IL-1 receptor accessory protein is essential for IL-33-induced activation of T N. Hayashi, T. Hoshino, J. Fujimoto, and K. Nakanishi. 2008. Administration of lymphocytes and mast cells. Proc. Natl. Acad. Sci. USA 104: 18660–18665. IL-33 induces airway hyperresponsiveness and hyperplasia in the 40. Kitamura, Y., S. Go, and K. Hatanaka. 1978. Decrease of mast cells in W/Wv by guest on September 25, 2021 lungs in the absence of adaptive immune system. Int. Immunol. 20: 791–800. mice and their increase by bone marrow transplantation. Blood 52: 447–452. 15. Cherry, W. B., J. Yoon, K. R. Bartemes, K. Iijima, and H. Kita. 2008. A novel IL- 41. Tajima, S., K. Oshikawa, S. Tominaga, and Y. Sugiyama. 2003. The increase in 1 family cytokine, IL-33, potently activates human eosinophils. J. Allergy Clin. serum soluble ST2 protein upon acute exacerbation of idiopathic pulmonary Immunol. 121: 1484–1490. fibrosis. Chest 124: 1206–1214. 16. Smithgall, M. D., M. R. Comeau, B. R. Yoon, D. Kaufman, R. Armitage, and 42. Marvie, P., M. Lisbonne, A. L’helgoualc’h, M. Rauch, B. Turlin, L. Preisser, D. E. Smith. 2008. IL-33 amplifies both Th1- and Th2-type responses through its K. Bourd-Boittin, N. The´ret, H. Gascan, C. Piquet-Pellorce, and M. Samson. activity on human basophils, -reactive Th2 cells, iNKT and NK cells. Int. 2009. Interleukin-33 overexpression is associated with liver fibrosis in mice and Immunol. 20: 1019–1030. humans. J. Cell. Mol. Med. . 17. Cayrol, C., and J. P. Girard. 2009. The IL-1-like cytokine IL-33 is inactivated 43. Lambert, E., E. Dasse´, B. Haye, and E. Petitfre`re. 2004. TIMPs as multifacial Proc. Natl. Acad. Sci. USA after maturation by caspase-1. 106: 9021–9026. . Crit. Rev. Oncol. Hematol. 49: 187–198. 18. Haraldsen, G., J. Balogh, J. Pollheimer, J. Sponheim, and A. M. Ku¨chler. 2009. 44. Syed, F., R. A. Panettieri, Jr., O. Tliba, C. Huang, K. Li, M. Bracht, Interleukin-33 - cytokine of dual function or novel alarmin? Trends Immunol. 30: B. Amegadzie, D. Griswold, L. Li, and Y. Amrani. 2005. The effect of IL-13 and 227–233. IL-13R130Q, a naturally occurring IL-13 polymorphism, on the gene expression 19. Lu¨thi, A. U., S. P. Cullen, E. A. McNeela, P. J. Duriez, I. S. Afonina, of human airway smooth muscle cells. Respir. Res. 6: 9 . C. Sheridan, G. Brumatti, R. C. Taylor, K. Kersse, P. Vandenabeele, et al. 2009. 45. Leonardi, A., R. Cortivo, I. Fregona, M. Plebani, A. G. Secchi, and G. Abatangelo. Suppression of interleukin-33 bioactivity through proteolysis by apoptotic cas- 2003. Effects of Th2 cytokines on expression of collagen, MMP-1, and TIMP-1 in pases. Immunity 31: 84–98. conjunctival fibroblasts. Invest. Ophthalmol. Vis. Sci. 44: 183–189. 20. Kuroiwa, K., T. Arai, H. Okazaki, S. Minota, and S. Tominaga. 2001. Identifi- 46. Zheng, T., M. H. Oh, S. Y. Oh, J. T. Schroeder, A. B. Glick, and Z. Zhu. 2009. cation of human ST2 protein in the sera of patients with autoimmune diseases. Transgenic expression of interleukin-13 in the skin induces a pruritic dermatitis Biochem. Biophys. Res. Commun. 284: 1104–1108. 21. Manetti, M., L. Ibba-Manneschi, V. Liakouli, S. Guiducci, A. Milia, G. Benelli, and skin remodeling. J. Invest. Dermatol. 129: 742–751. A. Marrelli, M. Conforti, E. Romano, R. Giacomelli, et al. 2009. The IL-1-like 47. Zhu, Z., R. J. Homer, Z. D. Wang, Q. S. Chen, G. P. Geba, J. M. Wang, Y. Zhang, cytokine IL-33 and its receptor ST2 are abnormally expressed in the affected and J. A. Elias. 1999. Pulmonary expression of interleukin-13 causes in- skin and visceral organs of patients with systemic sclerosis. Ann. Rheum. Dis. . flammation, mucus hypersecretion, subepithelial fibrosis, physiologic abnor- 22. Cullinan, E. B., L. Kwee, P. Nunes, D. J. Shuster, G. Ju, K. W. McIntyre, malities, and eotaxin production. J. Clin. Invest. 103: 779–788. R. A. Chizzonite, and M. A. Labow. 1998. IL-1 receptor accessory protein is an 48. Aliprantis, A. O., J. S. Wang, J. W. Fathman, R. Lemaire, D. M. Dorfman, essential component of the IL-1 receptor. J. Immunol. 161: 5614–5620. R. Lafyatis, and L. H. Glimcher. 2007. T-bet regulates skin 23. Glaccum, M. B., K. L. Stocking, K. Charrier, J. L. Smith, C. R. Willis, sclerosis through its function in innate immunity and via IL-13. Proc. Natl. Acad. C. Maliszewski, D. J. Livingston, J. J. Peschon, and P. J. Morrissey. 1997. Sci. USA 104: 2827–2830. Phenotypic and functional characterization of mice that lack the type I receptor 49. Liu, T. J., H. Jin, M. Ullenbruch, B. Hu, N. Hashimoto, B. Moore, A. McKenzie, for IL-1. J. Immunol. 159: 3364–3371. N. W. Lukacs, and S. H. Phan. 2004. Regulation of found in inflammatory zone 1 24. Mombaerts, P., J. Iacomini, R. S. Johnson, K. Herrup, S. Tonegawa, and expression in bleomycin-induced lung fibrosis: role of IL-4/IL-13 and mediation V. E. Papaioannou. 1992. RAG-1-deficient mice have no mature B and T lym- via STAT-6. J. Immunol. 173: 3425–3431. phocytes. Cell 68: 869–877. 50. Kolodsick, J. E., G. B. Toews, C. Jakubzick, C. Hogaboam, T. A. Moore, 25. McKenzie, G. J., A. Bancroft, R. K. Grencis, and A. N. J. McKenzie. 1998. A A. McKenzie, C. A. Wilke, C. J. Chrisman, and B. B. Moore. 2004. Protection distinct role for interleukin-13 in Th2-cell-mediated immune responses. Curr. from fluorescein isothiocyanate-induced fibrosis in IL-13-deficient, but not IL-4- Biol. 8: 339–342. deficient, mice results from impaired collagen synthesis by fibroblasts. J. Im- 26. Hoshino, K., S. Kashiwamura, K. Kuribayashi, T. Kodama, T. Tsujimura, munol. 172: 4068–4076. K. Nakanishi, T. Matsuyama, K. Takeda, and S. Akira. 1999. The absence of 51. Chiaramonte, M. G., D. D. Donaldson, A. W. Cheever, and T. A. Wynn. 1999. interleukin 1 receptor-related T1/ST2 does not affect type 2 de- An IL-13 inhibitor blocks the development of hepatic fibrosis during a T-helper velopment and its effector function. J. Exp. Med. 190: 1541–1548. type 2-dominated inflammatory response. J. Clin. Invest. 104: 777–785. The Journal of Immunology 1535

52. Andrews, A. L., J. W. Holloway, S. T. Holgate, and D. E. Davies. 2006. IL-4 re- 63. Asarnow, D. M., W. A. Kuziel, M. Bonyhadi, R. E. Tigelaar, P. W. Tucker, and ceptor a is an important modulator of IL-4 and IL-13 receptor binding: im- J. P. Allison. 1988. Limited diversity of gdantigen receptor genes of Thy-1+ plications for the development of therapeutic targets. J. Immunol. 176: 7456–7461. dendritic epidermal cells. Cell 55: 837–847. 53. Ramalingam, T. R., J. T. Pesce, F. Sheikh, A. W. Cheever, M. M. Mentink-Kane, 64. Jameson, J., K. Ugarte, N. Chen, P. Yachi, E. Fuchs, R. Boismenu, and M. S. Wilson, S. Stevens, D. M. Valenzuela, A. J. Murphy, G. D. Yancopoulos, W. L. Havran. 2002. A role for skin gammadelta T cells in wound repair. Science et al. 2008. Unique functions of the type II interleukin 4 receptor identified in 296: 747–749. mice lacking the interleukin 13 receptor a1 chain. Nat. Immunol. 9: 25–33. 65. Brint, E. K., D. Xu, H. Liu, A. Dunne, A. N. McKenzie, L. A. O’Neill, and 54. Fichtner-Feigl, S., W. Strober, K. Kawakami, R. K. Puri, and A. Kitani. 2006. IL- F. Y. Liew. 2004. ST2 is an inhibitor of interleukin 1 receptor and Toll-like re- 13 signaling through the IL-13a2 receptor is involved in induction of TGF-b1 ceptor 4 signaling and maintains endotoxin tolerance. Nat. Immunol. 5: 373–379. production and fibrosis. Nat. Med. 12: 99–106. 66. Espinassous, Q., E. Garcia-de-Paco, I. Garcia-Verdugo, M. Synguelakis, S. von 55. Andrews, A. L., T. Nasir, F. Bucchieri, J. W. Holloway, S. T. Holgate, and Aulock, J. M. Sallenave, A. N. McKenzie, and J. Kanellopoulos. 2009. IL-33 D. E. Davies. 2006. IL-13 receptor a 2: a regulator of IL-13 and IL-4 signal enhances lipopolysaccharide-induced inflammatory cytokine production from transduction in primary human fibroblasts. J. Allergy Clin. Immunol. 118: 858–865. mouse macrophages by regulating lipopolysaccharide receptor complex. J. Im- 56. Pohlers, D., J. Brenmoehl, I. Lo¨ffler, C. K. Mu¨ller, C. Leipner, S. Schultze- munol. 183: 1446–1455. Mosgau, A. Stallmach, R. W. Kinne, and G. Wolf. 2009. TGF-b and fibrosis in 67. Doherty, T. M., R. Kastelein, S. Menon, S. Andrade, and R. L. Coffman. 1993. different organs - molecular pathway imprints. Biochim. Biophys. Acta 1792: Modulation of murine macrophage function by IL-13. J. Immunol. 151: 7151– 746–756. 7160. 57. Lakos, G., S. Takagawa, S. J. Chen, A. M. Ferreira, G. Han, K. Masuda, 68. Herbert, D. R., C. Ho¨lscher, M. Mohrs, B. Arendse, A. Schwegmann, X. J. Wang, L. A. DiPietro, and J. Varga. 2004. Targeted disruption of TGF- M. Radwanska, M. Leeto, R. Kirsch, P. Hall, H. Mossmann, et al. 2004. Al- b/Smad3 signaling modulates skin fibrosis in a mouse model of scleroderma. ternative macrophage activation is essential for survival during schistosomiasis Am. J. Pathol. 165: 203–217. and downmodulates T helper 1 responses and immunopathology. Immunity 20: 58. Li, M. O., Y. Y. Wan, S. Sanjabi, A. K. Robertson, and R. A. Flavell. 2006. 623–635. Transforming -b regulation of immune responses. Annu. Rev. Im- 69. Taylor, M. D., A. Harris, M. G. Nair, R. M. Maizels, and J. E. Allen. 2006. F4/80+ munol. 24: 99–146. alternatively activated macrophages control CD4+ T cell hyporesponsiveness at 59. Taylor, A. W. 2009. Review of the activation of TGF-b in immunity. J. Leukoc. sites peripheral to filarial . J. Immunol. 176: 6918–6927. Biol. 85: 29–33. 70. Karlmark, K. R., R. Weiskirchen, H. W. Zimmermann, N. Gassler, F. Ginhoux, Downloaded from 60. Lee, C. G., R. J. Homer, Z. Zhu, S. Lanone, X. M. Wang, V. Koteliansky, C. Weber, M. Merad, T. Luedde, C. Trautwein, and F. Tacke. 2009. Hepatic J. M. Shipley, P. Gotwals, P. Noble, Q. S. Chen, et al. 2001. Interleukin-13 in- recruitment of the inflammatory Gr1+ monocyte subset upon liver injury pro- duces tissue fibrosis by selectively stimulating and activating transforming motes hepatic fibrosis. Hepatology 50: 261–274. growth factor b(1). J. Exp. Med. 194: 809–821. 71. Ishida, Y., A. Kimura, T. Kondo, T. Hayashi, M. Ueno, N. Takakura, 61. Fulkerson, P. C., C. A. Fischetti, and M. E. Rothenberg. 2006. Eosinophils and K. Matsushima, and N. Mukaida. 2007. Essential roles of the CC chemokine CCR3 regulate interleukin-13 transgene-induced pulmonary remodeling. Am. J. ligand 3-CC chemokine receptor 5 axis in bleomycin-induced pulmonary fibrosis Pathol. 169: 2117–2126. through regulation of macrophage and fibrocyte infiltration. Am. J. Pathol. 170:

62. Schmid-Grendelmeier, P., F. Altznauer, B. Fischer, C. Bizer, A. Straumann, 843–854. http://www.jimmunol.org/ G. Menz, K. Blaser, B. Wu¨thrich, and H. U. Simon. 2002. Eosinophils express 72. Stein, M., S. Keshav, N. Harris, and S. Gordon. 1992. Interleukin 4 potently functional IL-13 in eosinophilic inflammatory diseases. J. Immunol. 169: 1021– enhances murine macrophage mannose receptor activity: a marker of alternative 1027. immunologic macrophage activation. J. Exp. Med. 176: 287–292. by guest on September 25, 2021