cell adhesion molecules regulate fibrotic process via th1/th2/th17 cell balance in a bleomycin

of April 11, 2019.This information is current as

Bleomycin-Induced Scleroderma ModelProcess via Th1/Th2/Th17 Cell Balance in a Cell Adhesion Molecules Regulate Fibrotic

Thomas F. Tedder and Shinichi SatoKazuhiro Shimizu, Minoru Hasegawa, Manabu Fujimoto, Toshihide Hara, Fumihide Ogawa, Motoi Takenaka,Komura, Asako Ogawa, Yuichiro Akiyama, Eiji Muroi, Ayumi Yoshizaki, Koichi Yanaba, Yohei Iwata, Kazuhiro

http://www.jimmunol.org/content/185/4/2502doi: 10.4049/jimmunol.0901778July 2010;

2010; 185:2502-2515; Prepublished online 12J Immunol

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The Journal of Immunology

Cell Adhesion Molecules Regulate Fibrotic Process viaTh1/Th2/Th17 Cell Balance in a Bleomycin-InducedScleroderma Model

Ayumi Yoshizaki,* Koichi Yanaba,* Yohei Iwata,* Kazuhiro Komura,† Asako Ogawa,*

Yuichiro Akiyama,* Eiji Muroi,* Toshihide Hara,* Fumihide Ogawa,* Motoi Takenaka,*

Kazuhiro Shimizu,* Minoru Hasegawa,† Manabu Fujimoto,† Thomas F. Tedder,‡ and

Shinichi Satox

Mice s.c. injected with bleomycin, an experimental model for human systemic sclerosis, develop skin and lung fibrosis, which is

mediated by inflammatory cell infiltration. This process is highly regulated by multiple adhesion molecules and does not require Ag

sensitization. To assess the role of adhesion molecules in this pathogenetic process, bleomycin-induced fibrosis was examined in mice

lacking adhesion molecules. L-selectin and/or ICAM-1 deficiency inhibited skin and lung fibrosis with decreased Th2 and Th17

cytokines and increased Th1 cytokines. In contrast, P-selectin deficiency, E-selectin deficiency with or without P-selectin blockade,

or P-selectin glycoprotein ligand 1 (PSGL-1) deficiency augmented the fibrosis in parallel with increased Th2 and Th17 cytokines

and decreased Th1 cytokines. Furthermore, loss of L-selectin and/or ICAM-1 reduced Th2 and Th17 cell numbers in bronchoal-

veolar lavage fluid, whereas loss of P-selectin, E-selectin, or PSGL-1 reduced Th1 cell numbers.Moreover, Th1 cells exhibited higher

PSGL-1 expression and lower expression of LFA-1, a ligand for ICAM-1, whereas Th2 and Th17 cells showed higher LFA-1 and

lower PSGL-1 expression. This study suggests that L-selectin and ICAM-1 regulate Th2 and Th17 cell accumulation into the skin

and lung, leading to the development of fibrosis, and that P-selectin, E-selectin, and PSGL-1 regulate Th1 cell infiltration, resulting

in the inhibition of fibrosis. The Journal of Immunology, 2010, 185: 2502–2515.

Systemic sclerosis (SSc) is a connective tissue diseasecharacterized by excessive extracellular matrix depositionin the skin, lung, and other visceral organs with an auto-

immune background (1). The presence of autoantibodies is a cen-tral feature of SSc, since anti-nuclear Abs are detected in.90% ofpatients (2). SSc patients have autoantibodies that react to variousintracellular components, such as DNA topoisomerase I (topo I),centromeric protein B, U1-ribonucleoprotein (RNP), and histone(2). Furthermore, abnormal activation of immune cells, includingT cells, B cells, NK cells, and macrophages, has been identified inSSc (3). A recent study has shown that skin and lung fibrosis isameliorated by treatment with cyclophosphamide, an immunosup-pressive agent, indicating that immune activation leads to fibrosisthrough the stimulation of collagen production by fibroblasts (4).Indeed, SSc patients exhibit inflammatory cell infiltration, espe-cially CD4+ T cells, and elevated serum levels of various

cytokines, especially fibrogenic Th2 and Th17 cytokines and TGF-b1, a major fibrogenic growth factor, which positively correlatewith disease severity (5, 6).In general, leukocyte recruitment into inflammatory sites is

achieved using constitutive or inducible expression of multiple celladhesion molecules (7). L-selectin (CD62L), E-selectin (CD62E),and P-selectin (CD62P) primarily mediate leukocyte capture androlling on the endothelium (8). L-selectin is constitutively ex-pressed by most leukocytes (8). Whereas P-selectin is rapidlymobilized to the surface of activated endothelium or platelets,E-selectin expression is induced within several hours after activa-tion with inflammatory cytokines (8). The selectins share a highlyconserved N-terminal lectin domain that can interact with sialy-lated and fucosylated oligosaccharides such as sialyl Lewis X (9).Although various candidates have been identified as potentialligands for selectins, P-selectin glycoprotein ligand 1 (PSGL-1)is the best characterized ligand, which is recognized by all threeselectins (10). PSGL-1 is a mucin-like, disulfide-linked homo-dimer expressed by all subsets of leukocytes and is a high-affinity ligand for E- and P-selectins (11). PSGL-1 has also beenshown to bind to L-selectin, but its affinity is lower than E- andP-selectins (12). ICAM-1 (CD54) is a member of the Ig super-family that is constitutively expressed not only on endothelialcells, but also on fibroblasts and epithelial cells (13). It can be up-regulated transcriptionally by several proinflammatory cytokines,such as TNF-a, IFN-g, and IL-1 (13). ICAM-1 forms the counter-receptor for the lymphocyte b2 integrins, such as LFA-1 (7). TheICAM-1/LFA-1 interactions predominantly mediate firm adhesionand transmigration of leukocytes at sites of inflammation (7). In-hibition of LFA-1 attenuated intratracheal bleomycin treatment-induced pulmonary fibrosis. However, the studies investigating therole of L-selectin and ICAM-1 in fibrosis are limited. A recent

*Department of Dermatology, Nagasaki University Graduate School of BiomedicalSciences, Nagasaki; †Department of Dermatology, Kanazawa University GraduateSchool of Medical Science, Kanazawa; xDepartment of Dermatology, University ofTokyo Graduate School of Medicine, Tokyo, Japan; and ‡Department of Immunol-ogy, Duke University Medical Center, Durham, NC 27710

Received for publication June 5, 2009. Accepted for publication June 5, 2010.

This work was supported by a grant for research on intractable diseases from theMinistry of Health, Labor, and Welfare of Japan (to S.S. and Y.Y.) and NationalInstitutes of Health Grants CA96547, CA105001, and AI56363 (to T.F.T.).

Address correspondence and reprint requests to Dr. Shinichi Sato, Department ofDermatology, University of Tokyo Graduate School of Medicine, 7-3-1 Hongo, Bun-kyo-ku, Tokyo 113-8655, Japan. E-mail address: [email protected]

Abbreviations used in this paper: BAL, bronchoalveolar lavage; BLM, bleomycin;COL1A2, a2(I) collagen; CTL, control; HPF, high-power field; MFI, mean fluores-cence intensity; PSGL-1, P-selectin glycoprotein ligand 1; RNP, ribonucleoprotein;SSc, systemic sclerosis; topo I, topoisomerase I; WT, wild type.

Copyright� 2010 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/10/$16.00

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study has shown that intratracheal bleomycin treatment-inducedpulmonary fibrosis is inhibited in L-selectin2/2 mice and ICAM-12/2 mice (14). In contrast, another study has suggested that anantagonist of ICAM-1 does not attenuate intratracheal bleomycintreatment-induced pulmonary fibrosis, although the same treat-ment decreases leukocyte infiltration in the bronchoalveolar la-vage (BAL) (15). Thus, the in vivo contribution of L-selectinand ICAM-1 to fibrosis remains unclear.Although these cell adhesion molecules play important roles in

leukocyte transmigration, their association to inflammationremains controversial in several inflammatory models. Inhibition orloss of L-selectin, ICAM-1, P-selectin, E-selectin, or PSGL-1 leadsto a significant reduction in leukocyte rolling and emigration inmany inflammatory models, such as the tight-skin mouse model,which is a genetic model for human SSc, immune complex depo-sition-induced tissue injury, contact hypersensitivity, intratrachealbleomycin-induced pulmonary fibrosis, and peritonitis (14, 16–21).In contrast, some studies have suggested that loss of P-selectinand/or E-selectin increases inflammatory response, such as ex-perimental glomerulonephritis, collagen-induced arthritis, andbleomycin-induced pulmonary fibrosis models (22–25). Thus,E- and P-selectins regulate inflammatory response either posi-tively or negatively according to the inflammatory models. Pre-vious studies have shown that these cell adhesion molecules alsoregulate Th1, Th2, and Th17 cell migration. Inhibition or loss ofICAM-1 and/or L-selectin reduces Th1, Th2, and Th17 cell mi-gration (26–28), while some studies have suggested that Th1 andTh2 cell immigration is induced by L-selectin and/or ICAM-1deficiency or blockade (26, 27, 29). P-selectin, E-selectin, and/orPSGL-1 deficiency or inhibition reduces Th1 and Th2 cell infil-tration (28). Similarly, Th17 cell infiltration is significantly in-hibited by E- and P-selectin deficiency (29). Thus, these cell ad-hesion molecules regulate Th cell balance, according to the tissuesite and the nature of the inflammatory stimuli.Recently, Yamamoto et al. (30) established a new mouse model

of SSc using bleomycin treatment: the s.c. injection of bleomycininduces fibrosis in the dermis and lung, autoantibody production,and dermal and pulmonary inflammatory cell infiltration, all ofwhich closely mimic the features of human SSc. In this mousemodel, B cells play important roles in the development of fibrosis(31). However, in the absence of CD19, which is a critical signaltransduction molecule of B cells, bleomycin-induced fibrosis isnot completely inhibited, with an ∼30% reduction (31). Thisfinding suggests that other immune cells and molecules also playimportant roles in the development of fibrosis. However, the con-tribution of cell adhesion molecules to disease manifestations,including autoimmunity, and to the mechanism underlying Th1,Th2, and Th17 cell infiltration remains unknown in the bleomy-cin-induced SSc model. In this study, we investigated the role ofcell adhesion molecules in the development of fibrosis and auto-immunity induced by bleomycin using mice lacking L-selectin,ICAM-1, P-selectin, E-selectin, and PSGL-1. According to ourresults, L-selectin and/or ICAM-1 deficiency reduced dermalsclerosis, pulmonary fibrosis, and autoimmunity induced by bleo-mycin treatment with increased Th1 cell infiltration and de-creased Th2 and Th17 cell infiltration. In contrast, P-selectin,E-selectin, or PSGL-1 deficiency augments disease manifestationsinduced by bleomycin treatment in parallel with decreased Th1cell infiltration and increased Th2 and Th17 cell infiltration.These results suggest that L-selectin and ICAM-1 mainly regulateTh2 and Th17 cell infiltration, leading to the development offibrosis, whereas P-selectin, E-selectin, and PSGL-1 mainlyregulate Th1 cell infiltration, which results in the inhibition offibrosis.

Materials and MethodsMice

L-selectin-deficient (L-selectin2/2) mice were produced as describedelsewhere (32). ICAM-12/2, P-selectin2/2, E-selectin2/2, and PSGL-12/2

mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Micelacking both L-selectin and ICAM-1 (L-selectin/ICAM-12/2) weregenerated as described previously (18). All mice were backcrossed 10generations onto the C57BL/6 genetic background. Mice used forexperiments were 6 wk old. Both body size and lung size were similarfor mutant and wild-type (WT) mice (data not shown). All studies and pro-cedures were approved by the Committee on Animal Experimentation ofNagasaki University Graduate School of Medical Science.

Bleomycin treatment

Bleomycin (Nippon Kayaku, Tokyo, Japan) was dissolved in PBS ata concentration of 1 mg/ml and sterilized by filtration. Bleomycin or PBS(300 mg) was injected s.c. into a single location on the shaved back of themice daily for 4 wk with a 27-gauge needle, as described previously (30).For blocking study, mAbs to P-selectin (RB40.34, rat IgG1, 30 mg permouse; BD Biosciences, San Jose, CA) were injected i.v. 30 min be-fore first bleomycin or PBS treatment and three times per week intoE-selectin2/2 mice, as described previously (16).

Preparation of BAL fluid

BAL cells were prepared as described elsewhere (23). Briefly, bleomycin-or PBS-treated mice were sacrificed and both lungs were excised. BALfluid was collected as follows: 1 ml of saline was instilled three times andwithdrawn from the lung via an intratracheal cannula. Total leukocytecounts were performed using a hemocytometer in the presence of trypanblue. Cell differential counts were determined after cytospin centrifugationwith May-Giemsa staining. Neutrophils were identified morphologically inthe BAL cells, as described previously (23). A total of 200 cells werecounted from randomly chosen high-power microscopic fields for eachsample, and at least 10 mice of each group were examined.

Histopathologic assessment of dermal fibrosis

Morphologic characteristics of skin sections were assessed under a lightmicroscope. All skin sections were taken from the para-midline, lower backregion (the same anatomic site to minimize regional variations in thickness).Sections were stained with H&E. Dermal thickness, defined as the thick-ness of skin from the top of the granular layer to the junction between thedermis and s.c. fat, was examined. Ten random measurements were takenper section. All of the sections were examined independently by twoinvestigators in a blinded fashion. The skin from male mice was generallythicker than that from female mice despite the bleomycin or PBS treatment(data not shown). Since similar results were obtained when male or femalemice were analyzed separately, only data from female mice were presentedfor skin thickness in this study. Mast cells were identified by toluidine bluestaining. Cells containing metachromatic granules were counted in 10random grids under high-magnification (3400) power fields of a lightmicroscope.

Histopathologic assessment of lung fibrosis

Lungs were excised after 4 wk of treatment with bleomycin or PBS, pro-cessed as previously described, and stained by H&E and Van Gieson stain todetect collagen. The severity of fibrosis was semiquantitatively assessedaccording to Ashcroft et al. (33). Briefly, the lung fibrosis was graded ona scale of 0–8 by examining randomly chosen fields of the left middle lobeat a magnification of 3100. The grading criteria were as follows: grade 0,normal lung; grade 1, minimal fibrous thickening of alveolar or bronchiolarwalls; grade 3, moderate thickening of walls without obvious damage tolung architecture; grade 5, increased fibrosis with definite damage to lungstructure and formation of fibrous bands or small fibrous masses; grade 7,severe distortion of structure and large fibrous areas; and grade 8, totalfibrous obliteration of fields. Grades 2, 4, and 6 represent intermediatesbetween the aforementioned criteria. All of the sections were scored in-dependently by two investigators in a blinded fashion.

Immunohistochemical staining

Frozen tissue sections of skin and BAL cells after cytospin centrifugationwere incubated with rat mAb specific for macrophages (F4/80; Serotec,Oxford, U.K.), B220 (BD Pharmingen, San Diego, CA), and CD3 (clone145-2C11; BD Pharmingen). Rat IgG (SouthernBiotech, Birmingham, AL)was used as a control for nonspecific staining. Stained cells were counted in10 random grids under high-magnification (3400) power fields of a light

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microscope. Each section was examined independently by two investiga-tors in a blinded fashion.

Determination of hydroxyproline content in the skin and lungtissue

Hydroxyproline is a modified amino acid uniquely found at a high per-centage in collagen. Therefore, the skin and lung tissue hydroxyprolinecontent was measured as a quantitative measure of collagen deposition aspreviously described (18). The punch biopsy (6 mm) samples obtainedfrom shaved dorsal skin and the harvested right lung of each mouse wereanalyzed. A hydroxyproline standard solution of 0–6 mg/ml was used togenerate a standard curve.

Anti-nuclear Ab analysis

Anti-nuclear Abs were assessed by indirect immunofluorescence stainingusing sera diluted 1/50 and HEp-2 substrate cells (Medical and BiologicalLaboratories, Nagoya, Japan) as described (31). Anti-nuclear Abs weredetected using FITC-conjugated F(ab9)2 fragments specific for mouseIgG plus IgM plus IgA (SouthernBiotech).

ELISA for serum cytokines, Igs, and autoantibodies

Sera were obtained by a cardiac puncture after 4 wk of treatment withbleomycin or PBS and were stored at 280˚C. Serum levels of IL-4, IL-6, IL-10, IL-17, IFN-g, TGF-b1, and TNF-a were assessed usingspecific ELISA kits according to the manufacturer’s protocol (R&DSystems, Minneapolis, MN). Serum Ig concentrations were assessed asdescribed (31) using affinity-purified mouse IgM, IgG1, IgG2a, IgG2b,IgG3, and IgA (SouthernBiotech) to generate standard curves. The relativeIg concentration of each sample was calculated by comparing the meanOD obtained for duplicate wells to a semilog standard curve of titratedstandard Ab using linear regression analysis. The specific ELISA kits wereused to measure anti-topo I (Medical and Biological Laboratories), anti-centromeric protein B (Funakoshi, Tokyo, Japan), and anti–U1-RNP (Med-ical and Biological Laboratories). These ELISA plates were incubated withserum samples diluted 1/100. Relative levels of autoantibodies were de-termined for each group of mice using pooled serum samples. Sera werediluted at log intervals (1/10–1/105) and assessed for relative autoantibodylevels as above except that the results were plotted as OD versus dilution(log scale). The dilutions of sera giving half-maximal OD values weredetermined by linear regression analysis, thus generating arbitrary unitsper milliliter values for comparison between sets of sera.

RNA isolation and real-time PCR

Total RNA was isolated from lower back skin and lung with RNeasy spincolumns (Qiagen, Crawley, U.K.). Total RNA from each sample was re-verse-transcribed into cDNA. Expression of IL-4, IL-6, IL-10, IL-17, IFN-g,TGF-b1, and TNF-a was analyzed by an ABI Prism 7000 sequencedetector (Applied Biosystems, Foster City, CA) using a real-time PCRquantification method according to the manufacturer’s instructions. Se-quence-specific primers and probes were designed by Pre-Developed Taq-Man assay reagents or Assay-On-Demand (Applied Biosystems). GAPDHwas used to normalize mRNA. Relative expression of real-time PCR prod-ucts was determined by using the DDCT method (31) to compare targetgene and housekeeping gene mRNA expression.

Fibroblast culture and stimulation

Skin samples of 1 cm3 were taken from the para-middle, lower back region ofWT mice. To obtain fibroblasts, the tissue was cut into 1-mm3 pieces, placedin sterile plastic dishes, cultured in DMEM (Invitrogen, Gaithersburg, MD)containing 10% heat-inactivated FCS, 100 U/ml penicillin (Invitrogen), and100 mg/ml streptomycin (Invitrogen), and cultured at 37˚C in a 5% CO2

humidified atmosphere. After 2–3 wk of incubation, the outgrowth of fibro-blasts was detached by brief trypsin treatment and recultured in the medium.Confluent cultures of fibroblasts were serum-starved for 12 h and thencultured with or without 10 ng/ml murine rIL-4 and rIFN-g and 50ng/ml murine rIL-17 (R&D Systems) for 24 h. We used FITC-conjugatedanti-CD90.2 mAb (BD Biosciences) and PE-conjugated anti-CD45 mAb(Serotec) to differentiate fibroblasts and leukocytes (34). The CD452

CD90+ cells were recognized as fibroblasts. The purity of fibroblasts con-firmed with flow cytometry was .99%, with no leukocytes found in theharvested cells (data not shown). Furthermore, the purity of fibroblasts wasassessed by microscopic examination of parallel samples grown on cultureslides (BD Biosciences) and stained with H&E (data not shown), as pre-viously described (35). In each experiment, obtained fibroblasts were exam-ined at the same time and under the same conditions of cultures (e.g., celldensity, passage, and days after plating).

Fibroblast proliferation and collagen synthesis with IL-4, IFN-g,and IL-17 stimulation

Cultured dermal fibroblasts (1.2 3 104/well) were seeded into a 96-wellplate. Fibroblasts were serum-starved for 12 h and then cultured for 24 hwith or without 10 ng/ml murine rIL-4 and rIFN-g and 50 ng/ml murinerIL-17 (R&D Systems). Proliferation of cultured dermal fibroblasts wasquantified by a colorimetric BrdU cell proliferation ELISA kit (RocheApplied Science, Indianapolis, IN). After 24 h of incubation with or with-out rIL-4, rIFN-g, and rIL-17, BrdU (10 mM) was added to each well andincubated for 24 h. To analyze mRNA expression of pro-a2(I) collagen(COL1A2) and TGF-b1, total RNA was isolated from fibroblasts shortlyafter 24 h of incubation with or without murine rIL-4, rIFN-g, and rIL-17.

Isolation and polarization of splenic T cells

Splenocytes were obtained from 6- to 8-wk-old WT mice. Unless statedotherwise, all cell cultures were performed in RPMI 1640 supplementedwith 10% FCS, 2 mM glutamine, 100 U/ml penicillin, 100 mg/ml strep-tomycin, and 2 mM 2-ME. T cells were enriched with a mouse CD4+

T cell kit using an autoMACS isolator (Miltenyi Biotec, Bergisch Glad-bach, Germany). A total of .99% of these cells were CD4+ when testedwith anti-CD4 mAb (BD Biosciences; data not shown). Naive CD4+

T cells were obtained by surface staining with anti-CD62L, anti-CD44,and anti-CD25 mAbs. The CD62L+CD442CD252 population was isolatedby flow cytometry cell sorting with a FACSAria (BD Biosciences). Cellswere activated by plate-bound anti-CD3 (5 mg/ml) and anti-CD28 (5mg/ml) mAbs for 3 d. The Th0 condition indicated the neutral condition(no exogenous cytokines and anti-cytokine Abs). The Th1 condition indi-cates addition of IL-12 (10 ng/ml) and anti–IL-4 Ab (10 mg/ml). The Th2condition indicates addition of IL-4 (10 ng/ml) and anti–IL-12 Ab (10mg/ml). The Th17 condition indicates addition of IL-6 (10 ng/ml), TGF-b1(5 ng/ml), anti–IFN-g mAb (10 mg/ml), and anti–IL-4 mAb (10 mg/ml).All cytokines were from R&D Systems. All anti-cytokine Abs were fromBD Pharmingen.

Flow cytometry

Abs used in this study included FITC-conjugated mAbs to IL-4 (Imgenex,San Diego, CA), IFN-g (GeneTex, San Antonio, TX), and IL-17 (NovusBiologicals, Littleton, CO); PE-conjugated mAbs to IL-17, L-selectin,LFA-1, and PSGL-1 (all from BD Biosciences); and cyanine 5–PE anti-CD4 mAb (LifeSpan BioSciences, Seattle, WA). Single-cell suspensions ofBAL cells or CD4+ T cells were incubated with the Abs for 30 min at 4˚C.The cells were washed and fixed with 1% paraformaldehyde in PBS.IFN-g, IL-4, and IL-17 production of BAL lymphocytes and culturedCD4+ T cells was determined by flow cytometric intracellular cytokineanalysis, as previous described (36). Briefly, cells were suspended at 106

per milliliter in RPMI 1640 containing 2 mM glutamine and incubatedwith 10 mg/ml brefeldin A (Calbiochem, San Diego, CA) for 2 h at 37˚C.Samples were stained for cell surface markers PSGL-1, LFA-1, and CD4for 30 min at 4˚C. After permeabilizing with FACS permeabilizing solu-tion according to the manufacturer’s instructions (BD Pharmingen), thecells were then stained for intracellular IFN-g, IL-4, and IL-17. Cells werewashed and analyzed on a FACScan flow cytometer (BD Pharmingen),with data have been analyzed from 105 cells. Positive and negative pop-ulations of cells were determined using unreactive isotype-matched mAbs(Beckman Coulter, Brea, CA) as controls for background staining.

P-selectin, E-selectin, and ICAM-1 binding assay in polarizedTh cells

We performed P-selectin, E-selectin, and ICAM-1 binding assays, as de-scribed previously (37). Recombinant mouse P-selectin, E-selectin, orICAM-1/Fc chimera proteins were obtained from R&D Systems. Th0,Th1, Th2, or Th17 cells (5 3 104), which were isolated by flow cytometrycell sorting with a FACSAria (BD Biosciences), were resuspended in 100ml of RPMI 1640 medium. Cells were incubated with 0.3 mg of eachchimera protein for 30 min at 4˚C and resuspended in 100 ml of RPMI1640 medium containing FITC-labeled goat anti-mouse IgG (2 mg/ml;Jackson ImmunoResearch Laboratories, West Grove, PA). After incubationfor another 30 min at 4˚C, cells were analyzed on a FACScan flow cytom-eter (BD Pharmingen), with data having been analyzed from 105 cells.

Statistical analysis

All data are expressed as mean values6 SD. The Mann-WhitneyU test wasused to determine the level of significance of differences between samplemeans, and ANOVA followed by Bonferroni’s test was used for multiplecomparisons.

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ResultsL-selectin and/or ICAM-1 loss attenuated the development offibrosis induced by bleomycin, whereas P-selectin loss,E-selectin loss with or without P-selectin blockade, orPSGL-1 loss augmented it

Bleomycin was injected s.c. into the back of mice daily for4 wk. Previous studies have shown that skin and lung fibrosis,epithelial injury, and inflammatory cell infiltration develop duringthe first 4 wk of bleomycin treatment peak in the fourth week andbegin to resolve 6 wk after the cessation of treatment (30, 31). Inthis study, skin and lung fibrosis in mutant and WT mice treatedwith either bleomycin or PBS was histopathologically assessed1, 2, 3, and 4 wk after bleomycin treatment. The dermal thickness(the thickness from the top of the granular layer to the junctionbetween the dermis and s.c. fat) and lung fibrosis score showed

a time-dependent increase in bleomycin-treated mice (data notshown), which is consistent with previous studies (30, 31). After4 wk, bleomycin treatment induced significantly greater dermalthickness relative to PBS treatment in mutant and WT mice (p ,0.05; Fig. 1A, 1B). The dermal thickness was similar betweennontreated and PBS-treated mice (data not shown). The dermalthickness in bleomycin-treated WT mice significantly increasedby 2.3-fold compared with PBS-treated WT mice (p , 0.05; Fig.1A, 1B). Bleomycin-treated L-selectin2/2 mice, ICAM-12/2

mice, and L-selectin/ICAM-12/2 mice showed moderatethickening of dermal tissue that was 23, 26, and 49% thinner,respectively, than that found in bleomycin-treated WT mice(p , 0.05). Moreover, skin thickness of bleomycin-treated

L-selectin/ICAM-12/2 mice was thinner than that of bleomycin-treated L-selectin2/2 and ICAM-12/2 mice (p, 0.01). In contrast,the dermal thickness in bleomycin-treated P-selectin2/2 mice, E-selectin2/2 mice, E-selectin2/2 mice treated with anti–P-selectinmAb, and PSGL-12/2 mice was 15, 18, 28, and 31% thicker,respectively, than that in bleomycin-treated WT mice (p , 0.05;Fig. 1A, 1B). Bleomycin-treated E-selectin2/2 mice administratedwith anti–P-selectin mAb and PSGL-12/2 mice showed theincreased dermal thickness compared with bleomycin-treatedP-selectin2/2 and E-selectin2/2 mice (p , 0.05; Fig. 1A, 1B).Additionally, skin thickness of bleomycin-treated L-selectin2/2

mice, ICAM-12/2 mice, and L-selectin/ICAM-12/2 mice was

significantly thinner than that of bleomycin-treated P-selectin2/2

mice, E-selectin2/2 mice, E-selectin2/2 mice treated with anti–P-selectin mAb, and PSGL-12/2 mice (p , 0.01; Fig. 1A, 1B).Masson trichrome staining revealed thickened collagen bundles inthe skin from bleomycin-treated WT mice, which was reducedby L-selectin and/or ICAM-1 deficiency and was increased byP-selectin deficiency, E-selectin deficiency with or without P-selectin blockade, or PSGL-1 deficiency (data not shown). Cuta-neous fibrosis was also assessed by quantifying hydroxyprolinecontent of 10 mg of skin samples from mutant and WT mice(Fig. 1B). Although the hydroxyproline content in bleomycin-treated WT mice was increased by 2.9-fold relative to that inPBS-treated WT mice (p , 0.01), bleomycin-treated L-selectin2/2,

ICAM-12/2, and L-selectin/ICAM-12/2 mice reduced thehydroxyproline content by 29, 31, and 41%, respectively, inbleomycin-treated WT mice (p , 0.05). Moreover, the hydroxy-proline content of bleomycin-treated L-selectin/ICAM-12/2 micewas significantly reduced compared with bleomycin-treated L-selectin2/2 and ICAM-12/2 mice (p , 0.01). In contrast, thehydroxyproline content was increased in bleomycin-treated P-selectin2/2 mice (10% increase), E-selectin2/2 mice (9%), E-selectin2/2 mice treated with anti–P-selectin mAb (22%), andPSGL-12/2 mice (20%) compared with bleomycin-treated WT mice

(p , 0.05; Fig. 1B). Additionally, bleomycin-treated E-selectin2/2

mice administrated with anti–P-selectin mAb and PSGL-12/2 miceshowed further accumulation of the hydroxyproline comparedwith bleomycin-treated P-selectin2/2 and E-selectin2/2 mice (p ,0.05; Fig. 1B).Similar results were obtained for the lung fibrosis score and the

pulmonary hydroxyproline content (Fig. 2A, 2B). After 4 wk,bleomycin-treated WT mice exhibited extensive inflammatory cellinfiltration, fibrosis, granulomas, alveolar epithelial injury, and in-creased hydroxyproline content (Fig. 2A, 2B). L-selectin orICAM-1 deficiency reduced such histological changes and hydrox-yproline content, whereas P-selectin, E-selectin, or PSGL-1 defi-ciency augmented it. Thus, skin and lung fibrosis induced by s.c.bleomycin injection was inhibited by L-selectin or ICAM-1 defi-ciency and was exacerbated by P-selectin, E-selectin, or PSGL-1deficiency. Both L-selectin and ICAM-1 deficiency significantlyinhibited skin and lung fibrosis relative to L-selectin or ICAM-1deficiency alone. Furthermore, the deterioration effect of PSGL-1deficiency or E-selectin deficiency with P-selectin blockade on skinand lung fibrosis was greater than that of P-selectin or E-selectindeficiency alone.

Leukocyte infiltration into skin and lung was inhibited byL-selectin and/or ICAM-1 loss, whereas it was enhanced byP-selectin loss, E-selectin loss with or without P-selectinblockade, or PSGL-1 loss

The numbers of mast cells, macrophages, T cells, and B cells havebeen reported to increase in sclerotic skin and fibrotic lung fromhuman SSc patients and bleomycin-induced SSc mouse models(31, 38). Therefore, the numbers of these immune cells wereassessed in skin and BAL fluid after 4 wk of bleomycin treatmentin mutant and WT mice. The skin and BAL numbers of mast cells,neutrophils, macrophages, T cells, and B cells were greater inbleomycin-treated mice than in PBS-treated mice (p , 0.005;Figs. 1C, 2C). In skin tissue, bleomycin-treated L-selectin2/2,ICAM-12/2, and L-selectin/ICAM-12/2 mice exhibited lowernumbers of these cells compared with bleomycin-treated WT mice(p , 0.05), except for T cell numbers (Fig. 1C). In contrast, thenumbers of mast cells, T cells, and B cells were greater in bleo-mycin-treated P-selectin2/2mice, E-selectin2/2mice, E-selectin2/2

mice treated with anti–P-selectin mAb, and PSGL-12/2 mice thanin bleomycin-treated WT mice (p, 0.01; Fig. 1C).In BAL cells, bleomycin-treated L-selectin2/2, ICAM-12/2,

and L-selectin/ICAM-12/2 mice showed decreased numbers oftotal leukocytes, including neutrophils and macrophages,compared with bleomycin-treated WT mice (p , 0.05; Fig. 2C).Moreover, bleomycin-treated L-selectin/ICAM-12/2 mice showedlower numbers of total leukocytes and neutrophils than didbleomycin-treated L-selectin2/2 and ICAM-12/2 mice (p ,0.05; Fig. 2C). However, T cell and B cell numbers in BAL fluidwere similar among bleomycin-treated WT, L-selectin2/2, ICAM-12/2, and L-selectin/ICAM-12/2 mice. In contrast, the numbers oftotal leukocytes, including neutrophils, macrophages, T cells, andB cells, significantly increased in bleomycin-treated P-selectin2/2

mice, E-selectin2/2 mice, E-selectin2/2 mice treated with anti–P-selectin mAb, and PSGL-12/2 mice compared with those inbleomycin-treated WT mice (p , 0.05). E-selectin2/2 micetreated with anti–P-selectin mAb and PSGL-12/2 mice exhibitedfurther increased numbers of neutrophils relative to bleomycin-treated P-selectin2/2 and E-selectin2/2 mice (p , 0.05). Thus,inflammatory cell recruitment to BAL fluid was inhibited by L-selectin or ICAM-1 deficiency. Furthermore, both L-selectin andICAM-1 deficiency inhibited this recruitment more strongly thandid L-selectin or ICAM-1 deficiency alone. In contrast, P-selectin



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or E-selectin deficiency alone induced inflammatory cell recruit-ment to BAL fluid. Moreover, this increasing effect of E-selectindeficiency with P-selectin blockade and PSGL-1 deficiency wasgreater than that of P-selectin or E-selectin deficiency alone.

Effect of cell adhesion molecule deficiency or blockade oncytokine production

It has been suggested that IL-4, IL-6, IFN-g, IL-17, TNF-a, TGF-b1, and IL-10 production contributes to bleomycin-induced fibro-sis by regulating the collagen production by fibroblasts (30, 31, 39,40). Therefore, in the serum, sclerotic skin, and fibrotic lung, theproduction of these cytokines was assessed by ELISA and real-time PCR. In the serum, skin, and lung, bleomycin-treated mutantand WT mice had elevated levels of IL-4, IL-6, IFN-g, IL-17,TNF-a, TGF-b1, and IL-10 compared with PBS-treated mutantand WT mice (p , 0.01). Serum levels of IL-4, IL-6, IL-17, andTGF-b1 were reduced in bleomycin-treated L-selectin2/2, ICAM-

12/2, and L-selectin/ICAM-12/2 mice relative to bleomycin-treated WT mice, whereas serum levels of IFN-g were higherin these mice than in bleomycin-treated WT mice (p , 0.05;Fig. 3A). In contrast, bleomycin-treated P-selectin2/2 mice, E-selectin2/2 mice, E-selectin2/2 mice treated with anti–P-selectin mAb, and PSGL-12/2 mice exhibited elevated levels ofIL-4, IL-6, IL-17, and TGF-b1 compared with bleomycin-treatedWT mice (p , 0.05), whereas they showed lower levels of IFN-gthan did bleomycin-treated WT mice (p , 0.05; Fig. 3A). Therewere no significant differences in serum levels of TNF-a andIL-10 between bleomycin-treated mutant and WT mice. Similarresults were obtained for mRNA expression levels in the skin andlung (data not shown), except that bleomycin-treated L-selectin/ICAM-12/2 mice showed lower expression levels of IL-4, IL-17,and TGF-b1 in the skin and lung and IL-6 in the lung thandid L-selectin2/2 and ICAM-12/2 mice (p , 0.05). In lungtissue, ICAM-12/2 mice exhibited lower IL-17 expression than

FIGURE 1. Skin fibrosis (A, B) and the numbers of mast cells, macrophages, T cells, and B cells at the bleomycin-injected site of skin (C) from PBS-

treated (open bar) or BLM-treated (filled bar) WT mice, L-selectin2/2 mice (L2/2), ICAM-12/2 mice (ICAM-12/2), L-selectin/ICAM-12/2 mice (L/

ICAM-12/2), P-selectin2/2 mice (P2/2), E-selectin2/2 mice (E2/2), E-selectin2/2 mice treated with anti–P-selectin mAb (E2/2 + anti-P Ab), and PSGL-

12/2 mice (PSGL-12/2). A, Representative histological sections stained with H&E are shown (original magnification 340). Skin fibrosis was assessed by

quantitatively measuring dermal thickness and hydroxyproline content 4 wk after bleomycin treatment (B). Mast cells were identified by toluidine blue

staining; macrophages, T cells, and B cells were stained with F4/80, anti-CD3 mAb, and anti-B220 mAb, respectively (C). These results represent those

obtained with at least 10 mice in each group. The dermal thickness was measured under a light microscope. Cells were counted in 10 random grids under

magnification of3400 high-power fields. Each histogram shows the mean (6SD) results obtained for 10 mice in each group. pp, 0.05; ppp, 0.01 versus

PBS-treated each group of mice. †p , 0.05; ††p , 0.01 versus BLM-treated WT mice. BLM, bleomycin; HPF, high-power field.



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FIGURE 2. Lung fibrosis (A, B) and the influx numbers of total leukocytes, including neutrophils, macrophages, T cells, and B cells, into BAL fluid (C)

from PBS-treated (open bar) or bleomycin-treated (filled bar) WT mice, L-selectin2/2 mice (L2/2), ICAM-12/2mice (ICAM-12/2), L-selectin/ICAM-12/2

mice (L/ICAM-12/2), P-selectin2/2 mice (P2/2), E-selectin2/2 mice (E2/2), E-selectin2/2 mice treated with anti–P-selectin mAb (E2/2 + anti-PAb), and

PSGL-12/2 mice (PSGL-12/2). A, Representative histological sections stained with H&E are shown (original magnification 3100). Lung fibrosis was

assessed by quantitatively measuring lung fibrosis score and hydroxyproline content 4 wk after bleomycin treatment (B). The BAL cell counts were as

described in Materials and Methods. These results were obtained from at least 10 mice in each group. Lung fibrosis score was measured under a light mi-

croscope. The differential BAL cells were counted in 10 random grids under magnification of3400 high-power fields. Each histogram shows the mean (6SD)

results obtained for 10 mice of each group. pp, 0.05; ppp, 0.01 versus PBS-treated each group. †p, 0.05; ††p, 0.01 versus bleomycin-treated WT mice.

BLM, bleomycin; HPF, high-power field.



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did L-selectin2/2 mice (p , 0.05). Moreover, E-selectin2/2 micetreated with anti–P-selectin mAb and PSGL-12/2 mice exhibitedhigher expression levels of IL-4, IL-17, and TGF-b1 in the skin andlung and IL-6 in the lung than did P-selectin2/2 and E-selectin2/2

mice (p, 0.05). In contrast, E-selectin2/2 mice treated with anti–P-selectin mAb and PSGL-12/2 mice exhibited a reduction in skinIFN-g expression that was significantly lower than that found inP-selectin2/2 mice as well as in E-selectin2/2 mice (p , 0.05).Thus, in the serum, sclerotic skin, and fibrotic lung, bleomycintreatment induced the overexpression of various cytokines.L-selectin and/or ICAM-1 deficiency reduced expression levelsof fibrotic Th2 cytokines, such as IL-4 and IL-6, IL-17, andTGF-b1, a major fibrogenic growth factor, whereas P-selectindeficiency, E-selectin deficiency with or without P-selectin block-ade, and PSGL-1 deficiency increased it. In contrast, L-selectinand/or ICAM-1 deficiency increased expression of IFN-g, anti-fibrotic Th1 cytokine, whereas P-selectin deficiency, E-selectin de-ficiency with or without P-selectin blockade, and PSGL-1 deficiencydecreased it.

Effect of Th1, Th2, and Th17 cytokines on dermal fibroblasts

We investigated the effect of IL-4, IFN-g, and IL-17 on functionof fibroblasts obtained from WT mice (Fig. 4). Stimulation withIL-4 and IL-17 significantly increased fibroblast proliferation com-pared with media alone (p , 0.01). In contrast, IFN-g stimulationinhibited fibroblast proliferation compared with media alone (p ,0.05). COL1A2 and TGF-b1 mRNA expressions were quantifiedby real-time PCR in cultured dermal fibroblasts. COL1A2 andTGF-b1 mRNA levels in fibroblasts significantly increased byIL-4 and IL-17 stimulation, whereas they decreased by IFN-gstimulation compared with media alone (p, 0.01).

Infiltration of Th1, Th2, and Th17 cells was regulated by celladhesion molecules in the bleomycin-induced SSc mouse model

We investigated Th1, Th2, and Th17 cell frequencies in BAL fluidfrom bleomycin-treated mutant and WT mice (Fig. 5). Bleomycin-treated mutant and WT mice exhibited significantly increasedfrequencies of Th1, Th2, and Th17 cells compared with PBS-

FIGURE 3. Levels of IL-4, IL-6, IFN-g, IL-17, TNF-a, TGF-b1, and IL-10 in serum samples from WT mice, L-selectin2/2 mice (L2/2), ICAM-12/2

mice (ICAM-12/2), L-selectin/ICAM-12/2 mice (L/ICAM-12/2), P-selectin2/2 mice (P2/2), E-selectin2/2 mice (E2/2), E-selectin2/2 mice treated with

anti–P-selectin mAb (E2/2 + anti-P Ab), and PSGL-12/2 mice (PSGL-12/2) treated with either PBS (open bar) or bleomycin (filled bar). Serum samples

were obtained by a cardiac puncture 4 wk after treatment with either bleomycin or PBS. Serum cytokine levels were assessed using specific ELISA. Each

histogram shows the mean (6SD) results obtained for 10 mice of each group. ppp , 0.01 versus PBS-treated each group. †p , 0.05; ††p , 0.01 versus

BLM-treated WT mice.

FIGURE 4. Proliferation and collagen synthesis of dermal fibroblasts obtained from WT mice. Cultured fibroblasts were serum starved for 12 h and then

cultured for 24 h with murine rIL-4 (10 ng/ml), rIFN-g (10 ng/ml), and rIL-17 (50 ng/ml). Total RNA from fibroblasts was extracted and reverse transcribed

to cDNA, and mRNA expression of COL1A2 and TGF-b1 was analyzed by real-time PCR. In proliferation assays, after 24 h incubation, BrdU (10 mM)

was added to each well and incubated for 24 h. BrdU incorporation in proliferating cells was quantified by ELISA. Each histogram shows the mean (6SD)

results obtained for six mice of each group. pp , 0.05; ppp , 0.01 versus fibroblasts cultured with media alone.



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treated mutant and WT mice in the BAL fluid (p , 0.01; Fig. 5B)but not in homogenate lung parenchyma (data no shown). Therewere no significant differences in Th1, Th2, and Th17 cell fre-quencies between PBS-treated mutant and WT mice (Fig. 5B).The population expressing IFN-g, IL-4, or IL-17 did not over-lap (Fig. 5A), which is consistent with previous studies (36). Bleo-mycin-treated L-selectin2/2, ICAM-12/2, and L-selectin/ICAM-12/2 mice exhibited significantly reduced Th2 and Th17 cellfrequencies and significantly increased Th1 cell frequencies inBAL fluid compared with bleomycin-treated WT mice (p ,0.05; Fig. 5B). L-selectin/ICAM-12/2 mice displayed furtherreduction of Th2 and Th17 cell influx into BAL fluid relativeto L-selectin2/2 and ICAM-12/2 mice (p , 0.05). Th1 cell fre-quencies were significantly reduced in bleomycin-treated P-selectin2/2 and E-selectin2/2 mice compared with bleomycin-treated WT mice (p , 0.05). The reducing effect of E-selectinloss with P-selectin blockade and PSGL-1 loss on Th1 cell influxwas greater than that of P-selectin or E-selectin deficiency alone(p , 0.05). Bleomycin-treated P-selectin2/2 mice, E-selectin2/2

mice with or without P-selectin blockade, and PSGL-12/2 micealso exhibited significantly increased Th2 and Th17 cell fre-quencies in BAL fluid compared with bleomycin-treated WT mice(p , 0.05, respectively). Moreover, E-selectin2/2 mice treatedwith anti–P-selectin mAb and PSGL-12/2 mice displayed higherfrequencies of Th17 cells compared with P-selectin2/2 and E-selectin2/2 mice (p , 0.05). Thus, these results suggest thatL-selectin and/or ICAM-1 deficiency inhibits Th2 and Th17 cellinflux into BAL fluid, whereas Th1 cell infiltration is inducedby L-selectin and/or ICAM-1 deficiency. P-selectin loss, E-selectin loss with or without P-selectin blockade, and PSGL-1loss inhibit Th1 cell influx and induce Th2 and Th17 cell in-filtration.

Expression of LFA-1 and PSGL-1 on polarized Th1, Th2, andTh17 cells

To investigate how cell adhesion molecules regulate Th1, Th2, andTh17 cell infiltration, expression of LFA-1, a ligand of ICAM-1,and PSGL-1 in polarized Th1, Th2, and Th17 cells and nonpolar-ized Th0 cells were analyzed by flow cytometry (Fig. 6). In Th0cells obtained from the Th0 condition, IFN-g, IL-4, and IL-17expression was hardly detectable (Fig. 6A). In Th1, Th2, andTh17 cells obtained from the Th1, Th2, and Th17 conditions,respectively, the population expressing IFN-g, IL-4, or IL-17 didnot overlap (data not shown). Th1, Th2, and Th17 cells did notexpress L-selectin (data not shown). Th1 cells showed lowerLFA-1 expression and ICAM-1 binding ability compared withTh0 cells (p , 0.01 and p , 0.05, respectively; Fig. 6B, 6C).In contrast, Th1 cells exhibited higher PSGL-1 expression andP-selectin and E-selectin binding ability relative to Th0 cells(p , 0.01, p , 0.001, and p , 0.05, respectively). AlthoughLFA-1 expression and ICAM-1 binding ability increased in Th2and Th17 cells compared with Th0 cells (p, 0.01), Th2 and Th17cells exhibited lower expression levels of PSGL-1 and P-selectinand E-selectin binding ability (p , 0.01). Additionally,LFA-1 expression levels and ICAM-1 binding ability in Th17 cellswere greater than those in Th2 cells (p , 0.05). There was nosignificant difference in the frequencies of LFA-1+ or PSGL-1+

cells among Th0, Th1, Th2, and Th17 cells (Fig. 6A). Theseresults suggest that PSGL-1 is preferentially used to recruit Th1cells into inflammatory lesions, whereas Th2 and Th17 cells dom-inantly use LFA-1.

Contribution of cell adhesion molecules to Ig production

Serum Ig levels in bleomycin-treated mutant and WT mice werealso investigated (Fig. 7). PBS-treated mutant mice had similar

FIGURE 5. Th1, Th2, and Th17 cell frequencies of BAL in PBS-treated (open bar) or bleomycin-treated (filled bar) WT mice, L-selectin2/2 mice (L2/2),

ICAM-12/2 mice (ICAM-12/2), L-selectin/ICAM-12/2 mice (L/ICAM-12/2), P-selectin2/2 mice (P2/2), E-selectin2/2 mice (E2/2), E-selectin2/2 mice

treated with anti–P-selectin mAb (E2/2 + anti-P Ab), and PSGL-12/2 mice (PSGL-12/2). We determined Th1, Th2, and Th17 cells by surface

CD4 expression and intracellular expression of IFN-g, IL-4, and IL-17 as previously described (36). BAL fluid was analyzed by flow cytometry after

4 wk of PBS or bleomycin treatment. These data are representative of three independent experiments (A). Percentages of Th1, Th2, and Th17 cells are shown

in the each quadrant. We also show summaries of Th1, Th2, and Th17 cell frequencies in each group (B). Each histogram shows the mean (6SD) results

obtained for 10 mice of each group. pp , 0.005; ppp , 0.001 versus PBS-treated each group. †p , 0.05; ††p , 0.01 versus bleomycin-treated WT mice.

BLM, bleomycin.



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IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA levels to PBS-treatedWT mice (data not shown). Bleomycin treatment increased serumIgM, IgG1, IgG2a, IgG2b, IgG3, and IgA levels compared withPBS treatment (p , 0.05). L-selectin/ICAM-12/2 mice treatedwith bleomycin had decreased IgM and IgG3 levels compared withbleomycin-treated WT mice (p , 0.05), whereas the levels of otherisotypes were similar among bleomycin-treated L-selectin2/2,ICAM-12/2, L-selectin/ICAM-12/2, and WT mice. P-selectin2/2

and E-selectin2/2 mice administered bleomycin had increasedIgG2b levels compared with bleomycin-treated WT mice (p ,0.05), whereas there were no significant differences in the levels ofother isotypes among bleomycin-treated P-selectin2/2, E-selectin2/2,and WT mice. In E-selectin2/2 mice treated with anti–P-selectinmAb and in PSGL-12/2 mice, bleomycin administration increasedserum levels of all Ig isotypes, except IgA, compared withbleomycin-treated WT mice (p , 0.05). Thus, treatment with bleo-mycin induced hypergammaglobulinemia, which was inhibitedby L-selectin and/or ICAM-1 loss, whereas the loss of P-selectin,E-selectin with or without P-selectin blockade, or PSGL-1 aug-mented Ig production.

L-selectin and/or ICAM-1 loss inhibited autoantibodyproduction in bleomycin-treated mice, whereas P-selectin loss,E-selectin loss with or without P-selectin blockade, andPSGL-1 loss increased it

Anti-nuclear Abs were rarely detectable in PBS-treated mutant andWT mice (6%, 1/18). Anti-nuclear Abs with a homogeneous chro-mosomal staining pattern were detected in 47% (16/34) of bleomycin-treated WT mice, which was similar to that in bleomycin-treatedP-selectin2/2 mice (41%, 14/34), E-selectin2/2 mice (44%, 15/34), E-selectin2/2 mice with blockade of P-selectin (53%, 18/34),and PSGL-12/2 mice (53%, 18/34). In contrast, the frequenciesof anti-nuclear Ab positivity were lower in bleomycin-treated L-selectin2/2 (29%, 10/34), ICAM-12/2 (26%, 9/34), and L-selectin/ICAM-12/2 (12%, 4/34) mice. Autoantibody specificities werefurther assessed by ELISA (Fig. 8). The dilution of sera giving

half-maximal OD values in ELISA generated arbitrary units permilliliter that could be directly compared between groups (valuesin parentheses of Fig. 8). Bleomycin-treated L-selectin2/2 andICAM-12/2 mice had decreased IgM autoantibody levels to topo Iand U1-RNP and reduced IgG autoantibody levels to topo I relativeto bleomycin-treated WT mice (p, 0.05). L-selectin and ICAM-1deficiency decreased bleomycin-induced IgM autoantibody levelsto topo I and U1-RNP, as well as IgG autoantibody levels to topo Icompared with bleomycin-treatedWTmice (p, 0.05). In contrast,bleomycin-treated P-selectin2/2 and E-selectin2/2 mice showedincreased IgM autoantibody levels to topo I and increased IgGautoantibody levels to topo I relative to bleomycin-treatedWTmice(p , 0.05). Bleomycin-treated E-selectin2/2 mice treated withanti–P-selectin mAb and PSGL-12/2 mice had increased IgMautoantibody levels to topo I, as well as IgG autoantibody levelsto topo I compared with bleomycin-treated WT mice (p , 0.05).Furthermore, IgG anti-topo I Ab production in E-selectin2/2 micetreated with anti–P-selectin mAb and PSGL-12/2mice was greaterthan those in P-selectin2/2 and E-selectin2/2 mice (p , 0.05).Thus, bleomycin-induced production of various autoantibodies,especially SSc-specific anti-topo I Ab, decreased with L-selectinand/or ICAM-1 deficiency, whereas it increased by P-selectin loss,E-selectin loss with or without P-selectin blockade, or PSGL-1 loss.

DiscussionCell adhesion molecules play a critical role in the development ofseveral inflammatory diseases (7). In general, inhibition or loss ofcell adhesion molecules attenuates inflammatory response in manyin vivo experimental models (14, 16–18). Dermal sclerosis of thetight-skin mouse model, a genetic model for SSc, and pulmonaryfibrosis induced by intratracheal bleomycin treatment are almostcompletely eliminated by loss of both L-selectin and ICAM-1,whereas loss of L-selectin or ICAM-1 alone results in less inhibi-tion (14, 18). Similarly, cutaneous contact hypersensitivity responseand TNF-a–induced leukocyte rolling are almost completely inhi-bited by loss of both E-selectin and P-selectin, while loss of each

FIGURE 6. The LFA-1 and PSGL-1 expression levels (A, B) and ICAM-1, P-selectin, and E-selectin binding ability (C) in Th0, Th1, Th2, and Th17

cells. We determined Th0, Th1, Th2, and Th17 cells by surface CD4 expression and intracellular expression of IFN-g, IL-4, and IL-17 as previously

described (36). Polarized or nonpolarized splenic CD4+ T cells were analyzed by flow cytometry. These data are representative of three independent

experiments. Numbers indicate the percentage of cells in each quadrant. Histograms indicate MFI (6SD). MFI, mean fluorescence intensity.



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molecule alone leads to less inhibition (19, 20). In the presentstudy, L-selectin and/or ICAM-1 deficiency inhibited the develop-ment of dermal and pulmonary fibrosis with decreased inflamma-tory cell infiltration in the bleomycin-induced SSc model (Figs.1, 2). The inhibitory effect of both L-selectin and ICAM-1 defi-ciency was significantly greater than that of L-selectin or ICAM-1deficiency alone. Unexpectedly, P-selectin deficiency, E-selectindeficiency with or without P-selectin blockade, and PSGL-1 de-ficiency exacerbated inflammatory cell infiltration induced by bleo-mycin treatment, resulting in deteriorated dermal sclerosis andpulmonary fibrosis, more severe histologic change, and increaseddermal and pulmonary collagen deposition (Figs. 1, 2). The deteri-oration effect of E-selectin deficiency with P-selectin blockade andPSGL-1 deficiency was significantly greater than that of P-selectinor E-selectin deficiency alone. Collectively, the results of the presentstudy are the first to reveal cooperative and deterioration rolesof L-selectin and ICAM-1 and synergic and inhibitory rolesof P-selectin, E-selectin, and PSGL-1 in the development of bleo-mycin-induced fibrosis.Previous studies have demonstrated fibrotic effect of Th2 cyto-

kines, such as IL-4 and IL-6 on dermal sclerosis and pulmonary fi-brosis (14, 18, 31). Th17 cytokines, such as IL-17, also havea fibrogenic effect on dermal, pulmonary, and cardiac fibroblasts(41, 42). Indeed, SSc patients exhibit elevated serum levels of theseTh2 and Th17 cytokines (5, 6, 43, 44). These cytokines promotecollagen synthesis of fibroblasts via TGF-b1 and COL1A2signaling (41). Some studies have also shown that IFN-g, one ofthe Th1 cytokines, has an antifibrotic effect on skin and pulmonaryfibrosis (36, 45). Although these findings suggest that Th1, Th2,and Th17 responses are associated with disease activity in SScpatients, the mechanism of fibrosis in SSc remains unclear. Inthis study, we showed that IL-4 or IL-17 stimulation increasedproliferation and TGF-b1 and COL1A2 expression of dermalfibroblasts obtained from WT mice, whereas IFN-g inhibited it(Fig. 4). The results of this study indicate differential expression

levels of these cytokines in cell adhesion molecule-deficient micetreated with bleomycin (Fig. 3). Lack of L-selectin and/orICAM-1 reduced IL-4, IL-6, IL-17, and TGF-b1 expression andincreased IFN-g expression in parallel with inhibited dermalsclerosis and pulmonary fibrosis. P-selectin loss, E-selectin losswith or without P-selectin blockade, or PSGL-1 loss reduced IFN-gand increased IL-4, IL-6, IL-17, and TGF-b1, which wasaccompanied by increased dermal and pulmonary fibrosis. Thus,the expression of these cell adhesion molecules alters the cytokineproduction, which may contribute to the development of skin andlung fibrosis induced by bleomycin treatment.It is possible that loss of cell adhesion molecule function se-

lectively alters the trafficking pattern of fibrogenic Th2 and Th17cells and antifibrogenic Th1 cells to the skin and lung. This mayresult in differential production of cytokines, which may thendirectly or indirectly influence the development of dermal sclerosis,pulmonary fibrosis, and inflammatory cell infiltration. Previousstudies have demonstrated that loss of L-selectin and/or ICAM-1function inhibits lung fibrosis induced by intratracheal bleomycintreatment (14), whereas loss of P-selectin and/or E-selectin func-tion augments it (23). These studies suggest that the mechanismsby which cell adhesion molecules regulate tissue fibrosis could bethrough the regulation of Th1 and Th2 cell infiltration. Two stud-ies have demonstrated that loss of L-selectin and/or ICAM-1 func-tion inhibited Th2 cell immigration, inducing Th1 cell infiltrationin the allergic lung disease model (46, 47). Additionally, loss ofP-selectin, E-selectin, and/or PSGL-1 function prevents Th1 cellinfiltration and induces Th2 cell immigration in the cutaneousdelayed-type hypersensitivity and allergic lung disease models(11, 48). Consistent with these findings, the results of this studyshow that lack of L-selectin and/or ICAM-1 inhibited Th2 cellinfiltration and induced Th1 cell immigration into BAL fluid,whereas P-selectin loss, E-selectin loss with or without P-selectin blockade, or PSGL-1 loss decreased Th1 cell infiltrationand induced Th2 cell infiltration in the bleomycin-induced SSc

FIGURE 7. Serum Ig levels in bleomycin-treated WT mice, L-selectin2/2 mice (L2/2), ICAM-12/2 mice (ICAM-12/2), L-selectin/ICAM-12/2 mice

(L/ICAM-12/2), P-selectin2/2 mice (P2/2), E-selectin2/2 mice (E2/2), E-selectin2/2 mice treated with anti–P-selectin mAb (E2/2 + anti-P Ab), and

PSGL-12/2 mice (PSGL-12/2). Serum Ig levels in PBS-treated WT mice were used as control Ig levels. Serum Ig levels were determined by isotype-

specific ELISA. Horizontal bars represent mean Ig levels. pp , 0.05; ppp , 0.01 versus each CTL group. †p , 0.05; ††p , 0.01 versus bleomycin-treated

WT mice. CTL, control.



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model (Fig. 5). In contrast, other studies showed that L-selectinand/or ICAM-1 loss decreased Th1 cell infiltration and inducedTh2 cell infiltration on inflamed endothelium induced by Th2-delived factors (26, 27). Loss of P-selectin, E-selectin, and/orPSGL-1 function attenuated Th2 cell tethering and rolling onendothelium and epithelium in the intestinal inflammation model(28, 49). Thus, it is likely that each cell adhesion molecule regu-lates Th cell balance in several ways, according to the tissue siteand nature of the inflammation stimuli. Additionally, loss of L-selectin and/or ICAM-1 decreased Th17 cell infiltration,whereas P-selectin loss, E-selectin loss with or without P-

selectin blockade, and PSGL-1 loss induced Th17 cell migration(Fig. 5). Consistent with these findings, a recent study has sug-gested that loss of L-selectin and/or ICAM-1 function inhibitedTh17 cell infiltration to the inflammatory sites in the experimentalcolitis model (29, 50). Furthermore, P-selectin and E-selectin de-ficiency induces Th17 cells in the neutrophila condition inducedby G-CSF treatment (51). Collectively, in the bleomycin-inducedSSc model, loss of L-selectin and/or ICAM-1 function may lead todominant Th1 cell infiltration in parallel with decreased skin andlung fibrosis, whereas P-selectin loss, E-selectin loss with orwithout P-selectin blockade, and PSGL-1 loss may induce

FIGURE 8. Autoantibody levels in sera from bleomycin-treated WT mice, L-selectin2/2 mice (L2/2), ICAM-12/2 mice (ICAM-12/2), L-selectin/

ICAM-12/2 mice (L/ICAM-12/2), P-selectin2/2 mice (P2/2), E-selectin2/2 mice (E2/2), E-selectin2/2 mice treated with anti–P-selectin mAb (E2/2 +

anti-P Ab), and PSGL-12/2 mice (PSGL-12/2). Autoantibody levels in sera from PBS-treated WT mice were used as control. Relative autoantibody levels

were determined by Ig subclass-specific ELISA. Values in parentheses represent the dilutions of pooled sera giving half-maximal OD values in autoantigen-

specific ELISA, which were determined by linear regression analysis to generate arbitrary units per milliliter that could be directly compared between each

group of mice (n = 6 for each). Horizontal bars represent mean Ab levels. pp , 0.05; ppp , 0.01 versus each CTL group. †p , 0.05; ††p , 0.01 versus

bleomycin-treated WT mice. CTL, control.



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dominant Th2 and Th17 cell infiltration that is accompanied bydeteriorated skin and lung fibrosis.Our flow cytometric analysis of polarized CD4+ T cells

exhibited differential expression of LFA-1 and PSGL-1 and bind-ing ability for ICAM-1, P-selectin, and E-selectin, whereas thefrequencies of LFA-1+ or PSGL-1+ cells were similar amongTh0, Th1, Th2, and Th17 cells (Fig. 6). Previously, blockingstudies using anti–LFA-1 or PSGL-1 mAbs have suggested thatTh cell migration mainly depends on LFA-1 or PSGL-1, althoughvirtually all lymphocytes express LFA-1 and PSGL-1 (11, 46).Although the mechanism by which Th cell infiltration is regulatedby LFA-1 and PSGL-1 remains unclear, our present study is thefirst to suggest that expression intensity of LFA-1 and PSGL-1contributes to Th cell infiltration. LFA-1 was highly expressed byTh2 and Th17 cells compared with Th0 cells, whereas Th1 cellsshowed decreased intensity levels of LFA-1 expression. Th1 cellsexhibited increased expression intensity of PSGL-1, whereasTh2 and Th17 cells showed lower intensity levels of PSGL-1 ex-pression. Similarly, ICAM-1 binding ability of Th2 and Th17 cellswas higher than that of Th0 cells, while Th1 cells showed lowerbinding ability for ICAM-1. Th1 cells exhibited higher P-selectinand E-selectin binding ability, whereas Th2 and Th17 cells showedlower binding ability for P-selectin and E-selectin. These expressionand binding ability patterns may in part explain why Th2 andTh17 cell infiltration was inhibited by ICAM-1 deficiency andwhy Th1 cell migration was suppressed by E-selectin, P-selectin,or PSGL-1 deficiency (Fig. 5).L-selectin and LFA-1 play important roles in Ag sensitization,

because these adhesion molecules mediate naive T cell migrationinto the draining lymph nodes (52, 53). Furthermore, these adhe-sion molecules are one of the costimulatory molecules on thesurface of APCs (52–54). In this study, we used the bleomycin-induced SSc model, which does not require Ag sensitization (30,31). Therefore, we can exclude the possible role of these adhesionmolecules on the sensitization phase. During T cell differentiation,activated L-selectin is shed from the cell surface by proteolyticcleavage by an as yet unidentified membrane-bound metallopro-tease (“sheddase”) (55). Indeed, in this study, Th1, Th2, and Th17cells did not express L-selectin. However, previous studies haveshown that L-selectin plays important roles in passive Arthus re-action and intratracheal bleomycin-induced pulmonary fibrosis,which do not also require Ag sensitization (14, 21). Additionally,rapidly activated L-selectin shedding enhances LFA-1 expressionand the LFA-1/ICAM-1 binding ability (7, 55). Therefore,LFA-1 expression intensity also reflects L-selectin activity, whichcould account for the diminishing effect of L-selectin deficiencyon Th2 and Th17 immigration. Collectively, the results of thisstudy suggest that Th1 cell infiltration is mainly regulated byPSGL-1 and PSGL-1 counterreceptors, P-selectin and E-selectin,whereas Th2 and Th17 cell infiltration is mainly controlled byL-selectin, LFA-1, and an LFA-1 counterreceptor, ICAM-1. Fur-thermore, in bleomycin-treated L-selectin2/2, ICAM-12/2, andL-selectin/ICAM-12/2 mice, the reduced rate of Th17 cellinfiltration was greater than those of Th2 cell infiltration. Thismay reflect that Th17 cell infiltration strongly depends onLFA-1 compared with Th2 cell infiltration.As we reported previously, bleomycin treatment induced the

production of autoantibodies, especially SSc-specific anti-topo IAb, and hypergammaglobulinemia, both of which are central fea-tures of human SSc (31). In this study, L-selectin and/or ICAM-1deficiency reduced these autoimmune abnormalities, whereasP-selectin deficiency, E-selectin deficiency with or without P-selectin blockade, and PSGL-1 deficiency deteriorated it (Figs.7, 8). Recent studies showed that Th1 cells supported Ig isotype

switching to IgG2a. In contrast, Th2 cells provided efficient helpfor B cell activation and class switching to IgG1 (56, 57). IFN-gpromotes IgG2a class switching, and IL-4 promotes IgG1 classswitching (56–58). Therefore, concentrations of serum IgG2a andIgG1 reflect Th1 and Th2 responses in vivo (56–59). However, inthis study, serum levels of each Ig isotype were associated withtissue damage, such as inflammatory cell infiltration and fibrosis,rather than with Th1 and/or Th2 cytokine production. It has beenpreviously hypothesized that immune responses to autoantigensare induced by cryptic self-epitopes that are generated by themodification of self-Ags (60). The exposure of cryptic self-epitopes activates potentially autoreactive T cells that have notpreviously encountered the cryptic self, thereby breaking T celltolerance. In this regard, bleomycin-induced tissue injury has beenshown to induce modification of the self-Ags, such as Fas-dependent and/or oxygen species-induced metal-dependent cleav-age of topo I (61). Moreover, apoptosis is detected in the skin ofhuman SSc patients and bleomycin-induced SSc model mice,which were associated with the severity of tissue damage (61,62). Therefore, the production of anti-topo I Ab may be relatedto the modification of topo I by bleomycin-induced tissue injury.In this study, L-selectin and/or ICAM-1 deficiency reduced bleo-mycin-induced tissue injury, which paralleled with decreasedautoantibody and Ig production (Figs. 1, 2, 7, 8). In contrast,P-selectin deficiency, E-selectin deficiency with or without P-selectin blockade, and PSGL-1 deficiency exacerbated bleomycin-induced tissue injury, which was accompanied by increased antoan-tibody and Ig production (Figs. 1, 2, 7, 8). Thus, these regulations oftissue injury partially explain that cell adhesion molecule deficiencycontribute to autoantibody and Ig production in the bleomycin-induced SSc model.To date, there have been few studies addressing an in vivo role of

cell adhesion molecules in SSc models. This is the first systematicstudy to reveal relative contribution of adhesion molecules, L-selectin, ICAM-1, LFA-1, P-selectin, E-selectin, and PSGL-1 inthe development of dermal sclerosis and pulmonary fibrosis inthe bleomycin-induced SSc mouse model. These results provideadditional clues to understanding the complexity of the pathogen-esis of SSc.

AcknowledgmentsWe thank M. Yozaki, A. Usui, K. Shimoda, Y. Yamada, and M. Matsubara

for technical assistance.

DisclosuresThe authors have no financial conflicts of interest.

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cell adhesion molecules regulate fibrotic process via th1/th2/th17 cell balance in a bleomycin

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