Cross-Linking of CD44 on Rheumatoid Synovial Cells Up-Regulates VCAM-1 Koichi Fujii, Yoshiya Tanaka, Stefan Hubscher, Kazuyoshi Saito, Toshiyuki Ota and Sumiya Eto This information is current as of September 25, 2021. J Immunol 1999; 162:2391-2398; ; http://www.jimmunol.org/content/162/4/2391 Downloaded from References This article cites 51 articles, 22 of which you can access for free at: http://www.jimmunol.org/content/162/4/2391.full#ref-list-1

Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average by guest on September 25, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

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 © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Cross-Linking of CD44 on Rheumatoid Synovial Cells Up-Regulates VCAM-11

Koichi Fujii,* Yoshiya Tanaka,2* Stefan Hubscher,† Kazuyoshi Saito,* Toshiyuki Ota,‡ and Sumiya Eto*

CD44 is a ubiquitous molecule also known as or homing . However, the cellular functions and its role in inflammation, for example, rheumatoid synovitis, are currently unknown. In this study, we propose a novel function for CD44. Using synovial cells from rheumatoid arthritis (RA) patients, we demonstrated that CD44 cross-linking and binding to hyaluronan augmented VCAM-1 expression and subsequently VCAM-1-mediated adhesion. Briefly, we found that 1) rheumatoid synovial cells highly expressed CD44; 2) cross-linking of CD44 markedly but transiently augmented VCAM-1 expression and its mRNA much more than did IL-1␤ and TNF-␣; 3) hyaluronan, especially when fragmented, also up-regulated VCAM-1; 4) CD44 activated the transcription factor AP-1; and 5) the -dependent adhesive function of RA synovial cells to T cells was Downloaded from also amplified by CD44 cross-linking. These results indicate that the adhesion of RA synovial cells to matrices such as hyaluronic acid through CD44 could up-regulate VCAM-1 expression and VCAM-1-mediated adhesion to T cells, which might in turn cause activation of T cells and synovial cells in RA synovitis. We therefore propose that such cross-talking among distinct adhesion molecules may be involved in the pathogenesis of inflammation, including RA synovitis. The Journal of Immunology, 1999, 162: 2391–2398. http://www.jimmunol.org/ nflammation is characterized by the accumulation of leuko- LFA-1, VLA-4, CD2, CD28, and CD154, which induce costimu- cytes and other mesenchymal cells in the extravascular space latory signals in /APC binding via multiple cellular signaling I in response to multiple stimuli. Adhesion molecules play a molecules, including focal adhesion kinases, resulting in cell ac- fundamental role in inflammatory processes by mediating leuko- tivation and production (6–8). We have reported previ- cyte-endothelial , leukocyte migration, and T cell/ ously that the engagement of CD31 on T cell subsets amplified APC interactions. Ig superfamily members are involved in cell integrin-dependent adhesion to endothelial ligands, and also that adhesion in many cell types, and on endothelial cells, several Ig ICAM-1 cross-linking on rheumatoid synovial cells induced IL-1␤ family members, including ICAM-1, ICAM-2, ICAM-3, and

transcription by activation of a nuclear factor, AP-1 (9, 10). Thus, by guest on September 25, 2021 VCAM-1, are recognized by leukocyte such as LFA-1 in the new concept, adhesion molecules transduce certain activa- 3 and VLA-4 (1–3). VCAM-1 is highly inducible by several in- tion stimuli independent of cytokine stimulation. This has flammatory on endothelial cells and restricted subsets of prompted us to investigate whether stimulation of adhesion mol- APCs, compared with a lack of such induction by resting endo- ecules also up-regulates other adhesion molecule expression, es- thelial cells and noninflammatory tissues. VCAM-1 thereby plays pecially VCAM-1. From a survey of cross-linking of multiple ad- a pivotal role in migration/infiltration and lymphocyte hesion molecules, including LFA-1, VLA-4, ICAM-1, and CD44, activation by APC through its binding to the ligand VLA-4 and/or on rheumatoid synovial cells (a typical model of inflammation), we ␣ ␤ 4 7 in inflammatory processes (4, 5). found that one such molecule, CD44, was unique in its remarkable The expression of VCAM-1 is tightly regulated by locally pro- up-regulation of VCAM-1 expression on synovial cells and also of ␤ ␣ duced inflammatory cytokines such as IL-1 , and TNF- (4, 5). their adhesion to T cells. However, recent findings have indicated that certain adhesion mol- CD44 is a 90-kDa transmembrane widely dis- ecules not only function as a glue, but also regulate several cellular tributed on T , granulocytes, monocytes, fibro- functions by transducing signaling. The best-known molecules are blasts, keratinocytes, and epithelial cells (11). The principal known ligand of CD44 is hyaluronan (12). CD44 plays a major *First Department of Internal Medicine, and ‡Clinical Laboratory, University of Oc- role in multiple physiologic functions, including cell-cell adhe- cupational and Environmental Health, Japan, School of Medicine, Kitakyushu, Japan; and †Department of Pathology, University of Birmingham, Birmingham, United sion, cell-substrate interaction, lymphocyte recruitment to in- Kingdom flammatory sites, and tumor (13–16). Recently, the Received for publication March 17, 1998. Accepted for publication November function of CD44 as a signaling molecule has also been dem- 9, 1998. onstrated. Stimulation of CD44 with mAbs or hyaluronan trans- The costs of publication of this article were defrayed in part by the payment of page mits the signal into the cells, which leads to activation of T cells charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. and cytokine or release from monocytes (17, 18). 1 This work was supported in part by a Grant-in-Aid for Scientific Research from the CD44 is overexpressed in inflammatory sites in proportion to Ministry of Education, Science, and Culture of Japan. the intensity of inflammation, implicating CD44 in the patho- 2 Address correspondence and reprint requests to Dr. Yoshiya Tanaka, The First De- genesis of inflammation (19). partment of Internal Medicine, University of Occupational and Environmental Health, This study demonstrates a role for CD44 and the potent ligand Japan, School of Medicine, 1-1 Iseigaoka, Yanatanishi-ku, Kitakyusyu 807, Japan. E-mail address: [email protected] hyaluronan in VCAM-1 induction on rheumatoid synovial cells 3 Abbreviations used in this paper: VLA, very late antigen; EMSA, electrophoretic through activation of a nuclear transcription factor AP-1. We pro- mobility shift assay; OA, osteoarthritis; RA, rheumatoid arthritis. pose a model for the involvement of adhesion molecules per se (in

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 2392 VCAM-1 INDUCTION BY CD44

this study, CD44), in the induction or amplification of other ad- Immunohistochemistry of synovial tissue hesion molecules (in this study, VCAM-1) on the same cell and the Synovial tissue samples were obtained after total joint replacement from subsequent mediation of cellular adhesion in inflammatory five patients diagnosed with active RA according to the criteria of the processes. American College of Rheumatology, and from five patients with OA as controls. Tissues were snap frozen and later stained by a three-stage alka- line phosphatase/antialkaline phosphatase (APAAP) method with CD44 Materials and Methods mAb and CD3 mAb. Synovial tissues and culture of synoviocytes Northern blot analysis Synovial tissues were obtained from patients with active rheumatoid ar- thritis (RA) and osteoarthritis (OA), diagnosed according to the criteria of For Northern blot analysis, total cellular RNA was isolated from cultured ␮ the American College of Rheumatology, (Atlanta, GA), who were treated RA synovial cells by a single-step isolation procedure. The RNA (10 g) by joint replacement surgery or synovectomy. The synovial membrane was electrophoresed through a 1% agarose gel and blotted onto nylon fil- ␤ samples were snap frozen and later stained for immunohistochemical stud- ters (Amersham, Arlington Heights, IL). VCAM-1 cDNA and -actin cDNA (donated by Drs. A. Kitani and T. Matsuyama, Kagoshima, Japan) ies. Samples were also dissected under sterile conditions in PBS, and im- 32 mediately prepared for culture of fibroblast-like synovial cells. Briefly, the were labeled with [ P]dCTP (DuPont NEN, Boston, MA), and Northern tissue sample was minced into small pieces and digested with collagenase blot analysis was subsequently performed. (Sigma Aldrich Japan, Tokyo) in serum-free DMEM (Life Technologies, Preparation of nuclear extracts and electrophoretic mobility Grand Island, NY). After filtering through a nylon mesh, the cells were extensively washed and suspended in DMEM, supplemented with 10% shift assay (EMSA) FCS (Bio-Pro, Karlsruhe, Germany) and penicillin-streptomycin (10 U/ml; 2 Nuclear extracts were prepared by a previously reported method with some Sigma Aldrich). Finally, isolated cells were seeded in 25-cm culture flasks modifications. After harvest, 1 ϫ 107 cells were washed with cold PBS, Downloaded from (Falcon, Lincoln Park, NJ) and cultured in a humidified 5% carbon dioxide and incubated on ice for 15 min in 5 ml of buffer I (10 mM HEPES (pH atmosphere. After overnight culture, nonadherent cells were removed, and 7.9), at 4°C, 1.5 mM MgCl2, 10 mM KCl, 0.3 M sucrose, 0.1 mM EGTA, further incubation of adherent cells was continued in fresh medium. At 0.5 mM DTT, and 0.5 mM PMSF containing 1 ␮g/ml of each of the confluence, the cells were trypsinized, passaged at a 1:3 split ratio, and inhibitors (antipain, aprotinin, chymostatin, leupeptin, and pep- recultured. The medium was changed twice each week, and the cells were statin A)). After centrifugation, the cells were resuspended in 1 ml of buffer used after three to seven passages. I with protease inhibitors and then Dounce homogenized (20 strokes; Kon- tes, Vineland, NJ). The homogenate was microcentrifuged for 30 s, and Reagents and mAbs nuclei were resuspended in 0.3 ml of buffer II with protease inhibitors (20 http://www.jimmunol.org/ ␤ ␣ mM HEPES (pH 7.9), 1.5 mM MgCl2, 300 mM KCl, 0.1 mM EGTA, 0.2 IL-1 , IL-6, and TNF- (Cosmobio, Tokyo, Japan) were purchased. Frag- mM EDTA, 25% glycerol, 0.5 mM DTT, and 0.5 mM PMSF) and gently mented and native hyaluronan were kindly donated by the Tokyo Research rocked on a platform at 4°C for 30 min. After 30-min microcentrifugation Institute of Seikagaku. The following mAbs were used as purified Igs: at 4°C, supernatants were dialyzed against 50 vol of buffer III (20 mM MHC class I (mAb W6/32), CD14 (mAb 63D3), anti- (mAb ␣ HEPES (pH 7.9), 100 mM KCl, 0.2 mM EDTA, 20% glycerol, 0.5 mM 10F7), CD11a (LFA-1 ) (mAb TS1/22) (American Type Culture Collec- DTT, and 0.5 mM PMSF) for5hat4°C. After microcentrifugation for 30 tion, Manassas, VA), control (mAb Thy-1.2) (Becton Dickinson, San Jose, min at 4°C, concentrations in the supernatants were measured using CA), MHC class II (mAb IVA12, a gift from Dr. J. D. Capra, Dallas, TX), the Bradford assay and stored at Ϫ80°C. EMSA was performed essentially CD19 (mAb FMC63, from Dr. H. Zola, Bedford Park, Australia), CD16 as previously described. Briefly, 3 ␮g of nuclear extracts was preincubated (mAb 3G8; D. Siegal, Bethesda, MD), CD11b (mAb NIH11b-1), CD44 for 20 min at room temperature in 15 ␮l of a buffer (10 mM Tris-HCl (pH by guest on September 25, 2021 (mAb NIH 44-1), CD54 (ICAM-1) (mAb 84H10), CD49d (VLA-4) (mAb 7.5), 1 mM EDTA, 1 mM 2-ME, 4% glycerol, and 40 mM NaCl) contain- NIH 49d-1, gifts from Dr. S. Shaw, Bethesda, MD), and CD106 (mAb ing 0.5 ␮g of poly(dI-dC) and an oligonucleotide containing AP-1 or 2G7, Dr. W. Newman, Rockville, MD). NF-␬B binding sites (Promega, Madison, WI), which was labeled with T4 polynucleotide kinase (New England Biolabs, Beverly, MA). Consensus Purification of peripheral T cells oligonucleotides of AP-1 or NF-␬B were used as cold competitors. Highly purified T cells were prepared from PBMC of healthy volunteers by Adhesion assay exhaustive immunomagnetic negative selection, as previously described (9, 20, 21). We routinely used Dynabeads (Dynal, Fort Lee, NJ) and a mixture The adhesion assay was performed as previously described (9, 20). Briefly, of mAbs against MHC class II Ag, CD19, CD14, CD16, CD11b, and synovial cells were applied to 24-well culture plates (Nunc, Roskilde, Den- glycophorine. The purity of the CD3ϩ T cell subset was more than 99%, as mark) and cultured in DMEM (Life Technologies) with 10% heat- assayed by flow cytometry. inactivated FCS. T cells were labeled with sodium 51Cr (DuPont NEN). A total of 1 ϫ 106 T cells in 1 mM PMA (Sigma Aldrich) in the presence or Cross-linking of CD44 absence of relevant blocking mAb (10 ␮g/ml) was added, and the cells were incubated at 37°C for 30 min in PBS with 0.5% human serum albu- Cells were cultured until subconfluence and then incubated with NIH 44-1 min: nonadherent cells were washed off completely. Well contents were mAb (10 ␮g/ml) for 30 min at 37°C. After washing the cells three times, lysed with 1% Triton X-100 (Sigma Aldrich), and gamma emissions of 1 ␮g/ml of goat anti-mouse IgG-Fc was added as the second Ab for CD44 adherent wells were determined. cross-linking. Results Stimulation of CD44 by hyaluronan RA synovial cells highly expressed CD44 Cells were cultured until subconfluence and then incubated with frag- We initially characterized cultured synovial cells derived from the mented or native hyaluronan (0.1 mg/ml) for6hat37°C. synovium of RA patients. The cells were spindle shaped and grew in a cobblestone pattern. Flow-cytometric analysis of these cells Flow microfluorometry indicated that they lacked macrophage markers such as MHC class Staining and flow-cytometric analysis of synovial cells were conducted by II Ags, CD14 and CD11b, but spontaneously expressed CD44 and standard procedures, as already described, using a FACScan (Becton Dick- ICAM-1 (but not VCAM-1) with a monomodal pattern (Fig. 1). inson, Mountain View, CA). Briefly, cells (2 ϫ 105) were incubated with specific mAbs and subsequent FITC-conjugated anti-goat IgG Ab or FITC- Thus, RA synovial cells represented type B synovial fibroblast-like conjugated CD106 (VCAM-1) mAb at saturating concentrations in FACS cells. The vast majority of synovial cells highly expressed CD44. medium consisting of HBSS (Nissui, Tokyo, Japan), 0.5% human serum Two prominent differences between synovia of RA and OA were albumin (Green-cross, Osaka, Japan), and 0.2% NaN3 (Sigma Aldrich) for hyperplasia of the RA synovial lining layer and marked accumu- 30 min at 4°C. After three washes in FACS medium, the cells were ana- lyzed with FACScan. Amplification of the mAb binding was provided by lation of mononuclear inflammatory cells around both subsynovial a three-decade logarithmic amplifier. Quantification of the cell surface Ags and perivascular areas in RA tissue (22). Immunohistochemical on one cell was performed using beads, QIFKIT (Dako Japan, Kyoto). studies showed that CD44 was strongly expressed on synoviocytes The Journal of Immunology 2393

FIGURE 1. Phenotypic analysis of synovial cells. Cells were stained with CD11b mAb (NIH 11b-1), CD14 mAb (63D3), MHC class II mAb (IVA1), CD44 mAb (NIH 44-1), ICAM-1 mAb (84H10), and VCAM-1 mAb (2G7). Flow-cytometric analyses were performed using FACScan. Shaded area represents profiles of Thy-1.2 mAb as a negative control. Downloaded from Histograms are expressed from a representative experiment of five patients.

and weakly on the vessels of RA, whereas synoviocytes of OA only scarcely expressed CD44 (Fig. 2, A and B). Furthermore, a

marked accumulation of mononuclear cells, most of which were http://www.jimmunol.org/ CD3-positive T cells, was observed in RA synovium (Fig. 2C). These results suggest that the predominant difference in CD44 ex- pression between active RA and OA is observed on synoviocytes, and that CD44-positive cells are seen in areas rich in T cells in RA synovium.

Cross-linking of CD44 on RA synovial cells up-regulated the expression of VCAM-1

To characterize the function of CD44, we assayed the cell surface by guest on September 25, 2021 molecule expression effects of CD44 cross-linking using a specific mAb and second cross-linker Ab. Flow cytometry showed that VCAM-1 was markedly induced or augmented by the CD44 cross- linking on synovial cells. As shown in Figs. 3 and 4, VCAM-1 was only slightly expressed on nonstimulated synovial cells. However, CD44 cross-linking significantly up-regulated VCAM-1 expres- sion, whereas cross-linking of control anti-VLA-4 mAb had no effect. The results were consistent in five patients with RA. Time- FIGURE 2. Immunohistochemical studies of OA and RA synovium. course experiments showed that VCAM-1 expression on RA sy- Synovia from OA patient (A) and RA patient (B and C) were stained by a novial cells reached maximum levels (approximately a 10-fold in- three-stage alkaline phosphatase/antialkaline phosphatase (APAAP) crease compared with nonstimulated synovial cells), within6hof method with CD44 mAb (A and B) or CD3 mAb (C). CD44 cross-linking, but the expression returned to almost basal levels after 24 h of incubation (Fig. 5A). The results indicate that uli, including several cytokines and cell surface molecules expressed the CD44 cross-linking caused a marked but transient amplifica- on RA synovial cells. When ICAM-1, LFA-1, or VLA-4 Ag was tion of VCAM-1 expression on RA synovial cells. cross-linked by their specific mAb and second cross-linker Ab, the expression of VCAM-1 on RA synovial cells was unchanged or Cross-linking of CD44 triggered VCAM-1 mRNA transcription slightly increased, whereas CD44 cross-linking markedly augmented CD44 cross-linking also induced transcription of VCAM-1 mRNA VCAM-1 expression (Fig. 6A). It is noteworthy that multiple inflam- in RA synovial cells. After CD44 cross-linking, RNA was ex- matory cytokines such as IL-1␤ and TNF-␣, which are well known to tracted from RA synovial cells, and specific mRNA was detected exist in high concentrations in RA synovium and to be involved in the by Northern blot analysis using primers specific for human pathogenesis of RA synovitis, had much less effect on VCAM-1 ex- VCAM-1. RA synovial cells slightly expressed VCAM-1 mRNA pression than CD44 cross-linking at6hofstimulation of RA synovial without stimulation. CD44 cross-linking markedly augmented cells (Fig. 6B). Up-regulation of VCAM-1 induced by these cytokines VCAM-1 mRNA transcription, which was maximal within2hof took 24–48 h and remained still slight level at 6-h stimulation by stimulation, but subsequently gradually diminished (Fig. 5B). cytokines (data not shown). All of the kinetic studies were reproduc- ible among three different RA patients. Thus, CD44 stimulation and CD44 was the most potent stimulator of VCAM-1 expression cytokine stimulation require a clearly different kinetics, namely CD44 among surface molecules and cytokines functions much faster than cytokines, when they induce VCAM-1 In the next series of experiments, we compared the magnitude of expression. These results suggest that CD44 appears to play a pivotal CD44-induced VCAM-1 expression with that induced by other stim- role in the induction of VCAM-1 on the cell surface. 2394 VCAM-1 INDUCTION BY CD44

FIGURE 3. VCAM-1 up-regulation by CD44 cross-linking on RA sy- novial cells. Synovia were obtained from five patients with rheumatoid arthritis, and synovial cells were isolated. Cells were cross-linked with control medium (open bars); anti-CD44 mAb, NIH 44-1 (solid bars); and anti-VLA-4 mAb, NIH 49-d (hatched bars) at a concentration of 10 ␮g/ml Downloaded from for 6 h. VCAM-1 expression was analyzed by FACScan.

VCAM-1 expression was stimulated by fragmented hyaluronan Hyaluronic acids are major ligands for cell surface CD44. We assessed the biological activity of hyaluronan on VCAM-1 expres- http://www.jimmunol.org/ sion on RA synovial cells. As shown in Fig. 7, soluble full-length FIGURE 5. Time course of CD44-triggered VCAM-1 expression on hyaluronan markedly augmented VCAM-1 expression. Frag- RA synovial cells. A, RA fibroblast-like cells were cross-linked with 10 ␮g/ml of anti-CD44 mAb for the indicated duration. VCAM-1 expression mented hyaluronan is thought to be more relevant to inflammation was determined by FACScan. B, Time course of VCAM-1 mRNA expres- including RA synovitis. Of several hyaluronan fragments, the 6.9- sion by CD44 cross-linking. RA synovial cells were cross-linked with 10 kDa fragment had the most marked effect on VCAM-1 expression, ␮g/ml of anti-CD44 mAb for indicated time periods, and total RNA was approximately twice the effect of native hyaluronan. VCAM-1 up- collected. VCAM-1 mRNA expression was determined by Northern blot- regulation by hyaluronan also reached maximum level within 6 h. ting. Data are expressed from a representative experiment of three RA This suggests that hyaluronan, especially when fragmented, is a patients. possible ligand involved in CD44-mediated VCAM-1 expression by guest on September 25, 2021 on RA synovial cells.

AP-1 activation triggered by cross-linking of CD44 tracts with AP-1 oligonucleotides, but not with NF-␬B, was mark- We also examined the nuclear transcription factors involved in edly enhanced, which was completely competed away by excess CD44-mediated signaling by EMSA using a probe containing a amounts of unlabeled AP-1 oligonucleotide. Furthermore, mAb palindromic AP-1 and NF-␬B binding element. Nuclear extracts specific for c-jun produced clearly defined supershift complex in were prepared from RA synovial cells 60 min after CD44 cross- AP-1/DNA complex induced by CD44 stimulation (Fig. 8). These linking. As shown in Fig. 8, RA synovial cells exhibited low basal results were reproducible in three separate experiments. This result binding to AP-1 and NF-␬B oligonucleotides. However, after suggests that CD44 cross-linking results in a specific activation of CD44 cross-linking, the intensity of the complex of nuclear ex- the transcription factor AP-1.

FIGURE 4. Histogram of VCAM-1 expression induced by CD44 cross-link- ing on RA synovial cells. The figure shows one representative histogram of VCAM-1 (A–C) and ICAM-1 (D–F) ex- pression. RA synovial cells were cross- linked with the control medium (A and D), anti-CD44 mAb (B and E), or anti-VLA-4 mAb (C and F) for 6 h, and the expression of the indicated molecules was analyzed by FACScan. Representative histograms from one of five patients are shown. The Journal of Immunology 2395

FIGURE 8. EMSA for determination of transcription factors activated by cross-linking of CD44. RA synovial cells were incubated with control medium (lanes A and F), or cross-linked with anti-CD44 mAb (lanes B–E and G–J) for 1 h, and nuclear extracts were collected. Nuclear extracts were incubated with labeled AP-1 (lanes A–E) or labeled NF-␬B(lanes F–J) oligonucleotide in the presence of unlabeled AP-1 (lane C)orNF-␬B

(lane H) oligonucleotide for cold competition and mAbs specific for c-fos Downloaded from (lane D), c-jun (lane E), p50 (lane I), and p65 (lane J) for supershift analysis.

cross-linking for 12 h resulted in doubling the adhesion rate

relative to control (Fig. 9). Adhesion of both nonstimulated and http://www.jimmunol.org/ CD44-cross-linked RA synovial cells to PMA-activated T cells was significantly inhibited by either anti-VLA-4 or anti-LFA-1 mAbs alone or in combination. However, there was an interesting FIGURE 6. VCAM-1 up-regulation by cross-linking of adhesion mol- and consistent tendency for the anti-LFA-1 mAb to inhibit adhe- ecules and cytokine stimulation. RA synovial cells were cross-linked with sion of nonstimulated synovial cells better than the anti-VLA-4 10 ␮g/ml of the indicated mAb (A) or stimulated with 20 ng/ml of the mAb, whereas the anti-VLA-4 mAb more effectively reduced the indicated cytokine (B) for 6 h. VCAM-1 expression was analyzed by FAC- adhesion of CD44-stimulated synovial cells. Thus, the adhesion of Scan. Data are expressed from a representative experiment of three RA synovial cells to T cells is mediated by integrins LFA-1 and patients. VLA-4, but the VLA-4/VCAM-1 pathway appeared to be centrally by guest on September 25, 2021 involved in CD44-stimulated RA synovial cells.

CD44 cross-linking augmented integrin-mediated adhesion of Discussion RA synovial cell to T cells Adhesion molecules are fundamental to mechanisms of normal Finally, we investigated whether CD44-induced VCAM-1 up- immunity and also contribute to the pathogenesis of autoimmune regulation on RA synovial cells was involved in their adhesion to diseases. Definition of the molecular basis of cellular adhesion and T cells using three different RA patients. RA synovial cells ad- its importance in cell-cell and cell-matrix interactions has pro- hered to highly purified peripheral PMA-stimulated T cells without gressed during the last decade. The expression and function of any stimuli. However, stimulation of synovial cells by CD44

FIGURE 7. VCAM-1 up-regulation by fragmented hyaluronan stimula- FIGURE 9. Adhesion assay of CD44-cross-linked synovial cells and tion. RA synovial cells were incubated with 100 ng/ml of fragmented hya- PMA-activated T cells. RA synovial cells were incubated with control luronan for 6 h. The molecular mass of hyaluronan fragments were 1.7 medium (open bars), or cross-linked with CD44 mAb (solid bars) or MHC kDa, 6.9 kDa, 40 kDa, and native. VCAM-1 expression was determined by class I mAb (hatched bars) for 12 h, and sodium 51Cr-labeled T cells were FACScan. Data are expressed from a representative experiment of three added in the presence or absence of the indicated blocking mAb. Data are RA patients. representative of four different experiments. 2396 VCAM-1 INDUCTION BY CD44 adhesion molecules are tightly regulated by several cellular stim- in sustaining the inflammatory process. Our results also demon- uli. Cytokines are potent inducers of adhesion molecule expres- strated that CD44 cross-linking by anti-CD44 mAb was more ef- sion. Among them, IL-1␤ or TNF-␣, which are abundantly pro- fective in VCAM-1 up-regulation than hyaluronan stimulation. Re- duced at inflammatory sites, contributes to the up-regulation of cent reports suggest that epitope 1 of CD44 (NIH 44-1 mAb Ig-superfamily adhesion molecules, such as ICAM-1 and binding site) is more effectively involved in CD44-induced cell- VCAM-1 (23). However, on the basis of the results presented in cell adhesion than the epitope 2 (hyaluronan binding site) (42, 43). the current study, we propose a new concept, that stimulation of The different ability in VCAM-1 up-regulation between CD44 the adhesion molecule CD44 per se plays a pivotal role in the mAb and hyaluronan might result from the difference of stimulated regulation of VCAM-1 expression. We deduce this from the fol- epitope. lowing novel findings: 1) CD44 cross-linking on RA synovial fi- Adhesion molecules play a fundamental role in many diverse broblast-like cells up-regulated VCAM-1 expression, more and immunologic functions, especially Ag presentation, recognition of faster than did stimulation with inflammatory cytokines including target cells by effector cells, migration and retention of mononu- IL-1␤ and TNF-␣; 2) fragmented hyaluronan effectively increased clear cells, and leading to cellular activation or VCAM-1 expression compared with native hyaluronan; 3) proliferation (17, 19, 25, 44–46). VCAM-1 was originally re- integrin-dependent adhesion of RA synovial cells to PMA-acti- ported to be expressed on endothelium, but we and others have vated T cells was markedly increased by the CD44 cross-linking shown that VCAM-1 is also expressed on dendritic cells and ac- on synovial cells; and 4) CD44 stimulation activated the nuclear tivated macrophages, and that cellular adhesion mediated by transcription factor AP-1. VCAM-1 and ICAM-1 is involved in signal transduction in the Downloaded from The fact that stimulation of one adhesion molecule up-regulated immune system (47, 48). Increased expression of VCAM-1 has another prompted us to investigate how this phenomenon might been demonstrated in synovial tissue from RA patients: 1) contribute to the inflammatory process. We used RA synovial VCAM-1 is highly detected on synovial intimal fibroblast-like fibroblast-like cells, because RA synovium is one of the most typ- cells, vascular wall cells outside the endothelial layer, scattered ical models of inflammation, and because CD44 is predominantly stromal cells with cytoplasmic processes, and cells resembling fol- expressed on RA synovial cells and plays a major role in the patho- licular dendritic reticulum cells in lymphoid aggregates with ger- genesis of rheumatoid synovitis (19, 24). CD44 is found on the minal centers; 2) there is a marked accumulation of T cells in the http://www.jimmunol.org/ surface of most leukocytes, fibroblasts, keratinocytes, and epithe- perivascular area around VCAM-1-positive synoviocytes, includ- lial cells, and is implicated in multiple physiologic cellular func- ing both fibroblast-like and dendritic-like cells; and 3) the prom- tions, including cell-cell and cell-matrix adhesion, lymphocyte inent distinctive feature of the cells identified from a localized homing, and tumor metastasis, by binding to its ligand (16, region of pannus in and around large cartilage erosions from RA 24–27). However, the function of CD44 and relevance to the patients is their abundant surface display of VCAM-1 (49). These pathogenesis of inflammation are unknown at present. Recent re- findings indicate that the adhesion of T cells to synoviocytes or ports suggest that CD44 functions not just as an adhesion mole- endothelium through VCAM-1 and its ligand VLA-4, which is a cule, but also as a signaling molecule. For instance, T cell activa-

major T cell integrin, could contribute to the activation of inter- by guest on September 25, 2021 tion and cytokine release from monocytes are induced by the acting cells (both T cells and synoviocytes) and further release of CD44 stimulation (15, 17). cytokines and degradative enzymes from them. Furthermore, The best-known ligand for CD44 is hyaluronan (12). Hyaluro- CD44 is characteristically expressed on fibroblast-like and nan is a high m.w. linear repeating disaccharide, ␤-D-glucuronyl- dendritic-like cells as well as endothelium in rheumatoid ␤-D-N-acetylglucosamine, which is the major extracellular glycos- aminoglycan found in almost all types of , synovium (19). including rheumatoid synovium and synovial fluids (28–30). The The concomitant expression of CD44 and VCAM-1 in the in- nonaggregated form of hyaluronan is continuously secreted into flammatory pannus in RA and our current report that the stimula- the joint space by elements of the synovium. It comprises the ma- tion of CD44 by cross-linking and ligation by fragmented hyalu- jor macromolecular species of the synovial fluid and is responsible ronan up-regulated VCAM-1 expression on rheumatoid fibroblast- for the unique viscoelastic properties of what is otherwise effec- like cells and increased cell adhesion, implicate CD44 in an tively a simple plasma dialysate (30). Fibroblasts in the synovium important role in synovial cells. Taken together, these results im- mainly synthesize hyaluronan in response to stimulation with cy- plicate CD44 in the pathogenesis of rheumatoid synovial inflam- tokines, such as IL-1␤ and TNF-␣ (31). The presence of short mation and articular bone and cartilage destruction, which are chain molecules of hyaluronan in arthritic synovial fluid is due to characterized by the accumulation and proliferation of T cells and degradation after synthesis rather than to defective synthesis (32, synovial cells and the abundant production of cytokines and de- 33). Activated and reactive oxygen-derived radical generative enzymes. The VCAM-1 promoter possesses bind- species mediate damage to hyaluronan in inflammatory joint dis- ing sites for two major nuclear transcription factors, NF-␬B and orders, which leads to the accumulation of fragmented hyaluronan AP-1 (50). Initiation of VCAM-1 mRNA transcription induced by (34–36). Several studies have suggested that high molecular mass IL-1␤ and TNF-␣ has been reported to result from the activation of hyaluronan inhibits cellular proliferation of endothelial cells, fi- NF-␬B (51–53). However, we observed that CD44 stimulation ac- brocytes, and mitogen-stimulated lymphocytes, whereas low mo- tivated AP-1, but not NF-␬B, in nuclear extracts of RA synovial lecular mass fragments have a stimulatory effect. Low molecular cells, and confirmed this by EMSA. Thus, CD44 and these cyto- mass fragments have also been shown to stimulate , kines appear to differentially activate distinctive transcription fac- an important feature of early inflammation (37–41). Our results tors leading to transcription of VCAM-1 mRNA. Furthermore, a show that fragmented hyaluronan was more effective in up- clear difference of kinetics of VCAM-1 up-regulation was ob- regulation of VCAM-1 compared with native hyaluronan. These served between CD44 stimulation and cytokine stimulation, results demonstrate that hyaluronan is far from an inert space filler, namely its expression reached maximum level within6hbyCD44 but has an important role in biological activities, such as regulation stimulation, whereas its up-regulation by cytokines took at least of adhesion molecules, and that degraded products of the extra- 24 h, indicating that CD44 may play a pivotal role in VCAM-1 cellular matrix at inflammatory sites are as important as cytokines expression on synoviocytes. The Journal of Immunology 2397

Based on the findings presented in this work, we propose that 16. Gunthert, U., M. Hofmann, W. Rudy, S. Reber, M. Zoller, I. Haussmann, the stimulation of adhesion molecules may induce expression of S. Matzku, A. Wenzel, H. Ponta, and P. Herrlich. 1991. A new variant of glycoprotein CD44 confers metastatic potential to rat carcinoma cells. Cell other adhesion molecules on the same cell, i.e., we observed that 65:13. stimulation of CD44 by cross-linking or ligation with fragmented 17. Webb, D. S., Y. Shimizu, G. A. van Seventer, S. Shaw, and T. L. Gerrard. 1990. hyaluronan, as well as inflammatory cytokines, augmented LFA-3, CD44, and CD45: physiologic triggers of human monocyte TNF and IL-1 release. Science 249:1295. VCAM-1 expression (a representative inducible adhesion mole- 18. McKee, C. M., M. B. Penno, M. Cowman, M. D. Burdick, R. M. Strieter, C. Bao, cule). Furthermore, the concomitant expression of CD44 and and P. W. Noble. 1996. Hyaluronan (HA) fragments induce chemokine gene VCAM-1 on inflamed RA synovial cells and the more efficient expression in alveolar macrophages: the role of HA size and CD44. J. Clin. Invest. 98:2403. induction of VCAM-1 by CD44 stimulation than by several cyto- 19. Haynes, B. F., L. P. Hale, K. L. Patton, M. E. Martin, and R. M. McCallum. 1991. kines suggest that CD44 could play a pivotal role in the amplifi- Measurement of an adhesion molecule as an indicator of inflammatory disease cation of VCAM-1 and subsequent adhesion of syonovial cells to activity: up-regulation of the receptor for hyaluronate (CD44) in rheumatoid ar- T cells. Thus, CD44-mediated up-regulation of VCAM-1 appears thritis. Arthritis Rheum. 34:1434. 20. Tanaka, Y., D. H. Adams, S. Hubscher, H. Hirano, U. Siebenlist, and S. Shaw. to be very relevant to the pathogenesis of inflammatory diseases 1993. T-cell adhesion induced by -immobilized cytokine MIP-1␤. such as RA synovitis. Our findings warrant further studies into the Nature 361:79. stimulation of CD44, which might bring enormous flexibility to the 21. Tanaka, Y., K. Kimata, A. Wake, S. Mine, I. Morimoto, N. Yamakawa, H. Habuchi, S. Ashikari, H. Yamamoto, K. Sakurai, K. Yoshida, S. Suzuki, and processes of inflammation and introduce new pharmacologic ap- S. Eto. 1996. proteoglycan on leukemic cells is primarily in- proaches to control them. volved in integrin triggering and its mediated adhesion to endothelial cells. J. Exp. Med. 184:1987.

22. Nakatsuka, K., Y. Tanaka, S. Hubscher, M. Abe, A. Wake, K. Saito, I. Morimoto, Downloaded from Acknowledgments and S. Eto. 1997. Rheumatoid synovial fibroblasts are stimulated by the cellular adhesion to T cells through lymphocyte function associated antigen-1/intercellu- We thank Ms. T. Adachi for excellent technical assistance. We also lar adhesion molecule-1. J. Rheumatol. 24:458. thank the following investigators and an institute for providing mAbs, 23. Marlor, C. W., D. L. Webb, M. P. Bombara, J. M. Greve, and M. L. Blue. complementary deoxyribonucleic acid, and fragmented and native hya- 1992. Expression of vascular -1 in fibroblast-like sy- luronan: Drs. J. D. Capra, A. Kitani, T. Matsuyama, W. Newman, noviocytes after stimulation with . Am. J. Pathol. 140: S. Shaw, D. Siegal, and H. Zola, and the Tokyo Research Institute of 1055.

Seikagaku. 24. Johnson, B. A., G. K. Haines, L. A. Harlow, and A. E. Koch. 1993. Adhesion http://www.jimmunol.org/ molecule expression in human synovial tissue. Arthritis Rheum. 36:137. 25. Haynes, B. F., M. J. Telen, L. P. Hale, and S. M. Denning. 1989. CD44: a References molecule involved in leukocyte adherence and T-cell activation. [Published erratum appears in 1990 Immunol. Today 11:80.] Immunol. Today 10:423. 1. Elices, M. J., L. Osborn, Y. Takada, C. Crouse, S. Luhowskyj, M. E. Hemler, and 26. Haynes, B. F., E. A. Harden, M. J. Telen, M. E. Hemler, J. L. Strominger, R. R. Lobb. 1990. VCAM-1 on activated endothelium interacts with the leuko- T. J. Palker, R. M. Scearce, and G. S. Eisenbarth. 1983. Differentiation of human cyte integrin VLA-4 at a site distinct from the VLA-4/fibronectin binding site. T lymphocytes. I. Acquisition of a novel human cell surface protein (p80) during Cell 60:577. normal intrathymic T cell maturation. J. Immunol. 131:1195. 2. Szekanecz, Z., G. K. Haines, T. R. Lin, L. A. Harlow, S. Goerdt, G. Rayan, and A. E. Koch. 1994. Differential distribution of intercellular adhesion molecules 27. Denning, S. M., P. T. Le, K. H. Singer, and B. F. Haynes. 1990. (ICAM-1, ICAM-2, and ICAM-3) and the MS-1 antigen in normal and diseased against the CD44 p80, lymphocyte homing receptor molecule augment human peripheral blood T cell activation. J. Immunol. 144:7.

human synovia: their possible pathogenetic and clinical significance in rheuma- by guest on September 25, 2021 toid arthritis. Arthritis Rheum. 37:221. 28. Singla, S. K. 1987. Nomenclature of hyaluronic acid. Biochem. J. 242:623. 3. El-Gablrawy, G. H., M. Gallatin, R. Vazeux, G. Peterman, and J. Wilkins. 1994. 29. Laurent, T. C., and J. R. Fraser. 1986. The properties and turnover of hyaluronan. Expression of ICAM-R (ICAM-3), a novel counter-receptor for LFA-1, in rheu- Ciba Found. Symp. 124:9. matoid and nonrheumatoid synovium: comparison with other adhesion mole- 30. Ng, C. K., C. J. Handley, R. M. Mason, and H. C. Robinson. 1989. Synthesis of cules. Arthritis Rheum. 37:846. hyaluronate in cultured bovine articular cartilage. Biochem. J. 263:761. 4. Osborn, L., C. Hession, R. Tizard, C. Vassallo, S. Luhowskyj, R. G. Chi, and 31. Wells, A. F., L. Klareskog, S. Lindblad, and T. C. Laurent. 1992. Correlation R. Lobb. 1989. Direct expression cloning of vascular cell adhesion molecule between increased hyaluronan localized in arthritic synovium and the presence of 1, a cytokine-induced endothelial protein that binds to lymphocytes. Cell proliferating cells: a role for macrophage-derived factors. Arthritis Rheum. 35: 59:1203. 391. 5. Rice, G. E., and M. P. Bevilacqua. 1989. An inducible endothelial cell surface glycoprotein mediates melanoma adhesion. Science 246:1303. 32. Balazs, E. A., D. Watson, I. F. Duff, and S. Roseman. 1967. Hyaluronic acid in 6. Bednarczyk, J. L., and B. W. McIntyre. 1992. Expression and ligand-binding synovial fluid. I. Molecular parameters of hyaluronic acid in normal and arthritis ␣ ␤ human fluids. Arthritis Rheum. 10:357. function of the integrin 4 1 (VLA-4) on neural-crest-derived tumor cell lines. Clin. Exp. Metastasis 10:281. 33. Dahl, L. B., I. M. Dahl, L. A. Engstrom, and K. Granath. 1985. Concentration and 7. Ward, S. G., J. Westwick, N. D. Hall, and D. M. Sansom. 1993. Ligation of molecular weight of sodium hyaluronate in synovial fluid from patients with CD28 receptor by B7 induces formation of D-3 phosphoinositides in T lym- rheumatoid arthritis and other arthropathies. Ann. Rheum. Dis. 44:817. phocytes independently of T cell receptor/CD3 activation. Eur. J. Immunol. 34. Prehm, P. 1990. Release of hyaluronate from eukaryotic cells. Biochem. J. 267: 23:2572. 185. 8. Banchereau, J., F. Bazan, D. Blanchard, F. Briere, J. P. Galizzi, C. van Kooten, 35. Greenwald, R. A., and W. W. Moy. 1980. Effect of oxygen-derived free radicals Y. J. Liu, F. Rousset, and S. Saeland. 1994. The CD40 antigen and its ligand. on hyaluronic acid. Arthritis Rheum. 23:455. Annu. Rev. Immunol. 12:881. 36. McNeil, J. D., O. W. Wiebkin, W. H. Betts, and L. G. Cleland. 1985. Depoly- 9. Tanaka, Y., S. M. Albelda, K. J. Horgan, G. A. van Seventer, Y. Shimizu, merization products of hyaluronic acid after exposure to oxygen-derived free W. Newman, J. Hallam, P. J. Newman, C. A. Buck, and S. Shaw. 1992. CD31 ␤ radicals. Ann. Rheum. Dis. 44:780. expressed on distinctive T cell subsets is a preferential amplifier of 1 integrin- mediated adhesion. J. Exp. Med. 176:245. 37. Goldberg, R. L., and B. P. Toole. 1987. Hyaluronate inhibition of cell prolifer- 10. Koyama, Y., Y. Tanaka, K. Saito, M. Abe, K. Nakatsuka, I. Morimoto, ation. Arthritis Rheum. 30:769. P. E. Auron, and S. Eto. 1996. Cross-linking of intercellular adhesion molecule 38. Anastassiades, T., and W. Robertson. 1984. Modulation of mitogen-dependent 1 (CD54) induces AP-1 activation and IL-1␤ transcription. J. Immunol. 157: lymphocyte stimulation by hyaluronic acid. J. Rheumatol. 11:729. 5097. 39. Forrester, J. V., and E. A. Balazs. 1980. Inhibition of phagocytosis by high mo- 11. Underhill, C. 1992. CD44: the hyaluronan receptor. J. Cell Sci. 103:293. lecular weight hyaluronate. Immunology 40:435. 12. Aruffo, A., I. Stamenkovic, M. Melnick, C. B. Underhill, and B. Seed. 1990. 40. Ahlgren, T., and C. Jarstrand. 1984. Hyaluronic acid enhances phagocytosis of CD44 is the principal cell surface receptor for hyaluronate. Cell 61:1303. human monocytes in vitro. J. Clin. Immunol. 4:246. 13. Jalkanen, S., R. F. Bargatze, J. de los Toyos, and E. C. Butcher. 1987. Lympho- cyte recognition of high endothelium: antibodies to distinct epitopes of an 85–95 41. West, D. C., I. N. Hampson, F. Arnold, and S. Kumar. 1985. Angiogenesis in- kD glycoprotein antigen differentially inhibit lymphocyte binding to lymph node, duced by degradation products of hyaluronic acid. Science 228:1324. mucosal, or synovial endothelial cells. J. Cell Biol. 105:983. 42. Oostendorp, R. A. J., E. Spitzer, and P. Dormer. 1996. Adhesion of human he- 14. Shimizu, Y., G. A. van Seventer, R. Siraganian, L. Wahl, and S. Shaw. 1989. matopoietic progenitor cells to bone-marrow-derived stromal cells is enhanced by Dual role of the CD44 molecule in T cell adhesion and activation. J. Immunol. antibodies to CD44. Acta Haematol. 95:243. 143:2457. 43. Bendall, L. J., J. Kirkness, A. Hutchinson, A. Bianchi, V. Makrynikola, 15. Miyake, K., K. L. Medina, S. Hayashi, S. Ono, T. Hamaoka, and P. W. Kincade. K. F. Bradstock, and D. J. Gottlieb. 1997. Antibodies to CD44 enhance adhesion 1990. Monoclonal antibodies to Pgp-1/CD44 block lympho-hemopoiesis in long- of normal CD34ϩ cells and acute myeloblastic but not lymphoblastic leukemia term bone marrow cultures. J. Exp. Med. 171:477. cells to bone marrow stroma. Br. J. Haematol. 98:828. 2398 VCAM-1 INDUCTION BY CD44

44. Springer, T. A. 1990. Adhesion receptors of the immune system. Nature 346: to and invade normal human cartilage when engrafted into SCID mice. 425. Am. J. Pathol. 149:1607. 45. Haynes, B. F., L. P. Hale, S. M. Denning, P. T. Le, and K. H. Singer. 1989. 50. Cybulsky, M. I., J. W. Fries, A. J. Williams, P. Sultan, R. Eddy, M. Byers, The role of leukocyte adhesion molecules in cellular interactions: implica- T. Shows, M. J. Gimbrone, and T. Collins. 1991. Gene structure, chromosomal tions for the pathogenesis of inflammatory synovitis. Springer Semin. Immu- location, and basis for alternative mRNA splicing of the human VCAM-1 gene. nopathol. 11:163. Proc. Natl. Acad. Sci. USA 88:7859. 46. Hemler, M. E. 1988. Adhesive protein receptors on hematopoietic cells. Immunol. 51. Marui, N., M. K. Offermann, R. Swerlick, C. Kunsch, C. A. Rosen, M. Ahmad, Today 9:109. R. W. Alexander, and R. M. Medford. 1993. Vascular cell adhesion molecule-1 47. Damle, N. K., and A. Aruffo. 1991. Vascular cell adhesion molecule 1 induces (VCAM-1) gene transcription and expression are regulated through an antioxi- T-cell antigen receptor-dependent activation of CD4ϩ T lymphocytes. Proc. Natl. dant-sensitive mechanism in human vascular endothelial cells. J. Clin. Invest. Acad. Sci. USA 88:6403. 92:1866. 48. Van Seventer, G. A., W. Newman, Y. Shimizu, Y. Tanaka, K. J. Horgan, 52. Iademarco, M. F., J. J. McQuillan, G. D. Rosen, and D. C. Dean. 1992. Char- T. Nutman, and S. Shaw. 1991. Functional analysis of three activation regulated acterization of the promoter for vascular cell adhesion molecule-1 (VCAM-1). endothelial adhesion molecules ICAM-1, VCAM-1 and ELAM-1. J. Exp. Med. J. Biol. Chem. 267:16323. 174:901. 53. Neish, A. S., A. J. Williams, H. J. Palmer, M. Z. Whitley, and T. Collins. 1992. 49. Muller, L. U., J. Kriegsmann, B. N. Franklin, S. Matsumoto, T. Geiler, R. E. Gay, Functional analysis of the human vascular cell adhesion molecule 1 promoter. and S. Gay. 1996. Synovial fibroblasts of patients with rheumatoid arthritis attach J. Exp. Med. 176:1583. Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021