Functional Genomic Analysis in Arthritis-Affected Cartilage: Yin-Yang Regulation of Inflammatory Mediators by α5 β1 and αVβ3 This information is current as of September 29, 2021. Mukundan G. Attur, Mandar N. Dave, Robert M. Clancy, Indravadan R. Patel, Steven B. Abramson and Ashok R. Amin J Immunol 2000; 164:2684-2691; ; doi: 10.4049/jimmunol.164.5.2684 Downloaded from http://www.jimmunol.org/content/164/5/2684

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Functional Genomic Analysis in Arthritis-Affected Cartilage: ␣ ␤ Yin-Yang Regulation of Inflammatory Mediators by 5 1 and ␣ ␤ 1 V 3 Integrins

Mukundan G. Attur,* Mandar N. Dave,* Robert M. Clancy,* Indravadan R. Patel,* Steven B. Abramson,*† and Ashok R. Amin2*†‡

Osteoarthritis-affected cartilage exhibits enhanced expression of fibronectin (FN) and osteopontin (OPN) mRNA in differential ␣ ␤ ␣ ␤ display and bioinformatics screen. Functional genomic analysis shows that the engagement of the receptors 5 1 and v 3 ␣ ␤ of FN and OPN, respectively, have profound effects on chondrocyte functions. Ligation of 5 1 using activating mAb JBS5 (which acts as agonist similar to FN N-terminal fragment) up-regulates the inflammatory mediators such as NO and PGE2 as well as the

␣ ␤ Downloaded from , IL-6 and IL-8. Furthermore, up-regulation of these proinflammatory mediators by 5 1 integrin ligation is mediated via induction and autocrine production of IL-1␤, because type II soluble IL-1 decoy inhibits their production. In contrast, ␣ ␤ v 3 complex-specific function-blocking mAb (LM609), which acts as an agonist similar to OPN, attenuates the production of ␤ ␣ ␤ ␤ ␤ ␣ IL-1 , NO, and PGE2 (triggered by 5 1, IL-1 , IL-18, or IL-1 , TNF- , plus LPS) in a dominant negative fashion by osteo- arthritis-affected cartilage and activated bovine chondrocytes. These data demonstrate a cross-talk in signaling mechanisms among integrins and show that integrin-mediated “outside in” and “inside out” signaling very likely influences cartilage ho- meostasis, and its deregulation may play a role in the pathogenesis of osteoarthritis. The Journal of Immunology, 2000, 164: http://www.jimmunol.org/ 2684–2691.

␣ ␤ ␣ ␣ ␤ ϳ ntegrins are heterodimer receptors that contain and sub- of 4 and 6, whereas the 1-chain is expressed on 40% of the units that bind to extracellular matrix (ECM)3 components or cells (4). In addition to an altered display of integrins, there is an I other membrane-bound receptors (1). Ligands for integrins increased expression of selected ECM , such as FN are often large ECM proteins such as collagen, laminin, vitronec- and OPN. tin, osteopontin (OPN), and fibronectin (FN) (1). We and others have observed enhanced expression of FN and Articular cartilage is avascular, aneural, and alymphatic. The OPN mRNA in human OA-affected cartilage compared with nor- synthesis and degradation of cartilage matrix are regulated by mal cartilage (M. G. Attur, M. N. Dave, S. A. Stuchin, A. S. Ko- by guest on September 29, 2021 chondrocytes via mechanisms that depend in part upon the inter- walski, C. A. Lopez, J. Zhang, S. B. Abramson, D. T. Denhardt, action of chondrocytes with the ECM proteins. It is increasingly and A. R. Amin, manuscript in preparation) (5, 6). These ECM appreciated that there is specific induction and repression of chon- proteins may alter chondrocyte functions; FN and FN fragments drocyte gene expression, controlled through cellular contacts with have been reported to induce IL-1␤, IL-6, GM-CSF, matrix met- ECMs in the pericellular environment surrounding the chondro- alloproteinases (MMPs), and proteoglycan release in chondrocytes cytes (chondron), using a complex array of integrins on chondro- ␣ ␤ (5, 6). Engagement of 5 1 integrin by FN has also been impli- cytes that control cartilage homeostasis. cated in chondrocyte adhesion, spreading, and proliferation in vitro Normal human chondrocytes express various integrins, includ- (7, 8). ing ␣1␤ , ␣ ␤ , ␣ ␤ , and lesser quantities of ␣ ␤ and ␣ ␤ 1 5 1 v 5 v 3 3 1 OPN is a soluble secretory phosphoprotein with diverse func- heterodimers. The ␣ subunit-containing integrins are detected v tions, including a marker for commitment to endochondrial ossi- more readily on the superficial chondrocytes than on deep zone fication (9), inhibition of endotoxin-induced NO production in ep- chondrocytes (2, 3). Chondrocytes from osteoarthritis (OA)-af- ithelial cells (10), and regulation of the onset of osteoporosis (11). fected cartilage express ␣ , ␣ , ␣ , ␣ , and ␣ , with lesser amounts 1 3 5 2 v OPN is secreted by various cell types, including activated T cells, , osteoblasts, and hypertropic chondrocytes and is re- ␣ ␤ *Department of Rheumatology, Hospital for Joint Diseases, New York, NY 10003; ported to bind to various integrins, including v 3 (12). and Departments of †Pathology and ‡Medicine, Kaplan Center, New York Historically, OA has been a noninflammatory disease due to the University Medical Center, New York, NY 10016 absence of both infiltrating neutrophils and the characteristic signs Received for publication October 12, 1999. Accepted for publication December of inflammation, such as redness and swelling with heat and pain 16, 1999. (rubor et tumor cum colore et dolor). However, reports from our The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance own and other laboratories have demonstrated the superinduction with 18 U.S.C. Section 1734 solely to indicate this fact. of proinflammatory genes (which include NO synthase, COX-2, 1 This work was supported by The Joseph and Sophie Abeles Foundation. IL-1␤, IL-6, and IL-8) in osteoarthritic cartilage, which leads to 2 ␤ Address correspondence and reprint requests to Dr. Ashok R. Amin, Department of spontaneous production of NO, PGE2, IL-1 , IL-6, and IL-8 in ex Rheumatology, Hospital for Joint Diseases, 301 East 17th Street, Room 1600, New vivo conditions (13–20). The up-regulation of these cytokines and York, NY 10003. E-mail address: [email protected] mediators exerts detrimental effects on chondrocyte functions (13– 3 Abbreviations used in this paper: ECM, extracellular matrix; FN, fibronectin; OPN, osteopontin; OA, osteoarthritis; MMP, matrix metalloproteinase; MFI, median fluo- 20). This inflammatory response, which may be considered rescence intensity; sIL-1R II, soluble type II IL-1 receptor; FA, focal adhesion. molecular inflammation, is not reflected by clinical signs of

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 The Journal of Immunology 2685 inflammation, but leads to an imbalance of cartilage homeostasis (13). Briefly, 100 ␮l of culture supernatant was mixed with 100 ␮lof that results in progressive articular degeneration (15). The end Griess reagent (1% sulfanilamide in 2.5% on H3PO4 and 0.1% N-naphtyl- ethylenediamine dihydrochloride in H O) for 10 min in 96-well plates. The point of this disease is the loss of cartilage and chondrocytes, 2 optical density (OD550) was measured using a microplate reader. Nitrite which maintains cartilage homeostasis in the joints. concentrations were calculated with a standard curve of sodium nitrite In the present study we analyze one of the mechanisms by which ranging from 1–50 ␮M. ECM proteins regulate inflammatory mediators in cartilage (nor- PGE analysis mal and OA-affected) and primary bovine chondrocytes. We report 2 ␣ ␤ that 1) engagement of 5 1 induces NO, PGE2, IL-6, and IL-8 PGE2 was estimated by RIA using rabbit anti-PGE2-BSA as the immuno- production via autocrine IL-1␤ production; 2) engagement of ␣ ␤ gen (Sigma, St. Louis, MO) (14). The sample (100 ␮l) or standard (in v 3 dilution buffer: 0.01 M PBS, pH 7.4, containing 0.1% BSA and 0.1% inhibits the spontaneous production of NO, PGE2, IL-6, and IL-8 sodium azide) was mixed with 500 ␮l of the diluted antiserum (1/200, in production induced through autocrine IL-1␤; and 3) the function dilution buffer), vortex mixed, and incubated at 4°C for 1 h followed by the ␣ ␤ ␣ ␤ ␮ blocking anti- v 3 Ab (LM609) inhibits the function of anti- 5 1 addition of 200 l of cold dextran-coated charcoal (1% activated charcoal mAb (JBS5), IL-1␤, IL-18, and LPS (with respect to induction of suspended in dilution buffer containing 0.1% dextran sulfate). The tubes NO, PGE , and IL-1␤ production) in a dominant negative fashion. were vortex mixed vigorously and incubated for 10 min at 0°C in ice-water. 2 The mixture was centrifuged at 8000 rpm (room temperature) for 10 min. Supernatant (400 ␮l) was removed and added to the scintillation mixture Materials and Methods (Packard, Downers Grove, IL), and the amount of radioactivity was deter- Reagents mined. The standards were prepared by diluting the stock solution of PGE2 at concentrations of 15–1000 pg/100 ␮l. The standards were assayed for Recombinant IL-1␤, TNF-␣, and ELISA kits for IL-1␤, IL-6, and IL-8 every PGE analysis.

2 Downloaded from were purchased from R&D (Minneapolis, MN). IL-18 was purchased from PeproTech (Rocky Hill, NJ). Cyclic RGDN and RGEN were pro- Differential display analysis ␣ ␤ ␣ ␤ cured from Bachem (King of Prussia, PA). Anti- 5 1 (JBS5) and anti- v 3 (LM609) were purchased from Chemicon (Temecula, CA). Inhibitory anti- Total RNA was extracted from normal and OA-affected cartilage as pre- ␤ viously described (22) and was treated with DNase I to remove contami- 1 Ab (mAb13) was a gift from Dr. K. M. Yamada (National Institutes of Health, Bethesda, MD). nating genomic DNA using MessageClean kit (Gene Hunter, Nashville, TN). Differential display of mRNA from normal and OA-affected cartilage was performed using a Gene Hunter Kit (23). mRNA were amplified by

Procurement of human cartilage http://www.jimmunol.org/ RT-PCR using arbitrary primers as recommended by the manufacturer. Cartilage slices were taken from the tibial plateau and femoral condyle of Differentially expressed cDNA were isolated from the gels, cloned, and knees of patients (with the diagnosis of advanced OA; age, 50–70 years) sequenced on an automatic DNA Sequencer ( DNA Technology undergoing knee replacement surgery. Nonarthritic knee cartilage (normal Center, Rockefeller University, NY). control; age, 20–70 years) was obtained from patients with fractures or from accident victims after knee amputation. Preparation of cDNA subtraction libraries OA explant assay Human OA-specific subtracted cDNA libraries were prepared using PCR select cDNA subtraction kit as recommended by the manufacturer (Clon- Knee articular cartilage (from OA patients or normal individuals) were cut tech, Palo Alto, CA). The OA cartilage total RNA was used as a tester, and into 3-mm discs, and four to six discs were placed (in triplicate or qua- the normal cartilage total RNA was used as a driver. The subtracted cDNAs druplicate) in a 24-well plate containing 2 ml of Ham’s F-12 medium (with were cloned onto pTAdv vector using the Advantage PCR cloning kit by guest on September 29, 2021 ␣ ␤ 0.1% human albumin) in the presence or the absence of anti- 5 1 (JBS5) (Clontech, Palo Alto, CA), and the various clones thus obtained were ␣ ␤ or anti- v 3 (LM609) Abs and respective control isotype-specific mono- sequenced. clonal Ab along with various modulators as previously described (13, 16). Bioinformatics Isolation of bovine chondrocytes The sequences of various clones obtained from differential display and Bovine chondrocytes were isolated from young cow hooves as previously subtraction libraries were analyzed using the BLAST program against the described (21). Cartilage slices from bovine cartilage were minced finely National Center for Biotechnology Information (NCBI) database. and digested sequentially with 0.2% testicular hyaluronidase (5 min at ␤ 37°C), 0.2% trypsin (30 min at 37°C), and 0.2% collagenase (16 h at RT-PCR analysis of IL-1 37°C). After straining through sterile nylon mesh, cells were centrifuged at ϫ Total RNA from bovine chondrocytes was isolated using TRI reagent (Mo- 1200 g for 10 min. Cell pellets were washed twice with serum-free lecular Research Center, Cincinnati, OH) as described previously (22). The Ham’s F-12 medium, followed by resuspending them and centrifuging at ϫ total RNA was treated with DNase to remove contaminating genomic DNA 1000 g for 10 min. Finally, the pellet was suspended in complete F-12 and was purified using a Qiagen RNeasy Mini column (Qiagen, Valencia, medium before plating them in flasks. ␮ CA). Five micrograms of total RNA isolated from bovine chondrocytes Bovine chondrocytes treated with LM609 mAb (5 g/ml) did not show from various experiments were used for first-strand cDNA synthesis using significant difference in the viability count by trypan blue exclusion assay the Superscript Reverse Transcriptase II system (Life Technologies) and compared with control cells at the end of the experiment (72 h). PCR analysis. The sense primer 5Ј-GCGCCTGGTCACCAGGGCTGC-3Ј Ј Ј FACScan analysis and the antisense primer 5 -GGATCTCGCTCCTGGAAGATC-3 were used for amplification of GAPDH (for 30 cycles). PCR analysis of IL-1␤ Bovine chondrocytes were grown in 0.5% serum-containing medium. The was performed using the sense primer 5Ј-GAAGAGCTGCATCCAA- cells were gently released using 0.1% EDTA prepared in HBSS without CACC-3Ј and the antisense primer 5Ј-ATGCAGAACACCACTTCTCG-3Ј Ca2ϩ and Mg2ϩ, washed twice with PBS containing 1% BSA, and stained in a Perkin-Elmer thermal cycler (94°C for 30 s, 58°C for 1 min, and 72°C with primary mAb LM609 and secondary goat anti-mouse PE-labeled Ab for 2 min for 40 cycles). The signal was quantitated as previously described (Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 min using a densitometer (Molecular Dynamics, Sunnyvale, CA). For the each. As a control, cells were stained with secondary Ab alone for back- RT-PCR control, cDNA synthesis was conducted (from total RNA isolated ground fluorescence. Flow cytometric analysis of the level of FITC fluo- from JBS5- or LPS-treated cells) without the addition of reverse rescence on scatter-gated cells was begun immediately after addition of transcriptase. sheath fluid with or without unlabeled cells, and the median fluorescence intensity (MFI) of the cells acquired during each interval was determined Cloning and expression of soluble type II human IL-1R in with the use of FACScan-associated LYSYS II software (Becton Dickin- baculovirus son, Mountain View, CA). Full-length soluble type II IL-1R (sIL-1RII) was cloned from human neu- Nitrite assay trophils using RT-PCR. The forward primer 5Ј-CGGGATCCATGTT GCGCTTGTACGTGT-3Ј introduced a BamHI site at the 5Ј end of the NO production was determined spectrophotometrically by measuring the ATG, and the reverse primer 5Ј-TAAAGCGGCCGCTCACTTGGGATA Ј accumulation of nitrite (NO2) in culture supernatants by the Griess reaction GAATTG-3 introduced a NotI site immediately following the stop codon. 2686 ROLE OF INTEGRINS IN ARTHRITIS

␤ a Table I. Regulation of IL-1 , IL-6, IL-8, NO, and PGE2 by integrin in human OA-affected cartilage

Per g Cartilage

␮ ␤ Conditions Nitrite ( M) PGE2 (ng/ml) IL-1 (pg/ml) IL-6 (ng/ml) IL-8 (ng/ml) OA-affected cartilage control 12.0 Ϯ 2.0 13.0 Ϯ 0.9 4.0 Ϯ 1.0 7.3 Ϯ 3.0 9.3 Ϯ 2.7 ϩ JBS5 mAb 127.0 Ϯ 24.0* 19.1 Ϯ 3.2‡ 87.6 Ϯ 10.0* 178.8 Ϯ 4.4* 17.7 Ϯ 4.0† ϩ LM609 mAb 5.0 Ϯ 2.0† 8.2 Ϯ 2.2† 2.5 Ϯ 1.0† 2.9 Ϯ 1.9¶ 5.7 Ϯ 0.8† ϩ L-NMMA 8.0 Ϯ 1.0‡ 18.4 Ϯ 0.8‡ ND ND ND ϩ IL-1␤ 173.0 Ϯ 24.0* 296.0 Ϯ 72.0‡ ND ND ND a ϭ ␮ ␣ ␤ ␮ OA-affected cartilage (in triplicate for each modulator; n 3) was incubated in the medium with various modulators: 5 g/ml of 5 1 (JBS5), and 5 g/ml LM609 mAb, ␮ ␤ ␤ Ϯ L-NMMA (500 M), and IL-1 . The accumulation of IL-1 , IL-8, IL-6, nitrite, and PGE2 were estimated at 72 h poststimulation. Data represent mean SD. The p values Յ0.001; †, Յ0.01; ‡, Յ0.03; ¶, Յ0.05. ND, not done. The data ,ء :described are compared with the (control) unstimulated OA-affected cartilage. The p values are as follows represent one of the two independent experiments from two different patients.

The PCR-amplified DNA was digested with BamHI and NotI, and the sought to identify the roles of their respective receptors in cartilage fragment (1196 bp) was subcloned into pFAST BAC-1 BACMID. The inflammation. recombinant sIL-1RII was generated using the BAC to BAC system as described previously (24). The recombinant baculovirus-expressed sIL- 1RII was estimated by ELISA (R&D Systems). The biological activity of Role of integrin ligation on production of inflammatory Downloaded from sIL-1RII released from Sf9 insect cells was compared with that of the mediators in human OA-affected cartilage commercially available sIL-1RII from R&D Systems. We have recently observed that the autocrine production of IL-1␤ Statistical analysis may be regulated by OPN in cartilage. Addition of OPN inhibits ␤ All data are expressed as the mean Ϯ SD, and statistical analysis was IL-1 -mediated NO and PGE2 production in OA-affected carti- performed using GraphPad software (version 1.14, GraphPad, San Diego, lage, whereas neutralizing anti-OPN antiserum augments IL-1␤- CA). The t test or nonparametric test (Mann-Whitney or Wilcoxon test) mediated NO production (M. G. Attur et al., manuscript in prep- http://www.jimmunol.org/ was performed to analyze the data. aration). In view of the above observations and previous reports that inflammatory mediators are up-regulated and spontaneously Results and Discussion released from OA-affected cartilage, we examined whether the in- Differential display of mRNA was performed between normal and duction of these inflammatory mediators in human OA-affected OA-affected human cartilage (23). We observed up-regulation of cartilage is attributable to modulation of integrin functions. multiple genes in OA-affected cartilage compared with normal car- In the present study we avoided the use of natural ligands be- tilage (data not shown). Bioinformatic analysis of various cDNA cause of the multifunctional roles of FN and OPN, in particular,

sequences using BLAST search identified two known extracellular their ability to bind various receptors (1, 29). Furthermore, mAbs by guest on September 29, 2021 proteins, FN and OPN, that were up-regulated ϳ20-fold in OA- specifically induce high affinity binding and oligomerization of affected cartilage compared to normal cartilage (M. G. Attur et al., integrins without changes in integrin gene expression (1, 29). We manuscript in preparation). Similarly, sequencing of subtracted selected mAbs, which not only have been reported to inhibit the ␣ ␤ ␣ ␤ cDNA libraries expressing OA-specific genes also showed high binding of 5 1-or v 3-positive cells to plates coated with FN or frequency of FN and OPN mRNA (data not shown). Direct addi- vitronectin respectively, but which also act as agonists to mimic ␣ ␤ tion of FN fragments (25) or OPN (M. G. Attur et al., manuscript the functions of their respective ligands. JBS5 is an anti- 5 1 mAb in preparation) up-regulated or down-regulated inflammatory me- that inhibits the binding of FN and causes aggregation and homo- diators in chondrocytes, respectively. The present study is geared typic clustering of the receptors in Jurkat T cells (30). LM609 is an ␣ ␤ towards functional genomic analysis of FN and OPN receptors in anti- v 3 mAb that blocks the binding of its ligands to the receptor ␣ ␤ normal and OA-affected cartilage. and also inhibits v 3-mediated functions, such as , in ␤ Previous studies have shown that anti- 1 integrin mAb inhibit various experimental systems (31). ␤ ␣ ␤ ␣ ␤ attachment of FN to chondrocytes, whereas anti- 3 mAb inhibit The effects of anti- v 3 (LM609) and anti- 5 1 (JBS5) mAbs attachment on OPN (26). FN, like OPN, can bind to various inte- (32, 33) on the regulation of spontaneous release of NO, PGE2, ␣ ␤ ␤ grins, but preferentially binds to 5 1, whereas OPN binds pref- IL-6, IL-8, and IL-1 was evaluated in human OA-affected carti- ␣ ␤ erentially to v 3 (1, 27). In view of the proinflammatory role of lage in ex vivo conditions as described in Materials and Methods. FN fragments (28) and the anti-inflammatory role of OPN in hu- Human OA-affected cartilage (control) spontaneously released ␤ man cartilage (M. G. Attur et al., manuscript in preparation), we NO, PGE2, IL-6, IL-8, and IL-1 (14–16) (Table I). Addition of

␤ a Table II. Regulation of IL-1 , IL-6, IL-8, NO, and PGE2 by integrin in normal human cartilage

Per g Cartilage

␮ ␤ Conditions Nitrite ( M) PGE2 (ng/ml) IL-1 (pg/ml) IL-6 (ng/ml) IL-8 (ng/ml) Normal cartilage (control) 2.0 Ϯ 1.0 1.5 Ϯ 1.3 1.0 Ϯ 0.1 2.5 Ϯ 1.1 2.5 Ϯ 2.0 ϩ JBS5 mAb 154.0 Ϯ 28.0* 11.4 Ϯ 4.2† 37.7 Ϯ 3.2* 75.1 Ϯ 14.5* 17.8 Ϯ 5.0* ϩ LPS 326.0 Ϯ 66.0* 21.2 Ϯ 4.8* 262.1 Ϯ 15.4* ND ND ϩ IL-1␤ 355.0 Ϯ 22.0* 15.8 Ϯ 4.9† ND ND ND

a Normal human cartilage (in triplicate for each modulator; n ϭ 3) was incubated in the medium with JBS5 mAb (5 ␮g/ml), IL-1␤ (1 ng/ml), or LPS (100 ␮g/ml). The ␤ accumulation of nitrite, PGE2, IL-6, IL-8, and IL-1 was estimated as described in Materials and Methods. The p values described are compared with the (control) normal .Յ0.001; †, Յ0.01. ND, not done ,ء :cartilage. This is a representative experiment performed independently two times. The p values are as follows The Journal of Immunology 2687

␣ ␤ ␣ ␤ which shows greater ability to interact with 5 1 than v 3 (35) induced significant augmentation of NO (12.0 Ϯ 2.0 to 38.0 Ϯ 10.0 ␮M/g cartilage; p Ͻ 0.01) and IL-1␤ (4.0 Ϯ 1.0 to 11.0 Ϯ 2.0 pg/g cartilage; p Յ 0.01) accumulation over the uninduced control OA cartilage), whereas RGEN caused significant inhibi- tion of NO accumulation (12.0 Ϯ 2.0 to 4.0 Ϯ 2.0 ␮M/g cartilage; p Ͻ 0.03) compared with control cartilage. These experiments sug- gest that although RGDN and CCBD motifs are both required to ␣ ␤ modulate the 5 1 integrin by FN, these RGDN or RGEN peptides partially mimic the actions of these ligands and confirm that ECM- integrin interaction can modulate inflammatory mediators, such as NO production in OA-affected cartilage, similar to that observed with IL-1␤. These data support our hypothesis that increased accumulation/ synthesis of: 1) FN (and its proteolytic fragments) may interact ␣ ␤ with its prototypic receptor 5 1 integrin and induce NO, PGE2, IL-6, IL-8, and IL-1␤ production in human OA-affected cartilage; ␣ ␤ 2) OPN may interact with its prototypic receptor, v 3 integrin,

and inhibit NO and PGE2 production in OA-affected cartilage. Downloaded from It should be noted that OPN and LM609 Ab used in this study share the capacity to inhibit NO production. LM609 has also been reported to inhibit angiogenesis, tumor growth, and calcium-me- diated vasoconstriction (31, 35, 36). In contrast, other ligands ␣ ␤ (CYR6 or Del1) that engage v 3 promote angiogenesis and neo-

vascularization (37, 38). This is not surprising, because echistatin http://www.jimmunol.org/ ␣ ␤ (a snake venom protein) that also binds to v 3 (39) shows dif- FIGURE 1. A, Regulation of NO and PGE2 in primary bovine chondro- ferential regulation of inflammatory mediators distinct from those cytes by integrin receptor in the presence of sIL-1RII. Bovine chondrocytes observed with OPN (our unpublished observations). These results were seeded (in triplicate) in 24-well plates at a density of 0.5 ϫ 10 6 suggest that the type of or Ab and the epitope to which it cells/well in 2 ml of medium. After 48 h of postseeding, the cells were ␣ ␤ treated (in triplicate for each modulator; n ϭ 3) with either JBS5 mAb (5 binds in the binding pocket of v 3 are critical for the downstream ␮g/ml) or IL-1␤ (1 ng/ml) alone in the presence and the absence of sIL- signal relayed by this receptor. These observations suggest that ␣ ␤ 1RII (0. 5 ng/ml) for 72 h. NO (micromolar concentrations) and PGE2 v 3 integrin is a multifunctional receptor with discrete ligand (nanograms per milliliter) were estimated as described in Materials and binding regions that determines their function. Methods. The data represent one of the two similar experiments. Statistics by guest on September 29, 2021 ␤ ␣ ␤ were derived using unpaired Student’s t test. The p values are compared Up-regulation of NO, PGE2, IL-6, IL-8, and IL-1 by anti- 5 1 p Յ mAb in normal human cartilage ,ء .(with the respected control. Data represent the mean Ϯ SD (n ϭ 3 Յ ءءء Յ ءء 0.01; , p 0.001; , p 0.0001. B, Regulation of NO and PGE2 in We and others have previously shown that human OA-affected OA-affected cartilage by integrin receptors in the presence of sIL-1RII. cartilage releases various inflammatory cytokines (including IL- Human OA-affected cartilage organ cultures were set up as described in 1␤) that act in an autocrine fashion to affect chondrocyte functions Materials and Methods and Table I. The cultures were treated (in triplicate; n ϭ 3 for each modulator) with JBS5 mAb or IL-1␤ alone in the presence (16, 22, 40). In view of these observations, the possibility that the and the absence of sIL-1RII as described above. NO (micromolar concen- integrins may act cooperatively with IL-1R to amplify the effects ␤ trations per 100 mg) and PGE2 (nanograms per milliliter per gram) were of IL-1 cannot be ruled out. Lo et al. (41) have shown enrichment estimated at the end of experiment. The data represent one of two of IL-1R at focal adhesion (FA) sites (41). These FA sites are similar experiments and are the mean Ϯ SD. Statistics were derived enriched in signaling molecules that are used by integrins to trans- using unpaired Student’s t test. The p values are compared with duce signals. There is also evidence to indicate that binding of IL-1 spontaneously released, IL-1␤-induced, or JBS5 mAb-induced NO and to its receptor triggers activation of integrin-associated signaling Յ ءء Յ ء PGE2. , p 0.01; , p 0.001. components within the same FA complex (41). We therefore tested ␣ ␤ the effect of anti- 5 1 on normal human cartilage, which in the absence of stimulation produced significantly low, but detectable, ␤ ␣ ␤ JBS5 mAbs caused significant augmentation of NO, PGE2, IL-6, amounts of IL-1 , IL-6, or IL-8. Ligation of 5 1 by JBS5 mAb IL-8, and IL-1␤ production compared with the control cartilage. In in normal cartilage explants significantly stimulated the production ␤ contrast, addition of LM609 mAb inhibited spontaneous release of of NO, PGE2, IL-1 , IL-6, and IL-8 as observed in OA-affected ␤ NO, PGE2, IL-6, IL-8, and IL-1 production compared to that in cartilage (Table II). These experiments suggest that normal human ␣ ␤ the control cartilage (Table I). The activity of 5 1 mAb could be cartilage (like OA-affected cartilage) is also equally susceptible to ␣ ␤ absorbed by preincubating the Ab preparation with recombinant 5 1 integrin-mediated induction of inflammatory mediators. protein A coupled to agarose beads. These controls exclude the ␤ possibility of contaminating modulators in the Ab preparation used Autocrine production of IL-1 is required for induction of NO ␣ ␤ in this study. and PGE2 by 5 1 in chondrocytes Arner and Tortorella (34) have reported that cyclic peptides with We performed experiments to determine whether selected stimu- ␣ ␤ RGDN sequence (which are identified as minimal integrin-binding latory activities following 5 1 engagement were dependent upon recognition sequence motifs in various ECM proteins) induce IL-1␤ production. We therefore tested the role of autocrine IL-1␤

MMP production in bovine chondrocytes. We therefore tested the in the regulation of NO and PGE2 production in OA-affected car- effects of 1 mg/ml of cyclic RGDN and RGEN (negative control) tilage and primary bovine chondrocytes exposed to JBS5 or IL-1␤ on NO production in OA-affected cartilage. The RGDN peptide, mAb in the presence or the absence of sIL-1RII (Fig. 1, A and B). 2688 ROLE OF INTEGRINS IN ARTHRITIS

FIGURE 2. Regulation of IL-1␤ mRNA by integrins in bovine chondrocytes. Normal bovine chondrocytes were incubated with various modulators for 72 h and analyzed for IL-1␤ mRNA and GAPDH in the same experiments by RT-PCR as described in Materials and Methods. The 350-bp IL-1␤ and 200-bp GAPDH fragments were sequenced to ascertain their identity (not shown). In experiment I the cells were stimulated with LPS (100 ␮g/ml) or JBS5 mAb (5 ␮g/ml) in the presence or the absence of 5 ␮g/ml LM609. Lane 1, Standard marker (1-kb ladder); lane 2, uninduced cells; lane 3, LPS; lane 4, LM609 (5 ␮g/ml) mAb plus LPS; lane 5, JBS5 mAb (5 ␮g/ml); lane 6, LM609 and JBS5 mAbs. In experiment II lanes 7–11 are similar to lanes 2–6 in

experiment I. Lanes 12 and 13 are RT controls as described in Materials and Methods. LM609 mAb were preincubated for 30 min before inducing the cells Downloaded from with various modulators.

The rationale for using sIL-1RII for these studies was that sIL-1RII Ab alone in bovine chondrocytes was 62. Addition of primary has been reported to preferentially bind to both mature and pro- (LM609) and secondary mAb showed an increase in MFI to 244 IL-1␤ rather than IL-1Ra or IL-1RI (42). for chondrocytes. These experiments confirm the expression of ␤ ␣ ␤

Addition of IL-1 or JBS5 mAb induced the production of NO v 3 integrin (and cross-reactivity of LM609) in bovine chondro- http://www.jimmunol.org/ ␣ ␤ and PGE2 in primary bovine chondrocytes and OA-affected carti- cytes. Recently, Gibson et al. (44) reported the expression of v 3 ␣ ␤ ␣ ␤ lage. Preincubation of cells or cartilage (30 min) with sIL-1RII not in bovine chondrocytes. The expression of v 3 and 5 1 integrin ␤ only blocked the IL-1 -induced accumulation of NO and PGE2,as on human chondrocytes and cartilage has been shown by other expected, but also inhibited JBS5 mAb-induced production of investigators (2, 3, 45). these mediators. These experiments suggest that autocrine produc- We examined the role of LM609 mAb in the regulation of NO ␤ ␤ tion of IL-1 - and IL-1 -mediated signaling is required for induc- and PGE2 production mediated by other proinflammatory recep- ␣ ␤ tion of NO and PGE2 following ligation of the 5 1 integrin. tors. Normal bovine chondrocytes were preincubated (30 min) ␤ ␤ We also tested the effect of mAb13, which inhibits 1 function with LM609 mAb and stimulated with JBS5 mAb, IL-1 , LPS,

␤ ␣ by guest on September 29, 2021 (43), on the regulation of inflammatory mediators in bovine chon- and IL-1 , TNF- , plus LPS. The levels of NO and PGE2 were ␤ drocytes. mAb13, recognizes an epitope(s) on 1 that competes estimated. LM609 mAb inhibited the induction of NO and PGE2 with RGDN peptide and the CCBD fragment of FN and therefore by each of these stimuli (Fig. 3A) with cell viability of Ͼ95% at ␤ decreases the binding of FN (43). This 1 inhibitory Ab had no the end of 72 h as examined by trypan blue exclusion assay (data significant effect on the production of inflammatory mediators in not shown), thus excluding induced by both these mAb bovine chondrocytes or OA-affected cartilage (data not shown). in these cells. Similar results were observed in OA-affected carti- These experiments suggest that JBS5 engages an epitope in the lage that were induced with JBS5 mAb, IL-1␤, and IL-1␤, TNF-␣, ␣ ␤ 5 1 complex that is capable of generating an activation signal. and LPS (Fig. 3B). ␤ Regulation of IL-1 mRNA in bovine chondrocytes by integrins Regulation of NO and PGE2 production by IL-18 in human OA- In view of the above results, we examined the gene expression of affected cartilage IL-1␤ in chondrocytes stimulated with JBS5 mAb under various IL-18 is identified as an IFN-␥-inducing factor and is structurally conditions. Normal bovine chondrocytes were stimulated with similar to the IL-1 family of proteins (46). IL-18 is known to LPS, LM609 mAb plus LPS, JBS5 mAb, and LM609. Stimulation induce NO synthase, COX-2, IL-6, and MMPs (47), and its pro- with either LPS or JBS5 mAb induced up-regulation of IL-1␤ duction has been reported to be stimulated by IL-1 in chondro- mRNA accumulation (as analyzed by RT-PCR analysis), whereas cytes. Our preliminary data show that human OA-affected cartilage the addition of LM609 inhibited IL-1␤ mRNA accumulation in- in ex vivo conditions spontaneously released IL-18 (288.3 Ϯ 246.5 duced by LPS or JBS5 (Fig. 2). These experiments further indicate (n ϭ 5) pg/ml/g of cartilage within 72 h. In the present study we a role for integrins in the (negative and positive) regulation of also tested the effects of LM609 mAb on IL-18-induced NO and ␤ IL-1 gene expression in chondrocytes. These experiments also PGE2 production in human OA-affected cartilage as shown in Ta- ␣ ␤ ␤ Յ suggest that the v 3 integrin negatively modulates IL-1 expres- ble III. IL-18 significantly ( p 0.004) induced NO and PGE2 sion at the level of gene and thereby influences the production; prior incubation with LM609 inhibited both spontane- Յ Յ production of other inflammatory mediators; moreover, it indicates ous and IL-18-induced NO ( p 0.0002) and PGE2 ( p 0.01) ␣ ␤ ␣ ␤ ␣ ␤ that the v 3 integrin regulates 5 1 in a dominant negative production in these explant cultures. Thus, signaling through v 3 fashion. (i.e., ligation by LM609 mAb) results in the inhibition of inflam- matory mediator release in response to IL-1, IL-18, and ␣ ␤ .Itis ␣ ␤ ␣ ␤ ␤ 5 1 Anti- v 3 integrin mAb inhibit the actions of 5 1-, IL-1 -, known that LPS, IL-1, and IL-18 share selected signaling path- ␤ ␣ LPS-, and IL-1 -, TNF- -, and LPS-dependent NO and PGE2 ways, including the activation of NF-␬B (48–50). The pathway or production ␣ ␤ site at which the signal generated by ligation of v 3 interferes ␣ ␤ Expression of v 3 on bovine chondrocytes was confirmed by with IL-1 and IL-18 cellular activation is currently under FACS analysis. The background MFI for the secondary PE-labeled investigation. The Journal of Immunology 2689

␣ ␤ Table III. Regulation of NO and PGE2 by IL-18 and v 3 integrins in human OA-affected cartilagea

Per g Cartilage

␮ Conditions PGE2 (ng/ml) Nitrite ( M) Control 39.6 Ϯ 2.3 37.0 Ϯ 7.0 ϩ IL-18 (10 ng/ml) 106.3 Ϯ 5.9§ 73.0 Ϯ 9.0§ ϩ LM609 mAb 17.2 Ϯ 1.5* 9.0 Ϯ 6.0‡ ϩ IL-18 ϩ LM609 mAb 58.1 Ϯ 2.5† 3.0 Ϯ 2.0†

a Human OA-affected cartilage (in triplicate for each modulator; n ϭ 3) was in- cubated in ex vivo in the presence and absence of IL-18 (10 ng/ml) Ϯ LM609 (5 ␮ g/ml) for 72 h. The accumulation of PGE2 and nitrite was estimated. Data represent mean Ϯ SD. This is a representative experiment performed independently two times. ,‡ ;Յ0.01; †, Յ0.001 ,ء :The p value between control and experiments are as follows Յ0.002; §, Յ0.004. The p value between §/† for nitrite was Յ0.001.

we have performed most of our experiments under ex vivo con- ditions without exogenous growth factors, as dissection of integrin signaling pathways may be further complicated by the cellular re- Downloaded from sponses to growth factors (such as insulin-like and ), which can mediate similar intracellular responses as integrin-ligand interactions (1). ␣ ␤ Previous studies have shown that expression of v 3 in K562 ␣ ␤ cells inhibits phagocytic functions of the 5 1 through the sup-

pression of calcium/calmodulin-dependent protein kinase II (55). http://www.jimmunol.org/ ␣ ␤ Similarly, muscle-specific common receptor 7 1 negatively reg- ␣ ␤ ulates 5 1 receptor function by decreasing the ligand binding ␣ ␤ affinity (56). This intracellular trans-dominant inhibition of v 3 may be due to blockade of integrin signaling and/or conforma- ␤ tional changes in the extracellular domain induced by the 3 cy- ␣ ␤ toplasmic tail of the suppressive integrin as observed with IIb 3 integrin (57) or inhibition of intracellular signaling molecule(s) ␣ ␤ FIGURE 3. A, Regulation of NO and PGE2 by v 3 integrin in bovine

chondrocytes. Bovine chondrocytes were seeded in 24-well plates and by guest on September 29, 2021 stimulated with IL-1␤ (1 ng/ml), LPS (100 ␮g/ml), and LPS, IL-1␤, plus TNF-␣ (100 U/ml; LIT) in the presence or absence of LM609 mAb (5 ␮g/ml) or mouse IgG1 (mIgG1) in triplicate (n ϭ 3). The accumulation of

NO (micromolar concentrations) and PGE2 (nanograms per milliliter) was estimated at the end of 72 h. Statistics were derived using unpaired Stu- dent’s t test. The data represent the mean Ϯ SD from one of the two similar experiments. The p values are compared with the respective stimulated p Յ 0.0001. B, Regulation of ,ءءء ;p Յ 0.001 ,ءء ;p Յ 0.01 ,ء :conditions ␣ ␤ NO and PGE2 by v 3 integrin in human OA-affected cartilage. Human OA-affected explants were incubated in organ cultures as described in Ma- terials and Methods. The cultures were preincubated with LM609 mAb (5 ␮g/ml) and stimulated with IL-1␤ (1 ng/ml); LPS, IL-1␤, and TNF-␣ (LIT); or JBS5 mAb (5 ␮g/ml), and the levels of NO (micromolar con-

centrations per 100 mg) and PGE2 (nanograms per milliliter per gram) were estimated at the end of 72 h. The control represents an equivalent amount of isotype control (IgG1). The data are from one of the two similar exper- iments. Statistics were derived using unpaired Student’s t test. Values are the mean Ϯ SD. The p values compared with the respective stimulated .p Յ 0.0001 ,ءءء ;p Յ 0.001 ,ءء ;p Յ 0.01 ,ء ;conditions were FIGURE 4. Regulation of inflammatory mediators by integrins in car- tilage. The data show the hypothetical scheme for the mechanisms of action ␣ ␤ ␣ ␤ These experiments, on the whole, show trans-dominant inhibi- of anti- 5 1 and anti- v 3 mAb in cartilage and chondrocytes. The anti- ␣ ␤ ␤ ␤ ␣ ␣ ␤ ␣ ␤ ϩ Ϫ tion of 5 1-, IL-1 -, LPS-, and IL-1 -, TNF- -, plus LPS-in- 5 1 or v 3 mAb induced positive ( ) or negative ( ) signals, respec- ␣ ␤ tively, to up/down-regulate IL-1␤ gene expression in cartilage by “outside duced functions by v 3 in human and bovine chondrocytes. The possibility of ␣ ␤ and ␣ ␤ coassociating with IL-1R cannot be in” signaling. Similarly, an increase in the production of FN fragments or 5 1 v 3 ␤ ruled out, as ␣ ␤ receptors have been reported to associate with OPN may also induce/inhibit IL-1 synthesis in human OA-affected car- v 3 tilage. The IL-1␤ induces its own production in an autocrine/paracrine other receptors, such as CD47, in regulating proinflammatory cy- ␣ ␤ fashion, which, in turn, up-regulates other inflammatory mediators, such as tokines in human (51). The association of v 3 with NO, PGE , IL-6, IL-8, IL-18, and MMPs. It should be noted that IL-18 can ␤ 2 platelet-derived growth factor- and insulin receptor substrate-1 as independently induce NO, PGE , and MMPs (47). This “inside out” sig- ␣ ␤ 2 well as the dependence on v 3 for the biological effect of fibro- naling induced by ␣ ␤ integrin can be inhibited by addition of sIL-1RII, ␣ ␤ 5 1 blast growth factor-2 indicate that v 3 is linked with the cellular which neutralizes/inhibits the cascade induced by IL-1 responses of certain growth factors (52–54). It should be noted that and IL-1RI complex. 2690 ROLE OF INTEGRINS IN ARTHRITIS

␣ ␤ ␤ induced by 5 1, IL-1 , IL-18, and LPS receptors. These assump- 5. Yonezawa, I., K. Kato, H. Yagita, Y. Yamauchi, and K. Okumura. 1996. VLA- tions are supported by recent observations suggesting that 1) 5-mediated interaction with fibronectin induces production by human chondrocytes. Biochem. Biophys. Res. Commun. 219:261. INK4a ␣ ␤ p16 tumor suppressor protein inhibits v 3 integrin-mediated 6. Homandberg, G. A. 1999. Potential regulation of cartilage metabolism in osteo- cell spreading on vitronectin-coated plates by blocking PKC-de- arthritis by fibronectin fragments. Front. Biosci. 4:d713. ␣ ␤ 7. Enomoto-Iwamoto, M., M. Iwamoto, K. Nakashima, Y. Mukudai, D. Boettiger, pendent localization of v 3 to focal contacts (58); 2) tumor sup- ␣ ␤ M. Pacifici, K. Kurisu, and F. Suzuki. 1997. Involvement of 5 1 integrin in pressor PTEN (phosphatase and tensin homolog deleted on chro- matrix interactions and proliferation of chondrocytes. J. Bone Miner. Res. 12: mosome 10) inhibit FN-binding integrin and growth factor- 1124. 8. Johansson, S., G. Svineng, K. Wennerberg, A. Armulik, and L. Lohikangas. mediated -activated protein kinase signaling pathway (59); 1997. Fibronectin-integrin interactions. Front. Biosci. 2:d126. and 3) the ephrin B-1-induced increase in cell attachment is in- 9. McKee, M. D., M. J. Glimcher, and A. Nanci. 1992. High-resolution immuno- ␣ ␤ localization of osteopontin and osteocalcin in bone and cartilage during endo- volved in “inside out” activation of 5 1 integrin in HEK 293 and ␣ ␤ chondral ossification in the chicken tibia. Anat. Rec. 234:479. of v 3 integrin in endothelial cells (60). 10. Denhardt, D. T., C. A. Lopez, E. E. Rollo, S. M. Hwang, X. R. An, and In summary, these experiments show that two well-character- S. E. Walther. 1995. Osteopontin-induced modifications of cellular functions. ized mAbs that specifically bind to ␣ ␤ and ␣ ␤ integrins mimic Ann. NY Acad. Sci. 760:127. 5 1 v 3 11. Yoshitake, H., S. R. Rittling, D. T. Denhardt, and M. Noda. 1999. Osteopontin- the action of their ligands (FN fragments and OPN; Fig. 4). These deficient mice are resistant to ovariectomy-induced bone resorption. Proc. Natl. ␣ ␤ ␤ Acad. Sci. USA 96:8156. studies indicate that Abs to 5 1 induce or augment IL-1 pro- 12. Denhardt, D. T., and M. Noda. 1998. Osteopontin expression and function: role duction, which, in turn, is responsible for induction of NO, PGE2, in bone remodeling. J. Cell Biochem. 30–31(Suppl.):92. ␣ ␤ IL-6, and IL-8. In contrast, v 3, which is overexpressed in the 13. Amin, A. R., P. Di Cesare, P. Vyas, M. G. Attur, E. Tzeng, T. R. Billiar, superficial zone of OA-affected cartilage compared with normal S. A. Stuchin, and S. B. Abramson. 1995. The expression and regulation of synthase in human osteoarthritis-affected chondrocytes: evidence for up- ␤ Downloaded from cartilage (61), down-regulates IL-1 expression and the effects of regulated neuronal nitric oxide synthase. J. Exp. Med. 182:2097. IL-1␤ in a trans-dominant negative manner. Together, the data 14. Amin, A. R., M. G. Attur, R. N. Patel, G. D. Thakker, P. J. Marshall, J. Rediske, demonstrate that integrins and their ligands modulate chondrocyte and S. B. Abramson. 1997. Superinduction of cyclooxygenase-2 activity in hu- man osteoarthritis-affected cartilage: influence of nitric oxide. J. Clin. Invest. functions by influencing the synthesis of pleiotropic cytokines, 99:1231. such as IL-1␤, IL-6, and IL-8, and signaling molecules, such as 15. Amin, A. R., M. G. Attur, and S. B. Abramson. 1999. Regulation of nitric oxide NO and PGE , which have diverse catabolic functions. Overpro- and inflammatory mediators in human osteoarthritis-affected cartilage: implica- 2 tion for pharmacological intervention. In The Pathophysiology and Clinical Ap- duction of NO in chondrocytes inhibits collagen and proteoglycan plications of Nitric Oxide, Part B. G. M. Rubanyi, ed. Harwood Academic Pub- http://www.jimmunol.org/ synthesis and induces oxidant injury through peroxynitrite forma- lishers, Amsterdam, p. 397. 16. Attur, M. G., I. R. Patel, R. N. Patel, S. B. Abramson, and A. R. Amin. 1998. tion and apoptosis of chondrocytes (reviewed in Refs. 17 and 62). Autocrine production of IL-1␤ by human osteoarthritis-affected cartilage and There is also evidence that NO mediates the catabolic effects of differential regulation of endogenous nitric oxide, IL-6, E2 and IL-1 (63) and modulation of integrins (45, 64) in OA cartilage. The IL-8. Proc. Assoc. Am. Physicians 110:1. 17. Amin, A. R., and S. B. Abramson. 1998. The role of nitric oxide in articular overproduction of COX-2 and PGE2 in OA may contribute to the cartilage breakdown in osteoarthritis. Curr. Opin. Rheumatol. 10:263. pain experienced in this disease, because inhibitors of COX-2 pro- 18. Melchiorri, C., R. Meliconi, L. Frizziero, T. Silvestri, L. Pulsatelli, I. Mazzetti, vide symptomatic improvement in the clinic (65). Whether COX-2 R. M. Borzi, M. Uguccioni, and A. Facchini. 1998. Enhanced and coordinated in vivo expression of inflammatory cytokines and nitric oxide synthase by chon- up-regulation affects metalloproteinase activity or proteoglycan drocytes from patients with osteoarthritis. Arthritis Rheum. 41:2165. production in OA is as yet undetermined. 19. Moos, V., S. Fickert, B. Muller, U. Weber, J. Sieper, L. Bloom, K. C. Ingham, by guest on September 29, 2021 This study thus demonstrates the role of the ECM as a medium and R. O. Hynes. 1999. 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