The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Attur, M. G.
Right arrow Articles by Amin, A. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Attur, M. G.
Right arrow Articles by Amin, A. R.
The Journal of Immunology, 2000, 164: 2684-2691.
Copyright © 2000 by The American Association of Immunologists

Functional Genomic Analysis in Arthritis-Affected Cartilage: Yin-Yang Regulation of Inflammatory Mediators by {alpha}5ß1 and {alpha}Vß3 Integrins1

Mukundan G. Attur*, Mandar N. Dave*, Robert M. Clancy*, Indravadan R. Patel*, Steven B. Abramson*,{dagger} and Ashok R. Amin2,*,{dagger},{ddagger}

* Department of Rheumatology, Hospital for Joint Diseases, New York, NY 10003; and Departments of {dagger} Pathology and {ddagger} Medicine, Kaplan Cancer Center, New York University Medical Center, New York, NY 10016


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
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 integrin receptors {alpha}5ß1 and {alpha}vß3 of FN and OPN, respectively, have profound effects on chondrocyte functions. Ligation of {alpha}5ß1 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 cytokines, IL-6 and IL-8. Furthermore, up-regulation of these proinflammatory mediators by {alpha}5ß1 integrin ligation is mediated via induction and autocrine production of IL-1ß, because type II soluble IL-1 decoy receptor inhibits their production. In contrast, {alpha}vß3 complex-specific function-blocking mAb (LM609), which acts as an agonist similar to OPN, attenuates the production of IL-1ß, NO, and PGE2 (triggered by {alpha}5ß1, IL-1ß, IL-18, or IL-1ß, TNF-{alpha}, plus LPS) in a dominant negative fashion by osteoarthritis-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 homeostasis, and its deregulation may play a role in the pathogenesis of osteoarthritis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Integrins are heterodimer receptors that contain {alpha} and ß subunits that bind to extracellular matrix (ECM)3 components or other membrane-bound receptors (1). Ligands for integrins are often large ECM proteins such as collagen, laminin, vitronectin, osteopontin (OPN), and fibronectin (FN) (1).

Articular cartilage is avascular, aneural, and alymphatic. The synthesis and degradation of cartilage matrix are regulated by chondrocytes via mechanisms that depend in part upon the interaction of chondrocytes with the ECM proteins. It is increasingly appreciated that there is specific induction and repression of chondrocyte gene expression, controlled through cellular contacts with ECMs in the pericellular environment surrounding the chondrocytes (chondron), using a complex array of integrins on chondrocytes that control cartilage homeostasis.

Normal human chondrocytes express various integrins, including {alpha}1ß1, {alpha}5ß1, {alpha}vß5, and lesser quantities of {alpha}vß3 and {alpha}3ß1 heterodimers. The {alpha}v subunit-containing integrins are detected more readily on the superficial chondrocytes than on deep zone chondrocytes (2, 3). Chondrocytes from osteoarthritis (OA)-affected cartilage express {alpha}1, {alpha}3, {alpha}5, {alpha}2, and {alpha}v, with lesser amounts of {alpha}4 and {alpha}6, whereas the ß1-chain is expressed on ~40% of the cells (4). In addition to an altered display of integrins, there is an increased expression of selected ECM proteins, such as FN and OPN.

We and others have observed enhanced expression of FN and OPN mRNA in human OA-affected cartilage compared with normal cartilage (M. G. Attur, M. N. Dave, S. A. Stuchin, A. S. Kowalski, C. A. Lopez, J. Zhang, S. B. Abramson, D. T. Denhardt, and A. R. Amin, manuscript in preparation) (5, 6). These ECM proteins may alter chondrocyte functions; FN and FN fragments have been reported to induce IL-1ß, IL-6, GM-CSF, matrix metalloproteinases (MMPs), and proteoglycan release in chondrocytes (5, 6). Engagement of {alpha}5ß1 integrin by FN has also been implicated in chondrocyte adhesion, spreading, and proliferation in vitro (7, 8).

OPN is a soluble secretory phosphoprotein with diverse functions, including a marker for commitment to endochondrial ossification (9), inhibition of endotoxin-induced NO production in epithelial cells (10), and regulation of the onset of osteoporosis (11). OPN is secreted by various cell types, including activated T cells, macrophages, osteoblasts, and hypertropic chondrocytes and is reported to bind to various integrins, including {alpha}vß3 (12).

Historically, OA has been a noninflammatory disease due to the absence of both infiltrating neutrophils and the characteristic signs of inflammation, such as redness and swelling with heat and pain (rubor et tumor cum colore et dolor). However, reports from our own and other laboratories have demonstrated the superinduction of proinflammatory genes (which include NO synthase, COX-2, IL-1ß, IL-6, and IL-8) in osteoarthritic cartilage, which leads to spontaneous production of NO, PGE2, IL-1ß, IL-6, and IL-8 in ex vivo conditions (13, 14, 15, 16, 17, 18, 19, 20). The up-regulation of these cytokines and mediators exerts detrimental effects on chondrocyte functions (13, 14, 15, 16, 17, 18, 19, 20). This inflammatory response, which may be considered molecular inflammation, is not reflected by clinical signs of inflammation, but leads to an imbalance of cartilage homeostasis that results in progressive articular degeneration (15). The end point of this disease is the loss of cartilage and chondrocytes, which maintains cartilage homeostasis in the joints.

In the present study we analyze one of the mechanisms by which ECM proteins regulate inflammatory mediators in cartilage (normal and OA-affected) and primary bovine chondrocytes. We report that 1) engagement of {alpha}5ß1 induces NO, PGE2, IL-6, and IL-8 production via autocrine IL-1ß production; 2) engagement of {alpha}vß3 inhibits the spontaneous production of NO, PGE2, IL-6, and IL-8 production induced through autocrine IL-1ß; and 3) the function blocking anti-{alpha}vß3 Ab (LM609) inhibits the function of anti-{alpha}5ß1 mAb (JBS5), IL-1ß, IL-18, and LPS (with respect to induction of NO, PGE2, and IL-1ß production) in a dominant negative fashion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Reagents

Recombinant IL-1ß, TNF-{alpha}, and ELISA kits for IL-1ß, IL-6, and IL-8 were purchased from R&D (Minneapolis, MN). IL-18 was purchased from PeproTech (Rocky Hill, NJ). Cyclic RGDN and RGEN peptides were procured from Bachem (King of Prussia, PA). Anti-{alpha}5ß1 (JBS5) and anti-{alpha}vß3 (LM609) were purchased from Chemicon (Temecula, CA). Inhibitory anti-ß1 Ab (mAb13) was a gift from Dr. K. M. Yamada (National Institutes of Health, Bethesda, MD).

Procurement of human cartilage

Cartilage slices were taken from the tibial plateau and femoral condyle of knees of patients (with the diagnosis of advanced OA; age, 50–70 years) undergoing knee replacement surgery. Nonarthritic knee cartilage (normal control; age, 20–70 years) was obtained from patients with fractures or from accident victims after knee amputation.

OA explant assay

Knee articular cartilage (from OA patients or normal individuals) were cut into 3-mm discs, and four to six discs were placed (in triplicate or quadruplicate) in a 24-well plate containing 2 ml of Ham’s F-12 medium (with 0.1% human albumin) in the presence or the absence of anti-{alpha}5ß1 (JBS5) or anti-{alpha}vß3 (LM609) Abs and respective control isotype-specific monoclonal Ab along with various modulators as previously described (13, 16).

Isolation of bovine chondrocytes

Bovine chondrocytes were isolated from young cow hooves as previously described (21). Cartilage slices from bovine cartilage were minced finely 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 37°C). After straining through sterile nylon mesh, cells were centrifuged at 1200 x g for 10 min. Cell pellets were washed twice with serum-free Ham’s F-12 medium, followed by resuspending them and centrifuging at 1000 x g for 10 min. Finally, the pellet was suspended in complete F-12 medium before plating them in flasks.

Bovine chondrocytes treated with LM609 mAb (5 µg/ml) did not show significant difference in the viability count by trypan blue exclusion assay compared with control cells at the end of the experiment (72 h).

FACScan analysis

Bovine chondrocytes were grown in 0.5% serum-containing medium. The cells were gently released using 0.1% EDTA prepared in HBSS without Ca2+ and Mg2+, washed twice with PBS containing 1% BSA, and stained with primary mAb LM609 and secondary goat anti-mouse PE-labeled Ab (Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 min each. As a control, cells were stained with secondary Ab alone for background fluorescence. Flow cytometric analysis of the level of FITC fluorescence on scatter-gated cells was begun immediately after addition of sheath fluid with or without unlabeled cells, and the median fluorescence intensity (MFI) of the cells acquired during each interval was determined with the use of FACScan-associated LYSYS II software (Becton Dickinson, Mountain View, CA).

Nitrite assay

NO production was determined spectrophotometrically by measuring the accumulation of nitrite (NO2) in culture supernatants by the Griess reaction (13). Briefly, 100 µl of culture supernatant was mixed with 100 µl of Griess reagent (1% sulfanilamide in 2.5% on H3PO4 and 0.1% N-naphtylethylenediamine dihydrochloride in H2O) for 10 min in 96-well plates. The optical density (OD550) was measured using a microplate reader. Nitrite concentrations were calculated with a standard curve of sodium nitrite ranging from 1–50 µM.

PGE2 analysis

PGE2 was estimated by RIA using rabbit anti-PGE2-BSA as the immunogen (Sigma, St. Louis, MO) (14). The sample (100 µl) or standard (in dilution buffer: 0.01 M PBS, pH 7.4, containing 0.1% BSA and 0.1% sodium azide) was mixed with 500 µl of the diluted antiserum (1/200, in dilution buffer), vortex mixed, and incubated at 4°C for 1 h followed by the addition of 200 µl of cold dextran-coated charcoal (1% activated charcoal suspended in dilution buffer containing 0.1% dextran sulfate). The tubes were vortex mixed vigorously and incubated for 10 min at 0°C in ice-water. The mixture was centrifuged at 8000 rpm (room temperature) for 10 min. Supernatant (400 µl) was removed and added to the scintillation mixture (Packard, Downers Grove, IL), and the amount of radioactivity was determined. The standards were prepared by diluting the stock solution of PGE2 at concentrations of 15–1000 pg/100 µl. The standards were assayed for every PGE2 analysis.

Differential display analysis

Total RNA was extracted from normal and OA-affected cartilage as previously described (22) and was treated with DNase I to remove contaminating 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 RT-PCR using arbitrary primers as recommended by the manufacturer. Differentially expressed cDNA were isolated from the gels, cloned, and sequenced on an automatic DNA Sequencer (Protein DNA Technology Center, Rockefeller University, NY).

Preparation of cDNA subtraction libraries

Human OA-specific subtracted cDNA libraries were prepared using PCR select cDNA subtraction kit as recommended by the manufacturer (Clontech, Palo Alto, CA). The OA cartilage total RNA was used as a tester, and the normal cartilage total RNA was used as a driver. The subtracted cDNAs were cloned onto pTAdv vector using the Advantage PCR cloning kit (Clontech, Palo Alto, CA), and the various clones thus obtained were sequenced.

Bioinformatics

The sequences of various clones obtained from differential display and subtraction libraries were analyzed using the BLAST program against the National Center for Biotechnology Information (NCBI) database.

RT-PCR analysis of IL-1ß

Total RNA from bovine chondrocytes was isolated using TRI reagent (Molecular Research Center, Cincinnati, OH) as described previously (22). The total RNA was treated with DNase to remove contaminating genomic DNA and was purified using a Qiagen RNeasy Mini column (Qiagen, Valencia, CA). Five micrograms of total RNA isolated from bovine chondrocytes from various experiments were used for first-strand cDNA synthesis using the Superscript Reverse Transcriptase II system (Life Technologies) and PCR analysis. The sense primer 5'-GCGCCTGGTCACCAGGGCTGC-3' and the antisense primer 5'-GGATCTCGCTCCTGGAAGATC-3' were used for amplification of GAPDH (for 30 cycles). PCR analysis of IL-1ß was performed using the sense primer 5'-GAAGAGCTGCATCCAACACC-3' and the antisense primer 5'-ATGCAGAACACCACTTCTCG-3' in a Perkin-Elmer thermal cycler (94°C for 30 s, 58°C for 1 min, and 72°C for 2 min for 40 cycles). The signal was quantitated as previously described using a densitometer (Molecular Dynamics, Sunnyvale, CA). For the RT-PCR control, cDNA synthesis was conducted (from total RNA isolated from JBS5- or LPS-treated cells) without the addition of reverse transcriptase.

Cloning and expression of soluble type II human IL-1R in baculovirus

Full-length soluble type II IL-1R (sIL-1RII) was cloned from human neutrophils using RT-PCR. The forward primer 5'-CGGGATCCATGTTGCGCTTGTACGTGT-3' introduced a BamHI site at the 5' end of the ATG, and the reverse primer 5'-TAAAGCGGCCGCTCACTTGGGATAGAATTG-3' introduced a NotI site immediately following the stop codon. The PCR-amplified DNA was digested with BamHI and NotI, and the fragment (1196 bp) was subcloned into pFAST BAC-1 BACMID. The 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 sIL-1RII released from Sf9 insect cells was compared with that of the commercially available sIL-1RII from R&D Systems.

Statistical analysis

All data are expressed as the mean ± SD, and statistical analysis was performed using GraphPad software (version 1.14, GraphPad, San Diego, CA). The t test or nonparametric test (Mann-Whitney or Wilcoxon test) was performed to analyze the data.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Differential display of mRNA was performed between normal and OA-affected human cartilage (23). We observed up-regulation of multiple genes in OA-affected cartilage compared with normal cartilage (data not shown). Bioinformatic analysis of various cDNA sequences using BLAST search identified two known extracellular proteins, FN and OPN, that were up-regulated ~20-fold in OA-affected cartilage compared to normal cartilage (M. G. Attur et al., manuscript in preparation). Similarly, sequencing of subtracted cDNA libraries expressing OA-specific genes also showed high frequency of FN and OPN mRNA (data not shown). Direct addition of FN fragments (25) or OPN (M. G. Attur et al., manuscript in preparation) up-regulated or down-regulated inflammatory mediators in chondrocytes, respectively. The present study is geared towards functional genomic analysis of FN and OPN receptors in normal and OA-affected cartilage.

Previous studies have shown that anti-ß1 integrin mAb inhibit attachment of FN to chondrocytes, whereas anti-ß3 mAb inhibit attachment on OPN (26). FN, like OPN, can bind to various integrins, but preferentially binds to {alpha}5ß1, whereas OPN binds preferentially to {alpha}vß3 (1, 27). In view of the proinflammatory role of FN fragments (28) and the anti-inflammatory role of OPN in human cartilage (M. G. Attur et al., manuscript in preparation), we sought to identify the roles of their respective receptors in cartilage inflammation.

Role of integrin ligation on production of inflammatory mediators in human OA-affected cartilage

We have recently observed that the autocrine production of IL-1ß may be regulated by OPN in cartilage. Addition of OPN inhibits IL-1ß-mediated NO and PGE2 production in OA-affected cartilage, whereas neutralizing anti-OPN antiserum augments IL-1ß-mediated NO production (M. G. Attur et al., manuscript in preparation). In view of the above observations and previous reports that inflammatory mediators are up-regulated and spontaneously released from OA-affected cartilage, we examined whether the induction of these inflammatory mediators in human OA-affected cartilage is attributable to modulation of integrin functions.

In the present study we avoided the use of natural ligands because of the multifunctional roles of FN and OPN, in particular, their ability to bind various receptors (1, 29). Furthermore, mAbs specifically induce high affinity binding and oligomerization of integrins without changes in integrin gene expression (1, 29). We selected mAbs, which not only have been reported to inhibit the binding of {alpha}5ß1- or {alpha}vß3-positive cells to plates coated with FN or vitronectin respectively, but which also act as agonists to mimic the functions of their respective ligands. JBS5 is an anti-{alpha}5ß1 mAb that inhibits the binding of FN and causes aggregation and homotypic clustering of the receptors in Jurkat T cells (30). LM609 is an anti-{alpha}vß3 mAb that blocks the binding of its ligands to the receptor and also inhibits {alpha}vß3-mediated functions, such as angiogenesis, in various experimental systems (31).

The effects of anti-{alpha}vß3 (LM609) and anti-{alpha}5ß1 (JBS5) mAbs (32, 33) on the regulation of spontaneous release of NO, PGE2, IL-6, IL-8, and IL-1ß was evaluated in human OA-affected cartilage in ex vivo conditions as described in Materials and Methods. Human OA-affected cartilage (control) spontaneously released NO, PGE2, IL-6, IL-8, and IL-1ß (14, 15, 16) (Table IGo). Addition of JBS5 mAbs caused significant augmentation of NO, PGE2, IL-6, IL-8, and IL-1ß production compared with the control cartilage. In contrast, addition of LM609 mAb inhibited spontaneous release of NO, PGE2, IL-6, IL-8, and IL-1ß production compared to that in the control cartilage (Table IGo). The activity of {alpha}5ß1 mAb could be absorbed by preincubating the Ab preparation with recombinant protein A coupled to agarose beads. These controls exclude the possibility of contaminating modulators in the Ab preparation used in this study.


View this table:
[in this window]
[in a new window]
  		Table I. Regulation of IL-1ß, IL-6, IL-8, NO, and PGE2 by integrin in human OA-affected cartilage1

 
Arner and Tortorella (34) have reported that cyclic peptides with RGDN sequence (which are identified as minimal integrin-binding recognition sequence motifs in various ECM proteins) induce MMP production in bovine chondrocytes. We therefore tested the effects of 1 mg/ml of cyclic RGDN and RGEN (negative control) on NO production in OA-affected cartilage. The RGDN peptide, which shows greater ability to interact with {alpha}5ß1 than {alpha}vß3 (35) induced significant augmentation of NO (12.0 ± 2.0 to 38.0 ± 10.0 µM/g cartilage; p < 0.01) and IL-1ß (4.0 ± 1.0 to 11.0 ± 2.0 pg/g cartilage; p <= 0.01) accumulation over the uninduced control OA cartilage), whereas RGEN peptide caused significant inhibition of NO accumulation (12.0 ± 2.0 to 4.0 ± 2.0 µM/g cartilage; p < 0.03) compared with control cartilage. These experiments suggest that although RGDN and CCBD motifs are both required to modulate the {alpha}5ß1 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-1ß.

These data support our hypothesis that increased accumulation/synthesis of: 1) FN (and its proteolytic fragments) may interact with its prototypic receptor {alpha}5ß1 integrin and induce NO, PGE2, IL-6, IL-8, and IL-1ß production in human OA-affected cartilage; 2) OPN may interact with its prototypic receptor, {alpha}vß3 integrin, and inhibit NO and PGE2 production in OA-affected cartilage.

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-mediated vasoconstriction (31, 35, 36). In contrast, other ligands (CYR6 or Del1) that engage {alpha}vß3 promote angiogenesis and neovascularization (37, 38). This is not surprising, because echistatin (a snake venom protein) that also binds to {alpha}vß3 (39) shows differential regulation of inflammatory mediators distinct from those observed with OPN (our unpublished observations). These results suggest that the type of ligand or Ab and the epitope to which it binds in the binding pocket of {alpha}vß3 are critical for the downstream signal relayed by this receptor. These observations suggest that {alpha}vß3 integrin is a multifunctional receptor with discrete ligand binding regions that determines their function.

Up-regulation of NO, PGE2, IL-6, IL-8, and IL-1ß by anti-{alpha}5ß1 mAb in normal human cartilage

We and others have previously shown that human OA-affected cartilage releases various inflammatory cytokines (including IL-1ß) that act in an autocrine fashion to affect chondrocyte functions (16, 22, 40). In view of these observations, the possibility that the integrins may act cooperatively with IL-1R to amplify the effects of IL-1ß cannot be ruled out. Lo et al. (41) have shown enrichment of IL-1R at focal adhesion (FA) sites (41). These FA sites are enriched in signaling molecules that are used by integrins to transduce signals. There is also evidence to indicate that binding of IL-1 to its receptor triggers activation of integrin-associated signaling components within the same FA complex (41). We therefore tested the effect of anti-{alpha}5ß1 on normal human cartilage, which in the absence of stimulation produced significantly low, but detectable, amounts of IL-1ß, IL-6, or IL-8. Ligation of {alpha}5ß1 by JBS5 mAb in normal cartilage explants significantly stimulated the production of NO, PGE2, IL-1ß, IL-6, and IL-8 as observed in OA-affected cartilage (Table IIGo). These experiments suggest that normal human cartilage (like OA-affected cartilage) is also equally susceptible to {alpha}5ß1 integrin-mediated induction of inflammatory mediators.


View this table:
[in this window]
[in a new window]
  		Table II. Regulation of IL-1ß, IL-6, IL-8, NO, and PGE2 by integrin in normal human cartilage1

 
Autocrine production of IL-1ß is required for induction of NO and PGE2 by {alpha}5ß1 in chondrocytes

We performed experiments to determine whether selected stimulatory activities following {alpha}5ß1 engagement were dependent upon IL-1ß production. We therefore tested the role of autocrine IL-1ß in the regulation of NO and PGE2 production in OA-affected cartilage and primary bovine chondrocytes exposed to JBS5 or IL-1ß mAb in the presence or the absence of sIL-1RII (Fig. 1Go, A and B). The rationale for using sIL-1RII for these studies was that sIL-1RII has been reported to preferentially bind to both mature and pro-IL-1ß rather than IL-1Ra or IL-1RI (42).



View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 1. A, Regulation of NO and PGE2 in primary bovine chondrocytes by integrin receptor in the presence of sIL-1RII. Bovine chondrocytes were seeded (in triplicate) in 24-well plates at a density of 0.5 x 10 6 cells/well in 2 ml of medium. After 48 h of postseeding, the cells were treated (in triplicate for each modulator; n = 3) with either JBS5 mAb (5 µg/ml) or IL-1ß (1 ng/ml) alone in the presence and the absence of sIL-1RII (0. 5 ng/ml) for 72 h. NO (micromolar concentrations) and PGE2 (nanograms per milliliter) were estimated as described in Materials and Methods. The data represent one of the two similar experiments. Statistics were derived using unpaired Student’s t test. The p values are compared with the respected control. Data represent the mean ± SD (n = 3). *, p <= 0.01; **, p <= 0.001; ***, p <= 0.0001. B, Regulation of NO and PGE2 in OA-affected cartilage by integrin receptors in the presence of sIL-1RII. Human OA-affected cartilage organ cultures were set up as described in Materials and Methods and Table IGo. The cultures were treated (in triplicate; n = 3 for each modulator) with JBS5 mAb or IL-1ß alone in the presence and the absence of sIL-1RII as described above. NO (micromolar concentrations per 100 mg) and PGE2 (nanograms per milliliter per gram) were estimated at the end of experiment. The data represent one of two similar experiments and are the mean ± SD. Statistics were derived using unpaired Student’s t test. The p values are compared with spontaneously released, IL-1ß-induced, or JBS5 mAb-induced NO and PGE2. *, p <= 0.01; **, p <= 0.001.

 
Addition of IL-1ß or JBS5 mAb induced the production of NO and PGE2 in primary bovine chondrocytes and OA-affected cartilage. Preincubation of cells or cartilage (30 min) with sIL-1RII not only blocked the IL-1ß-induced accumulation of NO and PGE2, as expected, but also inhibited JBS5 mAb-induced production of these mediators. These experiments suggest that autocrine production of IL-1ß- and IL-1ß-mediated signaling is required for induction of NO and PGE2 following ligation of the {alpha}5ß1 integrin.

We also tested the effect of mAb13, which inhibits ß1 function (43), on the regulation of inflammatory mediators in bovine chondrocytes. mAb13, recognizes an epitope(s) on ß1 that competes with RGDN peptide and the CCBD fragment of FN and therefore decreases the binding of FN (43). This ß1 inhibitory Ab had no significant effect on the production of inflammatory mediators in bovine chondrocytes or OA-affected cartilage (data not shown). These experiments suggest that JBS5 engages an epitope in the {alpha}5ß1 complex that is capable of generating an activation signal.

Regulation of IL-1ß mRNA in bovine chondrocytes by integrins

In view of the above results, we examined the gene expression of IL-1ß in chondrocytes stimulated with JBS5 mAb under various conditions. Normal bovine chondrocytes were stimulated with LPS, LM609 mAb plus LPS, JBS5 mAb, and LM609. Stimulation with either LPS or JBS5 mAb induced up-regulation of IL-1ß mRNA accumulation (as analyzed by RT-PCR analysis), whereas the addition of LM609 inhibited IL-1ß mRNA accumulation induced by LPS or JBS5 (Fig. 2Go). These experiments further indicate a role for integrins in the (negative and positive) regulation of IL-1ß gene expression in chondrocytes. These experiments also suggest that the {alpha}vß3 integrin negatively modulates IL-1ß expression at the level of gene transcription and thereby influences the production of other inflammatory mediators; moreover, it indicates that the {alpha}vß3 integrin regulates {alpha}5ß1 in a dominant negative fashion.



View larger version (26K):
[in this window]
[in a new window]
  		FIGURE 2. Regulation of IL-1ß mRNA by integrins in bovine chondrocytes. Normal bovine chondrocytes were incubated with various modulators for 72 h and analyzed for IL-1ß mRNA and GAPDH in the same experiments by RT-PCR as described in Materials and Methods. The 350-bp IL-1ß and 200-bp GAPDH fragments were sequenced to ascertain their identity (not shown). In experiment I the cells were stimulated with LPS (100 µg/ml) or JBS5 mAb (5 µg/ml) in the presence or the absence of 5 µg/ml LM609. Lane 1, Standard marker (1-kb ladder); lane 2, uninduced cells; lane 3, LPS; lane 4, LM609 (5 µg/ml) mAb plus LPS; lane 5, JBS5 mAb (5 µg/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 with various modulators.

 
Anti-{alpha}vß3 integrin mAb inhibit the actions of {alpha}5ß1-, IL-1ß-, LPS-, and IL-1ß-, TNF-{alpha}-, and LPS-dependent NO and PGE2 production

Expression of {alpha}vß3 on bovine chondrocytes was confirmed by FACS analysis. The background MFI for the secondary PE-labeled Ab alone in bovine chondrocytes was 62. Addition of primary (LM609) and secondary mAb showed an increase in MFI to 244 for chondrocytes. These experiments confirm the expression of {alpha}vß3 integrin (and cross-reactivity of LM609) in bovine chondrocytes. Recently, Gibson et al. (44) reported the expression of {alpha}vß3 in bovine chondrocytes. The expression of {alpha}vß3 and {alpha}5ß1 integrin on human chondrocytes and cartilage has been shown by other investigators (2, 3, 45).

We examined the role of LM609 mAb in the regulation of NO and PGE2 production mediated by other proinflammatory receptors. Normal bovine chondrocytes were preincubated (30 min) with LM609 mAb and stimulated with JBS5 mAb, IL-1ß, LPS, and IL-1ß, TNF-{alpha}, plus LPS. The levels of NO and PGE2 were estimated. LM609 mAb inhibited the induction of NO and PGE2 by each of these stimuli (Fig. 3GoA) with cell viability of >95% at the end of 72 h as examined by trypan blue exclusion assay (data not shown), thus excluding apoptosis induced by both these mAb in these cells. Similar results were observed in OA-affected cartilage that were induced with JBS5 mAb, IL-1ß, and IL-1ß, TNF-{alpha}, and LPS (Fig. 3GoB).



View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 3. A, Regulation of NO and PGE2 by {alpha}vß3 integrin in bovine chondrocytes. Bovine chondrocytes were seeded in 24-well plates and stimulated with IL-1ß (1 ng/ml), LPS (100 µg/ml), and LPS, IL-1ß, plus TNF-{alpha} (100 U/ml; LIT) in the presence or absence of LM609 mAb (5 µg/ml) or mouse IgG1 (mIgG1) in triplicate (n = 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 Student’s t test. The data represent the mean ± SD from one of the two similar experiments. The p values are compared with the respective stimulated conditions: *, p <= 0.01; **, p <= 0.001; ***, p <= 0.0001. B, Regulation of NO and PGE2 by {alpha}vß3 integrin in human OA-affected cartilage. Human OA-affected explants were incubated in organ cultures as described in Materials and Methods. The cultures were preincubated with LM609 mAb (5 µg/ml) and stimulated with IL-1ß (1 ng/ml); LPS, IL-1ß, and TNF-{alpha} (LIT); or JBS5 mAb (5 µg/ml), and the levels of NO (micromolar concentrations 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 experiments. Statistics were derived using unpaired Student’s t test. Values are the mean ± SD. The p values compared with the respective stimulated conditions were; *, p <= 0.01; **, p <= 0.001; ***, p <= 0.0001.

 
Regulation of NO and PGE2 production by IL-18 in human OA-affected cartilage

IL-18 is identified as an IFN-{gamma}-inducing factor and is structurally similar to the IL-1 family of proteins (46). IL-18 is known to induce NO synthase, COX-2, IL-6, and MMPs (47), and its production has been reported to be stimulated by IL-1 in chondrocytes. Our preliminary data show that human OA-affected cartilage in ex vivo conditions spontaneously released IL-18 (288.3 ± 246.5 (n = 5) pg/ml/g of cartilage within 72 h. In the present study we also tested the effects of LM609 mAb on IL-18-induced NO and PGE2 production in human OA-affected cartilage as shown in Table IIIGo. IL-18 significantly (p <= 0.004) induced NO and PGE2 production; prior incubation with LM609 inhibited both spontaneous and IL-18-induced NO (p <= 0.0002) and PGE2 (p <= 0.01) production in these explant cultures. Thus, signaling through {alpha}vß3 (i.e., ligation by LM609 mAb) results in the inhibition of inflammatory mediator release in response to IL-1, IL-18, and {alpha}5ß1. It is known that LPS, IL-1, and IL-18 share selected signaling pathways, including the activation of NF-{kappa}B (48, 49, 50). The pathway or site at which the signal generated by ligation of {alpha}vß3 interferes with IL-1 and IL-18 cellular activation is currently under investigation.


View this table:
[in this window]
[in a new window]
  		Table III. Regulation of NO and PGE2 by IL-18 and {alpha}vß3 integrins in human OA-affected cartilage1

 
These experiments, on the whole, show trans-dominant inhibition of {alpha}5ß1-, IL-1ß-, LPS-, and IL-1ß-, TNF-{alpha}-, plus LPS-induced functions by {alpha}vß3 in human and bovine chondrocytes. The possibility of {alpha}5ß1 and {alpha}vß3 coassociating with IL-1R cannot be ruled out, as {alpha}vß3 receptors have been reported to associate with other receptors, such as CD47, in regulating proinflammatory cytokines in human monocytes (51). The association of {alpha}vß3 with platelet-derived growth factor-ß and insulin receptor substrate-1 as well as the dependence on {alpha}vß3 for the biological effect of fibroblast growth factor-2 indicate that {alpha}vß3 is linked with the cellular responses of certain growth factors (52, 53, 54). It should be noted that we have performed most of our experiments under ex vivo conditions without exogenous growth factors, as dissection of integrin signaling pathways may be further complicated by the cellular responses to growth factors (such as insulin-like growth factor and epidermal growth factor), which can mediate similar intracellular responses as integrin-ligand interactions (1).

Previous studies have shown that expression of {alpha}vß3 in K562 cells inhibits phagocytic functions of the {alpha}5ß1 through the suppression of calcium/calmodulin-dependent protein kinase II (55). Similarly, muscle-specific common receptor {alpha}7ß1 negatively regulates {alpha}5ß1 receptor function by decreasing the ligand binding affinity (56). This intracellular trans-dominant inhibition of {alpha}vß3 may be due to blockade of integrin signaling and/or conformational changes in the extracellular domain induced by the ß3 cytoplasmic tail of the suppressive integrin as observed with {alpha}IIbß3 integrin (57) or inhibition of intracellular signaling molecule(s) induced by {alpha}5ß1, IL-1ß, IL-18, and LPS receptors. These assumptions are supported by recent observations suggesting that 1) p16INK4a tumor suppressor protein inhibits {alpha}vß3 integrin-mediated cell spreading on vitronectin-coated plates by blocking PKC-dependent localization of {alpha}vß3 to focal contacts (58); 2) tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) inhibit FN-binding integrin and growth factor-mediated mitogen-activated protein kinase signaling pathway (59); and 3) the ephrin B-1-induced increase in cell attachment is involved in "inside out" activation of {alpha}5ß1 integrin in HEK 293 and of {alpha}vß3 integrin in endothelial cells (60).

In summary, these experiments show that two well-characterized mAbs that specifically bind to {alpha}5ß1 and {alpha}vß3 integrins mimic the action of their ligands (FN fragments and OPN; Fig. 4Go). These studies indicate that Abs to {alpha}5ß1 induce or augment IL-1ß production, which, in turn, is responsible for induction of NO, PGE2, IL-6, and IL-8. In contrast, {alpha}vß3, which is overexpressed in the superficial zone of OA-affected cartilage compared with normal cartilage (61), down-regulates IL-1ß expression and the effects of IL-1ß in a trans-dominant negative manner. Together, the data demonstrate that integrins and their ligands modulate chondrocyte functions by influencing the synthesis of pleiotropic cytokines, such as IL-1ß, IL-6, and IL-8, and signaling molecules, such as NO and PGE2, which have diverse catabolic functions. Overproduction of NO in chondrocytes inhibits collagen and proteoglycan synthesis and induces oxidant injury through peroxynitrite formation and apoptosis of chondrocytes (reviewed in Refs. 17 and 62). There is also evidence that NO mediates the catabolic effects of IL-1 (63) and modulation of integrins (45, 64) in OA cartilage. The overproduction of COX-2 and PGE2 in OA may contribute to the pain experienced in this disease, because inhibitors of COX-2 provide symptomatic improvement in the clinic (65). Whether COX-2 up-regulation affects metalloproteinase activity or proteoglycan production in OA is as yet undetermined.



View larger version (46K):
[in this window]
[in a new window]
  		FIGURE 4. Regulation of inflammatory mediators by integrins in cartilage. The data show the hypothetical scheme for the mechanisms of action of anti-{alpha}5ß1 and anti-{alpha}vß3 mAb in cartilage and chondrocytes. The anti-{alpha}5ß1 or {alpha}vß3 mAb induced positive (+) or negative (-) signals, respectively, to up/down-regulate IL-1ß gene expression in cartilage by "outside in" signaling. Similarly, an increase in the production of FN fragments or OPN may also induce/inhibit IL-1ß synthesis in human OA-affected cartilage. The IL-1ß induces its own production in an autocrine/paracrine fashion, which, in turn, up-regulates other inflammatory mediators, such as NO, PGE2, IL-6, IL-8, IL-18, and MMPs. It should be noted that IL-18 can independently induce NO, PGE2, and MMPs (47 ). This "inside out" signaling induced by {alpha}5ß1 integrin can be inhibited by addition of sIL-1RII, which neutralizes/inhibits the autocrine signaling cascade induced by IL-1 and IL-1RI complex.

 
This study thus demonstrates the role of the ECM as a medium for communication between the chondrocyte and the cartilage matrix. This hypothesis is supported by compelling evidence in the literature suggesting that mechanical strain and overloading of cartilage or chondrocytes may influence integrin (mechano-receptors) functions (66, 67, 68). This is the first observation that integrin can modulate synthesis of various proinflammatory mediators, such as NO and PGE2, which have profound effects on cartilage homeostasis. Inhibition of integrin-mediated functions could be a potential target for future therapeutic approaches.


   Acknowledgments
 
We thank Dr. Pat Mongini, Hospital for Joint Diseases (New York, NY), and Dr. K. M. Yamada, National Institutes of Health (Bethesda, MD), for generously providing various reagents, and Ms. Una Yearwood and Ms. Andrea L. Barrett for the preparation of the manuscript. We thank the National Disease Research Interchange (Philadelphia, PA) for some of the cartilage samples.


   Footnotes
 
1 This work was supported by The Joseph and Sophie Abeles Foundation. Back

2 Address correspondence and reprint requests to Dr. Ashok R. Amin, Department of Rheumatology, Hospital for Joint Diseases, 301 East 17th Street, Room 1600, New York, NY 10003. E-mail address: Back

3 Abbreviations used in this paper: ECM, extracellular matrix; FN, fibronectin; OPN, osteopontin; OA, osteoarthritis; MMP, matrix metalloproteinase; MFI, median fluorescence intensity; sIL-1R II, soluble type II IL-1 receptor; FA, focal adhesion. Back

Received for publication October 12, 1999. Accepted for publication December 16, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

  1. Aplin, A. E., A. Howe, S. K. Alahari, R. L. Juliano. 1998. Signal transduction and signal modulation by cell adhesion receptors: the role of integrins, cadherins, immunoglobulin-cell adhesion molecules, and selectins. Pharmacol. Rev. 50:197.[Abstract/Free Full Text]
  2. Woods, V. L. J., P. J. Schreck, D. S. Gesink, H. O. Pacheco, D. Amiel, W. H. Akeson, M. Lotz. 1994. Integrin expression by human articular chondrocytes. Arthritis Rheum. 37:537.[Medline]
  3. Salter, D. M., J. L. Godolphin, M. S. Gourlay. 1995. Chondrocyte heterogeneity: immunohistologically defined variation of integrin expression at different sites in human fetal knees. J. Histochem. Cytochem. 43:447.[Abstract]
  4. Lapadula, G., F. Iannone, C. Zuccaro, V. Grattagliano, M. Covelli, V. Patella, B. G. Lo, V. Pipitone. 1997. Integrin expression on chondrocytes: correlations with the degree of cartilage damage in human osteoarthritis. Clin. Exp. Rheumatol. 15:247.[Medline]
  5. Yonezawa, I., K. Kato, H. Yagita, Y. Yamauchi, K. Okumura. 1996. VLA-5-mediated interaction with fibronectin induces cytokine production by human chondrocytes. Biochem. Biophys. Res. Commun. 219:261.[Medline]
  6. Homandberg, G. A.. 1999. Potential regulation of cartilage metabolism in osteoarthritis by fibronectin fragments. Front. Biosci. 4:d713.
  7. Enomoto-Iwamoto, M., M. Iwamoto, K. Nakashima, Y. Mukudai, D. Boettiger, M. Pacifici, K. Kurisu, F. Suzuki. 1997. Involvement of {alpha}5ß1 integrin in matrix interactions and proliferation of chondrocytes. J. Bone Miner. Res. 12:1124.[Medline]
  8. Johansson, S., G. Svineng, K. Wennerberg, A. Armulik, L. Lohikangas. 1997. Fibronectin-integrin interactions. Front. Biosci. 2:d126.[Medline]
  9. McKee, M. D., M. J. Glimcher, A. Nanci. 1992. High-resolution immunolocalization of osteopontin and osteocalcin in bone and cartilage during endochondral ossification in the chicken tibia. Anat. Rec. 234:479.[Medline]
  10. Denhardt, D. T., C. A. Lopez, E. E. Rollo, S. M. Hwang, X. R. An, S. E. Walther. 1995. Osteopontin-induced modifications of cellular functions. Ann. NY Acad. Sci. 760:127.[Medline]
  11. Yoshitake, H., S. R. Rittling, D. T. Denhardt, M. Noda. 1999. Osteopontin-deficient mice are resistant to ovariectomy-induced bone resorption. Proc. Natl. Acad. Sci. USA 96:8156.[Abstract/Free Full Text]
  12. Denhardt, D. T., and M. Noda. 1998. Osteopontin expression and function: role in bone remodeling. J. Cell Biochem. 30–31(Suppl.):92.
  13. Amin, A. R., P. Di Cesare, P. Vyas, M. G. Attur, E. Tzeng, T. R. Billiar, S. A. Stuchin, S. B. Abramson. 1995. The expression and regulation of nitric oxide synthase in human osteoarthritis-affected chondrocytes: evidence for up-regulated neuronal nitric oxide synthase. J. Exp. Med. 182:2097.[Abstract/Free Full Text]
  14. Amin, A. R., M. G. Attur, R. N. Patel, G. D. Thakker, P. J. Marshall, J. Rediske, S. B. Abramson. 1997. Superinduction of cyclooxygenase-2 activity in human osteoarthritis-affected cartilage: influence of nitric oxide. J. Clin. Invest. 99:1231.[Medline]
  15. Amin, A. R., M. G. Attur, and S. B. Abramson. 1999. Regulation of nitric oxide and inflammatory mediators in human osteoarthritis-affected cartilage: implication for pharmacological intervention. In The Pathophysiology and Clinical Applications of Nitric Oxide, Part B. G. M. Rubanyi, ed. Harwood Academic Publishers, Amsterdam, p. 397.
  16. Attur, M. G., I. R. Patel, R. N. Patel, S. B. Abramson, A. R. Amin. 1998. Autocrine production of IL-1ß by human osteoarthritis-affected cartilage and differential regulation of endogenous nitric oxide, IL-6, prostaglandin E2 and IL-8. Proc. Assoc. Am. Physicians 110:1.[Medline]
  17. Amin, A. R., S. B. Abramson. 1998. The role of nitric oxide in articular cartilage breakdown in osteoarthritis. Curr. Opin. Rheumatol. 10:263.[Medline]
  18. Melchiorri, C., R. Meliconi, L. Frizziero, T. Silvestri, L. Pulsatelli, I. Mazzetti, R. M. Borzi, M. Uguccioni, A. Facchini. 1998. Enhanced and coordinated in vivo expression of inflammatory cytokines and nitric oxide synthase by chondrocytes from patients with osteoarthritis. Arthritis Rheum. 41:2165.[Medline]
  19. Moos, V., S. Fickert, B. Muller, U. Weber, J. Sieper, L. Bloom, K. C. Ingham, R. O. Hynes. 1999. Immunohistological analysis of cytokine expression in human osteoarthritic and healthy cartilage. J. Rheumatol. 26:870.[Medline]
  20. Pelletier, J. P., D. Jovanovic, J. C. Fernandes, P. T. Manning, J. R. I. Connor, M. G. Currie, J. A. Di Battista, J. Martel-Pelletier. 1998. Reduced progression of experimental osteoarthritis in vivo by selective inhibition of inducible nitric oxide synthase. Arthritis Rheum. 41:1275.[Medline]
  21. Attur, M. G., R. N. Patel, P. Di Cesare, G. Steiner, S. B. Abramson, A. R. Amin. 1998. Regulation of nitric oxide production by salicylates and tenidap in human OA-affected cartilage, rat chondrosarcomas and bovine chondrocytes. Osteoarthritis Cartilage 6:269.[Medline]
  22. Patel, I. R., M. G. Attur, R. N. Patel, S. A. Stuchin, R. A. Abagyan, S. B. Abramson, A. R. Amin. 1998. TNF-{alpha} convertase from human arthritis-affected cartilage: isolation of cDNA by differential display, expression of the active enzyme, and regulation of TNF-{alpha}. J. Immunol. 160:4570.[Abstract/Free Full Text]
  23. Liang, P., A. B. Pardee. 1992. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257:967.[Abstract/Free Full Text]
  24. Patel, I. R., P. Vyas, M. G. Attur, G. D. Thakker, S. B. Abramson, A. R. Amin. 1997. Cloning and expression of a functional, dimeric, multi-cofactor-requiring murine macrophage inducible nitric oxide synthase in bacteria-to-bacmid transposition-baculovirus system. Transgenics 2:161.
  25. Saito, S., N. Yamaji, K. Yasunaga, T. Saito, M. Si. Matsumoto, S. Kaboayashi Katoh, Y. Masuho. 1999. The fibronectin extra domain A activates matrix metalloproteinase gene expression by an interleukin-1-dependent mechanism. J. Biol. Chem. 274:30756.[Abstract/Free Full Text]
  26. Loeser, R. F.. 1993. Integrin-mediated attachment of articular chondrocytes to extracellular matrix proteins. Arthritis Rheum. 36:1103.[Medline]
  27. Miyauchi, A., J. Alvarez, E. M. Greenfield, A. Teti, M. Grano, S. Colucci, A. Zambonin-Zallone, F. P. Ross, S. L. Teitelbaum, D. Cheresh. 1991. Recognition of osteopontin and related peptides by an {alpha}vß3 integrin stimulates immediate cell signals in osteoclasts. J. Biol. Chem. 266:20369.[Abstract/Free Full Text]
  28. Homandberg, G. A., C. Wen. 1998. Exposure of cartilage to a fibronectin fragment amplifies catabolic processes while also enhancing anabolic processes to limit damage. J. Orthop. Res. 16:237.[Medline]
  29. Longhurst, C. M., L. K. Jennings. 1998. Integrin-mediated signal transduction. Cell. Mol. Life Sci. 54:514.[Medline]
  30. Shen, C. X., S. Stewart, E. Wayner, W. Carter, J. Wilkins. 1991. Antibodies to different members of the ß1 (CD29) integrins induce homotypic and heterotypic cellular aggregation. Cell. Immunol. 138:216.[Medline]
  31. Eliceiri, B. P., D. A. Cheresh. 1999. The role of {alpha}v integrins during angiogenesis: insights into potential mechanisms of action and clinical development. J. Clin. Invest. 103:1227.[Medline]
  32. Mould, A. P., J. A. Askari, S. Aota, K. M. Yamada, A. Irie, Y. Takada, H. J. Mardon, M. J. Humphries. 1997. Defining the topology of integrin {alpha}5ß1-fibronectin interactions using inhibitory anti-{alpha}5 and anti-ß1 monoclonal antibodies: evidence that the synergy sequence of fibronectin is recognized by the amino-terminal repeats of the {alpha}5 subunit. J. Biol. Chem. 272:17283.[Abstract/Free Full Text]
  33. Cheresh, D., R. C. Spiro. 1987. Biosynthetic and functional properties of an Arg-Gly-Asp-directed receptor involved in human melanoma cell attachment to vitronectin, fibrinogen, and von Willebrand factor. J. Biol. Chem. 262:17703.[Abstract/Free Full Text]
  34. Arner, E. C., M. D. Tortorella. 1995. Signal transduction through chondrocyte integrin receptors induces matrix metalloproteinase synthesis and synergizes with interleukin-1. Arthritis Rheum. 38:1304.[Medline]
  35. Mogford, J. E., G. E. Davis, G. A. Meininger. 1997. RGDN peptide interaction with endothelial {alpha}5ß1 integrin causes sustained endothelin-dependent vasoconstriction of rat skeletal muscle arterioles. J. Clin. Invest. 100:1647.[Medline]
  36. Friedlander, M., P. C. Brooks, R. W. Shaffer, C. M. Kincaid, J. A. Varner, D. A. Cheresh. 1995. Definition of two angiogenic pathways by distinct {alpha}v integrins. Science 270:1500.[Abstract/Free Full Text]
  37. Babic, A. M., M. L. Kireeva, T. V. Kolesnikova, L. F. Lau. 1998. CYR61, a product of a growth factor-inducible immediate early gene, promotes angiogenesis and tumor growth. Proc. Natl. Acad. Sci. USA 95:6355.[Abstract/Free Full Text]
  38. Penta, K., J. A. Varner, L. Liaw, C. Hidai, R. Schatzman, T. Quertermous. 1999. Del1 induces integrin signaling and angiogenesis by ligation of {alpha}vß3. J. Biol. Chem. 274:11101.[Abstract/Free Full Text]
  39. Zheng, B., D. R. Clemmons. 1998. Blocking ligand occupancy of the {alpha}vß3 integrin inhibits insulin- like growth factor I signaling in vascular smooth muscle cells. Proc. Natl. Acad. Sci. USA 95:11217.[Abstract/Free Full Text]
  40. Towle, C. A., H. H. Hung, L. J. Bonassar, B. V. Treadwell, D. C. Mangham. 1997. Detection of interleukin-1 in the cartilage of patients with osteoarthritis: a possible autocrine/paracrine role in pathogenesis. Osteoarthritis Cartilage 5:293.[Medline]
  41. Lo, Y. Y., L. Luo, C. A. McCulloch, T. F. Cruz. 1998. Requirements of focal adhesions and calcium fluxes for interleukin-1-induced ERK kinase activation and c-Fos expression in fibroblasts. J. Biol. Chem. 273:7059.[Abstract/Free Full Text]
  42. Symons, J. A., J. A. Eastgate, G. W. Duff. 1991. Purification and characterization of a novel soluble receptor for interleukin 1. J. Exp. Med. 174:1251.[Abstract/Free Full Text]
  43. Mould, A. P., S. K. Akiyama, M. J. Humphries. 1996. The inhibitory anti-ß1 integrin monoclonal antibody 13 recognizes an epitope that is attenuated by ligand occupancy: evidence for allosteric inhibition of integrin function. J. Biol. Chem. 271:20365.[Abstract/Free Full Text]
  44. Gibson, M. A., D. I. Leavesley, L. K. Ashman. 1999. Microfibril-associated glycoprotein-2 specifically interacts with a range of bovine and human cell types via {alpha}vß3 Integrin. J. Biol. Chem. 274:13060.[Abstract/Free Full Text]
  45. Clancy, R. M., J. Rediske, X. Tang, N. Nijher, S. Frenkel, M. Philips, S. B. Abramson. 1997. Outside-in signaling in the chondrocyte: nitric oxide disrupts fibronectin-induced assembly of a subplasmalemmal actin/Rho A/focal adhesion kinase signaling complex. J. Clin. Invest. 100:1789.[Medline]
  46. Dinarello, C. A., D. Novick, A. J. Puren, G. Fantuzzi, L. Shapiro, H. Muhl, D. Y. Yoon, L. L. Reznikov, S. H. Kim, M. Rubinstein. 1998. Overview of interleukin-18: more than an interferon-{gamma} inducing factor. J. Leukocyte Biol. 63:658.[Abstract]
  47. Olee, T., S. Hashimoto, J. Quach, M. Lotz. 1999. IL-18 is produced by articular chondrocytes and induces proinflammatory and catabolic responses. J. Immunol. 162:1096.[Abstract/Free Full Text]
  48. Hoshino, K., H. Tsutsui, T. Kawai, K. Takeda, K. Nakanishi, Y. Takeda, S. Akira. 1999. Cutting edge: generation of IL-18 receptor-deficient mice: evidence for IL-1 receptor-related protein as an essential IL-18 binding receptor. J. Immunol. 162:5041.[Abstract/Free Full Text]
  49. Zhang, F. X., C. J. Kirschning, R. Mancinelli, X. P. Xu, Y. Jin, E. Faure, A. Mantovani, M. Rothe, M. Muzio, M. Arditi. 1999. Bacterial lipopolysaccharide activates nuclear factor-{kappa}B through interleukin-1 signaling mediators in cultured human dermal endothelial cells and mononuclear phagocytes. J. Biol. Chem. 274:7611.[Abstract/Free Full Text]
  50. Lomaga, M. A., W. C. Yeh, I. Sarosi, G. S. Duncan, C. Furlonger, A. Ho, S. Morony, C. Capparelli, G. Van, S. Kaufman, et al 1999. TRAF6 deficiency results in osteopetrosis and defective interleukin-1, CD40, and LPS signaling. Genes Dev. 13:1015.[Abstract/Free Full Text]
  51. Hermann, P., M. Armant, E. Brown, M. Rubio, H. Ishihara, D. Ulrich, R. G. Caspary, F. P. Lindberg, R. Armitage, C. Maliszewski, et al 1999. The vitronectin receptor and its associated CD47 molecule mediates proinflammatory cytokine synthesis in human monocytes by interaction with soluble CD23. J. Cell Biol. 144:767.[Abstract/Free Full Text]
  52. Vuori, K., E. Ruoslahti. 1994. Association of insulin receptor substrate-1 with integrins. Science 266:1576.[Abstract/Free Full Text]
  53. Rusnati, M., E. Tanghetti, P. Dell’Era, A. Gualandris, M. Presta. 1997. {alpha}vß3 integrin mediates the cell-adhesive capacity and biological activity of basic fibroblast growth factor (FGF-2) in cultured endothelial cells. Mol. Biol. Cell 8:2449.[Abstract/Free Full Text]
  54. Schneller, M., K. Vuori, E. Ruoslahti. 1997. {alpha}vß3 integrin associates with activated insulin and PDGFß receptors and potentiates the biological activity of PDGF. EMBO J. 16:5600.[Medline]
  55. Blystone, S. D., S. E. Slater, M. P. Williams, M. T. Crow, E. J. Brown. 1999. A molecular mechanism of integrin crosstalk: {alpha}vß3 suppression of calcium/calmodulin-dependent protein kinase II regulates {alpha}5ß1 function. J. Cell Biol. 145:889.[Abstract/Free Full Text]
  56. Tomatis, D., F. Echtermayer, S. Schober, F. Balzac, S. F. Retta, L. Silengo, G. Tarone. 1999. The muscle-specific laminin receptor {alpha}7ß1 integrin negatively regulates {alpha}5ß1 fibronectin receptor function. Exp. Cell Res. 246:421.[Medline]
  57. Diaz-Gonzalez, F., J. Forsyth, B. Steiner, M. H. Ginsberg. 1996. Trans-dominant inhibition of integrin function. Mol. Biol. Cell 7:1939.[Abstract]
  58. Fahraeus, R., D. P. Lane. 1999. The p16(INK4a) tumor suppressor protein inhibits {alpha}vß3 integrin-mediated cell spreading on vitronectin by blocking PKC-dependent localization of {alpha}vß3 to focal contacts. EMBO J. 18:2106.[Medline]
  59. Gu, J., M. Tamura, K. M. Yamada. 1998. Tumor suppressor PTEN inhibits integrin- and growth factor-mediated mitogen-activated protein (MAP) kinase signaling pathways. J. Cell Biol. 143:1375.[Abstract/Free Full Text]
  60. Huynh-Do, U., E. Stein, A. A. Lane, H. Liu, D. P. Cerretti, T. O. Daniel. 1999. Surface densities of ephrin-B1 determine EphB1-coupled activation of cell attachment through {alpha}vß3 and {alpha}5ß1 integrins. EMBO J. 18:2165.[Medline]
  61. Ostergaard, K., D. M. Salter, J. Petersen, K. Bendtzen, J. Hvolris, C. B. Andersen. 1998. Expression of {alpha} and ß subunits of the integrin superfamily in articular cartilage from macroscopically normal and osteoarthritic human femoral heads. Ann. Rheum. Dis. 57:303.[Abstract/Free Full Text]
  62. Amin, A. R., M. Attur, S. B. Abramson. 1999. Nitric oxide synthase and cyclooxygenases: distribution, regulation, and intervention in arthritis. Curr. Opin. Rheumatol. 11:202.[Medline]
  63. Pelletier, J. M., N. Alaaeddine, J. P. Pelletier. 1999. Cytokines and their role in the pathophysiology of osteoarthritis. Front. Biosci. 4:d694.
  64. Milam, S. B., V. L. Magnuson, B. Steffensen, D. Chen, R. J. Klebe. 1991. IL-1ß and prostaglandins regulate integrin mRNA expression. J. Cell Physiol. 149:173.[Medline]
  65. Fort, J.. 1999. Celecoxib, a COX-2-specific inhibitor: the clinical data. Am. J. Orthop. 28:13.
  66. Fujisawa, T., T. Hattori, K. Takahashi, T. Kuboki, A. Yamashita, M. Takigawa. 1999. Cyclic mechanical stress induces extracellular matrix degradation in cultured chondrocytes via gene expression of matrix metalloproteinases and interleukin-1. J. Biochem. 125:966.[Abstract/Free Full Text]
  67. Wright, M. O., K. Nishida, C. Bavington, J. L. Godolphin, E. Dunne, S. Walmsley, P. Jobanputra, G. Nuki, D. M. Salter. 1997. Hyperpolarisation of cultured human chondrocytes following cyclical pressure-induced strain: evidence of a role for {alpha}5ß1 integrin as a chondrocyte mechanoreceptor. J. Orthop. Res. 15:742.[Medline]
  68. Millward-Sadler, S. J., M. O. Wright, H. Lee, K. Nishida, H. Caldwell, G. Nuki, D. M. Salter. 1999. Integrin-regulated secretion of interleukin 4: a novel pathway of mechanotransduction in human articular chondrocytes. J. Cell Biol. 145:183.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
A. Gigout, M. Jolicoeur, M. Nelea, N. Raynal, R. Farndale, and M. D. Buschmann
Chondrocyte Aggregation in Suspension Culture Is GFOGER-GPP- and {beta}1 Integrin-dependent
J. Biol. Chem., November 14, 2008; 283(46): 31522 - 31530.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
D. J. Caruso, A. J.K. Carmack, V. B. Lokeshwar, R. C. Duncan, M. S. Soloway, and B. L. Lokeshwar
Osteopontin and Interleukin-8 Expression is Independently Associated with Prostate Cancer Recurrence
Clin. Cancer Res., July 1, 2008; 14(13): 4111 - 4118.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
H. A. Arafat, A. K. Katakam, G. Chipitsyna, Q. Gong, A. R. Vancha, J. Gabbeta, and D. C. Dafoe
Osteopontin Protects the Islets and {beta}-Cells from Interleukin-1 {beta}-Mediated Cytotoxicity through Negative Feedback Regulation of Nitric Oxide
Endocrinology, February 1, 2007; 148(2): 575 - 584.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
F. H. Chen, A. O. Thomas, J. T. Hecht, M. B. Goldring, and J. Lawler
Cartilage Oligomeric Matrix Protein/Thrombospondin 5 Supports Chondrocyte Attachment through Interaction with Integrins
J. Biol. Chem., September 23, 2005; 280(38): 32655 - 32661.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
J. I. Pulai, H. Chen, H.-J. Im, S. Kumar, C. Hanning, P. S. Hegde, and R. F. Loeser
NF-{kappa}B Mediates the Stimulation of Cytokine and Chemokine Expression by Human Articular Chondrocytes in Response to Fibronectin Fragments
J. Immunol., May 1, 2005; 174(9): 5781 - 5788.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
J. Lammerding, A. R. Kazarov, H. Huang, R. T. Lee, and M. E. Hemler
Tetraspanin CD151 regulates {alpha}6{beta}1 integrin adhesion strengthening
PNAS, June 24, 2003; 100(13): 7616 - 7621.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. G. Attur, M. N. Dave, M. Y. Leung, C. Cipolletta, M. Meseck, S. L. C. Woo, and A. R. Amin
Functional Genomic Analysis of Type II IL-1{beta} Decoy Receptor: Potential for Gene Therapy in Human Arthritis and Inflammation
J. Immunol., February 15, 2002; 168(4): 2001 - 2010.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
C. Tan, A. Mui, and S. Dedhar
Integrin-linked Kinase Regulates Inducible Nitric Oxide Synthase and Cyclooxygenase-2 Expression in an NF-kappa B-dependent Manner
J. Biol. Chem., January 25, 2002; 277(5): 3109 - 3116.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
H. Guo, C. Q. Cai, R. A. Schroeder, and P. C. Kuo
Osteopontin Is a Negative Feedback Regulator of Nitric Oxide Synthesis in Murine Macrophages
J. Immunol., January 15, 2001; 166(2): 1079 - 1086.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. G. Attur, M. Dave, C. Cipolletta, P. Kang, M. B. Goldring, I. R. Patel, S. B. Abramson, and A. R. Amin
Reversal of Autocrine and Paracrine Effects of Interleukin 1 (IL-1) in Human Arthritis by Type II IL-1 Decoy Receptor. POTENTIAL FOR PHARMACOLOGICAL INTERVENTION
J. Biol. Chem., December 15, 2000; 275(51): 40307 - 40315.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Attur, M. G.
Right arrow Articles by Amin, A. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Attur, M. G.
Right arrow Articles by Amin, A. R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS