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1.
J Immunol ; 174(9): 5781-8, 2005 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-15843581

RESUMO

Fibronectin fragments (FN-f) that bind to the alpha(5)beta(1) integrin stimulate chondrocyte-mediated cartilage destruction and could play an important role in the progression of arthritis. The objective of this study was to identify potential cytokine mediators of cartilage inflammation and destruction induced by FN-f and to investigate the mechanism of their stimulation. Human articular chondrocytes, isolated from normal ankle cartilage obtained from tissue donors, were treated with a 110-kDa FN-f in serum-free culture, and expression of various cytokine genes was analyzed by cDNA microarray and by a cytokine protein array. Compared with untreated control cultures, stimulation by FN-f resulted in a >2-fold increase in IL-6, IL-8, MCP-1, and growth-related oncogene beta (GRO-beta). Constitutive and FN-f-inducible expression of GRO-alpha and GRO-gamma were also noted by RT-PCR and confirmed by immunoblotting. Previous reports of IL-1beta expression induced by FN-f were also confirmed, while TNF expression was found to be very low. Inhibitor studies revealed that FN-f-induced stimulation of chondrocyte chemokine expression was dependent on NF-kappaB activity, but independent of IL-1 autocrine signaling. The ability of FN-f to stimulate chondrocyte expression of multiple proinflammatory cytokines and chemokines suggests that damage to the cartilage matrix is capable of inducing a proinflammatory state responsible for further progressive matrix destruction, which also includes the chemoattraction of inflammatory cells. Targeting the signaling pathways activated by FN-f may be an effective means of inhibiting production of multiple mediators of cartilage destruction.


Assuntos
Cartilagem Articular/imunologia , Quimiocinas/biossíntese , Condrócitos/imunologia , Citocinas/biossíntese , Fibronectinas/fisiologia , NF-kappa B/fisiologia , Fragmentos de Peptídeos/fisiologia , Cartilagem Articular/citologia , Cartilagem Articular/enzimologia , Cartilagem Articular/metabolismo , Células Cultivadas , Quimiocinas/genética , Condrócitos/enzimologia , Condrócitos/metabolismo , Citocinas/genética , Perfilação da Expressão Gênica , Humanos , Integrina alfa5beta1/fisiologia , Interleucina-1/fisiologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Oligopeptídeos/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
2.
Arthritis Rheum ; 46(6): 1528-35, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12115183

RESUMO

OBJECTIVE: Chondrocyte cell death may play an important role in the development of arthritis. The goal of the present study was to evaluate the role of the extracellular matrix (ECM) in promoting chondrocyte survival via signals through the integrin family of ECM receptors. METHODS: Chondrocytes were isolated by sequential enzymatic digestion from normal ankle cartilage of organ donors and from osteoarthritic (OA) knee tissue obtained from patients undergoing total knee replacement. Cell survival in monolayer and in suspension culture was measured using fluorescent labels after treatment with specific integrin-blocking antibodies and echistatin, a disintegrin peptide. A quantitative enzyme-linked immunosorbent assay for histone-associated DNA fragments and morphologic evaluation by electron microscopy were used to evaluate apoptosis. RESULTS: Freshly isolated chondrocytes died when plated in serum-free media at low density on poly-L-lysine, but showed >95% survival on fibronectin (FN). A monoclonal blocking antibody to the alpha5-integrin subunit (FN receptor) significantly inhibited survival on FN, whereas control antibodies had no effect. Likewise, treatment of freshly isolated chondrocytes in serum-free alginate-suspension culture with the alpha5-blocking antibody resulted in cell death in a dose-dependent manner, with 20 microg/ml of the antibody reducing normal chondrocyte survival to 20% of that in controls, and OA chondrocyte survival to 23% of that in controls. Antibody inhibition of alphav and alpha1 integrins or treatment with echistatin did not cause cell death. Addition of insulin-like growth factor 1 (IGF-1; 100 ng/ ml) was not able to improve survival of alpha5-antibody-treated cells. However, treatment with 10% fetal bovine serum improved normal chondrocyte survival to 98% (a 5.1-fold increase) and OA chondrocyte survival to 64% (a 2.8-fold increase). Cell death due to alpha5 inhibition was associated with apoptosis. CONCLUSION: These results demonstrate that chondrocyte survival signals are transmitted via the alpha5beta1 FN receptor. Inhibition of matrix survival signals mediated by alpha5beta1 also inhibits the ability of IGF-1 to promote survival, suggesting that IGF-1-mediated survival signaling may require a cosignal from alpha5beta1.


Assuntos
Condrócitos/citologia , Condrócitos/metabolismo , Osteoartrite/metabolismo , Receptores de Fibronectina/metabolismo , Transdução de Sinais/fisiologia , Anticorpos Monoclonais/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proteínas Sanguíneas/farmacologia , Cartilagem Articular/citologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Matriz Extracelular/metabolismo , Proteínas Fetais/farmacologia , Humanos , Técnicas In Vitro , Fator de Crescimento Insulin-Like I/farmacologia , Osteoartrite/patologia , Receptores de Fibronectina/imunologia
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