Oncostatin M in combination with tumor necrosis factor alpha induces cartilage damage and matrix metalloproteinase expression in vitro and in vivo

OBJECTIVE: To determine the effects of the proinflammatory cytokine combination of oncostatin M (OSM) and tumor necrosis factor alpha (TNFalpha) on cartilage destruction in both in vitro and in vivo model systems. METHODS: The release of collagen and proteoglycan was assessed in bovine cartilage explant cultures, while messenger RNA (mRNA) from bovine chondrocytes was analyzed by Northern blotting. Immunohistochemistry was performed on sections prepared from murine joints following injection of adenovirus vectors encoding murine OSM and/or murine TNFalpha. RESULTS: The combination of OSM + TNFalpha induced significant collagen release from bovine cartilage, accompanied by high levels of active collagenolytic activity. Northern blot analysis indicated that this cytokine combination synergistically induced matrix metalloproteinase 1 (MMP-1), MMP-3, and MMP-13 mRNA. The in vivo data clearly indicated that OSM + TNFalpha overexpression increased MMP levels and decreased levels of tissue inhibitor of metalloproteinases 1 (TIMP-1). Specifically, OSM + TNFalpha induced marked synovial hyperplasia, inflammation, and cartilage and bone destruction with a concomitant increase in MMP expression in both cartilage and synovium and decreased TIMP-1 expression in the articular cartilage. These effects were markedly greater than those seen with either cytokine alone. CONCLUSION: This study demonstrates that OSM + TNFalpha represents a potent proinflammatory cytokine combination that markedly induces MMP production in both cartilage and synovium, thus promoting joint destruction.     

Staphylococcus aureus stimulates inducible nitric oxide synthase in articular cartilage.

OBJECTIVE: To determine if Staphylococcus aureus stimulates the L-arginine-nitric oxide (NO) synthase pathway in articular cartilage. METHODS: A heat-killed and sonicated (denatured) S. aureus preparation was added to cultures of bovine articular cartilage. NO production was measured as accumulated nitrite in the culture medium and by the NO synthase-dependent conversion of 3H-L-arginine to 3H-L-citrulline in cartilage homogenates. Inducible NO synthase (iNOS) messenger RNA (mRNA) expression was analyzed by Northern blot. Proteoglycan synthesis was measured by 35SO4 incorporation into glycosaminoglycan. RESULTS: Nitrite accumulation and 3H-L-citrulline formation in cartilage were elevated by denatured S. aureus (compared with unstimulated control cartilage) and inhibited by the NO synthase inhibitor N(G)-monomethyl-L-arginine. Northern blot analysis revealed increased iNOS mRNA expression in bovine chondrocytes in response to denatured S. aureus stimulation. Denatured S. aureus suppressed the accumulation of 35SO4-labeled macromolecules representing newly synthesized proteoglycans in bovine articular cartilage. The suppressed proteoglycan synthesis was due to the presence of NO. CONCLUSION: These findings support the hypothesis that a component of S. aureus can stimulate iNOS in articular cartilage, and that NO generated from this enzyme down-regulates cartilage matrix proteoglycan synthesis.     

Tumor necrosis factor alpha activation of the apoptotic cascade in murine articular chondrocytes is associated with the induction of metalloproteinases and specific pro-resorptive factors

OBJECTIVE: Tumor necrosis factor alpha (TNFalpha) blockade provides substantive reduction of the symptoms of rheumatoid arthritis (RA). While the biologic actions of TNFalpha have been well characterized in immune and synovial cells, which are known to be major contributors to the progression of cartilage destruction in RA, the current studies were designed to assess the direct effects of TNFalpha on chondrocytes. METHODS: We examined the expression of several groupings of messenger RNA (mRNA) that define key biologic pathways that have previously been associated with either the general actions of TNFalpha or cartilage destruction, in murine articular chondrocytes isolated from wild-type mice and TNFalpha receptor-null (p55/p75(-/-)) mice. RESULTS: TNFalpha induced the expression of multiple mRNA that facilitate apoptosis and lead to apoptosis-induced cell death. The induction of apoptosis was accompanied by the increased expression of several factors involved in the regulation of skeletal tissue proteolysis and resorption. Quantitative increases from 2-fold to >10-fold were seen for inducible nitric oxide synthase, matrix metalloproteinase 3, macrophage colony-stimulating factor, and osteoprotegerin mRNA expression. The dependence of the induction of these mRNA on TNFalpha was confirmed by comparison with the effects of TNFalpha on chondrocytes isolated from receptor-null mice. CONCLUSION: These findings demonstrate that TNFalpha alters the expression of a complex array of genes within murine chondrocytes that contribute to the destruction of joint surfaces, independent of its actions on synovial and immune cells. Further studies are needed to clarify the biologic actions of TNFalpha in human cartilage cells.     

In vivo expression of stromelysin in synovium and cartilage of rabbits injected intraarticularly with interleukin-1 beta.

OBJECTIVE. To examine the in vivo expression of the matrix metalloproteinase stromelysin in the synovium and articular cartilage of rabbits injected intraarticularly with recombinant human interleukin-1 beta (IL-1). METHODS. The direct isolation of messenger RNA (mRNA) from articular cartilage without the prior isolation of chondrocytes is described. The in vivo expression of stromelysin was examined at the mRNA level by Northern blot analysis, and at the protein level by in situ immunolocalization and by enzyme-linked immunosorbent assay. RESULTS. In the synovium of IL-1-injected joints, stromelysin mRNA levels were highest at 4 hours and declined to background levels within 24 hours. In the cartilage of IL-1-injected joints, stromelysin mRNA was elevated at 4 hours and continued to increase until 8 hours, before declining. Stromelysin mRNA expression preceded a similar increase in stromelysin protein levels in both synovium and cartilage. CONCLUSION. Intraarticular injection of IL-1 induced the endogenous expression of stromelysin mRNA and protein in both synovium and cartilage. The kinetics of stromelysin expression correlated well with the accumulation of stromelysin and proteoglycan in synovial fluids. Therefore, the de novo synthesis of stromelysin in cartilage may have contributed to the loss of proteoglycan from that tissue.     

Autocrine regulation of collagenase 3 (matrix metalloproteinase 13) during osteoarthritis

OBJECTIVE: To correlate the increased collagenase production previously seen in chondrocytes isolated from osteoarthritic (OA) lesions and the expression of cytokines and cytokine receptors. METHODS: Chondrocytes were isolated from OA cartilage and characterized for synthesis of collagenases, cytokines, and cytokine receptors by Northern and Western blot analyses, RNA protection assay, and flow cytometry. RESULTS: Chondrocytes located in cartilage proximal to the macroscopic OA lesions bound more tumor necrosis factor alpha (TNFalpha) and interleukin-1beta (IL-1beta) compared with chondrocytes isolated from morphologically normal cartilage from the same joint. In response to TNFalpha stimulation, messenger RNA (mRNA) levels for the IL-1 receptor I (IL-1RI), IL-1RII, TNF receptor II (TNFR II), and IL-6 receptor as well as the level of proinflammatory cytokines, such as IL-1alpha, IL-1beta, lymphotoxin beta, TNFalpha, and IL-6, also increased. In contrast, treatment with transforming growth factor beta1 (TGFbeta1) resulted in down-regulation of matrix metalloproteinase 1 (MMP-1) and MMP-13 concomitant with a reduction in the levels of mRNA for IL-1RI, IL-1RII, TNFRI, and TNFRII and proinflammatory cytokine levels. In contrast, the levels of mRNA for TGFbeta receptor I, TGFbeta1, and TGFbeta3 were up-regulated. CONCLUSION: These data show that TGFbeta1 has antagonistic effects upon OA chondrocytes, in contrast to the effects seen with TNFalpha. The cyclical course of OA, where a period of active disease is followed by a period of remission, can be explained by a sequential pattern of cytokine stimulation followed by a feedback inhibition of autocrine cytokine production and cytokine receptor expression, thus affecting collagenase synthesis.     

Differential effect of transforming growth factor beta on freshly isolated and cultured articular chondrocytes.

Transforming growth factor beta (TGF beta) is a multipotent regulator of cell proliferation and extracellular matrix synthesis. In our study we show that the effect of TGF beta on proliferation and proteoglycan synthesis of articular cartilage chondrocytes appears to be dependent on the period in culture of these chondrocytes. TGF beta inhibits DNA and proteoglycan synthesis of freshly isolated chondrocytes while the DNA and proteoglycan synthesis of chondrocytes cultured in monolayer was stimulated by TGF beta. Since TGF beta is present in high concentrations in synovial fluid of patients with osteoarthritis, TGF beta might play a role in the elevated proteoglycan synthesis and cell proliferation in osteoarthritic cartilage.     

Association of genotypes affecting the expression of interleukin-1beta or interleukin-1 receptor antagonist with osteoarthritis

OBJECTIVE: The majority of cytokines and growth factors known to be involved in cartilage metabolism are synthesized by the chondrocytes themselves. They are up-regulated in osteoarthritic (OA) cartilage, resulting in 2 opposite phenotypes, TNFalpha(high) and TNFalpha(low), that are characterized by an elevated number of tumor necrosis factor alpha (TNFalpha)-positive and interleukin-1beta (IL-1beta)-positive chondrocytes, respectively. To establish a hierarchy among the cytokines and growth factors expressed in articular chondrocytes, this study investigated cytokine genes for known polymorphisms that may contribute to the deregulated expression in OA cartilage. METHODS: Polymerase chain reaction techniques were performed either in a thermal cycler using standard methods or in a light cycler to analyze the frequencies of the TNFalpha (-308), IL-1 receptor antagonist (IL-1Ra) (intron 2), IL-1beta (exon 5), and IL-6 (-174) polymorphisms in 61 OA patients and 254 randomly chosen controls. RESULTS: For the TNFalpha(low) phenotype, a statistically significant association was found with the less frequent allele of IL-1beta, which carries a single-basepair substitution in exon 5 and may contribute to the characteristic increase in IL-beta-positive chondrocytes. In contrast, the TNFalpha(high) phenotype was significantly associated with the less frequent allele of IL-1Ra, which carries two 86-bp repeats in the second intron and is assumed to lead to an elevated expression of the antagonist. CONCLUSION: These results point to an association between the IL-1beta polymorphism and the TNFalpha(high) phenotype and between the IL-1Ra polymorphism and the TNFalpha(low) phenotype found in OA. Both associations suggest that IL-1beta may be more important than TNFalpha for the regulation of cytokine and growth factor expression in articular chondrocytes.     

In vivo expression of stromelysin in synovium and cartilage of rabbits injected intraarticularly with interleukin-1β

Objective. To examine the in vivo expression of the matrix metalloproteinase stromelysin in the synovium and articular cartilage of rabbits injected intraarticularly with recombinant human interleukin-1β (IL-1). Methods. The direct isolation of messenger RNA (mRNA) from articular cartilage without the prior isolation of chondrocytes is described. The in vivo expression of stromelysin was examined at the mRNA level by Northern blot analysis, and at the protein level by in situ immunolocalization and by enzyme-linked immunosorbent assay. Results. In the synovium of IL-1–injected joints, stromelysin mRNA levels were highest at 4 hours and declined to background levels within 24 hours. In the cartilage of IL-1–injected joints, stromelysin mRNA was elevated at 4 hours and continued to increase until 8 hours, before declining. Stromelysin mRNA expression preceded a similar increase in stromelysin protein levels in both synovium and cartilage. Conclusion. Intraarticular injection of IL-1 induced the endogenous expression of stromelysin mRNA and protein in both synovium and cartilage. The kinetics of stromelysin expression correlated well with the accumulation of stromelysin and proteoglycan in synovial fluids. Therefore, the de novo synthesis of stromelysin in cartilage may have contributed to the loss of proteoglycan from that tissue.     

Genetic enhancement of matrix synthesis by articular chondrocytes: comparison of different growth factor genes in the presence and absence of interleukin-1

OBJECTIVE: To determine whether articular chondrocytes express growth factor genes delivered by adenoviral vectors and whether expression of these genes influences matrix synthesis in the presence and absence of interleukin-1 (IL-1). METHODS: Monolayer cultures of rabbit articular chondrocytes were infected with recombinant adenovirus carrying genes encoding the following growth factors: insulin-like growth factor 1 (IGF-1), transforming growth factor beta1 (TGFbeta1), and bone morphogenetic protein 2 (BMP-2). As a control, cells were transduced with the lac Z gene. Cultures were also treated with each growth factor supplied as a protein. Levels of gene expression were noted, and the synthesis of proteoglycan, collagen, and noncollagenous proteins was measured by radiolabeling. Collagen was typed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The effects of growth factor gene transfer on proteoglycan synthesis in the presence of IL-1 were also measured. RESULTS: The expression of all transgenes was high following adenoviral transduction. Proteoglycan synthesis was stimulated approximately 8-fold by the BMP-2 gene and 2-3-fold by the IGF-1 gene. The effects of BMP-2 and IGF-1 genes were additive upon cotransduction. Synthesis of collagen and noncollagenous proteins, in contrast, was most strongly stimulated by the IGF-1 gene. In each case, collagen typing confirmed the synthesis of type II collagen. IL-1 suppressed proteoglycan synthesis by 50-60%. IGF-1 and TGFbeta genes restored proteoglycan synthesis to control levels in the presence of IL-1. The BMP-2 gene, in contrast, elevated proteoglycan synthesis beyond control levels in the presence of IL-1. CONCLUSION: Transfer of growth factor genes to articular chondrocytes can greatly increase matrix synthesis in vitro, even in the presence of the inflammatory cytokine IL-1. This result encourages the further development of gene therapy for the repair of damaged cartilage.