ABSTRACTcDNA microarray technology is used to profile complex diseases and discover novel disease-related genes. In inflammatory disease such as rheumatoid arthritis, expression patterns of diverse cell types contribute to the pathology. We have monitored gene expression in this disease state with a microarray of selected human genes of probable significance in inflammation as well as with genes expressed in peripheral human blood cells. Messenger RNA from cultured macrophages, chondrocyte cell lines, primary chondrocytes, and synoviocytes provided expression profiles for the selected cytokines, chemokines, DNA binding proteins, and matrix-degrading metalloproteinases. Comparisons between tissue samples of rheumatoid arthritis and inflammatory bowel disease verified the involvement of many genes and revealed novel participation of the cytokine interleukin 3, chemokine Gro␣ and the metalloproteinase matrix metallo-elastase in both diseases. From the peripheral blood library, tissue inhibitor of metalloproteinase 1, ferritin light chain, and manganese superoxide dismutase genes were identified as expressed differentially in rheumatoid arthritis compared with inflammatory bowel disease. These results successfully demonstrate the use of the cDNA microarray system as a general approach for dissecting human diseases.

Objective To examine by immunohistochemistry the relative distributions of 6 matrix metalloproteinases (MMPs 1, 2, 3, 8, 9, and 13) and the 2 proinflammatory cytokines interleukin‐1β (IL‐1β) and tumor necrosis factor α (TNFα) in osteoarthritic (OA) cartilage compared with normal, age‐matched articular cartilage. Methods Articular cartilage samples were obtained from the tibial plateau of OA knees removed at arthroplasty and from normal, nonarthritic, knees obtained at autopsy. Specimens were promptly fixed in Carnoy's fixative, processed, embedded in paraffin, sectioned, and examined by immunohistochemistry for MMP and cytokine production. In addition, human articular chondrocytes (HAC) were treated in vitro with either IL‐1β, TNFα, or phorbol myristate acetate (PMA) to assess their potential to produce each of the MMPs, as determined by Western blotting and gelatin zymography. Results Immunodetection of the collagenases (MMPs 1, 8, and 13) and stromelysin 1 (MMP‐3) was demonstrated in a proportion of chondrocytes in the superficial zone of almost all of the OA specimens that had degenerative matrix changes. The gelatinases (MMPs 2 and 9) were also demonstrated by immunohistochemistry but were not so prominent. IL‐1β– and TNFα‐positive chondrocytes were also observed in a proportion of cells in the superficial zones of OA specimens. Much less immunostaining for MMPs and cytokines was observed in the deep zone of all OA specimens, where the cartilage matrix and chondrocyte morphology appeared normal. In contrast, full‐thickness normal cartilage specimens showed virtually no immunostaining for these MMPs or cytokines. Confirmation that chondrocytes can produce these 6 MMPs was obtained from HAC cultures treated with either IL‐1β, TNFα, or PMA; conditioned medium from activated HAC contained all the MMPs demonstrated by immunohistochemistry. Dual immunolocalization studies of OA cartilage specimens demonstrated the coexpression of IL‐1 with MMP‐8 by individual chondrocytes in situ. Conclusion These results indicate that the superficial zone of OA cartilage specimens, which is characterized by fibrillations, chondrocyte clusters, and degenerative matrix changes, contains a variable proportion of cells that immunostain for IL‐1β, TNFα, and 6 different MMPs. These observations support the concept that cytokine–MMP associations reflect a modified chondrocyte phenotype and an intrinsic process of cartilage degradation in OA.

Objective.To examine by immunohistochemistry the relative distributions of 6 matrix metalloproteinases (MMPs 1, 2, 3, 8, 9, and 13) and the 2 proinflammatory cytokines interleukin-1␤ (IL-1␤) and tumor necrosis factor ␣ (TNF␣) in osteoarthritic (OA) cartilage compared with normal, age-matched articular cartilage.Methods. Articular cartilage samples were obtained from the tibial plateau of OA knees removed at arthroplasty and from normal, nonarthritic, knees obtained at autopsy. Specimens were promptly fixed in Carnoy's fixative, processed, embedded in paraffin, sectioned, and examined by immunohistochemistry for MMP and cytokine production. In addition, human articular chondrocytes (HAC) were treated in vitro with either IL-1␤, TNF␣, or phorbol myristate acetate (PMA) to assess their potential to produce each of the MMPs, as determined by Western blotting and gelatin zymography.Results. Immunodetection of the collagenases (MMPs 1, 8, and 13) and stromelysin 1 (MMP-3) was demonstrated in a proportion of chondrocytes in the superficial zone of almost all of the OA specimens that had degenerative matrix changes. The gelatinases (MMPs 2 and 9) were also demonstrated by immunohistochemistry but were not so prominent. IL-1␤-and TNF␣-positive chondrocytes were also observed in a proportion of cells in the superficial zones of OA specimens. Much less immunostaining for MMPs and cytokines was observed in the deep zone of all OA specimens, where the cartilage matrix and chondrocyte morphology appeared normal. In contrast, fullthickness normal cartilage specimens showed virtually no immunostaining for these MMPs or cytokines. Confirmation that chondrocytes can produce these 6 MMPs was obtained from HAC cultures treated with either IL-1␤, TNF␣, or PMA; conditioned medium from activated HAC contained all the MMPs demonstrated by immunohistochemistry. Dual immunolocalization studies of OA cartilage specimens demonstrated the coexpression of IL-1 with MMP-8 by individual chondrocytes in situ.Conclusion. These results indicate that the superficial zone of OA cartilage specimens, which is characterized by fibrillations, chondrocyte clusters, and degenerative matrix changes, contains a variable proportion of cells that immunostain for IL-1␤, TNF␣, and 6 different MMPs. These observations support the concept that cytokine-MMP associations reflect a modified chondrocyte phenotype and an intrinsic process of cartilage degradation in OA.Osteoarthritis (OA) is the most prevalent disease of articular joints and is the major cause of disability in the elderly (1). Degeneration and loss of articular cartilage are characteristic features of OA. The appearance of fibrillations, matrix depletion, cell clusters, and changes in matrix composition reflect the aberrant behavior of resident chondrocytes (2). Although biomechanical factors are strongly implicated, at present it is unclear which stimuli regulate the hyperactive pheno-

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Histochemical and ultrastructural studies of bone-cartilage junctions from 21 rheumatoid knee joints have demonstrated the presence of both osteoclasts and cbondroclasts. Significant erosions of bone and mineralized cartilage were observed in 15 specimens, and 6 showed localized erosions of unmineralized (hyaline) cartilage. Chondroclasts, defined by their close association with both mineralized and unmineralized cartilage, were morphologically and histochemically similar to osteoclasts. Our observations suggest that these multinucleate cells play a crucial role in subchondral tissue destruction, but that erosion of unmineralized cartilage is primarily the result of syaovial pannus tissue.The pathologic process of joint destruction in rheumatoid arthritis (RA) is complex. Cartilage erosion and bone erosion have often been considered separate entities. Cartilage degradation has been shown to be brought about by invasive, hypertrophic synovial pannus tissue (1-101, by cellular infiltrations from the subchondral bone region (7,111, and by chondrocytes themselves (5,121. Osteoclastic bone erosion is also a recognized feature of rheumatoid

Mast cell activation in vivo is often associated with areas of oedema and connective-tissue degradation. Tryptase and chymase are the major serine proteinases released by mast cells, but they appear to have little activity on most components of the extracellular matrix. The matrix metalloproteinases (MMP) are purported to degrade almost all connective tissue elements and are secreted by cells in the form of inactive precursors. Since the mechanisms of MMP activation in vivo are poorly understood we have examined the potential of mast cell proteinases to activate the precursor forms of human collagenase (MMP-l), stromelysin (MMP-3), gelatinase A (MMP-2) and gelatinase B (MMP-9).Mast cell proteinases prepared from purified dog mastocytoma cells were shown to process and activate purified precursor forms of both MMP-1 and MMP-3. Using antipain and chymostatin, inhibitors for tryptase and chymase, respectively, it was demonstrated that both pMMP-1 and pMMP-3 were effectively processed and activated by the chymase component. By contrast, tryptase activated only pMMP-3. The mast cell proteinases were unable to process or activate purified precursor forms of MMP-2 and MMP-9. However, MMP-3 previously activated by mast cell proteinases was shown to activate pMMP-9, but not pMMP-2. Since we have no evidence that mast cells express these four metalloenzymes, the release of mast cell serine proteinases following activatioddegranulation could contribute to local metalloproteinase activation and subsequent matrix degradation.Although numerous enzymes may contribute to the degradation of connective tissues, the family of metalloproteinases is of particular interest since they are purported to degrade almost all components of the extracellular matrix [l, 21. These enzymes are grouped into three main subclasses : interstitial [matrix metalloproteinase (MMP)-11 and polymorphonuclear (MMP-8) collagenases which degrade type I, I1 and I11 collagens; gelatinases A and B [MMP-2 (72 kDa) and MMP-9 (92 m a ) ] which degrade basement-membrane type IV collagen and gelatin; and stromelysins (MMP-3, MMP-10) with activity on a broad spectrum of substrates including proteoglycans, laminin, fibronectin and some collagen species [3]. While expressing different substrate specificities these metalloenzymes share some conserved sequence similarity and activation mechanisms [2]. All the metalloproteinases are secreted as precursor forms requiring extracellular activation prior to substrate attack. Activation of the pro- ~-enzymes has been demonstrated in vitro by proteolytic cleavage of their propeptide domains and also by organomercurial compounds such as aminophenyl mercuric acetate (H,NPhHgAc) [2, 4-61, but the activation mechanisms that function in vivo remain unclear.Since mast cells are commonly associated with sites of connective-tissue lysis, for example at sites of tumour invasion in melanoma and breast carcinoma [7, 81 and at cartilage erosion sites in rheumatoid arthritis [9], a potential role for this cell in matrix degradation has be...

Objectives-To examine the distribution and activation of mast cells (MCs)

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