ADAM-10 protein is present in human articular cartilage primarily in the membrane-bound form and is upregulated in osteoarthritis and in response to IL-1alpha in bovine nasal cartilage.

The objective of our study was to determine the tissue distribution and localization of ADAM-10 protein in human and bovine cartilage and the changes it undergoes with cartilage degeneration seen in osteoarthritis (OA) and under the influence of interleukin-1 (IL-1). Human normal and OA articular cartilage and bovine nasal cartilage cultured in the presence of IL-1alpha were processed for histology and immunohistochemistry. ADAM-10 protein was extracted from human cartilage and analyzed by Western blotting using anti-ADAM-10 antibodies. Fluor S Image analyzer and Quantity One software program were applied to quantify the total amount of ADAM-10. ADAM-10 protein was detected in both human and bovine cartilage. The strongest immunostaining was found in the cytoplasm and/or cell membranes of the superficial and upper middle layer of normal adult human cartilage, in the clusters and fibrillated areas of OA cartilage, and in IL-1alpha-stimulated bovine nasal cartilage. The distribution of ADAM-10 protein in bovine nasal cartilage was dependent on the length of exposure to IL-1alpha and corresponded to the areas of proteoglycan depletion. By Western blotting analysis of human cartilage, ADAM-10 was primarily detected in the membrane-enriched fraction and its levels were increased in degenerated and OA cartilage compared to normal cartilage. The results of this study suggest that ADAM-10 might be an important factor associated with cartilage degenerative processes. (J Histochem Cytochem 49:1165-1176, 2001)

Constitutive intra-articular expression of human IL-1 beta following gene transfer to rabbit synovium produces all major pathologies of human rheumatoid arthritis.

To investigate the pathophysiologic effects of chronically elevated intra-articular levels of IL-1 beta, we used an ex vivo gene transfer method to deliver and express human IL-1 beta (hIL-1 beta) in the knee joints of rabbits. Expression of hIL-1 beta resulted in a severe, highly aggressive form of arthritis analogous to chronic rheumatoid arthritis in humans. Intra-articular manifestations included intense inflammation, leukocytosis, synovial hypertrophy and hyperplasia, and highly aggressive pannus formation with erosion of the articular cartilage and periarticular bone. Systemic effects were also observed, including diarrhea, fever, weight loss, and an increased erythrocyte sedimentation rate. In addition, the hIL-1 beta was found to induce elevated levels of both rabbit IL-1 beta and TNF-alpha in synovial fluid. Following the loss of hIL-1 beta transgene expression between 14 and 28 days post-transplantation, many of these changes began to normalize. These results suggest that chronically elevated intra-articular levels of IL-1 beta alone are sufficient to produce virtually all the pathologies found in rheumatoid arthritis, and furthermore, demonstrate that gene transfer can be used to investigate the roles of specific gene products in the pathogenesis of arthritis.

Temporal pattern of cysteine endopeptidase (cathepsin B) expression in cartilage and synovium from rabbit knees with experimental osteoarthritis: gene expression in chondrocytes in response to interleukin-1 and matrix depletion.

OBJECTIVE: To determine the temporal pattern of expression of cathepsin-B in chondrocytes and synovium in experimental osteoarthritis, and to determine possible mechanisms for upregulation and secretion of cathepsin-B from chondrocytes. METHODS: Experimental osteoarthritis was induced with partial medial meniscectomy (PM); sham operated (SH) and normal (N) rabbits were used as controls. Cathepsin-B mRNA expression was assessed with northern blotting with a 32P labelled cDNA probe. Cathepsin-B was measured in conditioned media or cell extracts using a fluorogenic substrate Z-Arg-Arg-AMC. Chondrocyte monolayers were used to determine cathepsin-B expression in response to interleukin-1 beta (IL-1 beta). Cartilage explants were used to test the effect of matrix depletion on cathepsin-B release. RESULTS: Chondrocytes obtained from experimental osteoarthritis knees did not show cathepsin-B mRNA upregulation. However, isolated chondrocytes secreted cathepsin-B into the culture medium. Enzyme release was significantly higher at 8 weeks relative to controls, but not at 12 weeks or 4 weeks. Enzyme released from synovium was significantly higher in PM group compared with SH group at 4 and 8 weeks. IL-1 beta was ineffective in upregulating steady state cathepsin-B mRNA in chondrocytes; however, it upregulated the intracellular enzyme, and this was blocked with cycloheximide. Enzymatic depletion of cartilage matrix after exposure of explants to IL-1 resulted in release of significantly higher amounts of cathepsin-B into the medium by matrix depleted chondrocytes compared with intact explants. CONCLUSIONS: In experimental osteoarthritis, cathepsin-B is upregulated in synovial tissue during the early degenerative phase. Progression of experimental osteoarthritis is accompanied by upregulation of cathepsin-B in cartilage. Cartilage and synovial cathepsin-B levels decline as experimental osteoarthritis advances to more degenerative states. IL-1 upregulates intracellular cathepsin-B by increasing cathepsin-B protein synthesis; it is not an effective stimulus for enzyme secretion. Depletion of cartilage matrix during progression of experimental osteoarthritis may contribute to secretion of cathepsin-B and perpetuation of cartilage destruction.

Cloning, expression, and functional characterization of rat MIP-2: a neutrophil chemoattractant and epithelial cell mitogen.

Macrophage inflammatory protein-2 (MIP-2) is a member of a family of cytokines that play roles in inflammatory, immune, and wound healing responses. To clone the cDNA for rat MIP-2, RNA was isolated from the lungs of Fischer 344 rats after instillation of lipopolysaccharide. Reverse transcription-polymerase chain reaction was performed by using synthetic oligonucleotide primers designed from the mouse MIP-2 cDNA sequence. A cDNA containing the coding region of rat MIP-2 was cloned and sequenced. Comparison to the mouse MIP-2 cDNA demonstrated 90.3% homology at the nucleotide level and 86% homology at the amino acid level. The rat MIP-2 cDNA was expressed in Escherichia coli and protein evaluated for bioactivity. The recombinant rat MIP-2 was chemotactic for rat neutrophils but did not stimulate migration of rat alveolar macrophages or human peripheral blood eosinophils or lymphocytes. In addition, the recombinant rat MIP-2 and the related rat chemokine, KC/CINC stimulated proliferation of rat alveolar epithelial cells but not fibroblasts in vitro.

Suppression of intra-articular responses to interleukin-1 by transfer of the interleukin-1 receptor antagonist gene to synovium.

We have developed an ex vivo method for delivering genes to the synovial lining of joints and expressing them intra-articularly. The present studies were designed to determine whether transfer of a human interleukin-1 receptor antagonist protein (IRAP) gene by this method was able to antagonize the intra-articular actions of interleukin-1. Intra-articular injections of human recombinant interleukin-1 beta (hrIL-1 beta) into the knees of control rabbits provoked a marked leukocytic infiltrate into the joint space, severe synovial thickening and hypercellularity, and loss of proteoglycans from articular cartilage. Genetically modified knees contained several nanograms of human IRAP and inhibited each of these effects of IL-1 beta. These data demonstrate for the first time that delivery of an appropriate gene to joints can prevent intra-articular pathology. Such findings permit cautious optimism about the eventual development of a gene treatment for arthritis and other disorders of the joint.

Intraarticular expression of biologically active interleukin 1-receptor-antagonist protein by ex vivo gene transfer.

Gene therapy offers a radical different approach to the treatment of arthritis. Here we have demonstrated that two marker genes (lacZ and neo) and cDNA coding for a potentially therapeutic protein (human interleukin 1-receptor-antagonist protein; IRAP or IL-1ra) can be delivered, by ex vivo techniques, to the synovial lining of joints; intraarticular expression of IRAP inhibited intraarticular responses to interleukin 1. To achieve this, lapine synoviocytes were first transduced in culture by retroviral infection. The genetically modified synovial cells were then transplanted by intraarticular injection into the knee joints of rabbits, where they efficiently colonized the synovium. Assay of joint lavages confirmed the in vivo expression of biologically active human IRAP. With allografted cells, IRAP expression was lost by 12 days after transfer. In contrast, autografted synoviocytes continued to express IRAP for approximately 5 weeks. Knee joints expressing human IRAP were protected from the leukocytosis that otherwise follows the intraarticular injection of recombinant human interleukin 1 beta. Thus, we report the intraarticular expression and activity of a potentially therapeutic protein by gene-transfer technology; these experiments demonstrate the feasibility of treating arthritis and other joint disorders with gene therapy.

Structure of a multihormonally regulated rat gene.

Gene 33, a rat gene transcriptionally enhanced by glucocorticoids, insulin, or cyclic AMP, was isolated from a library of rat genomic DNA and characterized by sequence comparison to a full-length cDNA. The structural gene spans 13,500 bp encoding 2970 bp of exon sequences interrupted by three introns of about 9600, 101 and 811 bp, respectively. Exons (5' to 3') are 198, 194, 77 and 2501 bp in length; the first of these initiates at the transcriptional start point determined by S1 nuclease mapping. The 5'-flanking DNA contains several putative transcriptional control elements including TATA and CAAT boxes and a binding site for the Sp1 transcription factor in the usual locations proximal to the start point. Sequences resembling known glucocorticoid and cyclic AMP regulatory elements are also found upstream. A chimeric plasmid was constructed containing putative gene 33 regulatory elements fused to the Escherichia coli gene cat, encoding the enzyme chloramphenicol acetyltransferase, and transfected into cultured fibroblasts. Transient expression assays established that this gene 33 DNA is effective in promoting transcription.