OBJECTIVE: Cultured synovial fibroblast-like cells from 3 patients with rheumatoid arthritis (RA) and 3 patients with osteoarthritis (OA) were evaluated for their potential to secrete cysteine proteinases spontaneously and after stimulation by tumor necrosis factor alpha (TNF alpha) or interleukin-1 (IL-1). METHODS: Culture media and cell lysates were analyzed before and after high performance liquid chromatography (HPLC) using the enzymatic substrate, Z-Phe-Arg-AMC, and by immunoblotting with anti-cathepsin B antiserum. Immunolocalization of cathepsin B was studied on cell monolayers. RESULTS: Latent cysteine proteinase activity was found to be secreted spontaneously by cultured synovial fibroblast-like cells. This activity was increased after treatment with either TNF alpha or IL-1. Stimulated protease activity was eluted by HPLC at a peak coincident with that of purified cathepsin B. By immunoblot, cell supernatants contained a 43-kd form of cathepsin B, while cell lysates contained a 30-kd form, consistent, respectively, with cathepsin B before and after cleavage of its propeptide. An intracellular increase in cathepsin B after treatment with TNF alpha was also seen with immunohistochemical studies. CONCLUSION: TNF alpha (in the 6 cases studied) and IL-1 (in 4 cases) stimulated the secretion of a latent cysteine proteinase activity from synovial fibroblast-like cells, which appears to represent primarily cathepsin B.
OBJECTIVE: Cultured synovial fibroblast-like cells from 3 patients with rheumatoid arthritis (RA) and 3 patients with osteoarthritis (OA) were evaluated for their potential to secrete cysteine proteinases spontaneously and after stimulation by tumor necrosis factor alpha (TNF alpha) or interleukin-1 (IL-1). METHODS: Culture media and cell lysates were analyzed before and after high performance liquid chromatography (HPLC) using the enzymatic substrate, Z-Phe-Arg-AMC, and by immunoblotting with anti-cathepsin B antiserum. Immunolocalization of cathepsin B was studied on cell monolayers. RESULTS: Latent cysteine proteinase activity was found to be secreted spontaneously by cultured synovial fibroblast-like cells. This activity was increased after treatment with either TNF alpha or IL-1. Stimulated protease activity was eluted by HPLC at a peak coincident with that of purified cathepsin B. By immunoblot, cell supernatants contained a 43-kd form of cathepsin B, while cell lysates contained a 30-kd form, consistent, respectively, with cathepsin B before and after cleavage of its propeptide. An intracellular increase in cathepsin B after treatment with TNF alpha was also seen with immunohistochemical studies. CONCLUSION:TNF alpha (in the 6 cases studied) and IL-1 (in 4 cases) stimulated the secretion of a latent cysteine proteinase activity from synovial fibroblast-like cells, which appears to represent primarily cathepsin B.