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Based on findings which suggested the involvement of the neuropeptide substance P in the pathogenesis of rheumatoid arthritis (RA), we investigated the mechanism of synovial pannus formation in RA, and examined the interaction between the cytokine production of synovial tissues and the concentration of substance P in the cartilage-pannus junction (CPJ). The CPJ and other peripheral synovial tissues were separately obtained from each part of the synovium from the knee joints of seven RA patients. The concentrations of substance P and the cytokines interleukin (IL)-1 and IL-6 in the CPJ and peripheral synovial tissues were determined by enzyme-linked immunosorbent assays. In addition, synovial cells were isolated from the CPJ and peripheral synovial tissues and treated with substance P or neurokinin-1 receptor antagonist to analyze the changes in cytokine production. The substance P levels were 211.2 and 50.5 pg/mg protein in the CPJ and the peripheral synovium, respectively. The IL-1 and IL-6 levels in the CPJ were 24.6 and 12.8 pg/mg protein, respectively. In the peripheral synovium, these levels were 4.3 and 2.5 pg/mg protein, respectively. In the CPJ, the IL-1 and IL-6 levels in tissue containing a high concentration of substance P (Ͼ200 pg/mg protein) were 39.4 and 21.6 pg/mg protein, respectively, and those in tissue containing a low concentration of substance P (р200 pg/mg protein) were 11.6 and 5.1 pg/mg protein, respectively. Synovial cells from the CPJ produced higher levels of IL-1 and IL-6 than those from peripheral tissues. In addition, treatment of the cells with an NK-1 antagonist significantly reduced the production of these cytokines by the synovial cells. The theory that substance P plays a role in the pathogenesis of RA via the upregulation of cytokine production should be considered in further studies on the immunomodulatory properties of substance P in arthritis.

Abstract--We detected and analyzed an intracellular mechanism of a substance P-induced priming effect on cytokine production using human synovial cells. The synovial tissues were isolated from the knee joints of osteoarthritis patients. After the administration of a low dose of substance P (1 nM) without significant effect alone, the synovial cells were stimulated with substance P (30/zM), phorbol 12-myristate 13-acetate (PMA) (100 nM), and calcium ionophore (A23187) (1/zM). The total interleukin (IL)-l/3 and IL-6 levels in the supernatant was measured by an enzymelinked immunosorbent assay (ELISA) kit, and the changes in the intracellular calcium concentration ([Ca2+]i) and protein kinase C (PKC) activation were measured by the fura 2-AM fluorescence method and a radioimmunoassay, respectively. The substance P-induced cytokine production was accompanied by an elevation of [Ca 2+ ]i and PKC activation. The amounts of cytokines produced from the substance P (1 nM)-primed synovial cells stimulated with 30 #M substance P were approximately 4 times as much as that observed in non-primed cells. In addition, the priming treatment with 1 nM substance P enhanced not only the subsequent substance P-induced cytokine production, but also the PMA-induced response. However, substance P (1 nM) priming treatment did not affect the A23187-induced response. Furthermore, in substance P-primed cells, substance P (30/zM) induced a significant activation of PKC without changing the [Ca2+]i elevation response. These results suggest that the substance P-priming effect on synovial cells contributed to changes in intracellular mechanisms such as PKC activation.