Substance P-induced priming effects on synovial cells

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 μM), phorbol 12-myristate 13-acetate (PMA) (100 nM), and calcium ionophore (A23187), (1 μM). The total interleukin (IL)-1β 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 [Ca2+]i and PKC activation. The amounts of cytokines produced from the substance P (1 nM)-primed synovial cells stimulated with 30 μM 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 μM) 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.