Effects of Substance P on the Release of Cytokines from Immune Cell Lines

Lee, Jin Yong; Kim, Soo Ah; Seo, Seok Ran; Kim, Hyong Seop

Abstract

The neuropeptide substance P(SP) has been implicated in the mediation of inflammation and immune-mediated disease such as arthritis. Recently, it was reported that SP was markedly increased around the blood vessels in inflamed gingiva as well as in close association with the inflammatory cell infiltrate. These results support that SP may contribute to the pathophysiology of neuronal inflammation in human periodontal tissues. SP may regulate inflammatory/immune responses by stimulating the proliferation of human T cells, differentiation and antibody-secreting potential of B cells, macrophage respiratory burst, connective tissue proliferation, and the secretion of cytokines from monocytes and T cells. Here, I studied potential role of SP as a costimulatory chemical signal in inflammatory/immune responses, by determining the released proinflammatory cytokines such as MIP-1alpha, IL-1beta, and IL-6 from culture supernatants of homogeneous immune cell lines. Serum free cell supernatants were concentrated with TCA precipitation, fractionated with SDS-PAGE, and subjected into western blot analysis. Among 15 cell lines tested, macrophage/monocyte cell line RAW264.7 and WR19m.1 showed the highest level of induction of MIP-1alphawhen stimulated with LPS. Discrete IL-6 bands with multiple forms of molecular mass were detected from supernatants of B cell lines A20(32kDa), Daudi(32, 35kDa), and SKW6.4(29kDa), which were expressed constitutively. IL-1beta could not be detected by the method of western blot analysis from supernatants of all cell lines tested except RAW264.7, WR19m.1, and erythroid cell line K562 which showed the least amount of IL-1betasecretion. SP 10(-9)M with suboptimal dose of LPS treatment showed synergistic induction of MIP-1alpharelease from RAW264.7 or WR19m.1, and also IL-6 release from A20, but this synergism is not the case in costimulation of RAW264.7 or WR19m.1 with SP 10(-9)M and TPA. Although treatment of T cell line CTLL-R8 with SP 10(-7)M or PHA+TPA induced modest level of MIP-1alpha secretion, synergism was not observed when they are applied together. These findings all together suggest the possibility of a regulatory role of SP in inflammatory/immune reaction through differential modulation of bioactivities of other chemical cosignals.