Synergism of TNF and IL-1 in the induction of matrix metalloproteinase-3 in trabecular meshwork

PURPOSE. TNF and 1L-1 increase matrix metalloproteinase-3 (MMP-3) expression in the trabecular meshwork (TM). TNF-a, in combination with IL-la or 1L-1 beta, produces highly synergistic MMP-3 increases. Possible mechanisms for this synergism in TM cells were investigated. METHODS. Porcine and human TM cells were treated with TNF-alpha, IL-1 alpha, IL-1 beta and their combinations. Western immunoblots were used to evaluate MMP-3, MMP-9, MMP-12, TNF-alpha, IL-1 alpha, 1L-1 beta, IL-6, TNF receptor I(RI), IL-1 RI and IL-1 RII levels and the phosphorylation of Erk, JNK, and p38 MAP kinases. Dose-response effects for TNF-alpha, IL-1 alpha and IL-1 beta on MMP-3 were evaluated. Microarray and quantitative RT-PCR were used to determine mRNA levels. MMP-3 transcription rate was assessed by transfecting TM cells with an MMP-3 promoter/ reporter construct. Combined cytokine effects on outflow facility were appraised in perfused anterior segment organ culture. RESULTS. TNF-alpha, IL-1 alpha, and IL-1 beta each individually increased MMP-3 levels, whereas TNF-alpha in combination with IL-1 alpha or IL-1 beta produced highly synergistic increases. MMP-9 and MMP-12 levels were also elevated, but only MMP-12 showed synergism. IL-1 alpha, IL-1 beta, and IL-6, but not TNF-alpha mRNA or protein level, were elevated by these cytokines. Maximum MMP-3 production for individual cytokines, even at high doses was far less than with dual cytokine doses. Erk 1 and 2, JNK and 2, and p38 alpha and beta phosphorylation increased, but not synergistically. However, phosphorylation of novel isoforms of JNK and p38 5 and gamma did show synergism. MMP-3 mRNA levels and transcription rates also demonstrated synergism. TNF-alpha significantly increased IL-1 RI levels. Synergism in outflow facility was observed with TNF-alpha and IL-1 alpha. CONCLUSIONS. TNF-alpha, in combination with IL-1 alpha or 1L-1 beta, produced intense synergistic increases in MMP-3 and MMP-12 but not in MMP-9. Induction of IL-1 RI by TNF-alpha partially explains the synergism. Responses of novel JNK and p38 MAP kinase 5 and gamma isoforms also partially account for the synergism. Understanding t, is strong synergistic effect may provide useful insight into optimizing therapeutic regulation of intraocular pressure in glaucoma.