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A.Y. Rose, K. Song, Y. Chen, T.S. Acott, M.J. Kelley; Synergistic Effects of TNF, IL–1 and IL–1ß on MMP–3 Production by Trabecular Meshwork (TM) Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1862.
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TNFα, IL–1α and IL–1ß are strong modulators of the expression of some matrix metalloproteinases (MMPs). Increased activity of MMPs restores normal aqueous humor outflow facility and normalizes intraocular pressure after laser trabeculoplasty treatment for glaucoma. To determine possible regulatory mechanisms involved in MMP–3 production, studies were conducted with single and dual cytokine regimens.
Porcine and human TM cells were treated with recombinant human TNF–α, recombinant porcine or human IL–1α or IL–1ß at 2, 10, 25 or 50 ng/ml for 15 min and for 24, 48 or 72 hrs. Western immunoblot analysis was used to examine MMP–3, MMP–9, and MMP–12 protein levels and JNK1/2 (Thr183/Tyr185), ERK1/2 (Thr202/Tyr204) and p38 MAP kinase (Thr180/Tyr182) phosphorylation levels. The effects of these cytokines separately and in combination on MMP–12 and MMP–3 were analyzed by real time quantitative RT–PCR. To analyze MMP–3 promoter activity, a secreted alkaline phosphotase (SEAP) reporter construct containing a 2.3kb MMP–3 promoter was transfected into TM cells. SEAP levels in the media were determined by a chemiluminenscent assay following cytokine treatment.
Individually, TNFα, IL–1α and IL–1ß induce MMP–3, MMP–9 and MMP–12 expression by 24 hrs after treatment. In combination, TNF–α and IL–1α induce MMP–3 promoter activity and protein levels much more than separately. Similar synergistic effects were observed on MMP–3 and MMP–12 mRNA levels. MMP–3 and MMP–12 but not MMP–9 protein levels were synergistically increased by several fold by combining TNFα (10ng/ml) with IL–1α (10ng/ml) or IL–1ß (50ng/ml). Separately, even at 5 fold higher concentrations, these cytokines were less effective than in combination. TNFα and IL–1α significantly increased JNK, ERK and p38 phosphorylation at 15 minutes, however, the cytokine combination did not have synergistic effects on phosphorylation. Synergism was observed at 24 hrs affecting JNK and p38 phosphorylation.
The downstream effects of TNF–α, IL–1α and IL–1ß treatments are increased expression of MMPs that are responsible for regulation of intraocular pressure. The combination of TNFα with IL–1α or IL–1ß has dramatic synergistic effects on MMP–3 and MMP–12 but not MMP–9 production. The synergetic cytokine effect can be only partially explained by the MAP kinase phosphorylation increases and is unlikely to be completely transcriptional.
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