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J.M. Bradley, A. Rose, M.J. Kelley, Y. Chen, K. Song, T.S. Acott; Interactions Between IL–1 and TNF: Their Effects on Trabecular Meshwork Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1865.
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The efficacy of laser trabeculoplasty, a common glaucoma treatment, is due, in part to the induction of TNFα and IL–1. Prior evidence showed that these cytokines initiated extracellular matrix turnover in the trabecular meshwork (TM) via enhanced production of matrix metalloproteinase–3 (MMP–3). To further understand this process, we evaluated the effects of exogenous TNFα, IL–1α and IL–1ß, both separately and in combination, on outflow facility and on the activation and cellular distribution of p38 MAP kinase. We also measured the levels of specific mRNAs known, from microarray studies, to be regulated by these cytokines.
Cytokine treated cultured porcine TM cells were examined for changes in mRNA levels of tenascin C, MMP–3, MMP–12, VCAM–1 using quantitative RT–PCR. Relative p38 activation level and cellular location was determined at various times via confocal microscopy using both pan and phospho–specific antibodies. The microfilament pattern was similarly evaluated with labeled phallodin. Outflow facility and MMP–3 levels were assessed using perfused porcine anterior segments and Western blot analysis. Transcriptional analysis of MMP–3 was accomplished by using a reporter vector driven by the MMP–3 promoter.
All of the cytokine treatments caused increases in RNA for the MMP–3 with the dual IL–1/TNF treatments having the largest effects. These dual treatments also initiated major changes in the microfilament patterns. At early treatment time points phospho p38 was seen in the nucleus/nucleolus and also colocalized along the ends of actin filaments. The colocalization was most pronounced in the IL–1/TNF dual treatments and IL–1α. At a later time point, this effect was diminished and predominantly nuclear staining was seen. Increases in the outflow facility in the explants matched well with the increases seen in MMP mRNA from cultured cells and total MMP–3 protein increases as shown by western blot analysis. TNFα treatment by itself caused the largest increases in mRNA for tenascin C and VCAM–1 with the dual treatments showing little additive effect on them but synergistic effects on MMP–12.
There is a strong correlation between the total amounts of MMP–3 mRNA/ protein produced by these treatments and the change in outflow facility. The dual treatments and IL–1α alone produced the greatest effects. The colocalization of phospho p38 to microfilaments as well as the change in the microfiliment patterning noted with some of these treatments points to an important cytoskeletal involvement in the IL–1/TNF transduction pathway.
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