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(Investigative Ophthalmology and Visual Science. 2000;41:2922-2929.)
© 2000 by The Association for Research in Vision and Ophthalmology, Inc.

Regulation of Collagenase, Stromelysin, and Gelatinase B in Human Conjunctival and Conjunctivochalasis Fibroblasts by Interleukin-1ß and Tumor Necrosis Factor-{alpha}

Daniel Meller1, De–Quan Li1 and Scheffer C. G. Tseng1,2

1 From the Ocular Surface and Tear Center, Department of Ophthalmology, Bascom Palmer Eye Institute; and the 2 Department of Cell Biology and Anatomy, University of Miami School of Medicine, Miami.

PURPOSE. Overexpression and increased activities of matrix metalloproteinases (MMPs) have recently been reported in cultured conjunctival fibroblasts from patients with conjunctivochalasis. The role of inflammatory cytokines in modulating expression of MMPs, their tissue inhibitors (TIMPs), and urokinase plasminogen activator (uPA) as potential contributors to the pathogenesis of conjunctivochalasis was investigated.

METHODS. Interleukin-1ß (IL-1ß) or tumor necrosis factor-{alpha} (TNF-{alpha}) was added at 10 ng/ml to a serum-free medium. Expression of transcripts and proteins of MMPs, TIMPs, and uPA by cultured normal human conjunctival and conjunctivochalasis fibroblasts was determined by Northern hybridization, enzyme-linked immunosorbent assay (ELISA) and Western blot analysis, respectively. Gelatin and casein zymographies were performed in serum-free conditioned media with and without the respective enzyme inhibitors.

RESULTS. Without challenging the cells, conjunctivochalasis fibroblasts showed mRNA and protein overexpression of MMP-1 and MMP-3 compared with normal conjunctival fibroblasts, which showed minor or no expression of these enzymes. IL-1ß markedly and TNF-{alpha} to lesser extent increased mRNA and protein expression of MMP-1 and MMP-3 in conjunctivochalasis fibroblasts from 2 subjects when compared with normal conjunctival fibroblasts from 2 subjects and with their nonstimulated counterparts. In conjunctivochalasis fibroblasts and normal conjunctival fibroblasts, TNF-{alpha}, but not IL-1ß, induced a gelatinolytic activity of MMP-9, which was further confirmed by Western blot analysis and ELISA. Expression of MMP-2, TIMP-1, and TIMP-2 mRNA and protein was not influenced by IL-1ß or TNF-{alpha}, and no difference was found in the gelatinolytic activity of MMP-2 between both cell types.

CONCLUSIONS. Inflammatory cytokines such as IL-1ß and TNF-{alpha}, which can potentially be derived from the ocular surface and tears, may be responsible for increased expression of MMPs in cultured conjunctivochalasis fibroblasts. Ocular inflammation might be one important denominator in the pathogenesis of conjunctivochalasis.




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