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(Investigative Ophthalmology and Visual Science. 2002;43:3190-3195.)
© 2002 by The Association for Research in Vision and Ophthalmology, Inc.

Suppression of the TNF{alpha}-Induced Increase in IL-1{alpha} Expression by Hypochlorite in Human Corneal Epithelial Cells

Mayumi Mohri1, Peter S. Reinach2, Atsuhiro Kanayama3, Makoto Shimizu3, Jackob Moskovitz4, Tatsuhiro Hisatsune1 and Yusei Miyamoto1

1 From the Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Chiba, Japan; the 2 Department of Biological Sciences, College of Optometry, State University of New York, New York, New York; the 3 Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, Japan; and the 4 Laboratory of Biochemistry, National Heart Lung and Blood Institute, Bethesda, Maryland.

PURPOSE. In response to injury, activated neutrophils release tumor necrosis factor (TNF)-{alpha} and myeloperoxidase (MPO). TNF{alpha} in turn causes human corneal epithelial cells to secrete interleukin (IL)-1{alpha}, whereas MPO results in formation of HClO/OCl-. The effect of HClO/OCl- on the expression of the IL-1{alpha} gene and protein is unknown. The current study was undertaken to examine in immortalized human corneal epithelial cells whether NaOCl alters TNF{alpha}-induced increases in expression of IL-1{alpha} gene and protein.

METHODS. Semiquantitative RT-PCR and ELISA characterized IL-1{alpha} gene and protein expression, respectively. TNF{alpha}-induced nuclear transfer of nuclear factor (NF)-{kappa}B was measured by electrophoretic mobility shift assay (EMSA). The {alpha} isoform of inhibitory protein {kappa}B (I{kappa}B{alpha}) was identified by Western blot analysis.

RESULTS. Exposure to NaOCl (0.75 mM) for 10 minutes caused suppression of TNF{alpha}-induced increases in IL-1{alpha} mRNA and protein, declines in NF{kappa}B nuclear transfer, and a modification of I{kappa}B{alpha}, based on a bandshift detected by Western blot analysis. Modified I{kappa}B{alpha} became resistant to TNF{alpha}-induced proteolysis. Methionine sulfoxide reductase A (MsrA, 10 µM) eliminated the NaOCl-induced I{kappa}B{alpha} bandshift.

CONCLUSIONS. NaOCl oxidizes I{kappa}B{alpha} at methionine residues and thereby suppresses dissociation of I{kappa}B{alpha} from NF{kappa}B. Decreased dissociation could in turn suppress TNF{alpha}-induced activation of NF{kappa}B, resulting in declines in expression of IL-1{alpha} gene and protein. These effects suggest that release of HClO/OCl- in vivo by activated neutrophils may counterbalance TNF{alpha}-induced NF{kappa}B-dependent secretion if IL-1{alpha} and suppress an excessive inflammatory reaction.




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