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


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


RESULTS
Exposure to NaOCl (0.75 mM) for 10 minutes caused suppression of TNFalpha-induced increases in IL-1alpha mRNA and protein, declines in NFkappaB nuclear transfer, and a modification of IkappaBalpha, based on a bandshift detected by Western blot analysis. Modified IkappaBalpha became resistant to TNFalpha-induced proteolysis. Methionine sulfoxide reductase A (MsrA, 10 micro M) eliminated the NaOCl-induced IkappaBalpha bandshift. CONCLUSIONS; NaOCl oxidizes IkappaBalpha at methionine residues and thereby suppresses dissociation of IkappaBalpha from NFkappaB. Decreased dissociation could in turn suppress TNFalpha-induced activation of NFkappaB, resulting in declines in expression of IL-1alpha gene and protein. These effects suggest that release of HClO/OCl(-) in vivo by activated neutrophils may counterbalance TNFalpha-induced NFkappaB-dependent secretion if IL-1alpha and suppress an excessive inflammatory reaction.
D000469:Alkaline Phosphatase / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002461:Cell Line, Transformed; D002467:Cell Nucleus / Q000378:metabolism; D019573:Epithelium, Corneal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D006997:Hypochlorous Acid / Q000494:pharmacology*; D020672:I-kappa B Proteins / Q000378:metabolism; D007375:Interleukin-1 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000494:pharmacology; D051150:Methionine Sulfoxide Reductases; D016328:NF-kappa B / Q000378:metabolism; D016877:Oxidants / Q000494:pharmacology*; D010088:Oxidoreductases / Q000494:pharmacology; D012973:Sodium Hypochlorite / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D020672:I-kappa B Proteins; D007375:Interleukin-1; D007527:Isoenzymes; D016328:NF-kappa B; D016877:Oxidants; D014409:Tumor Necrosis Factor-alpha; D006997:Hypochlorous Acid; D012973:Sodium Hypochlorite; D010088:Oxidoreductases; D051150:Methionine Sulfoxide Reductases; C027219:methionine sulfoxide reductase; D000469:Alkaline Phosphatase