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The British Journal of Rheumatology, Vol 36, 522-529, Copyright © 1997 by British Society for Rheumatology

ORIGINAL PAPERS

Detection of protein and mRNA of various components of the NADPH oxidase complex in an immortalized human chondrocyte line

PJ Moulton, TS Hiran, MB Goldring and JT Hancock
Faculty of Applied Sciences, University of the West of England, Bristol.

The immortalized human chondrocyte cell line C-20/A4 has the ability to produce superoxide constitutively at low levels of 5.4 x 10(-2) nmol/min/10(6) cells (S.E.M. = +/-0.5, n = 30) and at raised levels upon stimulation with ionomycin and phorbol 12-myristate 13-acetate. Priming and anti-priming effects of interleukin (IL)-1 beta and IL-4, respectively, are also demonstrated. Reverse transcriptase polymerase chain reaction (RT-PCR) amplification using oligonucleotide primers to components of the NADPH oxidase enzyme complex showed mRNA expression of p22-phox, p40-phox and p47-phox. Western blot analysis using polyclonal antisera indicated the presence of the p47-phox p67-phox polypeptide components. These results show that the C-20/A4 cells contain an NADPH oxidase-like complex, similar to that found in other cell types, which produces superoxide anions.
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