DETECTION OF PROTEIN AND MESSENGER-RNA OF VARIOUS COMPONENTS OF THE NADPH OXIDASE COMPLEX IN AN IMMORTALIZED HUMAN CHONDROCYTE LINE

The immortalized human chondrocyte cell line C-20/A4 has the ability t o 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 s timulation with ionomycin and phorbol 12-myristate 13-acetate. Priming and anti-priming effects of interleukin (IL)-1 beta and IL-4, respect ively, are also demonstrated. Reverse transcriptase polymerase chain r eaction (RT-PCR) amplification using oligonucleotide primers to compon ents of the NADPH oxidase enzyme complex showed mRNA expression of p22 -phox, p40-phox, p47-phox and p67-phox. Western blot analysis using po lyclonal antisera indicated the presence of the p47-phox and p67-phox polypeptide components. These results show that the C-20/A4 cells cont ain an NADPH oxidase-like complex, similar to that found in other cell types, which produces superoxide anions.