Diphenyleneiodium chloride blocks inflammatory cytokine-induced up-regulation of group IIA phospholipase A(2) in rat mesangial cells

Inflammatory cytokines, interleukin 1 beta and tumor necrosis factor-alpha, potently stimulate rat mesangial cells to express and secrete group IIA ph ospholipase A(2) (PLA(2)). Cytokine-induced up-regulation of PLA(2) has bee n blocked by inhibitors (antioxidants) of the transcription factor, nuclear factor-kappa B (NF-kappa B), suggesting a role for NF-kappa B in the regul ation of group IIA PLA(2) expression. Reactive oxygen species such as H2O2, which are elevated in mesangial cells after cytokine activation, can mimic cytokine-induced NF-kappa B activation. However, the source of reactive ox ygen species generation in mesangial cells, produced by cytokine stimulatio n, has yet to be clarified. Recently, tumor necrosis factor-alpha has been demonstrated to increase superoxide radical generation in mesangial cells. Therefore, we hypothesized that a selective NADPH oxidase inhibitor, diphen yleneiodium chloride (DPI), could block cytokine-induced group IIA PLA(2) u p-regulation by attenuating NF-kappa B binding. To test this hypothesis, we isolated rat mesangial cells and characterized them by ultrastructural and immunochemical methods. This homogeneous mesangial cell population was res ponsive to cytokine as evidenced by an increase in steady-state levels of g roup IIA PLA(2) mRNA and extracellular enzymatic activity over time. DPI (0 .02-20 mu M), added 90 min before cytokine activation, inhibited both group IIA PLA(2) mRNA and enzymatic activity in a concentration-dependent manner . By electrophoretic mobility shift analysis, cytokine activation also incr eased specific NF-kappa B binding to one of two NF-kappa B consensus elemen ts in the rat group IIA PLA(2) promoter and also was suppressed by DPI pret reatment. Antibodies to NF-kappa B p65 (Rel A) and p50 (but not normal rabb it IgG) supershifted this retardation signal and verified the type of NF-ka ppa B species as the classical p50/p65 heterodimer.