Cooperative effects of interferon-gamma on the induction of NADPH oxidase by retinoic acid or 1,25(OH)2-vitamin D3 in monocytic U937 cells.

The effect of retinoic acid (RA), 1,25-dihydroxyvitamin D3 (1,25-D3) or human recombinant interferon-gamma (IFN-gamma) on the induction of NADPH oxidase was studied in premonocytic U937 cells. Differentiation with the combination of either RA (1 microM) or 1,25-D3 (10 nM) with IFN-gamma (100 IU/ml) induced NADPH oxidase activity as demonstrated by increased superoxide anion (O2-) generation in response to stimulation with phorbol myristate acetate (PMA, 100 nM). Induction of NADPH oxidase activity was preceded by increases in mRNA levels of p47-phox, p67-phox and gp91-phox, which encode three subunits of the enzyme, and immunoblot analysis of the p47-phox and p67-phox proteins revealed that the increases in mRNA levels were equally reflected by increases in protein levels. In contrast, RA, 1,25-D3 or IFN-gamma alone did not induce NADPH oxidase activity which correlated with their failure to increase p67-phox and gp91-phox mRNA levels. The mRNA of p21 rac1, a GTP-binding protein that regulates NADPH oxidase activity in macrophages, was constitutively expressed in undifferentiated cells and was not affected by differentiation. These data indicate that induction of a functional NADPH oxidase in premonocytic U937 cells requires the cooperative actions of IFN-gamma plus RA or 1,25-D3 and is reflected in the increased expression of p67-phox and gp91-phox.