COOPERATIVE EFFECTS OF INTERFERON-GAMMA ON THE INDUCTION OF NADPH OXIDASE BY RETINOIC ACID OR 1,25(OH)(2)-VITAMIN-D-3 IN MONOCYTIC U937 CELLS

The effect of retinoic acid (RA), 1,25-dihydroxyvitamin D-3 (1,25-D-3) 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 mu M) or 1,25-D-3 (10 nM) with I FN-gamma (100 IU/ml) induced NADPH oxidase activity as demonstrated by increased superoxide anion (O-2(-)) generation in response to stimula tion 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, an d immunoblot analysis of the p47-phox and p67-phox proteins revealed t hat the increases in mRNA levels were equally reflected by increases i n protein levels. In contrast, RA, 1,25-D-3 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 p21rac1, a GT P-binding protein that regulates NADPH oxidase activity in macrophages , was constitutively expressed in undifferentiated cells and was not a ffected by differentiation. These data indicate that induction of a fu nctional NADPH oxidase in premonocytic U937 cells requires the coopera tive actions of IFN-gamma plus RA or 1,25-D-3 and is reflected in the increased expression of p67-phox and gp91-phox.