Effects of all-trans-retinoic acid on superoxide generation in intact neutrophils and a cell-free system

To determine the pathophysiology of the retinoic acid syndrome which occurs during all-trans-retinoic acid (ATRA) treatment of acute promyelocytic leukaemia patients, we investigated the direct effects of ATRA on the function of human neutrophils. We found that ATRA (10-200 microM) dose-dependently stimulated superoxide (O2-) generation in intact neutrophils. The maximal activity of ATRA-stimulated O2- generation was 3.0 nmol/min/10(6) cells. Adding EGTA to the assay mixture did not affect the activity nor was the intracellular free calcium concentration changed upon stimulation. The treatment of neutrophils with 0.1 microM staurosporine, an antagonist of protein kinase C, for 10 min, enhanced the activity of ATRA-stimulated O2- generation up to 186% of that for control samples. Wortmannin (1 microM), an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), reduced this stimulatory activity by 67%. These results suggest that ATRA activates the signalling pathway related to PI 3-kinase rather than that utilizing calcium and protein kinase C. ATRA enhanced the O2- generated in a sodium-dodecyl-sulphate (SDS) cell-free system, resulting in rates up to 288% higher than that seen with SDS alone. This enhancement was not affected by pretreatment with staurosporine or wortmannin. ATRA may thus directly activate and/or enhance the function of neutrophils.