1α,25-dihydroxyvitamin D3-induced monocyte antimycobacterial activity is regulated by phosphatidylinositol 3-kinase and mediated by the NADPH-dependent phagocyte oxidase

We investigated the basis for the induction of monocyte antimycobacterial activity by 1 alpha ,25-dihydroxyvitamin D-3 (D-3). As expected, incubation of Mycobacterium tuberculosis-infected THP-1 cells or human peripheral blood, monocyte-derived macrophages with hormone resulted in the induction of antimycobacterial activity. This effect was significantly abrogated by pretreatment of cells with either of the phosphatidylinositol 3-kinase (PI 3-K) inhibitors, wortmannin or LY294002, or with antisense oligonueleotides to the p110 subunit of PI 3-K alpha. Cells infected with M. tuberculosis alone or incu- bated with D-3 alone produced little or undetectable amounts of superoxide anion (O . (-)(2)). In contrast, exposure of M. tuberculosis-infected cells to D-3 led to significant production of O . (-)(2) and this response was eliminated by either wortmannin, LY294002, or p110 antisense oligonucleotides. As was observed for PI 3-K inactivation, the reactive oxygen intermediate scavenger, 4-hydroxy-TEMPO, and degradative enzymes, polyethylene glycol coupled to either superoxide dismutase or catalase, also abrogated D-3-induced antimycobacterial activity. Superoxide production by THP-1 cells in response to D3 required prior infection with live Al. tuberculosis, since exposure of cells to either killed M. tuberculosis or latex beads did not prime for an oxidative burst in response to subsequent hormone treatment. Consistent with these findings, redistribution of the cytosolic oxidase components p47(phox) and p67(phox) to the membrane fraction was observed in cells incubated with live M. tuberculosis and D-3 but not in response to combined treatment with heat-killed M. tuberculosis followed by D-3. Redistribution of p47(phox) and p67(phox) to the membrane fraction in response to live M. tuberculosis and D-3 was also abrogated under conditions where PI 3-K was inactivated. Taken together, these results indicate that D-3-induced, human monocyte antimycobacterial activity is regulated by PI 3-K and mediated by the NADPH-dependent phagocyte oxidase.