Superoxide is formed by human mononuclear phagocytes in response to leishmahia chagasipromastigotes using spin trapping techniques

Leisfmanit chegasi (Lc), a cause of visceral leishmaniasls, is an obligative Intracellular parasite of mammalian macrophages. Macrophage infection with the promastigote form of Lc Is initiated by phagocytosis, during which the promastigote must avoid killing by Superoxide (Oand hydrogen peroxide (HbOJ generated by the macrophage NADPHdependent oxidase. Early studies suggested that macrophages and peripheral blood monocytes (MC) generate O2VH2O2 upon phagocytosis of Lc promastlgotes. More recent data suggest that the process may not occur due to the ability of Lc llpophosphoglycan (LPG) to inhibit phagocyte protein kinase C, a key enzyme in the signal transduction process which initiates (V/HjOj formation. In the present study, we used spin trapping techniques In conjunction with EPR to Investigate the ability of human MC and monocyte-dertved macrophages (MOM) to form 02' In response to Lc promastlgotes. When MC or MDM were incubated with Lc promastlgotes at 37°C for 30 mln in the presence of the spin trap DMPO, we did not detect OMPO-derived spin adducts indicative of CY generation. Different results were obtained when Lc promastigotes were opsonized with 0.5% pooled human serum or 5% CS-deficient serum. Incubation of MC or MDM with opsonized promastigotes in the presence of DMPO yielded EPR detectable formation of the DMPOrOH spin adduct. DMPCVOH production was inhibited by SOD, but not catalase indicating the spin trapping of O2. Promastigotes harvested during log phase of In vitro growth are less virulent In animal models than those in stationary growth phase. No differences were observed between the magnitude of Oj~ spin trapped when MC or MDM were incubated with opsonized log vs stationary phase promastigotes. Opsonized virulent or attenuated (long-term in vitro passage) promastigotes also did not differ In their Induction of MC or MDM O2' formation. Although MC and MDM phagocytose both opsonized and non-opsonized Lc promastigotes, only opsonized promastigotes result in MC/MDM CV production. We conclude that promastigote LPG does not prevent MC/MDM O2' generation during early Infection. Future work will identify the receptor ligand interactions which initiate phagocyte Cb formation.