Effect of iron and phagocytosis on murine macrophage activation in vitro

Iron-exposed murine macrophages have a modified bactericidal activity as shown by previous observations. In order to assess the role of iron in macrophage activation, as measured by free radical production and by intracellular bacterial killing, murine peritoneal macrophages were cultivated in the presence of various sources of iron, human iron-saturated transferrin and ammonium ferric citrate, or iron chelators, Desferal, and human Apo-transferrin, and were infected with an enteropathogenic strain ofE. coli. The release of nitrite (NO₂ ^?), and the production of superoxide anion (O₂ ^?) and hydrogen peroxide (H₂O₂) by the phagocytes were measured and compared to the production by uninfected macrophages. The synergistic action with murine r.IFN-γ was also studied in the radical production reaction and for the bactericidal activity of macrophages. Our results show that in vitro phagocytosis ofE. coli induced elevated production of NO₂ ^? and H₂O₂ by macrophages, and that oxygen derivatives were released independently of the presence of added iron or chelator. Despite a phagocytosis-related enhancement of NO₂ ^? release, reactive nitrogen intermediates (RNI) are not directly involved in the bactericidal mechanism, as revealed by increased intracellular killing owing to RNI inhibitors. Moreover, bacterial killing may depend on oxygen derivatives, as suggested by the effect of the antioxidant sodium ascorbate leading to both a diminished H₂O₂ production and a decreased bactericidal activity of macrophages.