Relationship of tumor necrosis factor alpha, the nitric oxide synthase pathway, and lipopolysaccharide to the killing of gamma interferon-treated macrophage-like RAW264.7 cells by Rickettsia prowazekii

Abstract

Macrophage-like RAW264.7 cells are killed by the combination of gamma interferon (IFN-gamma) treatment and infection with Rickettsia prowazekii. The roles of tumor necrosis factor alpha (TNF-alpha), the nitric oxide synthase pathway, and lipopolysaccharide (LPS) in this killing were investigated. R. prowazekii, both the Breinl and Madrid E strains, induced RAW264.7 cells to produce TNF-alpha. However, dead rickettsiae (which cannot kill the IFN-gamma-treated RAW264.7 cells) induced the production of as much TNF-alpha as viable rickettsiae. Inhibition of the production of TNF-alpha (by the addition of actinomycin D or emetine during the rickettsial infection) or neutralization of TNF-alpha (by the addition of polyclonal rabbit anti-mouse TNF-alpha serum both during the IFN-gamma treatment and during the rickettsial infection) did not inhibit the killing of the RAW264.7 cells. Addition of polymyxin B (which inhibits many effects of LPS) during the IFN-gamma treatment did not inhibit the ability of IFN-gamma to prepare the RAW264.7 cells to be killed by R. prowazekii. Suppression of nitrite production by addition of the nitric oxide synthase inhibitor aminoguanidine both during the IFN-gamma treatment and during the rickettsial infection also did not inhibit the killing of the RAW264.7 cells. R. prowazekii-mediated killing of the RAW264.7 cells was dramatically suppressed in cultures treated with IFN-gamma plus LPS compared with that in cultures treated with IFN-gamma alone, and inhibition of nitric oxide synthase restored the rickettsia-induced killing of the RAW264.7 cells in cultures treated with IFN-gamma plus LPS. These data indicate that (i) TNF-alpha, LPS, and the nitric oxide synthase pathway are not required in order for IFN-gamma to prepare RAW264.7 cells to be killed by R. prowazekii; (ii) neither TNF-alpha nor the nitric oxide synthase pathway is responsible for the killing of the IFN-gamma-treated RAW264.7 cells by R. prowazekii; and (iii) in cultures treated with IFN-gamma plus LPS and then incubated with rickettsiae, a nitric oxide synthase pathway-dependent mechanism inhibits the killing of the RAW264.7 cells.