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

Macrophagelike RAW264.7 cells are killed by the combination of gamma interferon (IFN--y) treatment and infection with Rickettsia prowazekii. The roles of tumor necrosis factor alpha (TNF-at), 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-ot. However, dead rickettsiae (which cannot kill the IFN-y-treated RAW264.7 cells) induced the production of as much TNF-o as viable rickettsiae. Inhibition of the production of TNF-ax (by the addition of actinomycin D or emetine during the rickettsial infection) or neutralization of TNF-a (by the addition of polyclonal rabbit anti-mouse TNF-at serum both during the IFN--y 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--y treatment did not inhibit the ability of IFN-y 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-y 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-y plus LPS compared with that in cultures treated with IFN-'y alone, and inhibition of nitric oxide synthase restored the rickettsia-induced killing of the RAW264.7 cells in cultures treated with IFN-y plus LPS. These data indicate that (i) TNF-a, LPS, and the nitric oxide synthase pathway are not required in order for IFN-y to prepare RAW264.7 cells to be killed by R. prowazekii; (ii) neither TNF-ot nor the nitric oxide synthase pathway is responsible for the killing of the IFN-y-treated RAW264.7 cells by R. prowazekii; and (iii) in cultures treated with IFN-y plus LPS and then incubated with rickettsiae, a nitric oxide synthase pathway-dependent mechanism inhibits the killing of the RAW264.7 cells. * Corresponding author. Phone: (205) 460-6925. Fax: (205) 460-7269. on July 15, 2020 by guest http://iai.asm.org/ Downloaded from

Nitric Oxide-Mediated Inhibition of the Ability ofRickettsia prowazekiiTo Infect Mouse Fibroblasts and Mouse Macrophagelike Cells

“…Our results demonstrate that suppression of the initial R. prowazekii infection in appropriately treated L929 cells (Table 1) and RAW264.7 cells (Fig. 2) and protection of IFN-␥-plus-LPS-treated RAW264.7 cells from R. prowazekii-mediated, rapid killing (29) are both dependent on the NOS pathway. It is likely that another cytokine in the LK acts together with IFN-␥ to induce NOS in L929 cells.…”

“…NOS pathway-dependent suppression of the initial rickettsial infection in RAW264.7 cell cultures treated with IFN-␥ plus LPS. We previously determined that substantial concentrations of nitrite (averaging about 50 M) are present in culture media collected from RAW264.7 cells treated for 21 h with IFN-␥ (25 U/ml) plus LPS (80 ng/ml), whereas much lower concentrations are detected in media collected from RAW264.7 cell cultures treated with IFN-␥ alone or LPS alone (29). To determine if NOS pathway-dependent suppression of the initial rickettsial infection occurs in macrophagelike RAW264.7 cell cultures treated with IFN-␥ plus LPS (and to begin evaluating the hypothesis that suppression of the rapid killing of the RAW264.7 cells occurs in these cultures [29] because the rickettsiae are prevented from infecting the RAW264.7 cells), the initial rickettsial infections in untreated RAW264.7 cell cultures, cultures treated with IFN-␥ plus LPS, and cultures treated with IFN-␥ plus LPS plus aminoguanidine were compared (Fig.…”

“…The mixture was exposed to air, and the entire volume (300 l) was added to the well of RAW264.7 cells. The 24-well plates were centrifuged (500 ϫ g for 15 min at room temperature) and then incubated at 34°C as previously described (29). The initial rickettsial infection and viability of the RAW264.7 cells were determined as described above.…”

“…In addition, such investigations determined that a lack of the amino acid tryptophan does not explain the ability of IFN-␥ to limit the growth of R. prowazekii in either mouse L929 cells (in which tryptophan is not depleted in response to IFN-␥) or human fibroblasts (in which reconstitution of the depleted tryptophan pool in IFN-␥-treated cells does not alleviate the inhibition of rickettsial growth) (25). Furthermore, such studies revealed (i) that both NOS-independent and NOS-dependent mechanisms are involved in suppressing the growth of R. prowazekii in mouse L929 cells treated with IFN-␥ plus TNF-␣ (28), (ii) that the inhibition of growth of R. prowazekii observed in L929 cells treated with either IFN-␥ alone or TNF-␣ alone is largely NOS independent (28), (iii) that the mechanism responsible for IFN-␥-induced, NOS-independent inhibition of growth of R. prowazekii in mouse L929 cells is likely to exist also in IFN-␥-treated human fibroblasts (30), and (iv) that the cytotoxic effects on host cells brought about by the combination of cytokine treatment and R. prowazekii infection are not dependent on NOS (28,29). These effects of cytokine treatment on R. prowazekii-host cell interactions are observed in host cells infected with either the virulent Breinl strain or the avirulent Madrid E strain of R. prowazekii.…”

“…In RAW264.7 cell cultures pretreated with a low concentration of IFN-␥ plus lipopolysaccharide (LPS), decreased R. prowazekii (Madrid E)-mediated, rapid killing of the RAW264.7 cells is associated with increased production of nitric oxide (NO), and addition of NOS inhibitors restores the R. prowazekii-mediated killing of the RAW264.7 cells in such cultures to a level comparable to that observed in RAW264.7 cultures pretreated with a low concentration of IFN-␥ alone (29). Because the initial rickettsial infections were not monitored in these experiments, it is not known if the decreased R. prowazekii-mediated, rapid killing of the RAW264.7 cells observed in cultures treated with IFN-␥ plus LPS is associated with lower initial rickettsial infection.…”