Francisella tularensis live vaccine strain (LVS) was grown in culture with nonadherent resident, starchelicited, or Proteose Peptone-elicited peritoneal cells. Numbers of bacteria increased 4 logs over the input inoculum in 48 to 72 h. Growth rates were faster in inflammatory cells than in resident cells: generation times for the bacterium were 3 h in inflammatory cells and 6 h in resident macrophages. LVS-infected macrophage cultures treated with lymphokines did not support growth of the bacterium, although lymphokines alone had no inhibitory effects on replication of LVS in culture medium devoid of cells. Removal of gamma interferon (IFN-y) by immunoaffinity precipitation rendered lymphokines ineffective for induction of macrophage anti-LVS activity, and recombinant IFN-y stimulated both resident and inflammatory macrophage populations to inhibit LVS growth in vitro. Inflammatory macrophages were more sensitive to effects of IFN-y: half-maximal activity was achieved at 5 U/ml for inflammatory macrophages and 20 U/mI for resident macrophages. IFN-'y-induced anti-LVS activity correlated with the production of nitrite (NO2-), an oxidative end product of L-arginine-derived nitric oxide (NO). Anti-LVS activity and nitrite production were both completely inhibited by the addition of either the L-arginine analog NG-monomethyl-L-arginine or anti-tumor necrosis factor antibodies to activated macrophage cultures. Thus, macrophages can be activated by IFN-'y to suppress the growth of F. tularensis by generation of toxic levels of NO, and inflammatory macrophages are substantially more sensitive to activation activities of IFN-y for this effector reaction than are more differentiated resident cells.
We previously showed that a purE mutant (⌬purE201) of Brucella melitensis 16M is attenuated for growth in cultured human monocytes (E.
Alveolar macrophages (AMs) were analyzed for ability to support replication of the intracellular bacterium Francisella tularensis live vaccine strain (LVS). AM supported in vitro growth (2 to 3 logs over 5 days) of LVS with a doubling time of 8 +/- 0.8 h. AMs were analyzed for responsiveness to rIFN-gamma for destruction of this lung pathogen. AM treated with 50 U/ml rIFN-gamma allowed early growth of bacteria (six doublings over 48 h) but between 48 and 96 h rIFN-gamma-treated AM eliminated 1.5 logs of LVS. AMs were sensitive to effects of rIFN-gamma; as little as 5 U/ml rIFN-gamma stimulated AM antimicrobial activity, with half-maximal activity 0.3 U/ml. rIFN-gamma-induced antimicrobial effects in AM correlated with amount of nitrites produced, but nitric oxide played only a minimal role in antibacterial effects induced in AM, because NG-MMLA (specific inhibitor of L-arginine-dependent nitric oxide production) failed to block antimicrobial activity of IFN-gamma-stimulated AM. IL-10, TGF-beta 1, and IFN-alpha (cytokines known to regulate effector functions of activated macrophages) also did not block anti-F. tularensis activity of IFN-gamma-stimulated AM. Reactive oxygen metabolites, depletion of tryptophan, and sequestration of iron did not contribute to anti-F. tularensis activity because addition of superoxide dismutase or catalase, excess iron, or tryptophan to IFN-gamma-treated AM did not reverse the anti-F. tularensis activity observed in these cells. Regulation of AM effector activity differed from that of other macrophage populations, in that while rIFN-gamma-stimulated AM produced TNF-alpha (100 U/ml at 72 h), TNF-alpha was not required as a costimulator for induction of antimicrobial activities by rIFN-gamma because anti-TNF-alpha treatment of rIFN-gamma-stimulated AM blocked TNF-alpha but had no effect on either production of nitrites or anti-F. tularensis activity.
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