Stimulation of Nitric Oxide Production in Macrophages by Babesia bovis

Gamma interferon (IFN-γ)-activated macrophages are believed to play a key role in resistance to Babesia bovis through parasite suppression by macrophage secretory products. However, relatively little is known about interactions between this intraerythrocytic parasite and the macrophages of its bovine host. In this study, we examined the in vitro effect of intact and fractionated B. bovis merozoites on bovine macrophage nitric oxide (NO) production. In the presence of IFN-γ, B. bovis merozoites stimulated NO production, as indicated by the presence of increased l-arginine-dependent nitrite (NO₂⁻) levels in culture supernatants of macrophages isolated from several cattle. The merozoite crude membrane (CM) fraction stimulated greater production of NO, in a dose-dependent manner, than did the merozoite homogenate or the soluble, cytosolic high-speed supernatant fraction. Stimulation of NO production by CM was enhanced by as little as 1 U of IFN-γ per ml of culture medium. Upregulation of inducible NO synthase mRNA in bovine macrophages by either B. bovis-parasitized erythrocytes and IFN-γ or CM was also observed. B. bovis-specific T-helper lymphocyte culture supernatants, all of which contained IFN-γ, were also found to induce l-arginine-dependent NO₂⁻ production. Supernatants that induced the highest levels of NO also contained biologically active TNF. These results show that B. bovis merozoites and antigen-stimulated B. bovis-immune T cells can induce the production of NO, a molecule implicated in both protection and pathologic changes associated with hemoprotozoan parasite infections.Bovine babesiosis is an economically important tick-borne disease of cattle that is caused by intraerythrocytic apicomplexan parasites of the genus Babesia. Babesia bovis-infected erythrocytes undergo sequestration by attachment to capillary endothelium in a manner reminiscent of the most severe form of human malaria, caused by Plasmodium falciparum; this results in organ damage, cerebral dysfunction, and pulmonary edema (58). It has been hypothesized that the severe organ abnormalities that occur during acute B. bovis infection, similar to those observed during experimental malaria, are mediated in part by inflammatory cytokines, including gamma interferon (IFN-γ) and tumor necrosis factor (TNF), and nitric oxide (NO) (58).Although activation of macrophages could lead to immunopathological consequences, macrophages are also believed to be important for immunity to B. bovis and other intraerythrocytic parasites via removal of parasitized erythrocytes by phagocytosis and as antigen-presenting cells (APC) for T-helper (Th) lymphocytes. Furthermore, macrophage secretory products have been shown to inhibit the growth of P. falciparum and B. bovis in vitro (24, 34). When generated by chemical donors or activated macrophages in vitro, NO and its reactive nitrogen intermediate derivatives were shown to inhibit intracellular parasites including Leishmania major, P. falciparum, and B. bovis (22, 23, 27, 40, 53, 54).Studies in mice with B. microti, P. yoelii, P. vinckei, and P. chabaudi demonstrated that IFN-γ, TNF-α, and TNF-β are important components of immunity to these parasites (3, 15, 32, 44, 47). IFN-γ facilitates the phagocytosis of P. falciparum-infected erythrocytes by human macrophages (33, 35). In addition, parasite-specific, IFN-γ-producing human CD4⁺ Th-cell clones inhibited the growth of P. falciparum in the presence of adherent peripheral blood mononuclear cells (PBMC) in vitro (18, 37). The protective role of TNF-α appears to depend on the timing of its appearance, which may partially explain the paradoxical roles of TNF in protection and immunopathology. In resistant C57BL/6 mice, TNF-α appeared early during P. chabaudi infection, whereas in susceptible A/J mice, high levels of TNF-α were observed only late in infection, just preceding death (25).Experiments performed during the past decade showed that malarial parasites and secreted toxins induced TNF-α in human and murine macrophages and inducible NO synthase (iNOS) in murine macrophages. Macrophages exposed in vitro to P. yoelii, P. berghei, or P. falciparum produced TNF-α, which was enhanced by IFN-γ (4, 36, 50, 51). Similarly, P. falciparum extract, in the presence of IFN-γ, stimulated murine macrophages to produce both TNF-α and NO (31, 41). However, the interpretation of these results has been questioned by the recent discovery that many continuously cultured strains of P. falciparum are contaminated with Mycoplasma species (56). Mycoplasma organisms induce TNF-α and other inflammatory mediators in murine, human, and bovine macrophages (28–30, 56), and experiments with Mycoplasma-free P. falciparum are being repeated to verify that the induction of inflammatory cytokines and NO was due to the parasite itself (43).Several observations support the hypothesis that IFN-γ produced by effector Th cells plays a key role in protection against babesiosis (10). In cattle, IFN-γ regulates B-cell synthesis of the opsonizing immunoglobulin G2 subclass (17). Parasite-specific Th-cell clones isolated from B. bovis-immune cattle produce IFN-γ and TNF (11, 14). Bovine macrophages have upregulated expression of iNOS when activated by IFN-γ in the presence of bacterial LPS (2) or TNF-α (20). Thus, the induction of macrophage iNOS by either B. bovis extracts or antigen-activated T cells would be indicative of macrophage activation and a potential babesiacidal effector mechanism. Conversely, overproduction of NO could also contribute to the pathologic changes associated with infection, including cerebral babesiosis.This study was undertaken to determine if bovine macrophages produce NO when exposed to B. bovis in the presence or absence of IFN-γ. Potential Mycoplasma contamination of B. bovis cultures was ruled out by a PCR-based assay. We report, for the first time, that bovine macrophages produce NO following in vitro exposure to B. bovis merozoites or a membrane-enriched fraction. The effect of different concentrations of either IFN-γ or B. bovis on NO production was also examined. Furthermore, the functional relevance of B. bovis-specific Th cells that produce IFN-γ and TNF was evaluated by the determining the ability of Th-cell supernatants to stimulate NO production. Induction of iNOS was confirmed by demonstrating reduced nitrite (NO₂⁻) levels in the presence of N^G-monomethyl-l-arginine (l-NMMA) and enhanced levels of iNOS steady-state mRNA in macrophages cultured with B. bovis.