Stimulation of nitric oxide production in macrophages by Babesia bovis

Abstract

Gamma interferon (IFN-gamma)-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-gamma, 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-gamma per ml of culture medium. Upregulation of inducible NO synthase mRNA in bovine macrophages by either B. bovis-parasitized erythrocytes and IFN-gamma or CM was also observed. B. bovis-specific T-helper lymphocyte culture supernatants, all of which contained IFN-gamma, 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.

FIG. 1

l-Arginine-dependent production of NO₂⁻ by macrophages exposed to different B. bovis preparations and IFN-γ. Bovine macrophages were exposed to 100 μg of protein per ml of H, HSS, or CM fractions of B. bovis merozoites or a CM fraction of uninfected RBC, either alone (open bars), with 500 U of IFN-γ per ml (solid bars), or with IFN-γ and 250 μM l-NMMA (hatched bars). Cells treated with medium or LPS (2 μg per ml) served as negative and positive controls, respectively. NO₂⁻ levels were determined by the Griess assay. The results are presented as the mean NO₂⁻ concentrations for quadruplicate cultures and 1 SD.

FIG. 2

NO₂⁻ production by macrophages is dependent on the concentration of B. bovis CM and IFN-γ. (A) Macrophages were stimulated with 6.25 to 200 μg of CM per ml alone (diamonds) or in the presence of 100 (circles), 500 (triangles), or 1,000 (squares) U of IFN-γ per ml. (B) Macrophages were incubated with B. bovis CM (100 μg per ml) and 0 to 1,000 U of IFN-γ per ml (diamonds), IFN-γ alone (circles), or CM plus IFN-γ in the presence of 250 μM l-NMMA (squares). (C) Macrophages were stimulated with B. bovis CM (100 μg per ml) in the presence of 0.03 to 8 U of IFN-γ per ml. The results are presented as the mean NO₂⁻ concentrations for quadruplicate cultures.

FIG. 3

Expression of iNOS mRNA after exposure to B. bovis. Total RNA was isolated from macrophages, reverse transcribed, and amplified by PCR with iNOS- or actin-specific primers. (A) Macrophages from animal G6 were cultured for 6 h with medium (lane 1), 500 U of IFN-γ per ml (lane 2), 2 μg of LPS per ml plus IFN-γ (lane 3), 100 μg of B. bovis CM per ml (lane 4), or B. bovis CM plus IFN-γ (lane 5). (B) Macrophages from animal G5 were cultured for 24 h with medium (lane 1), 500 U of IFN-γ per ml (lane 2), 2 μg of LPS per ml (lane 3), LPS plus IFN-γ (lane 4), 100 μg of B. bovis CM per ml (lane 5), or B. bovis CM plus IFN-γ (lane 6). (C) Macrophages from animal G4 were cultured for 6 h with medium (lane 1), uninfected RBC (10% PCV) plus 50 U of IFN-γ per ml (lane 2), B. bovis-infected RBC (10% PCV, 10% PPE) (lane 3), or B. bovis-infected RBC plus IFN-γ (lane 4). (D) Macrophages from animal G6 were cultured for 24 h with uninfected RBC (2.5% PCV) (lane 1), uninfected RBC plus 500 U of IFN-γ per ml (lane 2), B. bovis-infected RBC (2.5% PCV, 4% PPE) (lane 3), or B. bovis-infected RBC plus IFN-γ (lane 4). The reverse transcription-PCR products for iNOS (372 bp) and actin (890 bp) were visualized on ethidium bromide-stained agarose gels, and the relative band intensities were derived by densitometry image analysis and normalization to actin.

FIG. 4

NO production by macrophages exposed to culture supernatants of B. bovis-specific Th-cell lines. NO₂⁻ levels were determined by the Griess assay of culture supernatants from macrophages that were cultured for 96 h with B. bovis-specific T-cell culture supernatants (75%, vol/vol) obtained from C97, G3, and G6 cell lines. Controls consisted of complete RPMI 1640 culture medium or 1,000 U of IFN-γ per ml, and each treatment was performed in the absence (open bars) or presence (solid bars) of 250 μM l-NMMA. Results are shown as the mean NO₂⁻ levels for quadruplicate cultures and 1 SD.