Platelet-activating factor induces nitric oxide synthesis in Trypanosoma cruzi-infected macrophages and mediates resistance to parasite infection in mice

Abstract

Trypanosoma cruzi replicates in nucleated cells and is susceptible to being killed by gamma interferon-activated macrophages through a mechanism dependent upon NO biosynthesis. In the present study, the role of platelet-activating factor (PAF) in the induction of NO synthesis and in the activation of the trypanocidal activity of macrophages was investigated. In vitro, PAF induced NO secretion by T. cruzi-infected macrophages and the secreted NO inhibited intracellular parasite growth. The addition of a PAF antagonist, WEB 2170, inhibited both NO biosynthesis and trypanocidal activity. The inducible NO synthase/L-arginine pathway mediated trypanocidal activity, since it was inhibited by treatment with L-N-monomethyl arginine (L-NMMA), an L-arginine analog. PAF-mediated NO production in infected macrophages appears to be dependent on tumor necrosis alpha (TNF-alpha) production, since the addition of a neutralizing anti-TNF-alpha monoclonal antibody mAb inhibited NO synthesis. To test the role of PAF in mediating resistance or susceptibility to T. cruzi infection, infected mice were treated with WEB 2170, a PAF antagonist. These animals had higher parasitemia and earlier mortality than did vehicle-treated mice. Taken together, our results suggest that PAF belongs to a group of mediators that coordinate the mechanisms of resistance to infections with intracellular parasites.

FIG. 1

PAF induces NO production by T. cruzi-infected macrophages. C3H/HeJ-derived thioglycolate-elicited peritoneal macrophages were cultured with T. cruzi trypomastigotes (Y strain) at a parasite-to-host cell ratio of 1:1 for 48 h in the presence of PAF (A) or IFN-γ (B) at 37°C in a humidified chamber containing 5% CO₂. The supernatants were harvested, and the nitrite concentration was assayed by the Griess method. Bars represent the mean and standard deviation (SD) of triplicate samples from one of three independent experiments. ∗, P < 0.05 compared with the values obtained with infected cells cultured in medium (Med) alone.

FIG. 2

Anti-TNF-α MAb inhibits PAF-induced NO production. C3H/HeJ-derived peritoneal macrophages were cultured with T. cruzi trypomastigotes (Tc) at a parasite-to-cell ratio of 1:1 with or without PAF (10⁻⁷ M) or PAF plus anti-TNF-α MAb (aTNF) (50 μg/ml). After 48 h, the supernatants were harvested and the nitrite concentration was assayed by the Griess method. Bars represent mean ± SD of triplicate samples. The results are representative of three independent experiments. ∗, P < 0.05 compared with the values obtained with cells cultured in the presence of medium (Med) alone.

FIG. 3

WEB 2170 and l-NMMA inhibit PAF-induced NO production. C3H/HeJ-derived peritoneal macrophages were cultured with T. cruzi trypomastigotes (Tc) at a parasite-to-cell ratio of 1:1 with or without PAF (10⁻⁷ M) plus WEB 2170 (WEB) (10⁻⁵ M), PAF plus l-NMMA (LN) (200 mM), or lyso-PAF (Lyso) (10⁻⁷ M). After 48 h, the supernatants were harvested and the nitrite concentration was assayed. Bars represent the mean and SD of triplicate samples. The results are representative of three independent experiments. ∗, P < 0.05 compared with the values obtained with cells cultured in the presence of medium alone.

FIG. 4

Parasite growth is inhibited in PAF-treated macrophages. C3H/HeJ-derived peritoneal adherent cells were cultured with T. cruzi trypomastigotes at a parasite-to-host-cell ratio of 1:1 and with various concentrations of PAF (A) or IFN-γ (B) in a humidified chamber containing 5% CO₂ at 37°C. The number of intracellular parasites in at least 500 cells was determined by light microscopy. Symbols represent the mean and SD of triplicate counts of 100 cells. The data are representative of three independent experiments. ∗, P < 0.05 compared to the values obtained with infected cells cultured in medium alone.

FIG. 5

PAF-induced microbicidal activity is decreased by inhibition of the PAF receptor or iNOS activation. C3H/HeJ-derived peritoneal adherent cells were infected with T. cruzi trypomastigotes at a parasite-to-host-cell ratio of 1:1 in the absence of stimulus (Med) or with lyso-PAF (L-PAF) (10⁻⁷ M) or PAF (10⁻⁷ M) with or without WEB 2170 (10⁻⁵ M) (WEB) or l-NMMA (200 mM) (LN) and cultured for 48 h in a humidified chamber containing 5% CO₂ at 37°C. Bars represent the mean and SD of triplicate counts of the number of intracellular parasites found in 100 macrophages. The data are representative of three independent experiments. ∗, P < 0.05 compared to the values obtained with infected cells cultured in medium alone.

FIG. 6

The presence of PAF decreases the release of parasites from infected macrophages. C3H/HeJ-derived peritoneal macrophages were infected with T. cruzi trypomastigotes (Y strain) at a parasite-to-host-cell ratio of 1:1 in a humidified chamber containing 5% CO₂ at 37°C. Two hours later, extracellular parasites were removed and the infected cells were incubated with medium alone (Med), PAF (10⁻⁷ M), or IFN-γ (100 U/ml). On day 5 postinfection, the parasites released into the medium were counted. Bars represent the mean and SD of triplicate samples and are representative of three independent experiments. ∗, P < 0.05 compared to the values obtained with infected cells cultured in medium alone.

FIG. 7

PAF mediates resistance in mice infected with T. cruzi. BALB/c mice were infected with 10⁴ (A) or 10³ (B) blood trypomastigotes and treated with WEB 2170 (●) or with vehicle (○) 4 h before and again once daily after infection with T. cruzi. Parasitemia (A) and survival rates (B) were evaluated during the acute phase. Lines represent means and SD of data obtained with 10 mice per group in one of three independent experiments. ∗, P < 0.05 compared to the values obtained for parasitemia in vehicle-treated mice.