Leishmania amazonensis fails to induce the release of reactive oxygen intermediates by CBA macrophages

Abstract

CBA mouse macrophages effectively control Leishmania major infection, yet are permissive to Leishmania amazonensis. It has been established that some Leishmania species are destroyed by reactive oxygen species (ROS). However, other species of Leishmania exhibit resistance to ROS or even down-modulate ROS production. We hypothesized that L. amazonensis-infected macrophages reduce ROS production soon after parasite-cell interaction. Employing a highly sensitive analysis technique based on chemiluminescence, the production of superoxide (O(·-)(2)) and hydrogen peroxide (H(2)O(2)) by L. major- or L. amazonensis-infected CBA macrophages were measured. L. major induces macrophages to release levels of (O(·-)(2)) 3·5 times higher than in uninfected cells. This (O(·-)(2)) production is partially dependent on NADPH oxidase (NOX) type 2. The level of accumulated H(2)O(2) is 20 times higher in L. major-than in L. amazonensis-infected cells. Furthermore, macrophages stimulated with L. amazonensis release amounts of ROS similar to uninfected cells. These findings support previous studies showing that CBA macrophages are effective in controlling L. major infection by a mechanism dependent on both (O(·-)(2)) production and H(2)O(2) generation. Furthermore, these data reinforce the notion that L. amazonensis survive inside CBA macrophages by reducing ROS production during the phagocytic process.

Figure 1

Leishmania major promastigotes induce NOX-dependent production. Thioglycolate-elicited peritoneal macrophages were incubated with L. major or L. amazonensis promastigotes at a 10:1 ratio at 37°C for 30 min in the presence of lucigenin (25 μm). Control cells were incubated with dead L. major or dead L. amazonensis promastigotes, as well as zymosan, under the same conditions. production was measured using lucigenin-based chemiluminescence (CL), expressed in photon counts. L. major promastigotes induce the release of significantly higher amounts of in comparison with L. amazonensis (n = 11, P<0·001, One-way anova and Newman–Keuls), but these levels did not differ significantly from those produced by control macrophage cultures stimulated with dead parasites (a). Lucigenin-based CL decreased in stimulated cell cultures in response to superoxide dismutase (SOD). SOD (2·5 U/mL) was added at the end of each assay, which confirms that photon released in response to L. major or zymosan is dependent on production (one representative experiment out of eight similar experiments) (b). NOX inhibition by apocynin cause partial reduction in lucigenin-based CL. L. major-infected cells were pretreated for 18 h with apocynin (500 μm) prior to the addition of parasites. production was detected at 37°C for 10 min and was partially reduced by apocynin. Results are expressed as the percentage of the number of photons emitted by apocynin-treated cells (ranging from 57·6 to 193·9 photons) in relation to untreated macrophages considered as 100% (ranging from 126·2 to 318·7 photons) (n = 4, P=0·02, Mann–Whitney U-test) (c).

Figure 2

production by macrophages sequentially stimulated with L. major or L. amazonensis promastigotes. Phagocytic assays were performed by incubating cells with L. major (green) or L. amazonensis (red) promastigotes for 30 min. Next, the parasite stimuli were switched, and the cells were incubated for a second 30-min period, with either L. amazonensis or L. major promastigote (10:1), respectively. These sequential stimulations were performed in the presence of lucigenin (25 μm) at 37°C and production was measured via lucigenin-based chemiluminescence emitted by cells. L. amazonensis did not revert the production induced by L. major in macrophage cultures, yet incubation with L. major in cultures previously stimulated with L. amazonensis did revert relative low levels of production (one representative experiment out of three similar experiments).

Figure 3

L. major promastigotes induce higher levels of H₂O₂ accumulation. Thioglycolate-elicited peritoneal macrophages were incubated with L. amazonensis or L. major promastigotes (10:1) for 30 min at 37°C. H₂O₂ accumulation was measured in cell supernatants by chemiluminescence decay in the presence of luminol (25 μm) and microperoxidase (80 nm) for an additional two min. The highest amount of H₂O₂ was detected in supernatants collected from live L. major-stimulated macrophages (one representative experiment out of five similar experiments) (a). Differences in H₂O₂ accumulation between L. major- and L. amazonensis-infected cells are expressed as the maximal oxidative responses for a given time interval [R] (as described in Materials and Methods). L. major-infected macrophages accumulated twenty times more H₂O₂ than L. amazonensis-infected cells (n = 3, P=0·04, Student’s t-test with Welch’s correction) (b).