Objective: To analyze the macrophage activation pattern and its implications in T. cruzi infection resistance after immunization of C57BL/6 with cruzipain. Methods and Results: Mice (n=16) were id immunized with 3-doses weekly of 10ug/ml of cruzipain (immune) or OVA (control) plus CFA. F4/80+ splenocytes showed an augmented CD80 and MHCII expression. Spleen adherent cells (SAC) were a macrophage enriched population (40% F4/80+, 27%CD3+ and 27%CD19+ cells).
Immune SAC produced higher levels of nitric oxide (NO) than controls (16.9±2 vs 3±0.5 uM, p<0.05), and exhibited enhanced iNOS expression at protein and RNA levels. Immune SAC secreted higher levels of IFNg and IL-12 than controls (pg/ml), 5238±297 vs 1636±228 and 374±61 vs 215±20, respectively p<0.05. Intracellular cytokine analysis revealed an increase in percentage of immune splenocytes F4/80+IL-12+, CD19+IL-12+, CD3+IFNg+ and CD19+IFNg+. Although B-cells had intracellular IFNg, cytokine concentration determined by ELISA was under the detection limit when total spleen cells were T-cells depleted. Immune SAC infected with T. cruzi had higher anti-parasite activity than controls. This result was reverted by NOS inhibitor addition to the cultures or by IFNg neutralization. Specific T-cells were essential for NO production, since their depletion diminished IFNg, TNFa and NO concentrations. Conclusion: Cells and cytokines from innate and adaptative immunity induced by cruzipain are critical for macrophage effector response.
