Research Article

Ecotin-Like ISP of L. major Promastigotes Fine-Tunes Macrophage Phagocytosis by Limiting the Pericellular Release of Bradykinin from Surface-Bound Kininogens: A Survival Strategy Based on the Silencing of Proinflammatory G-Protein Coupled Kinin B2 and B1 Receptors

Figure 1

Microvascular plasma leakage and leukocyte accumulation in the HCP sensitized with Leishmania. The data represent relative fluorescence units (RFU: mean ± SD) induced by the topical application of L. major promastigotes (MHOM/JL/80/Friedlin, 500 μL of 1,5 × 107/mL) on hamster cheek pouch preparations (HCPs) after 30 min of stabilization period without significant increase in RFU. Applications of promastigotes were made during 10 min of interrupted superfusion of the HCPs. (a) Pharmacological interventions. Four groups of HCP were sensitized with L. major WT promastigotes, whereas one group (saline control; ) served as untreated control. The first group () corresponds to the positive controls, that is, profile of HCP exposed to L. major alone; the second group () received HOE-140 (0.5 μM) 5 min prior to promastigote application; the third group () received the antagonist of histamine receptor (H1R) mepyramine (10 μM) 5 min prior to challenge with promastigotes; and the fourth group () was pretreated (i.v.) with dextran sulfate 500 (DXS-500; 2 mg/kg) at time of FITC-dextran injection. Plasma leakage was significantly reduced () in all experimental groups subjected to pharmacological interventions. (b) Effect of the mast cell stabilizer cromoglycate. Data represent mean values ± SD obtained in HCP sensitized by L. major WT () and a saline control group (). Two hamsters were given cromoglycate 40 mg/kg i.p. at time of anesthesia induction, and this treatment resulted in a complete inhibition of plasma leakage and leukocyte accumulation elicited by L. major despite the fact that the HCPs responded to histamine stimuli (4 μM) at the end of the experiment, that is, 60 min after topical application of L. major promastigotes. As an internal control, one hamster from the DXS-treated group (, Figure 1(a)) received rhodamine i.v. prior to parasite challenge. Measurements of leukocyte accumulation showed that DXS-500 reduced the Leishmania response to levels below the saline control group while plasma leakage decreased to the level of controls depicted in Figure 1(a) (data not shown). (c) Comparative analysis of kinin/B2R-driven microvascular plasma leakage induced by different Leishmania species. The graph depicts responses evoked by L. major WT and L. chagasi promastigotes (500 μL de 1,5 × 107/mL). L. major WT (blue filled circles, ); L. chagasi (brown filled circles, ); L. chagasi + captopril 1 μM (black crosses, ); L. chagasi + o.5 μM HOE-140 (red squares, ); L. chagasi + 10 μM mepyramine (orange triangles, ); and saline control (grey diamonds, ). The maximal microvascular response to L. major was 5-fold higher than L. chagasi at 50 min after parasite application. The tests involving pharmacological interventions in HCP sensitized with L. chagasi groups were different () from the L. chagasi control at 40 min. (d) Microvascular plasma leakage elicited by L. major . The data represent mean values ± SD. One group represents the microvascular responses evoked by WT L. major (MHOM/JL/80/Friedlin, ) whereas the second group represents responses induced by L. major (). The plasma leakage induced by WT versus promastigotes was significantly different () between 25 and 60 min after topical application of the pathogens.