Kinins Released by Erythrocytic Stages of Plasmodium falciparum Enhance Adhesion of Infected Erythrocytes to Endothelial Cells and Increase Blood Brain Barrier Permeability via Activation of Bradykinin Receptors

Figures

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  Figure 1. Adhesion of Pf-iRBCs to BMEC monolayers is increased by the supernatant from P. falciparum culture. BMEC were cultured in 24-well plates (4 × 105 cells/well) for 24 h and incubated with Pf-iRBCs (5 × 104 erythrocytes/well, 5% parasitemia). When indicated, BMEC were treated with different compounds before incubation with Pf-iRBCs. The adhesion index was determined by direct counting of adhered erythrocytes per BMEC, as described in the Materials and methods. (A) Representative images of adhesion of Pf-iRBCs to BMEC monolayers. Arrows indicates adhered Pf-iRBCs. Scale bar = 50 μm. (B) Adhesion index was determined in BMEC preincubated or not with increasing concentrations of Pf-iRBC conditioned medium (CM), overnight, before incubation with Pf-iRBCs. (C) Adhesion index was determined in BMECs preincubated or not with 20% control medium (CT-M), 20% Pf-iRBC conditioned medium (CM) or 10−7 M BK before incubation with Pf-iRBCs. (D,E) Effect of 10−7 M DALBK or 10−7 M HOE-140 on the adhesion of Pf-iRBCs to BMEC. BMECs were pretreated with drugs for 30 min prior to overnight incubation with conditioned medium (D) or BK (E). CT-M, control medium, obtained from a suspension of non-infected…

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  Figure 2. P. falciparum infected erythrocytes uptake high molecular weight kininogen from the extracellular medium. Schizont-enriched P. falciparum cultures were incubated overnight with 70 μg/mL high molecular weight kininogen conjugated to fluorescein isothiocyanate (HK-AF488) at 37°C. The cells were harvested, plated in poly-lysine-coated microscope dishes, and analyzed by fluorescence microscopy. Images were analyzed using green (internalized HK-AF488) and blue (parasite nucleus) filters or DIC (erythrocytes). RBC, non-infected erythrocytes; Pf-iRBC, infected erythrocytes. Scale bar = 5 μm.

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  Figure 3. P. falciparum conditioned medium induces endothelial barrier disruption in a BKR-dependent manner. Confluent BMEC monolayers, cultivated in the upper chamber of a transwell system, were pretreated or not with 10−7 M HOE140 or 10−7 M DALBK, for 30 min, before overnight incubation with 20% conditioned medium or 10−7 M BK. The next day, cells were washed and incubated with 15 μg/mL BSA-FITC, for 15 min. The supernatant from the lower chamber was collected for fluorimetry analysis. Bar graph representing the amount of fluorescence detected in the lower chamber for each experimental setup. Albumin permeability was expressed in arbitrary units as the mean ± SE of at least three independent experiments. Statistical significance compared with *control; #BK or conditioned medium (P < 0.05).

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  Figure 4. P. falciparum conditioned medium induces disruption of the endothelial barrier by reducing β-catenin and ZO-1 expression. Sub-confluent BMEC monolayers, cultivated in coverslips, were pretreated or not with 10−7 M HOE140, for 30 min, before overnight incubation with 20% Plasmodium falciparum conditioned medium (CM) (A) or 10−7 M BK (B). The next day, cells were washed and prepared for immunofluorescence as described in Materials and Methods section. Representative photomicrographs of ZO-1 (green), β-catenin (red), DAPI (Blue), and merged channels are shown. Magnification = 630x, Scale bar = 20 μm. The figures are representative of three independent experiments with different cell suspensions. At least three different slides were prepared for each experimental condition and at least five fields per slide, randomly chosen, were analyzed.

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  Figure 5. Proposed model for the effect of kinins liberated in the supernatant of cultures of P. falciparum-infected erythrocytes in the sequestration of iRBCs and endothelial permeability. Erythrocytes that freely traffic in the blood flow acquire adherent properties during P. falciparum malaria infection. PfEMP-1 expressed in the surface of infected erythrocytes is recognized by receptors in the endothelium promoting parasite sequestration. Kinins accumulated in the plasma, through B2R activation, enhances Pf-iRBC sequestration within the microvasculature and induce disruption of the endothelial junctions culminating in leakage.