Cytoadherence of Plasmodium berghei-infected red blood cells to murine brain and lung microvascular endothelial cells in vitro

Abstract

Sequestration of infected red blood cells (iRBC) within the cerebral and pulmonary microvasculature is a hallmark of human cerebral malaria (hCM). The interaction between iRBC and the endothelium in hCM has been studied extensively and is linked to the severity of malaria. Experimental CM (eCM) caused by Plasmodium berghei ANKA reproduces most features of hCM, although the sequestration of RBC infected by P. berghei ANKA (PbA-iRBC) has not been completely delineated. The role of PbA-iRBC sequestration in the severity of eCM is not well characterized. Using static and flow cytoadherence assays, we provide the first direct in vitro evidence for the binding of PbA-iRBC to murine brain and lung microvascular endothelial cells (MVEC). We found that basal PbA-iRBC cytoadherence to MVECs was significantly higher than that of normal red blood cells (NRBC) and of RBC infected with P. berghei K173 (PbK173-iRBC), a strain that causes noncerebral malaria (NCM). MVEC prestimulation with tumor necrosis factor (TNF) failed to promote any further significant increase in mixed-stage iRBC adherence. Interestingly, enrichment of the blood for mature parasites significantly increased PbA-iRBC binding to the MVECs prestimulated with TNF, while blockade of VCAM-1 reduced this adhesion. Our study provides evidence for the firm, flow-resistant binding to endothelial cells of iRBC from strain ANKA-infected mice, which develop CM, and for less binding of iRBC from strain K173-infected mice, which develop NCM. An understanding of P. berghei cytoadherence may help elucidate the importance of sequestration in the development of CM and aid the development of antibinding therapies to help reduce the burden of this syndrome.

Fig 1

(A) Adhesion of Plasmodium berghei ANKA- and K173-infected red blood cells to murine brain and lung microvascular endothelial cells. Static assays were performed as described in Materials and Methods; notably, mixed-stage iRBC were incubated for 90 min on resting or activated (TNF, 50 ng/ml) endothelial cells. More PbA-iRBC than NRBC and PbK173-iRBC bound to both B3 and L2 MVECs. Data shown are the mean numbers ± SEM of RBC per field of view from at least 15 fields from 3 separate experiments (*, P < 0.05). (B) Adhesion of Plasmodium berghei ANKA-infected red blood cells on TNF-stimulated murine brain microvascular endothelial monolayers. Representative bright-field image with fluorescent Calcein-labeled RBC on TNF-activated (50 ng/ml) brain MVEC (×400 magnification; Olympus IX71 inverted microscope). In the magnified inset, arrows indicate bound RBC.

Fig 2

Firm, flow-resistant binding of PbA-iRBC to murine brain and lung microvascular endothelial cells. RBC were resuspended at 1% hematocrit and perfused over endothelial monolayers for 15 min at a flow rate of 0.227 ml/min in a flow chamber. More PbA-iRBC adhered than did NRBC, despite a shear stress of 0.05 Pa being maintained in the flow chamber. Data shown are the mean numbers ± SEM of RBC per field of view from at least 15 fields from 3 separate experiments (*, P < 0.05).

Fig 3

Phenotyping of B3 and L2 cells under resting and inflammatory conditions. Endothelial cells were activated with TNF (10 ng/ml) or allowed to rest for 24 h prior to direct immunolabeling and subsequent flow cytometry analysis. The cell surface expression levels of ICAM-1 and VCAM-1 on resting (red line) and TNF-activated (blue line) B3 and L2 cell lines are demonstrated. Histograms are representative of three experiments and show the number of cells positively labeled with these antibodies and their corresponding isotype controls (shaded gray).

Fig 4

Enrichment for mature parasites increases the number of RBC bound to TNF-stimulated endothelium, and this binding is VCAM-1 dependent. Prestimulation with TNF significantly increased the number of mature PbA-iRBC bound compared to that for resting monolayers. Binding of mature PbA-iRBC was reduced in the presence of monoclonal antibody raised against VCAM-1 but not ICAM-1. Data shown are the mean numbers ± SEM of RBC per field of view (magnification, ×400) from at least 15 fields from 3 separate experiments (*, P < 0.05). ctrl, control.