Neural cell adhesion molecule, a new cytoadhesion receptor for Plasmodium falciparum-infected erythrocytes capable of aggregation

Abstract

The cytoadhesion of Plasmodium falciparum-infected erythrocytes (IEs) to the endothelial cells lining the microvasculature, clogging the microvessels of various organs, is a key event in the pathogenesis of certain severe forms of malaria, such as cerebral malaria and pulmonary edema. Studies aiming to identify possible correlations between the severity of clinical cases and the presence of particular cytoadhesion phenotypes have been largely unsuccessful. One of the possible reasons for this failure is that some of the key receptors and/or mechanisms involved have yet to be identified. By combining IE selection, cell transfection, and adhesion inhibition assays, we identified a new cytoadhesion receptor, neural cell adhesion molecule (NCAM). NCAM is a member of the immunoglobulin superfamily and has nonpolysialylated and polysialylated isoforms, the latter being rare in adults. The nonpolysialylated form is present on the surfaces of endothelial cells in the microvessels of various organs in which IE sequestration occurs. We found that multiphenotypic IEs interacted with nonpolysialylated NCAM and with another, as yet unidentified receptor. These IEs also displayed cytoadhesion in flow conditions, presenting the unique ability to form adherent macroaggregates composed of hundreds of IEs. These features may act as virulence factors, increasing the capacity of IEs to clog microvessels via receptor synergy and macroaggregate formation, thereby facilitating the pathogenesis of severe forms of malaria.

FIG. 1.

Inhibition of the cytoadhesion of FCR3^NCAMPSA− IEs to CHO-745 cells by anti-NCAM antibodies. We assessed the ability of anti-NCAM 123C3 monoclonal antibody and H300 and goat anti-NCAM polyclonal antibodies to inhibit FCR3^NCAMPSA− IE cytoadhesion to CHO-745 cells, when added to the cells concomitantly with the IEs (light gray bars) or after incubation of the antibodies for 2 hours with the CHO745 cells before the addition of IEs (dark gray bars). Values correspond to the means ± SD of two experiments for 123C3 and goat-anti-NCAM antibodies without prior incubation (eight values from two experiments performed on two different days) and at least three experiments for the other conditions.

FIG. 2.

Immunocytochemistry of NCAM on NCAM-transfected COS-7, SBEC 1D, and CHO-745 cells. (A) Confocal images of NCAM-transfected COS-7 cells (a and b), SBEC 1D cells (c and d), and CHO-745 cells (e and f) immunostained with anti-PSA (a, c, and e) and anti-NCAM antibodies (b, d, and f). The absence of visible labeling of the SBEC 1D cells in confocal images is due to low levels of expression of the 140-kDa NCAM lacking the PSA chain, as revealed by overexposure of the immunoblot. Scale bar, 10 μm. (B) Immunoblot of PSA-NCAM and NCAM on SBEC 1D and CHO-745 cells and endoneuraminidase (EndoN) digestion profile. Note the shift in molecular size of CHO-745 NCAM induced by EndoN digestion.

FIG. 3.

Cytoadhesion of FCR3^NCAMPSA− IEs to NCAM-transfected COS-7 cells. Controls were set up in which adhesion to Mock-COS-7 cells was assessed without (COS; considered as 100%) or with (COS + Case) prior treatment of the cells with 1 U/ml of chondroitinase ABC. The level of cytoadhesion of IEs per mm² of the control cells is indicated. The cytoadhesion of IEs to NCAM-transfected COS-7 cells without (NCAM-COS) or with (NCAM-COS + Case) prior treatment of the cells with 1 U/ml of chondroitinase ABC is expressed as a percentage of control. Values correspond to the means ± SD of three experiments, except for COS + Case (two experiments).

FIG. 4.

Cytoadhesion of FCR3^NCAMPSA− IEs under flow conditions. FCR3^NCAMPSA− IEs cytoadhered individually (I) or as macroaggregates (MA) to SBEC 1D (A and B) and CHO-745 (C) cells under flow conditions similar to those observed in microvessels (0.05 Pa; A and B). Macroaggregates of less than 100 IEs (A) or thousands of IEs (B) were observed up to a wall shear stress of 0.4 Pa, and numerous individual cytoadherent IEs resisted wall shear stresses of up to 1.6 Pa (C).