Evaluating specific adhesion of Plasmodium falciparum-infected erythrocytes to immobilised hyaluronic acid with comparison to binding of mammalian cells James G. Beeson * , Stephen J. Rogerson, Graham V. Brown Department of Medicine, University of Melbourne, Royal Melbourne Hospital, Melbourne, Vic. 3050, Australia Received 17 April 2002; received in revised form 16 May 2002; accepted 16 May 2002 Abstract A feature of infection with Plasmodium falciparum is the ability of parasite-infected erythrocytes to adhere to vascular endothelial cells and accumulate in vital organs, associated with severe clinical disease. Hyaluronic acid was recently identified as a receptor for adhesion and has been implicated in mediating the accumulation of parasites in the placenta. Here, we report in vitro assays to measure specific adhesion of infected erythrocytes to hyaluronic acid that is distinct from binding to chondroitin sulphate A, another glycosaminoglycan implicated as a receptor in placental malaria. In this study, specific adhesion of mature stage infected erythrocytes to hyaluronic acid of high purity immobilised on plastic surfaces was abolished by pre-treating hyaluronic acid with a specific hyaluronate lyase from Streptomyces, whereas the same treatment of chondroitin sulphate A had little effect. Adhesion to hyaluronic acid could not be explained by the presence of chondroitin sulphate A or other glycosaminoglycans in the hyaluronic acid preparations. Chinese hamster ovary cells bound in a similar manner in the assays and confirmed that hyaluronic acid was appropriately immobilised for cell adhesion. In contrast to parasites, these cells did not adhere to chondroitin sulphate A. The adsorption of hyaluronic acid onto plastic surfaces was also confirmed by the use of a specific hyaluronic acid-binding protein. Fixing cells with glutaraldehyde at the completion of adhesion assays reduced the number of parasites remaining adherent to hyaluronic acid, but not to chondroitin sulphate A or CD36. These findings have important implications for under- standing and evaluating interactions between P. falciparum and hyaluronic acid that may be involved in disease pathogenesis. q 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. Keywords: Plasmodium falciparum; Cell adhesion; Hyaluronic acid; Chondroitin sulphate; Placenta; Ovarian cell 1. Introduction The pathogenesis of malaria caused by Plasmodium falci- parum involves the adhesion of parasite-infected erythro- cytes to the vascular endothelium (reviewed in Beeson and Brown, 2002). Adhesion may contribute to parasite survival through avoidance of immune-mediated destruc- tion and splenic clearance from the circulation. However, the accumulation or sequestration of large numbers of para- sites in the vascular beds of organs such as the brain, lung, and placenta can lead to severe disease in the host, with substantial mortality. Infected erythrocytes can adhere to a range of receptors on the endothelial surface, such as CD36, intercellular adhesion molecule 1, and chondroitin sulphate A (Barnwell et al., 1989; Berendt et al., 1989; Ockenhouse et al., 1989; Rogerson et al., 1995). We have recently iden- tified hyaluronic acid (or hyaluronan) as an additional recep- tor for parasite adhesion, in the context of placental malaria (Beeson et al., 2000). Hyaluronic acid is a non-sulphated high molecular weight glycosaminoglycan composed of repeating disaccharide units of N-acetylglucosamine linked to glucuronic acid. It is abundant in the extracellular matrix of many tissues, but is also expressed on the surface of endothelial cells and syncy- tiotrophoblasts, which line the placental blood spaces (Matejevic et al., 2001; Mohamadzadeh et al., 1998; Sunderland et al., 1985). Infected erythrocytes can bind to immobilised purified hyaluronic acid, in static assays and under conditions of physiologically-relevant flow, and the minimal length of hyaluronic acid for parasite interaction appears to be 12 monosaccharide units (Beeson et al., 2000; Chai et al., 2001). Plasmodium falciparum-infected erythro- cytes are phenotypically diverse and can undergo clonal antigenic variation (Beeson and Brown, 2002). As for adhe- sion to other receptors, parasite adhesion to hyaluronic acid International Journal for Parasitology 32 (2002) 1245–1252 0020-7519/02/$20.00 q 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. PII: S0020-7519(02)00097-8 www.parasitology-online.com * Corresponding author. Tel.: 161-3-8344-6252; fax: 161-3-9347-1863. E-mail address: beeson@unimelb.edu.au (J.G. Beeson).