Sialic Acid Mediates the Initial Binding of Positively Charged Inorganic Particles to Alveolar Macrophage Membranes1,2

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Abstract
Pulmonary macrophages phagocytize inhaled particles and are postulated to play a role in the development of pulmonary interstitial fibrogenesis. The basic biologic mechanisms through which inhaled particles bind to macrophage membranes and subsequently are phagocytized remain unclear. We hypothesize that positively charged particles bind to negatively charged sialic acid (SA) residues on macrophage membranes. Alveolar Macrophages (AM) were collected by saline lavage from normal rat lungs. The cells adhered to plastic coverslips in serum-free phosphate buffered saline at 37° C for 45 min and then were maintained at 4° C for the binding experiments. Even distribution of SA groups on AM surfaces was demonstrated by scanning electron microscopy of wheat germ agglutinin (WGA) conjugated to 50 nm gold spheres. The WGA is a lectin that binds specifically to sialic acid, and pretreatment of AM with this lectin prevented the binding of positively charged carbonyl iron (C-Fe) spheres, aluminum (Al) spheres, and chrysotile asbestos fibers to AM surfaces. Limulus protein, another lectin with binding specificity for SA, similarly blocked the binding of positively charged spheres and chrysotile asbestos fibers but not negatively charged glass spheres or crocidolite asbestos fibers. Con A and ricin, lectins that bind to mannose and galactose residues, respectively, did not block particle binding. When both positively charged iron spheres and negatively charged glass spheres were prebound to AM membranes, subsequent treatment with WGA displaced only the positively charged spheres from macrophage surfaces. Con A and ricin had no effect on prebound positively charged C-Fe and Al spheres. In competition experiments, addition of the competing sugar N-acetylglucosamine to the incubation medium containing WGA and C-Fe or Al spheres reversed the inhibitory effect seen with WGA alone. In contrast, the noncompeting sugars mannose and galactosamine did not reverse the inhibitory effects of WGA on carbonyl iron and Al sphere binding. Finally, WGA prebound to AM was displaced by the subsequent addition of both a competing sugar and C-Fe spheres. In contrast, addition of noncompeting sugars and C-Fe spheres did not reverse the inhibitory effect of WGA on positively charged particle binding. In addition, we found that increasing the charge density of Al spheres significantly enhanced the number of particles binding to negatively charged moieties on alveolar macrophage membranes. Considered together, these data support our hypothesis that negatively charged SA groups on macrophage membranes mediate the initial binding of positively charged particles in vitro. This event could precede phagocytosis and be essential for expression of the toxicity that occurs after particle-induced cell injury.