Immunochemical studies of organ and tumor lipids . XIX . Cytolytic

Rabbit antisera to rat lymphosarcoma contain antibodies that are cytolytic for rat erythrocytes in the presence of complement. The reaction can be inhibited completely by pure cytolipin R showing that (u) immune hemolysis can be mediated through lipid determinants in the membrane, and ( b ) that cytolipin R determinants are present in the intact erythrocyte membrane and exposed on the surface. Optimal conditions for measurement of cytolysis in this system based on release of 51Cr are described. Degrees of specificity of a number of different antilymphosarcoma sera are shown, based on inhibition of cytolysis by cytolipin R, cytolipin K, cytolipin H, cytolipin F (F-hapten), glucocerebroside, galactocerebroside, ceramide trisaccharide (cer-glu-gal-gal), and a mixed brain ganglioside preparation. The data suggest that cytolytic antibodies and agglutinating antibodies in these antisera are distinctive despite their common specificity for cytolipin R. Lymphosarcoma cells are more effective than erythrocytes in absorbing cytolytic antibodies. Supplementary key words haptens . rat erythrocytes hemagglutinin . 61Cr . cytotoxicity . lymphosarcoma membrane CYTOLIPIN R is a ceramide tetrasaccharide containing four carbohydrate residues in the linear sequence glucosegalactose-galactose-N-acetylgalactosamine (Ref. 1 and unpublished studies). I t is therefore very similar in structure to two other naturally occurring ceramide tetrasaccharides, cytolipin K or globoside I (2) and cytolipin F or F-hapten (3), but can be distinguished from them by physical, chemical, and immunological properties. Although cytolipin R was first detected in and isolated from rat lymphosarcoma, it was recently shown that cytolipin R is present in rat erythrocytes (4). This evidence was based on the observation that trypsin-treated erythrocytes could be agglutinated by antisera to rat lymphosarcoma and that the reaction could be completely and specifically inhibited by small quantities of pure cytolipin R. Although untreated erythrocytes were not particularly susceptible to agglutination by these antisera, the presence and availability for reaction of cytolipin R determinants in the intact erythrocyte membrane was deduced from the capacity of such cells to absorb the hemagglutinin. These studies have now been extended. We wish tu describe a cytolytic reaction produced by antibodies directed against cytolipin R determinants, and mediated through these determinants in intact membranes of rat erythrocytes. This reaction is of interest for several reasons. First, it confirms by an independent method the presence and availability for reaction with antibody of cytolipin R determinants in the intact rat erythrocyte membrane. Second, it permits evaluation of the specificity of glycosphingolipid-antibody reactions in a completely new system. And finally, it provides a new model for studying alterations in cell membrane structure through immunochemical reactions involving specific, chemically defined, membrane determinants. In the studies to be described, the antisera were prepared against rat lymphosarcoma, because such antisera always contain antibodies directed against cytolipin R. The cytolytic reaction is complement-dependent, and the cytolytic activities of these antisera were determined using normal rabbit serum as a source of complement. Antibody specificity to various glycosphingolipids (galactorerebroside, glucocerebroside, cytolipin H, cytolipin K, cytolipin F, cytolipin R, ceramide trisaccharide [cer-glu-gal-gal], and a preparation of mixed brain Journal of Lipid Research Volume 13,1922 119 by gest, on O cber 9, 2017 w w w .j.org D ow nladed fom gangliosides) was studied by determining the degree of inhibition of cytolysis with different quantities of pure lipid. MATERIALS AND METHODS