Ceramide-Enriched Membrane Domains in Red Blood Cells and the Mechanism of Sphingomyelinase-Induced Hot−Cold Hemolysis

Abstract

Hot−cold hemolysis is the phenomenon whereby red blood cells, preincubated at 37 °C in the presence of certain agents, undergo rapid hemolysis when transferred to 4 °C. The mechanism of this phenomenon is not understood. PlcHR2, a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa, that is the prototype of a new phosphatase superfamily, induces hot−cold hemolysis. We found that the sphingomyelinase, but not the phospholipase C activity, is essential for hot−cold hemolysis because the phenomenon occurs not only in human erythrocytes that contain both phosphatidylcholine (PC) and sphingomyelin (SM) but also in goat erythrocytes, which lack PC. However, in horse erythrocytes, with a large proportion of PC and almost no SM, hot−cold hemolysis induced by PlcHR2 is not observed. Fluorescence microscopy observations confirm the formation of ceramide-enriched domains as a result of PlcHR2 activity. After cooling down to 4 °C, the erythrocyte ghost membranes arising from hemolysis contain large, ceramide-rich domains. We suggest that formation of these rigid domains in the originally flexible cell makes it fragile, thus highly susceptible to hemolysis. We also interpret the slow hemolysis observed at 37 °C as a phenomenon of gradual release of aqueous contents, induced by the sphingomyelinase activity, as described by Ruiz-Argüello et al. [(1996) J. Biol. Chem. 271, 26616]. These hypotheses are supported by the fact that ceramidase, which is known to facilitate slow hemolysis at 37 °C, actually hinders hot−cold hemolysis. Differential scanning calorimetry of erytrocyte membranes treated with PlcHR2 demonstrates the presence of ceramide-rich domains that are rigid at 4 °C but fluid at 37 °C. Ceramidase treatment causes the disapperance of the calorimetric signal assigned to ceramide-rich domains. Finally, in liposomes composed of SM, PC, and cholesterol, which exhibit slow release of aqueous contents at 37 °C, addition of 10 mol % ceramide and transfer to 4 °C cause a large increase in the rate of solute efflux.