Inflammation enhances the secretion of sphingomyelinases (SMases). SMases catalyze the hydrolysis of sphingomyelin into phosphocholine and ceramide. In erythrocytes, ceramide formation leads to exposure of the removal signal phosphatidylserine (PS), creating a potential link between SMase activity and anemia of inflammation. Therefore, we studied the effects of SMase on various pathophysiologically relevant parameters of erythrocyte homeostasis. Time-lapse confocal microscopy revealed a SMase-induced transition from the discoid to a spherical shape, followed by PS exposure, and finally loss of cytoplasmic content. Also, SMase treatment resulted in ceramide-associated alterations in membrane–cytoskeleton interactions and membrane organization, including microdomain formation. Furthermore, we observed increases in membrane fragility, vesiculation and invagination, and large protein clusters. These changes were associated with enhanced erythrocyte retention in a spleen-mimicking model. Erythrocyte storage under blood bank conditions and during physiological aging increased the sensitivity to SMase. A low SMase activity already induced morphological and structural changes, demonstrating the potential of SMase to disturb erythrocyte homeostasis. Our analyses provide a comprehensive picture in which ceramide-induced changes in membrane microdomain organization disrupt the membrane–cytoskeleton interaction and membrane integrity, leading to vesiculation, reduced deformability, and finally loss of erythrocyte content. Understanding these processes is highly relevant for understanding anemia during chronic inflammation, especially in critically ill patients receiving blood transfusions.

1032 Sphingomyelinases (SMases) catalyze the hydrolysis of sphingomyelin, a major lipid component of cell membranes, generating phosphocholine and ceramide. Inflammation arising from various diseases including sepsis is known to enhance SMase secretion in the circulation. Also, chronic inflammation typically induces anemia through reduced production and enhanced clearance of red cells, which is often associated with poor disease outcome. Incubation of red cells with plasma of septic patients with enhanced SMase activity has been described to result in ceramide formation and exposure of phosphatidylserine (PS). Therefore, we studied the effect of SMase on various pathophysiologically relevant parameters of red cell homeostasis in fresh and stored erythrocytes. Using time-lapse confocal microscopy we observed a typical sequence of events during SMase treatment of erythrocytes. The cells first lost their discoid shape, PS became externalized, and ultimately there was a sudden loss of cytoplasmic content. Additional confocal microscopy, flow cytometry and immunoblot analyses showed that SMase treatment resulted in ceramide-induced alterations in red cell membrane-cytoskeleton interaction and organization, including lipid raft formation. These changes probably underlie the increase in osmotic fragility, membrane vesiculation and invagination, as well as large hemoglobin aggregates that we also observed. The membrane lipid scrambling, altered morphology, enhanced fragility and presence of rigid membrane patches may all contribute to enhanced removal of sphingomyelinase-exposed red cells. Generation of very low ceramide levels in the membrane by SMase, as detected by Positive-Ion MALDI-TOF MS, already affected red cell shape, membrane rearrangement and osmotic responsiveness. Combined with the observation that 24 hour incubation of red cells with pathological SMase levels induced shape change and PS exposure, these data suggest that red cell homeostasis could well be disturbed by SMase during chronic inflammation. Of note, red cell storage greatly increased the sensitivity to SMase-induced changes. This may well have implications for red cell transfusion in critically ill patients. Disclosures: No relevant conflicts of interest to declare.