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. PlcHR 2 , 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 PlcHR 2 is not observed. Fluorescence microscopy observations confirm the formation of ceramide-enriched domains as a result of PlcHR 2 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 PlcHR 2 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 ceramiderich 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.Hemolysis consists of the release of hemoglobin from red blood cells (RBC) 1 to the surrounding medium. In vivo hemolysis is a pathologic event that occurs in various forms of anemia, as well as in response to intoxications and infections. In addition, in vitro hemolysis is often used as a sensitive and reliable method to assay the lytic action on cell membranes of any physical or chemical agent. In this context RBC membranes are used as a membrane model. It was found long ago that erythrocytes become more sensitive to hemolysis induced by certain drugs or toxins when, after incubation at 37 °C, the cell suspension is cooled to 4 °C (1-3). This is known as "hot-cold hemolysis". The molecular basis for this phenomenon is not known. Different explanations have been proposed, but none has gained wide acceptance.PlcHR 2 toxin from Pseudomonas aeruginosa is an enzyme with both phospholipase C and sphingomyelinase activities. Stonehouse et al. (4) found that PlcHR 2 was highly hemolytic when the RB...

Ceramide formation mediated by acid sphingomyelinase facilitates endosomal escape of caliciviruses

“…Ceramide has emerged as an important mediator of diverse cellular effects from various stress stimuli including bacterial or viral infection, as well as ionizing radiation, UV light and heat (Gulbins and Kolesnick, 2002, 2003; He et al, 2003; Montes et al, 2008; Stancevic and Kolesnick, 2010). Modulation of the biophysical properties of membranes by ceramide has been reported to be associated with formation of small rafts that fuse together to form large ceramide-enriched membrane platforms, changes in membrane fluidity and permeability, facilitation of membrane fusion, or promotion of macropinocytosis (Basáñez et al, 1997; Gulbins et al, 2004; Gulbins and Kolesnick, 2002; Montes et al, 2002; Siskind and Colombini, 2000), formation of channels large enough for proteins to cross membranes or cause lipid flip-flop (Contreras et al, 2009; Samanta et al, 2011).…”

Bacterial Sphingomyelinases and Phospholipases as Virulence Factors

“…Treatment of sheep erythrocytes with B. cereus SMase C reduces cell membrane fluidity and leads to the self-assembly of Cer-rich domains that exclude cholesterol (25). In erythrocytes, the coalescence of these domains and the formation of interfaces between rigid and fluid domains causes fragility of the plasma membrane, leading to lysis (25,26). In the apical brush border membrane, Cer domain formation induced by SMase Cs from commensal bacteria likely modulates the epithelial barrier and affects intestinal homeostasis (27).…”

“…PlcN hydrolyzes PC and PS, but not SM, and is nonhemolytic (247), whereas PlcH hydrolyzes PC, SM, and lyso-PC at equal rates (248) and also acts on cardiolipin, PE, and PG (249). P. aeruginosa PlcH, which is expressed during lung infections in humans (250), is hemolytic to human erythrocytes (26) and selectively cytotoxic to endothelial cells (248). Furthermore, it suppresses the neutrophil respiratory burst and induces a strong chemokine response in mice and human granulocytes (248,251).…”

Membrane-damaging and cytotoxic sphingomyelinases and phospholipases

“…The mechanism of hot-cold hemolysis is attributed to the generation of fragile erythrocytes after the cleavage of plasma membrane SM. Thus, when the membrane lipids cool, the phase change causes stress, resulting in lysis [31,40]. Treatment of sheep erythrocytes with B. cereus SMase C leads to the formation of Cer-rich domains and a reduction in the fluidity of the cell membrane [31].…”

“…The PLases C PlcB, PlcH, and PlcN could play a role facilitating bacterial transit through the mucus layer and liberating nutrients for the bacteria. PlcH is hemolytic for human erythrocytes [40] and cytotoxic to endothelial cells [160]. Furthermore, it induces a strong chemokine response in mice and human granulocytes and suppresses neutrophil respiratory burst by interfering with a PKC-dependent pathway [160].…”