Ligation of complement receptor 1 increases erythrocyte membrane deformability

Abstract

Microbes as well as immune complexes and other continuously generated inflammatory particles are efficiently removed from the human circulation by red blood cells (RBCs) through a process called immune-adherence clearance. During this process, RBCs use complement receptor 1 (CR1, CD35) to bind circulating complement-opsonized particles and transfer them to resident macrophages in the liver and spleen for removal. We here show that ligation of RBC CR1 by antibody and complement-opsonized particles induces a transient Ca ؉؉ influx that is proportional to the RBC CR1 levels and is inhibited by T1E3 pAb, a specific inhibitor of TRPC1 channels. The CR1-elicited RBC Ca ؉؉ influx is accompanied by an increase in RBC membrane deformability that positively correlates with the number of preexisting CR1 molecules on RBC membranes. Biochemically, ligation of RBC CR1 causes a significant increase in phosphorylation levels of ␤-spectrin that is inhibited by preincubation of RBCs with DMAT, a specific casein kinase II inhibitor. We hypothesize that the CR1-dependent increase in membrane deformability could be relevant for facilitating the transfer of CR1-bound particles from the RBCs to the hepatic and splenic phagocytes.(Blood. 2010;116(26): 6063-6071) IntroductionIn primates, in contrast to other vertebrates, clearing the intravascular space of complement-opsonized inflammatory particles (eg, microbes and immune complexes) is mediated by circulating red blood cells (RBCs) using complement receptor 1 (CR1, CD35). 1,2 During this process, known as immune-adherence clearance, RBCs immobilize complement-tagged particles and transport them to the liver and spleen where resident macrophages remove the complement-tagged particles and leave the RBCs intact. Immuneadherence clearance acts as a "buffer system," preventing deposition of circulating immune complexes in susceptible organs, such as the kidney, and preventing activation of circulating leukocytes by inflammatory particles. 3,4 We and others have also shown that CR1-mediated immune-adherence promotes more efficient phagocytosis and intracellular killing of complement-opsonized pathogens compared with opsonized pathogens that are free-floating in plasma and not RBC-bound. 5,6 We have previously found that, in circulating human RBCs, CR1 is disperse in RBC plasma membranes, and, after ligation by immune particles, interacts with Fas-associated phosphatase-1 and rearranges into large clusters. 7 Beneath the plasma membrane of RBCs, the spectrin cytoskeleton defines a series of "corrals" that are critical for maintaining RBC shape and deformability and for regulating the range and magnitude of lateral diffusion of most transmembrane proteins. 8 The mechanical attributes of the spectrin meshwork depend critically on the transient phosphorylation of ␤-spectrin, adducin, and protein 4.1R. 9-11 Therefore, we hypothesized that ligation-mediated CR1 clustering is an active process with CR1 directly affecting the phosphorylation status of cytoskeletal proteins and thus the mechanical p...

CR1-mediated ATP Release by Human Red Blood Cells Promotes CR1 Clustering and Modulates the Immune Transfer Process

“…Our results have also shown that although the spectrin phosphorylation promoted by CR1 ligation lasted less than 15 min, the effect on membrane deformability persisted for several hours suggesting that other mechanisms have critical roles in membrane deformability (6). New data demonstrate that the interaction between spectrin and plasma membrane lipids as well as rearrangement of lipid subdomains in RBC membranes play a considerable role in RBC membrane stability and deformability (22).…”

“…Measurement of RBC Membrane Deformability Using Twodimensional Filters-The ability of RBC to undergo capillarylike deformations was measured using a two-dimensional microchannel array as previously described (6). The device uses hydrostatic pressure to drive the RBC through the microchannel array.…”

“…microbeads were prepared with minor modifications from the protocol previously described (6). In brief, 0.2-m microbeads or 1-m FluoSpheres were briefly sonicated and reacted with BSA or Alexa Fluor 647-conjugated BSA.…”

“…Using pharmacological inhibitors, we found that CR1-mediated ATP release is mediated by multidrug resistance-associated proteins (MRPs) and does not depend on CK2 or PKA activity, enzymes previously shown to mediate the increase in membrane deformability triggered by CR1 ligation (6). By using fluorescence recovery after photobleaching (FRAP) and fluorescence microscopy we demonstrated that extracellular ATP increases the mobility of the lipid fraction of RBC membranes, which in turn facilitates CR1 clustering, enhancing the binding avidity of complement-opsonized particles to the RBC CR1.…”

“…Functionally, binding of complement-opsonized microbeads to CR1 triggers a significant Ca 2ϩ influx, the magnitude of which depends directly on the genetically determined expression levels of CR1 on RBC membranes. Subsequent activation of PKA and protein kinase CK2 (formerly known as casein kinase II) triggers a signaling cascade that results in increased phosphorylation of ␤-spectrin and increased RBC membrane deformability (6). Upon binding complement-opsonized particles, RBC themselves become "opsonized" by IgG and complement fragments.…”