Role of the CD47/SIRPα-interaction in regulation of macrophage phagocytosis

Sammanfattning: CD47 is a cell surface glycoprotein that is expressed by virtually all cells in the body. Binding of CD47 to the macrophage receptor Signal Regulatory Protein alpha (SIRPα) yields an inhibitory signal that counteracts phagocytosis. Red blood cells (RBCs) that lack CD47 are rapidly cleared from the circulation, whereas CD47 expressing cells have a normal turnover rate. CD47 has therefore been proposed to function as a marker of self, enabling the immune system to discriminate between self and foreign. Thus, the studies of the present thesis aimed at further investigating the role of CD47 as a marker of self in regulating phagocytosis of platelets, phagocytosis of viable or senescent RBCs, and the mechanisms involved.CD47 on platelets was found to regulate their turnover in vivo, since platelets from CD47-/- mice transfused into wild type recipients were cleared more rapidly from the circulation than wild type platelets. In addition, CD47-/- mice were found to suffer from a mild spontaneous thrombocytopenia, without any signs of accelerated platelet apoptosis or increased platelet activation. CD47-/- mice were more sensitive to experimental immune thrombocytopenia (ITP), as compared with wild type mice. In vitro phagocytosis experiments proved that platelet CD47 was responsible for this effect, since blocking antibodies to macrophage SIRPα increased phagocytosis of wild type platelets to the levels seen for CD47-/- platelets. When unopsonized platelets or RBCs from CD47+/- mice (expressing about 50 % less CD47 than wild type cells) were transfused into wild type recipients, they were cleared from the circulation at virtually the same rate as wild type cells. However, CD47+/- cells were cleared more rapidly than wild type cells when transfused animals were challenged with an antibody directed against the transfused cell type. In vitro, IgG-opsonized CD47+/- platelets and RBCs were ingested to a higher extent than wild type cells, but less than CD47-/- cells, suggesting that CD47 dose-dependently regulates phagocytosis in macrophages. It was also investigated if inhibitory SIRPα signaling is localized to the site of contact with the cell that is to be ingested, or whether the inhibition of phagocytosis is more general in the whole macrophage. Experiments with a mix of IgG-opsonized wild type and CD47-/- RBCs showed that the effect of inhibitory CD47-SIRPα signaling was local in the macrophage and limited to the site of contact with a specific target cell. Thus, contact with one or several wild type RBCs did not affect the increased phagocytosis of CD47-/- RBCs by the same macrophage.RBC senescence involves oxidation of membrane lipids and proteins, as well as exposure of phosphatidylserine (PS) on the cell surface, and clearance of senescent RBCs is believed to be regulated by several different factors. To investigate the role of CD47 in uptake of experimentally senescent RBCs, RBCs were oxidized with CuSO4/ascorbic acid (Ox-RBCs). Phagocytosis of Ox-RBCs required recognition of PS on the RBCs, recognition by scavenger receptors on the macrophages, and was strongly dependent on serum. CD47 did not inhibit serum-dependent phagocytosis of experimentally senescent unopsonized RBCs, since phagocytosis of senescent wild type or CD47-/- RBCs was virtually similar. The ability of CD47 to cluster in the plasma membrane upon cross-linking with antibodies was reduced in senescent RBCs. Despite this, CD47 inhibited phagocytosis of IgG-opsonized viable or senescent RBCs to the same extent.In summary, CD47 can function as a marker of self on both RBCs and platelets. The phagocytosis-inhibitory effect is dependent on the CD47 expression level, and CD47-SIRPα signaling acts locally in the macrophage at the contact with a target cell. In experimentally senescent RBCs, CD47 does not inhibit serum-dependent phagocytosis in the absence of opsonization, but still inhibits FcγR-mediated phagocytosis.Key words: CD47, SIRPα, platelets, red blood cells, macrophages, phagocytosis, Fcγ receptor, senescence