Myelin down-regulates myelin phagocytosis by microglia and macrophages through interactions between CD47 on myelin and SIRPα (signal regulatory protein-α) on phagocytes

<p>Abstract</p> <p>Background</p> <p>Traumatic injury to axons produces breakdown of axons and myelin at the site of the lesion and then further distal to this where Wallerian degeneration develops. The rapid removal of degenerated myelin by phagocytosis is advantageous for repair since molecules in myelin impede regeneration of severed axons. Thus, revealing mechanisms that regulate myelin phagocytosis by macrophages and microglia is important. We hypothesize that myelin regulates its own phagocytosis by simultaneous activation and down-regulation of microglial and macrophage responses. Activation follows myelin binding to receptors that mediate its phagocytosis (e.g. complement receptor-3), which has been previously studied. Down-regulation, which we test here, follows binding of myelin CD47 to the immune inhibitory receptor SIRPα (signal regulatory protein-α) on macrophages and microglia.</p> <p>Methods</p> <p>CD47 and SIRPα expression was studied by confocal immunofluorescence microscopy, and myelin phagocytosis by ELISA.</p> <p>Results</p> <p>We first document that myelin, oligodendrocytes and Schwann cells express CD47 without SIRPα and further confirm that microglia and macrophages express both CD47 and SIRPα. Thus, CD47 on myelin can bind to and subsequently activate SIRPα on phagocytes, a prerequisite for CD47/SIRPα-dependent down-regulation of CD47^+/+myelin phagocytosis by itself. We then demonstrate that phagocytosis of CD47^+/+myelin is augmented when binding between myelin CD47 and SIRPα on phagocytes is blocked by mAbs against CD47 and SIRPα, indicating that down-regulation of phagocytosis indeed depends on CD47-SIRPα binding. Further, phagocytosis in serum-free medium of CD47^+/+myelin is augmented after knocking down SIRPα levels (SIRPα-KD) in phagocytes by lentiviral infection with SIRPα-shRNA, whereas phagocytosis of myelin that lacks CD47 (CD47^-/-) is not. Thus, myelin CD47 produces SIRPα-dependent down-regulation of CD47^+/+myelin phagocytosis in phagocytes. Unexpectedly, phagocytosis of CD47^-/-myelin by SIRPα-KD phagocytes, which is not altered from normal when tested in serum-free medium, is augmented when serum is present. Therefore, both myelin CD47 and serum may each promote SIRPα-dependent down-regulation of myelin phagocytosis irrespective of the other.</p> <p>Conclusions</p> <p>Myelin down-regulates its own phagocytosis through CD47-SIRPα interactions. It may further be argued that CD47 functions normally as a marker of "self" that helps protect intact myelin and myelin-forming oligodendrocytes and Schwann cells from activated microglia and macrophages. However, the very same mechanism that impedes phagocytosis may turn disadvantageous when rapid clearance of degenerated myelin is helpful.</p

CD47 : a cell surface glycoprotein which regulates multiple functions of hematopoietic cells in health and disease.

Interactions between cells and their surroundings are important for proper function and homeostasis in a multicellular organism. These interactions can either be established between the cells and molecules in their extracellular milieu, but also involve interactions between cells. In all these situations, proteins in the plasma membranes are critically involved to relay information obtained from the exterior of the cell. The cell surface glycoprotein CD47 (integrin-associated protein (IAP)) was first identified as an important regulator of integrin function, but later also was shown to function in ways that do not necessarily involve integrins. Ligation of CD47 can induce intracellular signaling resulting in cell activation or cell death depending on the exact context. By binding to another cell surface glycoprotein, signal regulatory protein alpha (SIRPα), CD47 can regulate the function of cells in the monocyte/macrophage lineage. In this spotlight paper, several functions of CD47 will be reviewed, although some functions may be more briefly mentioned. Focus will be on the ways CD47 regulates hematopoietic cells and functions such as CD47 signaling, induction of apoptosis, and regulation of phagocytosis or cell-cell fusion

Hyperglycemia-Induced Protein Kinase C Activation Inhibits Phagocytosis of C3b- and Immunoglobulin G–Opsonized Yeast Particles in Normal Human Neutrophils

The aim of this study was to investigate the effects of elevated glucose concentrations on complement receptor– and Fcγ receptor–mediated phagocytosis in normal human neutrophils. D-Glucose at 15 or 25 mM dose-dependently inhibited both complement receptor– and Fcγ receptor– mediated phagocytosis, as compared to that at a normal physiological glucose concentration. The protein kinase C (PKC) inhibitors GF109203X and Go6976 both dose dependently and completely reversed the inhibitory effect of 25 mM D-glucose on phagocytosis. Complement receptor– mediated phagocytosis was dose-dependently inhibited by the cell permeable diacylglycerol analogue 1,2-dioctanoylsn- glycerol (DAG), an effect that was abolished by PKC inhibitors. Furthermore, suboptimal inhibitory concentrations of DAG and glucose showed an additive inhibitory effect on complement receptor–mediated phagocytosis. The authors conclude that elevated glucose concentrations can inhibit complement receptor and Fcγ receptor–mediated phagocytosis in normal human neutrophils by activating PKCα and/or PKCβ, an effect possibly mediated by DAG

In vitro phagocytosis of liquid-stored red blood cells requires serum and can be inhibited with fucoidan and dextran sulphate

BACKGROUND AND OBJECTIVES: Red-blood-cells (RBCs) undergo structural and metabolic changes with prolonged storage, which ultimately may decrease their survival after transfusion. Although the storage-induced damage to RBCs has been rather well described biochemically, little is known about the mechanisms underlying the recognition and rapid clearance of the damaged cells by macrophages. MATERIALS AND METHODS: We, here, used a murine model for cold (+4°C) RBC storage and transfusion. Phagocytosis of human or murine RBCs, liquid stored for 6-8 weeks or 10-14 days, respectively, was investigated in murine peritoneal macrophages. RESULTS: The effects of storage on murine RBCs resembled that described for stored human RBCs with regard to decreased adenosine triphosphate (ATP) levels, accumulation of microparticles (MPs) during storage, and RBC recovery kinetics after transfusion. Under serum-free conditions, phagocytosis of stored human or murine RBCs in vitro was reduced by 70-75%, as compared with that in the presence of heat-inactivated fetal calf serum (FCS). Human serum promoted phagocytosis of stored human RBCs similar to that seen with FCS. By adding fucoidan or dextran sulphate (blockers of scavenger receptors class A (SR-A)), phagocytosis of human or murine RBCs was reduced by more than 90%. Phagocytosis of stored human RBCs was also sensitive to inhibition by the phosphatidylinositol 3 kinase-inhibitor LY294002, the ERK1/2-inhibitor PD98059, or the p38 MAPK-inhibitor SB203580. CONCLUSION: RBCs damaged during liquid storage may be recognized by macrophage SR-A and serum-dependent mechanisms. This species-independent recognition mechanism may help to further understand the rapid clearance of stored RBCs shortly after transfusion

CD47 Regulates Collagen I-Induced Cyclooxygenase-2 Expression and Intestinal Epithelial Cell Migration

Increased epithelial cell expression of the cyclooxgenase-2 (COX-2) enzyme is a characteristic event of both inflammatory bowel disease and colon cancer. We here report the novel findings that collagen I-induced de novo synthesis of COX-2 in intestinal epithelial cells is inhibited by pertussis toxin (PTX) and by an inhibitory peptide selective for the heterotrimeric Gai3-protein. These findings could be explained by a regulatory involvement of the G-protein-dependent integrinassociated protein CD47. In support of this notion, we observed a collagen I-induced association between CD47 and a2 integrins. This association was reduced by a blocking anti-CD47 antibody but not by PTX or a control anti-b2 antibody. Furthermore, a blocking antibody against CD47, dominant negative CD47 or specific siRNA knock down of CD47, significantly reduced collagen I-induced COX-2 expression. COX-2 has previously been shown to regulate intestinal epithelial cell adhesion and migration. Morphological analysis of intestinal cells adhering to collagen I revealed a colocalisation of CD47 and a2 integrins to non-apoptotic membrane blebs enriched in Rho A and F-actin. The blocking CD47 antibody, PTX and a selective COX-2 inhibitor, dramatically inhibited the formation of these blebs. In accordance, migration of these cells on a collagen I-coated surface or through a collagen I gel were significantly reduced by the CD47 blocking antibody, siRNA knock down of CD47 and the COX-2 inhibitor NS-398. In conclusion, we present novel data that identifies the G-protein-dependent CD47 protein as a key regulator of collagen I-induced COX-2 expression and a promoter o