The Role of CD47 in Neutrophil Transmigration

CD47 (integrin-associated protein) is an integral membrane protein that is required for granulocyte and T cell recruitment to sites of infection (1, 2), and its absence on red blood cells leads to their rapid macrophage-mediated clearance (3). CD47 may also function as a costimulator to regulate T cell activation, survival, and Th1 versus Th2 differentiation (4, 5). Endogenous ligands for the extracellular domain of CD47 include the secreted protein thrombospondin-1 (TSP1) 1 and potentially other members of the thrombospondin family, several integrins (6 -9), and some members of the signal-regulatory protein family (3, 10 -13). Engagement of CD47 by soluble ligands or signal-regulatory protein counter receptors modulates several cell signaling pathways, including activation of a heterotrimeric G protein (14).Although some signal transduction through CD47 is integrin-independent (4, 15, 16), association of CD47 with certain integrins was found to modulate their function to mediate cell adhesion or motility (6,8,9,17). In addition to its known functional and physical interactions with ␣ v ␤ 3 , ␣ IIb ␤ 3 , and ␣ 2 ␤ 1 integrins, several publications have suggested an association of CD47 with ␣ 4 ␤ 1 integrin. CD47 and ␣ 4 ␤ 1 were colocalized on microvilli of K562 erythroleukemia cells (18), and CD47-dependent arrest of T cells on inflammatory endothelium could be blocked by antibodies that prevent ␣ 4 ␤ 1 integrin binding to VCAM-1 (2). In the latter study, ligation of CD47 by TSP1 or signal-regulatory protein 1␣ was inferred to activate ␣ 4 ␤ 1 integrin on T cells, although this was not demonstrated either functionally or biochemically.We recently found that CD47 expression is required for stimulation of T cell motility and expression of MMP-2 stimulated by ␣ 4 ␤ 1 integrin ligands (19). An antibody to CD47 also blocked the motility response to an ␣ 4 ␤ 1 integrin ligand (19). Therefore, at least a functional interaction occurs between CD47 and ␣ 4 ␤ 1 integrin.We identified a binding site for ␣ 4 ␤ 1 integrin in the Nterminal domains of TSP1 and TSP2 (19), whereas two binding sites for CD47 have been localized to the C-terminal domain of TSP1 (for review, see Ref. 20). Thus, TSP1 could potentially regulate its own interaction with ␣ 4 ␤ 1 integrin through simultaneous interactions with CD47 and ␣ 4 ␤ 1 integrin on a T cell, analogous to the ability of soluble TSP1 to stimulate ␣ v ␤ 3 integrin-dependent melanoma cell adhesion to immobilized TSP1 (21).To define a molecular basis for functional cross-talk between these two receptors, we have examined the effect of CD47 ligands on the function of ␣ 4 ␤ 1 integrin in T cells and melanoma cells. We confirmed that ligation of CD47 can activate ␣ 4 ␤ 1 integrin function but unexpectedly found the mechanism of this activity to be CD47 ligand-dependent. We further show that CD47 ligation differentially activates and inactivates ␣ v ␤ 3 and ␣ 4 ␤ 1 in melanoma cells and ␣ 4 ␤ 1 and ␣ 5 ␤ 1 in Jurkat cells. Unexpectedly, but consistent with a recent report demonstrating C...

Section: A Cd47-binding Peptide Stimulatesmentioning

confidence: 95%

Regulation of Integrin Function by CD47 Ligands

Barazi

Cashel

et al. 2002

“…Rabbit polyclonal antibody against FcR␥ was obtained from Upstate Biotechnology, Inc. As a noninhibitory antibody binding control for PMN transmigration assays, we used anti-JAM-A mAb J10.4 (22). As an inhibitory control for PMN transmigration assays, we used anti-CD47 mAb C5D5 as described previously (23).…”

Section: Methodsmentioning

confidence: 99%

“…Unstimulated and stimulated PMN (2 ϫ 10 8 cells per condition) were then resuspended in 5 ml of cavitation buffer (0.34 M sucrose, 10 mM HEPES, 1 mM EDTA, 0.1 mM MgCl 2 , 1 mM Na 2 ATP (pH 7.4)) and disrupted by nitrogen cavitation (15 min, 400 p.s.i., 4°C). The lysates were centrifuged at low speed (1000 rpm), and the supernatants were subjected to isopycnic sucrose density gradient fractionation on linear 20 -55% sucrose gradients in a Beckman SW-28 swinging bucket rotor (100,000 ϫ g, 3 h, 4°C) as described previously (23). From each gradient, 1.5-ml fractions were collected and were analyzed for sucrose density and protein.…”

Section: Site-directed Mutagenesis Of Cysteine Residues In Sirp␤1-mentioning

confidence: 99%

“…To detect SIRP␤1 in leukocytes, immunoprecipitation was performed using mAb B4B6 or B1D5 followed by immunoblotting with the same mAb or with rabbit anti-SIRP␣1.ex. To confirm SIRP␤1 existing as a disulfide-bonded dimer, total cell lysates or SIRP␤1 immunoprecipitates were treated with 30 -300 mM iodoacetamide for 30 , and CD11b were detected by immunoblotting of the cell lysates using mAb B4B6, mouse anti-DAP12 antibody, and rabbit anti-CD11b antibody R7928A (23). SIRP␣ Co-precipitation Experiments-A recombinant fusion protein consisting of the putative extracellular domain of CD47 and alkaline phosphatase (CD47-AP) was generated as described previously (17).…”

Section: Pmn Peripheral Blood Mononuclear Cells (Pbmc)mentioning

confidence: 99%

See 1 more Smart Citation

SIRPβ1 Is Expressed as a Disulfide-linked Homodimer in Leukocytes and Positively Regulates Neutrophil Transepithelial Migration

Liu

Soto

Qiao

et al. 2005

Self Cite

Signal regulatory proteins (SIRPs) comprise a family of cell surface signaling receptors differentially expressed in leukocytes and the central nervous system. Although the extracellular domains of SIRPs are highly similar, classical motifs in the cytoplasmic or transmembrane domains distinguish them as either activating (␤) or inhibitory (␣) isoforms. We reported previously that human neutrophils (polymorphonuclear leukocytes (PMN)) express multiple SIRP isoforms and that SIRP␣ binding to its ligand CD47 regulates PMN transmigration. Here we further characterized the expression of PMN SIRPs, and we reported that the major SIRP␣ and SIRP␤ isoforms expressed in PMN include Bit/PTPNS-1 and SIRP␤1, respectively. Furthermore, although SIRP␣ (Bit/PTPNS-1) is expressed as a monomer, we showed that SIRP␤1 is expressed on the cell surface as a disulfide-linked homodimer with bond formation mediated by Cys-320 in the membrane-proximal Ig loop. Subcellular fractionation studies revealed a major pool of SIRP␤1 within the plasma membrane fractions of PMN. In contrast, the majority of SIRP␣ (Bit/PTPNS-1) is present in fractions enriched in secondary granules and is translocated to the cell surface after chemoattractant (formylmethionylleucylphenylalanine) stimulation. Functional studies revealed that antibody-mediated ligation of SIRP␤1 enhanced formylmethionylleucylphenylalanine-driven PMN transepithelial migration. Co-immunoprecipitation experiments to identify associated adaptor proteins revealed a 10 -12-kDa protein associated with SIRP␤1 that was tyrosine-phosphorylated after PMN stimulation and is not DAP10/12 or Fc receptor ␥ chain. These results provide new insights into the structure and function of SIRPs in leukocytes and their potential role(s) in fine-tuning responses to inflammatory stimuli. Signal regulatory proteins (SIRPs)3 are a family of transmembrane receptor-like signaling proteins that are abundantly expressed in hematopoietic cells, including granulocytes, monocytes, dendritic cells, and lymphocytes (1-3). In addition, SIRPs are expressed in neuronal cells (4 -6) and certain types of cancer cells (7-10). SIRPs can be divided into two subfamilies, SIRP␣ and SIRP␤, based on the putative structures of their C-terminal intracellular domains (11). SIRPs share typical immunoglobulin superfamily structures with an N-terminal extracellular domain containing three cysteine-bound Ig-like loops, a single membrane-spanning transmembrane domain, and a C-terminal intracellular domain (11). The C-terminal intracellular domains of the SIRP␣ subfamily contain a relatively long amino acid sequence (110 amino acids for SIRP␣1) that includes four tyrosine residues to form two immunoreceptor tyrosine-based inhibition motifs (ITIM). Conversely, SIRP␤ subfamily members have a short intracellular domain containing only a few amino acids (4 amino acids for SIRP␤1). Despite a short cytoplasmic tail, SIRP␤1 contains a positively charged lysine in the transmembrane domain that can mediate interactions with an immunoreceptor tyrosi...

“…We have recently shown that the CD47-SHPS-1 system and SHP-2 contribute to the inhibition of cell migration by cell-cell contact (22). Neutrophil migration to sites of inflammation is markedly impaired in CD47 knockout mice (23) and monoclonal antibodies (mAbs) to CD47 inhibit neutrophil transmigration (24), suggesting that the CD47-SHPS-1 system might mediate bidirectional inhibitory regulation of cell migration. The binding of CD47 on red blood cells to SHPS-1 on macrophages also inhibits phagocytosis of the red blood cells by the macrophages (25).…”

mentioning

confidence: 99%

Ectodomain Shedding of SHPS-1 and Its Role in Regulation of Cell Migration

Ohnishi

Kobayashi

Okazawa

et al. 2004

SHPS-1 is a transmembrane protein whose cytoplasmic region undergoes tyrosine phosphorylation and then binds the protein-tyrosine phosphatase SHP-2. Formation of the SHPS-1-SHP-2 complex is implicated in regulation of cell migration. In addition, SHPS-1 and its ligand CD47 constitute an intercellular recognition system that contributes to inhibition of cell migration by cell-cell contact. The ectodomain of SHPS-1 has now been shown to be shed from cells in a reaction likely mediated by a metalloproteinase. This process was promoted by activation of protein kinase C or of Ras, and the released ectodomain exhibited minimal CD47-binding activity. Metalloproteinases catalyzed the cleavage of a recombinant SHPS-1-Fc fusion protein in vitro, and the primary cleavage site was localized to the juxtamembrane region of SHPS-1. Forced expression of an SHPS-1 mutant resistant to ectodomain shedding impaired cell migration, cell spreading, and reorganization of the actin cytoskeleton. It also increased the tyrosine phosphorylation of paxillin and FAK triggered by cell adhesion. These results suggest that shedding of the ectodomain of SHPS-1 plays an important role in regulation of cell migration and spreading by this protein.The extracellular region of various transmembrane proteins is cleaved by proteases, such as metalloproteinases, and released as a soluble protein fragment (1, 2). This process, known as "ectodomain shedding," often influences the mode of action or biological activity of the affected protein. For instance, ectodomain shedding of heparin-binding epidermal growth factor (EGF) 1 -like growth factor (HB-EGF), a membrane-anchored EGF-related protein (3), results in its activation, in that the released fragment binds to EGF receptors and thereby stimulates cell proliferation (3, 4). In contrast, the membraneanchored forms of c-kit ligand (5) and of ephrins, the latter of which are ligands of Eph receptor tyrosine kinases (6), are fully functional, whereas their soluble forms generated by ectodomain shedding exhibit little or no biological activity. Ectodomain shedding is thus an important regulator of the function of certain membrane proteins that contribute to cellcell communication.SHPS-1 (SHP substrate-1) (7), also known as SIRP␣ (8), BIT (9), and p84 neural adhesion molecule (10), is a receptor-like transmembrane protein that is particularly abundant in neurons and macrophages (10, 11), although other cell types, such as fibroblasts, also express this protein (7). The putative extracellular region of SHPS-1 comprises three immunoglobulin (Ig)-like domains with multiple N-linked glycosylation sites, whereas the cytoplasmic region of the protein contains four YXX(L/V/I) motifs, which are putative tyrosine phosphorylation sites and binding sites for the Src homology 2 (SH2) domains of the protein-tyrosine phosphatases SHP-2 and SHP-1 (7, 8). Tyrosine phosphorylation of SHPS-1 is regulated by various growth factors, including insulin and EGF, as well as by cell adhesion to extracellular matrix proteins (12). SH...