Signal-regulatory proteins (SIRPs) represent a new family of inhibitory/activating receptor pairs. They consist of 3 highly homologous immunoglobulin (Ig)-like domains in their extracellular regions, but differ in their cytoplasmic regions by the presence (SIRP␣) or absence (SIRP␤) of immunoreceptor tyrosine-based inhibitory motifs (ITIMs). To analyze the differential expression on hematopoietic cells, function and ligand binding capacity of SIRP␣ and SIRP␤ molecules, soluble fusion proteins consisting of the extracellular domains of SIRP␣1, SIRP␣2, and SIRP␤1, as well as SIRP␣/␤-specific and SIRP␤-specific monoclonal antibodies (MoAbs) were generated. In contrast to SIRP␣1 and SIRP␣2, no adhesion of SIRP␤1 to CD47 could be detected by cell attachment assays and flow cytometry. Using deletion constructs of SIRP␣1, the epitope responsible for SIRP␣1 binding to CD47 could be confined to the Nterminal Ig-like loop. Flow cytometry analysis with SIRP␣/␤-and SIRP␤-specific MoAbs revealed that SIRP␣ but not SIRP␤ is expressed on CD34 ؉ CD38 ؊ hematopoietic cells. In addition, a strong SIRP␣ expression was also observed on primary myeloid dendritic cells (DCs) from peripheral blood as well as on in vitro generated DCs. Analysis of the T-cell stimulatory capacity of in vitro generated DCs in the presence of soluble SIRP␣1 fusion proteins as well as SIRP␣/␤-specific and CD47-specific MoAbs revealed a significant reduction of T-cell proliferation in mixed lymphocyte reaction and inhibition of induction of primary T-cell responses under these conditions. In contrast, soluble SIRP␣ or SIRP␤-specific antibodies had no effect. The data suggest that the interaction of SIRP␣ with CD47 plays an important role during T-cell activation and induction of antigen-specific cytotoxic T-lymphocyte responses by DCs. IntroductionSignal-regulatory proteins (SIRPs) comprise a novel transmembrane glycoprotein family involved in receptor tyrosine kinasecoupled signaling pathways. 1 These molecules are also called SHPS-1 (src homology 2 domain-containing phosphatase substrate-1), 2 BIT (brain immunoglobulin [Ig]-like molecule with a tyrosine-based activation motif), 3 P84, 4 and MFR (macrophage fusion receptor). 5 Structurally, all SIRP members share a large extracellular region with 3 Ig-like loops. 1 The cytoplasmic domains of SIRP␣ subfamily members display 2 immunoreceptor tyrosinebased inhibitory motifs (ITIMs). 1 These ITIM regions, which are also present on other inhibitory receptor molecules, recruit src homology 2 domain-containing phosphatases SHP-2 1,2 and SHP-1 6,7 and negatively regulate signal transduction cascades. Although SIRP␣1 is known to inhibit receptor tyrosine kinase-coupled signaling pathways, 1 it can also positively regulate the mitogenactivated protein kinase (MAPK) pathway in response to insulin and potentiate integrin-induced MAPK activation. 8,9 Thus, under certain circumstances SIRP␣1 is involved in activating rather than in inhibiting processes.In contrast to SIRP␣ molecules, members of the SIRP␤ subfamily expre...

Signal-regulatory proteins (SIRPs) comprise a novel transmembrane glycoprotein family involved in the negative regulation of receptor tyrosine kinase-coupled signaling pathways. To analyze the expression and function of SIRPs, we prepared soluble recombinant fusion proteins of the extracellular regions of SIRP1 and SIRP2, as well as a variety of monoclonal antibodies (MoAbs) against these domains. The antibodies reacted predominantly with monocytes, granulocytes, dendritic cells, and their precursors, as well as with bone marrow CD34+, AC133+, CD90+hematopoietic stem/progenitor cells. In contrast, SIRP expression was absent or significantly reduced on the majority of myeloid blasts from patients with acute myeloid leukemia (AML) or chronic myeloid leukemia (CML). Functional studies showed that the extracellular domains of SIRP1 and SIRP2 support adhesion of a number of primary hematopoietic cells and cell lines. This interaction could be blocked by 4 of 7 SIRP1-reactive MoAbs. In addition, SIRP1 and SIRP2 competed for the same cell binding site, suggesting a common widely expressed SIRP ligand. In an approach to identify this molecule, MoAbs were generated against the SIRP-binding cell line CCRF-CEM, and MoAb CC2C6 was selected because of its capacity to inhibit cell binding to SIRP1. Further analysis showed that this antibody recognized CD47, a ubiquitously expressed plasma membrane protein previously implicated in integrin function, host defense action, and neutrophil migration. In this study, we identify CD47 as the extracellular ligand for human SIRP and show that these two counterreceptors are involved in cellular adhesion.

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Signal-regulatory proteins (SIRPs) comprise a novel transmembrane glycoprotein family involved in the negative regulation of receptor tyrosine kinase-coupled signaling pathways. To analyze the expression and function of SIRPs, we prepared soluble recombinant fusion proteins of the extracellular regions of SIRP1 and SIRP2, as well as a variety of monoclonal antibodies (MoAbs) against these domains. The antibodies reacted predominantly with monocytes, granulocytes, dendritic cells, and their precursors, as well as with bone marrow CD34+, AC133+, CD90+hematopoietic stem/progenitor cells. In contrast, SIRP expression was absent or significantly reduced on the majority of myeloid blasts from patients with acute myeloid leukemia (AML) or chronic myeloid leukemia (CML). Functional studies showed that the extracellular domains of SIRP1 and SIRP2 support adhesion of a number of primary hematopoietic cells and cell lines. This interaction could be blocked by 4 of 7 SIRP1-reactive MoAbs. In addition, SIRP1 and SIRP2 competed for the same cell binding site, suggesting a common widely expressed SIRP ligand. In an approach to identify this molecule, MoAbs were generated against the SIRP-binding cell line CCRF-CEM, and MoAb CC2C6 was selected because of its capacity to inhibit cell binding to SIRP1. Further analysis showed that this antibody recognized CD47, a ubiquitously expressed plasma membrane protein previously implicated in integrin function, host defense action, and neutrophil migration. In this study, we identify CD47 as the extracellular ligand for human SIRP and show that these two counterreceptors are involved in cellular adhesion.