The immune system recognizes invaders as foreign because they express determinants that are absent on host cells or because they lack "markers of self" that are normally present. Here we show that CD47 (integrin-associated protein) functions as a marker of self on murine red blood cells. Red blood cells that lacked CD47 were rapidly cleared from the bloodstream by splenic red pulp macrophages. CD47 on normal red blood cells prevented this elimination by binding to the inhibitory receptor signal regulatory protein alpha (SIRPalpha). Thus, macrophages may use a number of nonspecific activating receptors and rely on the presence or absence of CD47 to distinguish self from foreign. CD47-SIRPalpha may represent a potential pathway for the control of hemolytic anemia.

Neutrophils have long been regarded as essential for host defense against Staphylococcus aureus infection. However, survival of the pathogen inside various cells, including phagocytes, has been proposed as a mechanism for persistence of this microorganism in certain infections. Therefore, we investigated whether survival of the pathogen inside polymorphonuclear neutrophils (PMN) contributes to the pathogenesis of S. aureus infection. Our data demonstrate that PMN isolated from the site of infection contain viable intracellular organisms and that these infected PMN are sufficient to establish infection in a naive animal. In addition, we show that limiting, but not ablating, PMN migration into the site of infection enhances host defense and that repletion of PMN, as well as promoting PMN influx by CXC chemokine administration, leads to decreased survival of the mice and an increased bacterial burden. Moreover, a global regulator mutant of S. aureus (sar−) that lacks the expression of several virulence factors is less able to survive and/or avoid clearance in the presence of PMN. These data suggest that the ability of S. aureus to exploit the inflammatory response of the host by surviving inside PMN is a virulence mechanism for this pathogen and that modulation of the inflammatory response is sufficient to significantly alter morbidity and mortality induced by S. aureus infection.

In autoimmune hemolytic anemia (AIHA), circulating red blood cells (RBCs) opsonized with autoantibody are recognized by macrophage Fcγ and complement receptors. This triggers phagocytosis and elimination of RBCs from the circulation by splenic macrophages. We recently found that CD47 on unopsonized RBCs binds macrophage signal regulatory protein α (SIRPα), generating a negative signal that prevents phagocytosis of the unopsonized RBCs. We show here that clearance and phagocytosis of opsonized RBCs is also regulated by CD47-SIRPα. The inhibition generated by CD47-SIRPα interaction is strongly attenuated but not absent in mice with only residual activity of the phosphatase Src homology 2 domain–containing protein tyrosine phosphatase (SHP)-1, suggesting that most SIRPα signaling in this system is mediated by SHP-1 phosphatase activity. The macrophage phagocytic response is controlled by an integration of the inhibitory SIRPα signal with prophagocytic signals such as from Fcγ and complement receptor activation. Thus, augmentation of inhibitory CD47-SIRPα signaling may prevent or attenuate RBC clearance in AIHA.

The C-terminal "cell-binding domain" (CBD) of thrombospondin-1 (TS1) is a binding site for many cell types. Cell-binding peptides based on the sequence RFYVVM from the CBD of TS1 affinity label a 52-kDa cell surface glycoprotein, which we show is integrin-associated protein (IAP or CD47). IAP associates with alpha v beta 3 and thereby modulates the activity of several integrins. Cells that express IAP bind strongly to TS1, the CBD, and its active cell-binding peptides while IAP negative cells do not. The 52-kDa protein is affinity labeled on IAP-positive but not IAP-negative cells, and monoclonal antibodies against IAP specifically immunoprecipitate the affinity-labeled 52-kDa protein from lysates of IAP-positive cells. Consistent with the association of IAP with alpha v beta 3 integrin, the labeled 52-kDa protein is immunoprecipitated by an anti-alpha v beta 3 antibody. Endothelial cells exhibit chemotaxis toward TS1 (at concentrations above 10 nM) and RFYVVM peptides. Chemotaxis to both agents is specifically inhibited by a function blocking anti-IAP monoclonal antibody. These data establish IAP (CD47) as a receptor for the CBD of TS1 and suggest a mechanism for the well established effects of the CBD on cell motility.

Granulocyte [polymorphonuclear leucocyte (PMN)] migration to sites of infection and subsequent activation is essential for host defense. Gene-targeted mice deficient for integrin-associated protein (IAP, also termed CD47) succumbed to Escherichia coli peritonitis at inoccula survived by heterozygous littermates. In vivo, they had an early defect in PMN accumulation at the site of infection. In vitro, IAP-/- PMNs were deficient in beta3 integrin-dependent ligand binding, activation of an oxidative burst, and Fc receptor-mediated phagocytosis. Thus, IAP plays a key role in host defense by participating both in PMN migration in response to bacterial infection and in PMN activation at extravascular sites.

Abstract. Integrin Associated Protein (LAP) is a 50-kD membrane protein which copurifies with the integrin a4~3 from placenta and coimmunoprecipitates with 153 from platelets. IAP also is functionally associated with signal transduction from the Leukocyte Response Integrin. Using tryptic peptide sequence, human and murine IAP cDNAs have been isolated. The protein has an extracellular amino-terminal immunoglobulin domain that binds all monoclonal anti-LAP antibodies. The carboxy-terminal region is highly hydrophobic with three or five membrane-spanning segments and a short hydrophilic tail. Immunofluorescence microscopy suggests that this hydrophilic tail is located on the inside of the cytoplasmic membrane. Monoclonal anti-IAP antibody inhibits the binding of vitronectin-coated beads to c~vfl3 on human erythroleukemia cells, and polyclonal anti-IAP recognizes hamster LAP on CHO cells and inhibits vitronectin bead binding. When CHO cells are transfected with human LAP, monoclonal anti-human antibody completely inhibits vitronectin bead binding. These data suggest a model in which ligand binding by otv~3 is regulated by IAE HESION receptors of the integrin family recognize several proteins of the extracellular matrix and appear to be critical for the induction of normal morphology and migration of many cell types (2,4,6,22,31). While the interaction of integrins with their ligands has been the subject of intense study over the past several years, much less information has been gathered about the molecular mechanisms by which integrins affect cell phenotype and function. Recently, we described a cell surface protein which we copurified with the integrin avfl3 from placenta and which also coimmunoprecipitated with f13 integrins from platelets (5). This coprecipitating protein had a Mr of ~50 kD on SDS-PAGE, was highly hydrophobic, and was widely expressed. The 50-kD protein itself did not recognize the Arg-Gly-Asp peptides with which ot453 was purified (5), but mAb recognizing the 50-kD protein could inhibit several neutrophil and monocyte functions mediated by a leukocyte fl3-1ike integrin, including ligand binding (5,16,17,35). Therefore, we hypothesized that the 50-kD protein, which we called Integrin-associated Protein (IAP), ~ could physically associate with some integrins and, without directly binding the integrin ligands, could regulate integrin function. Now, we have cloned IAP eDNA from both human and mouse cells and examined its structure and role in binding vitronectin (Vn) ligand in native and transfected cells.

Abstract. Integrin-associated protein (IAP) is a receptor for the carboxyl-terminal "cell-binding domain" (CBD) of thrombospondin 1 (TS1). lAP associates with av[33 integrin and mAbs against lAP inhibit certain integrin functions. Here we examine the effects of the TS1 CBD and 4N1K (KRFYVVMWKK), a cell-binding peptide derived from it, on the adhesion and spreading on vitronectin (VN) of C32 human melanoma cells which express lAP, 0tv133 , and avl35. Cells adhere to VN at low surface densities via etv[35 and spread very slowly while adhesion to higher density VN involves both etv135 and Otv[33 and results in rapid spreading. Spreading of the cells, but not adhesion, on sparse VN coatings is markedly enhanced by the presence of soluble TS1, the recombinant CBD and 4N1K, but not the "mutant" peptide 4NGG, KRFYGGMWKK, which fails to bind IAP. This enhanced spreading is completely blocked by mAb LM609 against 0tv133 and the anti-lAP mAb B6H12. Correlated with this enhanced spreading is increased tyrosine phosphorylation of focal adhesion kinase (FAK), paxillin, and a protein of ca. 90 kD. The enhanced spreading induced by TS1 and 4N1K and the constitutive spreading on higher density VN are both blocked by calphostin C (100 nM), wortmannin (10 nM), and tyrosine kinase inhibitors. In contrast, pertussis toxin specifically blocks only the TS1 stimulated spreading on low density VN, indicating that lAP exerts its effects on signal transduction via a heterotrimeric Gi protein acting upstream of a common cell spreading pathway which includes PI-3 kinase, PKC, and tyrosine kinases.T HE family of thrombospondins has been implicated in acute regulation of a number of diverse physiological processes such as platelet aggregation, inflammation, angiogenesis, cell adhesion and migration, and other developmental processes (5,6,16,61). The prototypic member of this family is platelet thrombospondin or thrombospondin-1 (TS1). 1 Many cell interaction sites and peptides have been identified within the modular TS1 structure and as many as a dozen different receptors, including several 131 and 133 integrins have been proposed to mediate the effects of TS1 on cells (12,16,33,70). Of this plethora of putative receptors, the direct binding of TS1 to molecularly characterized receptors has been demonstrated only for HSPGs (40), sulfatides (49), CD36 (1), and the integrins Otllb133 and etv133 (33). In the case of the two integrins, binding of native TS1 has been demonstrated for some cell types, while for other cells, the TS1 RGD se- quence appears to be cryptic (62). A number of peptide epitopes within the complex TS1 structure have been identified with specific functions of the protein. For example, sequences within the procollagen-like domain, and two of the type 1 (properdin-or malaria-like) repeats inhibit angiogenesis in vivo and endothelial cell (EC) motility in vitro (65). Also, a peptide from the amino-terminal heparin-binding domain reverses focal adhesions in fibroblasts and EC (41, 42) and a tripeptide in one of the type 1 repeat...

Abstract. The plasma protein fibronectin is an important opsonin in wound repair and host defense. To better understand the process of fibronectin-mediated phagocytosis, we have transfectod K562 cells, which endogenously express 0~5fll, with a,f13. In these transfectants, antibodies to t~fl3 block phagocytosis of fibronectin-opsonized beads completely, even though half the ingestion occurs through endogenous 0t5/3~ receptors, c~5/31-mediated adhesion to fibronectincoated surfaces is unaffected by c~j33 ligation. Neither av/$5 nor O/M/~2 ligation affects O~5/~1 phagocytic function in transfectants expressing these receptors. Pharmacologic data suggest that o~v/~3 ligation suppresses the phagocytic competence of high affinity ot5/$1 receptors through a signal transduction pathway, perhaps involving protein kinase C. In addition to its significance for phagocytosis, otv/33 regulation of o~5/31 function may be significant for its roles in cell migration, metastasis, and angiogenesis. MACROPHAGE interaction with fibronectin (Fn) 1 is recognized as an important aspect of host defense and wound repair. Fibronectin opsonlzation is necessary for macrophage recognition and phagocytosis of particulate debris released from tissues after bum and trauma (23,41,53,58), resolution of bacteremia during sepsis (48, 53), and clearance of fibrin during disseminated intravascular coagulation (9,11,60). In addition, macrophage adhesion to fibronectin-coated surfaces affects a variety of macrophage functions including chemotaxis (25, 50), differentiation (8), secretion (7,45), and phagocytosis via immunologic receptors (12,52,66). Nonetheless, the moleculax nature of the interactions leading to these critical macrophage functions is unknown.Studies which have examined fibronectin receptors on macrophages in detail have demonstrated an unexpectedly large number of integrin and non-integrin fibronectin-binding proteins (10,13,16,17,29,42,59,63). Four integrin receptors with fibronectin binding capability have been identified on mononuclear phagocytes. Fibronectin binding to VLA-5 (ot5/~1), the vitronectin receptor (otJ~3) and the Leukocyte Response Integrin (LRI) appears dependent on the RGD adhesion sequence (13, 16, 29); (VLA-4 (~,) binds fibronectin independent of this sequence (28). Additional macrophage integrins ~J~5, VLA-3 (ot3/~,), and OtM/~2, may also Please address all correspondence to Dr. Eric J. Brown, Infectious Diseases, Campus Box 8051, Washington University School of Medicine, St. Louis, MO 63110. bind fibronectin (1%42,59,63). To add to the complexity of fibronectin binding by macrophages, several of these receptors can recognize alternative, potentially competing ligands. Moreover, a~/31 and perhaps other of these integrins can assume two affinity states for fibronectin (26). The existence of these distinct but related receptors for the same ligand suggest that they may have different roles in macrophage function. The purpose of the present work was to begin to determine how these various receptors contributed to adhe...