Frederik P. Lindberg

Decreased Resistance to Bacterial Infection and Granulocyte Defects in IAP-Deficient Mice

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 β3 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.

Survival of Staphylococcus aureus Inside Neutrophils Contributes to Infection

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.

Bidirectional Negative Regulation of Human T and Dendritic Cells by CD47 and Its Cognate Receptor Signal-Regulator Protein-α: Down-Regulation of IL-12 Responsiveness and Inhibition of Dendritic Cell Activation

Proinflammatory molecules, including IFN-γ and IL-12, play a crucial role in the elimination of causative agents. To allow healing, potent anti-inflammatory processes are required to down-regulate the inflammatory response. In this study, we first show that CD47/integrin-associated protein, a ubiquitous multispan transmembrane protein highly expressed on T cells, interacts with signal-regulator protein (SIRP)-α, an immunoreceptor tyrosine-based inhibition motif-containing molecule selectively expressed on myelomonocytic cells, and next demonstrate that this pair of molecules negatively regulates human T and dendritic cell (DC) function. CD47 ligation by CD47 mAb or L-SIRP-α transfectants inhibits IL-12R expression and down-regulates IL-12 responsiveness of activated CD4+ and CD8+ adult T cells without affecting their response to IL-2. Human CD47-Fc fusion protein binds SIRP-α expressed on immature DC and mature DC. SIRP-α engagement by CD47-Fc prevents the phenotypic and functional maturation of immature DC and still inhibits cytokine production by mature DC. Finally, in allogeneic MLR between mDC and naive T cells, CD47-Fc decreases IFN-γ production after priming and impairs the development of a Th1 response. Therefore, CD47 on T cells and its cognate receptor SIRP-α on DC define a novel regulatory pathway that may be involved in the maintenance of homeostasis by preventing the escalation of the inflammatory immune response.

The Thrombospondin Receptor Integrin-associated Protein (CD47) Functionally Couples to Heterotrimeric Gi

Integrin-associated protein (IAP; CD47) is a thrombospondin receptor that forms a signaling complex with β3 integrins resulting in enhanced αvβ3-dependent cell spreading and chemotaxis and, in platelets, αIIbβ3-dependent spreading and aggregation. These actions of CD47 are all specifically abrogated by pertussis toxin treatment of cells. Here we report that CD47, its β3 integrin partner, and Gi proteins form a stable, detergent-soluble complex that can be recovered by immunoprecipitation and affinity chromatography. Giα is released from this complex by treatment with GTP or AlF4. GTP and AlF4 also reduce the binding of CD47 to its agonist peptide (4N1K) derived from thrombospondin, indicating a direct association of CD47 with Gi. 4N1K peptide causes a rapid decrease in intraplatelet cyclic AMP levels, a Gi-dependent event necessary for aggregation. Finally, 4N1K stimulates the binding of GTPγ35S to membranes from cells expressing IAP and αvβ3. This functional coupling of CD47 to heterotrimeric G proteins provides a mechanistic explanation for the biological effects of CD47 in a wide variety of systems.

Nonphlogistic Clearance of Late Apoptotic Neutrophils by Macrophages: Efficient Phagocytosis Independent of β2 Integrins

Neutrophils undergo constitutive death by apoptosis, leading to safe nonphlogistic phagocytosis and clearance by macrophages. Recent work has shown that before secondary necrosis, neutrophils exhibiting classical features of apoptosis can progress to a morphologically defined late apoptotic state. However, whether such neutrophils could be safely cleared was unknown. We now report that human late apoptotic neutrophils could be purified from cultured neutrophil populations undergoing constitutive death and were subsequently ingested by human monocyte-derived macrophages by serum-independent mechanisms that did not trigger the release of IL-8 or TNF-α. Such ingestion was specifically inhibited by Abs to thrombospondin-1 and the αvβ3 vitronectin receptor. Murine bone marrow-derived macrophage phagocytosis of late and early apoptotic neutrophils occurred by similar mechanisms, proceeding with the same efficiency as that observed for wild-type controls when macrophages from αm−/− or β2−/− mice were used. We conclude that specific nonphlogistic, β2 integrin-independent mechanisms involving thrombospondin-1 and αvβ3 allow macrophages to ingest late apoptotic neutrophils without eliciting inflammatory cytokine secretion.

Inducible Tyrosine Phosphorylation of the β3 Integrin Requires the αv Integrin Cytoplasmic Tail

We have found that the integrin β3 chain can be phosphorylated on tyrosine residues in K562 cells transfected with αvβ3. Tyrosine phosphorylation of the β3 cytoplasmic tail is induced by adhesion to αvβ3-specific ligand or antibody or by incubation in manganese-containing buffer. Under the same conditions, β5 does not become tyrosine-phosphorylated in K562 transfected with αvβ5. Phosphorylation of the β3 subunit requires the simultaneous presence of the αv subunit cytoplasmic tail, because neither the αIIb subunit nor a truncated αv subunit is sufficient to permit phosphorylation of β3 when coexpressed as a heterodimer with β3. Finally, tyrosine phosphorylation of the β3 cytoplasmic tail occurs on both human and murine β3 and is inducible in the ovarian carcinoma OV10 as well, independent of expression of integrin-associated protein (CD47). Tyrosine phosphorylation of the β3 integrin subunit facilitates association of Grb-2, an adaptor protein leading to activation of the Ras signaling pathway, and may contribute to the unique functional and signaling capabilities of αvβ3.

Expression of a synapse-associated membrane protein, P84/SHPS-1, and its ligand, IAP/CD47, in mouse retina

P84 and integrin associated protein (IAP) are heterophilic binding partners that are expressed in the central nervous system in addition to a variety of other tissues. Both molecules are known to be involved in cell signaling in nonneural tissues. In the retina, both molecules are expressed prominently in plexiform layers, suggesting a possible association with synapses. Here, we examined the cellular expression and ultrastructural localization of the two molecules in the developing mouse retina. Both appeared to be expressed at one or both sides of synaptic sites, although the expression of IAP in the retina precedes that of P84. Examination of transgenic IAP‐null retinae revealed a failure of P84 to become associated with synaptic sites, suggesting the interaction of P84 with IAP was necessary for P84s synaptic localization. These findings suggest that the signaling activities of P84 and IAP are localized to sites of synaptic contact in the retina. Thus this pair of synapse‐associated molecules represents a bidirectional signaling system that could function to modify synaptic activity or possibly trophic interactions between central neurons. J. Comp. Neurol. 416:335–344, 2000.

SHPS-1 Induces Aggregation of Ba/F3 Pro-B Cells Via an Interaction with CD47

SHPS-1 (SH2-domain bearing protein tyrosine phosphatase (SHP) substrate-1), a member of the inhibitory-receptor superfamily that is abundantly expressed in macrophages and neural tissue, appears to regulate intracellular signaling events downstream of receptor protein-tyrosine kinases and integrin-extracellular matrix molecule interactions. To investigate the function of SHPS-1 in a hematopoietic cell line, SHPS-1 was expressed in Ba/F3 cells, an IL-3-dependent pro-B-cell line that lacks endogenous SHPS-1 protein. Interestingly, expression of either SHPS-1, or a mutant lacking the intracellular domain of SHPS-1 (ΔCT SHPS-1), resulted in the rapid formation of macroscopic Ba/F3 cell aggregates. As the integrin-associated protein/CD47 was shown to be a SHPS-1 ligand in neural cells, we investigated whether CD47 played a role in the aggregation of SHPS-1-expressing Ba/F3 cells. In support of this idea, aggregate formation was inhibited by an anti-CD47 Ab. Furthermore, erythrocytes from control, but not from CD47-deficient mice, were able to form rosettes on SHPS-1-expressing Ba/F3 cells. Because erythrocytes do not express integrins, this result suggested that SHPS-1-CD47 interactions can take place in the absence of a CD47-integrin association. We also present evidence that the amino-terminal Ig domain of SHPS-1 mediates the interaction with CD47. Although SHPS-1-CD47 binding likely triggers bidirectional intracellular signaling processes, these results demonstrate that this interaction can also mediate cell-cell adhesion.

Leucocyte adhesion molecules in host defence against infection

The destructive potential of leucocytes needs to be fully held in check in the circulation, while being released at the site of tissue destruction and infection. Leucocyte adhesion molecules are the key to this regulation, the recognition by the leucocytes of areas of tissue damage, and to transendothelial migration of leucocytes into these areas. The three classes of leucocyte adhesion molecules-the integrins, selectins and immunoglobulin superfamily members-are reviewed with their ligands and presented in the context of leucocyte migration, activation and host defence.

CD47 associates with alpha 5 integrin and regulates responses of human articular chondrocytes to mechanical stimulation in an in vitro model.

BackgroundRecent studies provide evidence of roles for integrins in mechanical signalling in bone and cartilage. Integrin signalling is modulated by various mechanisms, including interaction with other transmembrane proteins. We aimed to identify whether one such protein, integrin-associated protein (CD47/IAP), is expressed by chondrocytes and whether it may regulate integrin-dependent mechanotransduction.MethodsChondrocytes, isolated from macroscopically normal and osteoarthritic articular cartilage of human knee joints, were studied in a resting state or following mechanical stimulation at 0.33 Hz. CD47/IAP expression and associations were confirmed by immunohistology, reverse transcription-polymerase chain reaction, Western blotting, and immunoprecipitation. Roles in mechanotransduction were studied by assessing effects of function-blocking antibodies on a range of electrophysiological, cellular, and molecular responses of primary chondrocytes and responses of CD47/IAP-null cell lines transfected with CD47/IAP.ResultsHuman articular chondrocytes were shown to express CD47/IAP, predominantly the type 2 isoform. Immunoprecipitation showed association of CD47/IAP with α5 integrin and thrombospondin but not SIRPα (signal-regulatory protein-alpha). The function-blocking anti-CD47/IAP antibody Bric 126 inhibited changes in membrane potential, tyrosine phosphorylation, and elevation of relative levels of aggrecan mRNA induced by mechanical stimulation, whereas in the presence of B6H12, an antibody that has partial agonist activity, a membrane depolarisation rather than a membrane hyperpolarisation response was induced by mechanical stimulation. CD47-null cell lines did not show changes in cell membrane potential following mechanical stimulation. Changes in cell membrane potential following mechanical stimulation were seen when CD47-null cells were transfected with CD47/IAP expression vectors but were not seen following mechanical stimulation of cells transfected with vectors for the extracellular immunoglobulin variable (IgV) domain of CD47/IAP in the absence of the transmembrane and intracellular domains.ConclusionCD47/IAP is necessary for chondrocyte mechanotransduction. Through interactions with α5β1 integrin and thrombospondin, CD47/IAP may modulate chondrocyte responses to mechanical signals.