David M. Rose

A receptor for phosphatidylserine-specific clearance of apoptotic cells

The culmination of apoptosis in vivo is phagocytosis of cellular corpses. During apoptosis, the asymmetry of plasma membrane phospholipids is lost, which exposes phosphatidylserine externally. The phagocytosis of apoptotic cells can be inhibited stereospecifically by phosphatidylserine and its structural analogues, but not by other anionic phospholipids, suggesting that phosphatidylserine is specifically recognized. Using phage display, we have cloned a gene that appears to recognize phosphatidylserine on apoptotic cells. Here we show that this gene, when transfected into B and T lymphocytes, enables them to recognize and engulf apoptotic cells in a phosphatidylserine-specific manner. Flow cytometric analysis using a monoclonal antibody suggested that the protein is expressed on the surface of macrophages, fibroblasts and epithelial cells; this antibody, like phosphatidylserine liposomes, inhibited the phagocytosis of apoptotic cells and, in macrophages, induced an anti-inflammatory state. This candidate phosphatidylserine receptor is highly homologous to genes of unknown function in Caenorhabditis elegans and Drosophila melanogaster, suggesting that phosphatidylserine recognition on apoptotic cells during their removal by phagocytes is highly conserved throughout phylogeny.

Binding of paxillin to α4 integrins modifies integrin-dependent biological responses

The α4 integrins are indispensable for embryogenesis, haematopoiesis and immune responses, possibly because α4 regulates cellular functions differently from other integrins through its cytoplasmic tail. We used novel mimics of the α4 tail to identify molecules that could account for α4-specific signalling. Here we report that the α4 tail, but not several other α-subunit tails, binds tightly to the signalling adaptor paxillin. Paxillin physically associated with α4 integrins in Jurkat T cells at high stoichiometry, and joining the α4 tail to αIIb resulted in a complex of integrin αIIbβ3 with paxillin. This association markedly enhanced the rates of αIIbβ3-dependent phosphorylation of focal adhesion kinase and cell migration. It also reduced cell spreading, focal adhesion and stress fibre formation. A point mutation within the α4 tail that disrupts paxillin binding reversed all of these effects. Furthermore, α4β1-dependent adhesion to VCAM-1 led to spreading of mouse embryonic fibroblasts derived from paxillin-null but not from wild-type mice. Thus, the tight association of paxillin with the α4 tail leads to distinct biochemical and biological responses to integrin-mediated cell adhesion.

Increased filamin binding to β-integrin cytoplasmic domains inhibits cell migration

Multicellular animal development depends on integrins. These adhesion receptors link to the actin cytoskeleton, transmitting biochemical signals and force during cell migration and interactions with the extracellular matrix. Many integrin–cytoskeleton connections are formed by filamins and talin. The β7 integrin tail binds strongly to filamin and supports less migration, fibronectin matrix assembly and focal adhesion formation than either the β1D tail, which binds strongly to talin, or the β1A tail, which binds modestly to both filamin and talin. To probe the role of filamin binding, we mapped the filamin-binding site of integrin tails and identified amino acid substitutions that led to selective loss of filamin binding to the β7 tail and gain of filamin binding to the β1A tail. These changes affected cell migration and membrane protrusions but not fibronectin matrix assembly or focal adhesion formation. Thus, tight filamin binding restricts integrin-dependent cell migration by inhibiting transient membrane protrusion and cell polarization.

Integrins in the Immune System

Publisher Summary This chapter discusses the principles governing the integrins, their capacity to recognize ligands, cellular regulation of integrin function, and aspects of integrin function in the immune system. The chapter brings these general principles into focus to understand the immune system and to describe how perturbation of these receptors can be used to modulate immunological responses. Integrins are heterodimers formed by combination of 17 α and 8 β subunits in humans and are important because they play key roles in the cell migration, cell adhesion, control of differentiation, and critical decisions specifying cell proliferation and programmed cell death. The remarkable information storage and retrieval capacities of the immune system employ the general machinery involved in vertebrate development. Thus, integrins play crucial roles in modulating signals generated by clonotypic receptors and as effectors in processes such as cytolytic killing and phagocytosis.

α4β1-dependent adhesion strengthening under mechanical strain is regulated by paxillin association with the α4-cytoplasmic domain

The capacity of integrins to mediate adhesiveness is modulated by their cytoplasmic associations. In this study, we describe a novel mechanism by which α4-integrin adhesiveness is regulated by the cytoskeletal adaptor paxillin. A mutation of the α4 tail that disrupts paxillin binding, α4(Y991A), reduced talin association to the α4β1 heterodimer, impaired integrin anchorage to the cytoskeleton, and suppressed α4β1-dependent capture and adhesion strengthening of Jurkat T cells to VCAM-1 under shear stress. The mutant retained intrinsic avidity to soluble or bead-immobilized VCAM-1, supported normal cell spreading at short-lived contacts, had normal α4-microvillar distribution, and responded to inside-out signals. This is the first demonstration that cytoskeletal anchorage of an integrin enhances the mechanical stability of its adhesive bonds under strain and, thereby, promotes its ability to mediate leukocyte adhesion under physiological shear stress conditions.

The Affinity of Integrin α4β1 Governs Lymphocyte Migration

The interaction of integrin α4β1 with endothelial VCAM-1 controls the trafficking of lymphocytes from blood into peripheral tissues. Cells actively regulate the affinity of α4β1 for VCAM-1 (activation). To investigate the biological function of α4β1 activation, we isolated Jurkat T cell lines with defective α4β1 activation. Using these cells, we found that α4β1-stimulated αLβ2-dependent cell migration was dramatically reduced in cells with defects in α4β1 activation. These cells required 20 times more VCAM-1 to promote αLβ2-dependent cell migration. This defect was at the level of α4β1 affinity as an activating α4β1 Ab rescued α4β1-stimulated αLβ2-dependent migration. In contrast, migration of α4β1 activation-defective cells on VCAM-1 alone was enhanced at higher VCAM-1 densities. Thus, α4β1 activation determines a set point or threshold at which VCAM-1 can regulate αLβ2-dependent as well as α4β1-dependent cell migration. Changes in this set point may specify preferred anatomical sites of integrin-dependent leukocyte emigration from the bloodstream.

Blocking the α4 integrin–paxillin interaction selectively impairs mononuclear leukocyte recruitment to an inflammatory site

Antagonists to alpha4 integrin show promise for several autoimmune and inflammatory diseases but may exhibit mechanism-based toxicities. We tested the capacity of blockade of alpha4 integrin signaling to perturb functions involved in inflammation, while limiting potential adverse effects. We generated and characterized mice bearing a Y991A mutation in alpha4 integrin [alpha4(Y991A) mice], which blocks paxillin binding and inhibits alpha4 integrin signals that support leukocyte migration. In contrast to the embryonic-lethal phenotype of alpha4 integrin-null mice, mice bearing the alpha4(Y991A) mutation were viable and fertile; however, they exhibited defective recruitment of mononuclear leukocytes into thioglycollate-induced peritonitis. Alpha4 integrins are essential for definitive hematopoiesis; however, the alpha4(Y991A) mice had intact lymphohematopoiesis and, with the exception of reduced Peyers patches, normal architecture and cellularity of secondary lymphoid tissues. We conclude that interference with alpha4 integrin signaling can selectively impair mononuclear leukocyte recruitment to sites of inflammation while sparing vital functions of alpha4 integrins in development and hematopoiesis.

α4 Integrins in Cardiovascular Development and Diseases

Abstract α4 integrins (α4β1 and α4β7) have a restricted distribution pattern and are critical for the development and diseases of the cardiovascular system. α4 integrins support unique biological properties such as promoting cell migration and inhibiting cell spreading and focal adhesion formation. We have found that the α4 integrin subunit directly and tightly binds to a signaling adapter molecule, paxillin, and disruption of the α4-paxillin interaction interferes with many of α4-dependent biological functions. Consequently, the interaction of α4 integrins with paxillin may play an important role in regulating α4-mediated functions. This review focuses on what we have known about the α4-paxillin interaction and discusses the possible mechanism of regulation for this interaction.

A Small Molecule That Inhibits the Interaction of Paxillin and α4 Integrin Inhibits Accumulation of Mononuclear Leukocytes at a Site of Inflammation

Extracellular antagonists of α4 integrin are an effective therapy for several autoimmune and inflammatory diseases; however, these agents that directly block ligand binding may exhibit mechanism-based toxicities. Inhibition of α4 integrin signaling by mutations of α4 that block paxillin binding inhibits inflammation while limiting mechanism-based toxicities. Here, we test a pharmacological approach by identifying small molecules that inhibit the α4 integrin-paxillin interaction. By screening a large (∼40,000-compound) chemical library, we identified a noncytotoxic inhibitor of this interaction that impaired integrin α4-mediated but not αLβ2-mediated Jurkat T cell migration. The identified compound had no effect on α4-mediated migration in cells bearing the α4(Y991A) mutation that disrupts the α4-paxillin interaction, establishing the specificity of its action. Administration of this compound to mice led to impaired recruitment of mononuclear leukocytes to a site of inflammation in vivo, whereas an isomer that does not inhibit the α4-paxillin interaction had no effect on α4-mediated cell migration, cell spreading, or recruitment of leukocytes to an inflammatory site. Thus, a small molecule inhibitor that interferes with α4 integrin signaling reduces α4-mediated T cell migration in vivo, thus providing proof of principle for inhibition of α4 integrin signaling as a target for the pharmacological reduction of inflammation.

Integrins as therapeutic targets

Integrins are widely expressed receptors that primarily function as mediators of cell-extracellular matrix interactions. They have multiple physiological roles, and alterations in their structure and function result in pathological conditions in multicellular organisms. Selective targeting of integrins is a therapeutic modality that can impact disease processes which are major causes of morbidity and mortality. This is most clearly seen in vascular disease, where anti-integrin agents are routinely used for treating acute ischaemic myocardial conditions. In the future, therapeutic targeting of integrins may also play a role in the treatment of inflammatory and neoplastic conditions. In this review we discuss the mechanisms whereby integrins are involved in thrombotic vascular conditions, inflammation and neoplasia, and outline how they can be targeted as therapeutic modalities.