The insider's guide to leukocyte integrin signalling and function

The activation of leukocyte integrins through diverse receptors results in transformation of the integrin from a bent, resting form to an extended conformation, which has at least two states of ligand-binding activity. This highly regulated activation process is essential for T cell migration and the formation of an immunological synapse. The signalling events that drive integrin activation are complex. Some key players have been well-characterized, but other aspects of the signalling mechanisms involved are still unclear. This Review focuses on the integrin lymphocyte function-associated antigen 1 (LFA1; also known as αLβ2 integrin), which is expressed by T cells, and explores how disparate signalling pathways synergize to regulate LFA1 activity.     

A Small Molecule That Inhibits the Interaction of Paxillin and ��4 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.     

Novel inhibitors of leukocyte transendothelial migration

Leukocyte transendothelial migration is one of the most important step in launching an inflammatory immune response and chronic inflammation can lead to devastating diseases. Leukocyte migration inhibitors are considered as promising and potentially effective therapeutic agents to treat inflammatory and auto-immune disorders. In this study, based on previous trioxotetrahydropyrimidin based integrin inhibitors that suboptimally blocked leukocyte adhesion, twelve molecules with a modified scaffold were designed, synthesized, and tested in vitro for their capacity to block the transendothelial migration of immune cells. One of the molecules, namely, methyl 4-((2-(tert-butyl)-6-((2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene) methyl) phenoxy) methyl) benzoate, (compound 12), completely blocked leukocyte transendothelial migration, without any toxic effects on immune or endothelial cells (IC₅₀ = 2.4 µM). In vivo, compound 12 exhibited significant therapeutic effects in inflammatory bowel disease (IBD)/Crohn’s disease, multiple sclerosis, fatty liver disease, and rheumatoid arthritis models. A detailed acute and chronic toxicity profile of the lead compound in vivo did not reveal any toxic effects. Such a type of molecule might therefore provide a unique starting point for designing a novel class of leukocyte transmigration blocking agents with broad therapeutic applications in inflammatory and auto-immune pathologies.     

Paxillin binding to the alpha 4 integrin subunit stimulates LFA-1 (integrin alpha L beta 2)-dependent T cell migration by augmenting the activation of focal adhesion kinase/proline-rich tyrosine kinase-2

Engagement of very late Ag-4 (integrin alpha(4)beta(1)) by ligands such as VCAM-1 markedly stimulates leukocyte migration mediated by LFA-1 (integrin alpha(L)beta(2)). This form of integrin trans-regulation in T cells requires the binding of paxillin to the alpha(4) integrin cytoplasmic domain. This conclusion is based on the abolition of trans-regulation in Jurkat T cells by an alpha(4) mutation (alpha(4)(Y991A)) that disrupts paxillin binding. Furthermore, cellular expression of an alpha(4)-binding fragment of paxillin that blocks the alpha(4)-paxillin interaction, selectively blocked VCAM-1 stimulation of alpha(L)beta(2)-dependent cell migration. The alpha(4)-paxillin association mediates trans-regulation by enhancing the activation of tyrosine kinases, focal adhesion kinase (FAK) and/or proline-rich tyrosine kinase-2 (Pyk2), based on two lines of evidence. First, disruption of the paxillin-binding site in the alpha(4) tail resulted in much less alpha(4)beta(1)-mediated phosphorylation of Pyk2 and FAK. Second, transfection with cDNAs encoding C-terminal fragments of Pyk2 and FAK, which block the function of the intact kinases, blocked alpha(4)beta(1) stimulation of alpha(L)beta(2)-dependent migration. These results define a proximal protein-protein interaction of an integrin cytoplasmic domain required for trans-regulation between integrins, and establish that augmented activation of Pyk2 and/or FAK is an immediate signaling event required for the trans-regulation of integrin alpha(L)beta(2) by alpha(4)beta(1).     

Competition for talin results in trans-dominant inhibition of integrin activation

The ability of integrin adhesion receptors to undergo rapid changes in affinity for their extracellular ligands (integrin activation) is essential for the development and function of multicellular animals and is dependent on interactions between the integrin beta subunit-cytoplasmic tail and the cytoskeletal protein talin. Cross-talk among different integrins and between integrins and other receptors impacts many cellular processes including adhesion, spreading, migration, clot retraction, proliferation, and differentiation. One form of integrin cross-talk, transdominant inhibition of integrin activation, occurs when ligand binding to one integrin inhibits the activation of a second integrin. This may be relevant clinically in a number of settings such as during platelet adhesion, leukocyte trans-migration, and angiogenesis. Here we report that competition for talin underlies the trans-dominant inhibition of integrin activation. This conclusion is based on our observations that (i). beta tails selectively defective in talin binding are unable to mediate trans-dominant inhibition, (ii). trans-dominant inhibition can be reversed by overexpression of integrin binding and activating fragments of talin, and (iii). expression of another non-integrin talin-binding protein, phosphatidylinositol phosphate kinase type Igamma-90, also inhibits integrin activation. Thus, the sequestration of talin by the suppressive species is both necessary and sufficient for trans-dominant inhibition of integrin activation.     

Leukocyte transendothelial migration: orchestrating the underlying molecular machinery.

Transendothelial migration of leukocytes involves the spatiotemporal regulation of adhesion molecules, chemokines and cytoskeletal regulators. Recent results show that distinct steps of leukocyte transendothelial migration are regulated by sequential integrin activation and coordinated Rho family GTPase activity. Progress has been made in understanding how the dynamic regulation of these molecules translates into leukocyte transmigration.     

Cells on the run: shear-regulated integrin activation in leukocyte rolling and arrest on endothelial cells

The arrest of rolling leukocytes on various target vascular beds is mediated by specialized leukocyte integrins and their endothelial immunoglobulin superfamily (IgSF) ligands. These integrins are kept in largely inactive states and undergo in situ activation upon leukocyte-endothelial contact by both biochemical and mechanical signals from flow-derived shear forces. In vivo and in vitro studies suggest that leukocyte integrin activation involves conformational alterations through inside-out signaling followed by ligand-induced rearrangements accelerated by external forces. This activation process takes place within fractions of seconds by in situ signals transduced to the rolling leukocyte as it encounters specialized endothelial-displayed chemoattractants, collectively termed arrest chemokines. In neutrophils, selectin rolling engagements trigger intermediate affinity integrins to support reversible adhesions before chemokine-triggered arrest. Different leukocyte subsets appear to use different modalities of integrin activation during rolling and arrest at distinct endothelial sites.     

Integrin activation by chemokines: relevance to inflammatory adhesion cascade during T cell migration

The adhesive function of integrins is regulated through cytoplasmic signaling induced by several stimuli, whose process is designated "inside-out signaling". A large number of leukocytes are rapidly recruited to the sites of inflammation where they form an essential component of the response to infection, injury, autoimmune disorders, allergy, tumor invasion, atherosclerosis and so on. The recruitment of leukocytes into tissue is regulated by a sequence of interactions between the circulating leukocytes and the endothelial cells. Leukocyte integrins play a pivotal role in leukocyte adhesion to endothelial cells. During the process, the activation of integrins by various chemoattractants, especially chemokines, is essential for integrin-mediated adhesion in which a signal transduced to the leukocyte converts the functionally inactive integrin to an active adhesive configuration. We have proposed that H-Ras-sensitive activation of phosphoinositide 3 (PI 3)-kinase and subsequent profilin-mediated actin polymerization, can be involved in chemokine-induced integrin-dependent adhesion of T cells. The present review documents the relevance of cytoplasmic signaling and cytoskeletal assembly to integrin-mediated adhesion induced by chemoattractants including chemokines during inflammatory processes. In contrast, various adhesion molecules are known to transduce extracellular information into cytoplasm, which leads to T cell activation and cytokine production from the cells, designated "outside-in signaling". Such a bi-directional "cross-talking" among adhesion molecules and cytokines is most relevant to inflammatory processes by augmenting immune cell migration from circulation into inflamed tissue such as rheumatoid arthritis, tumor invasion, Beh?et's disease and atherosclerosis.     

Effect of posttranslational processing on the in vitro and in vivo activity of chemokines

The CXC and CC chemokine gene clusters provide an abundant number of chemotactic factors selectively binding to shared G protein-coupled receptors (GPCR). Hence, chemokines function in a complex network to mediate migration of the various leukocyte subsets, expressing specific GPCRs during the immune response. Further fine-tuning of the chemokine system is reached through specific posttranslational modifications of the mature proteins. Indeed, enzymatic processing of chemokines during an early phase of inflammation leads to activation of precursor molecules or cleavage into even more active or receptor specific chemokine isoforms. At a further stage, proteolytic processing leads to loss of GPCR signaling, thereby providing natural chemokine receptor antagonists. Finally, further NH₂-terminal cleavage results in complete inactivation to dampen the inflammatory response. During inflammatory responses, the two chemokines which exist in a membrane-bound form may be released by proteases from the cellular surface. In addition to proteolytic processing, citrullination and glycosylation of chemokines is also important for their biological activity. In particular, citrullination of arginine residues seems to reduce the inflammatory activity of chemokines in vivo. This goes along with other positive and negative regulatory mechanisms for leukocyte migration, such as chemokine synergy and scavenging by decoy receptors.     

KIF14 negatively regulates Rap1a–Radil signaling during breast cancer progression

The small GTPase Rap1 regulates inside-out integrin activation and thereby influences cell adhesion, migration, and polarity. Several Rap1 effectors have been described to mediate the cellular effects of Rap1 in a context-dependent manner. Radil is emerging as an important Rap effector implicated in cell spreading and migration, but the molecular mechanisms underlying its functions are unclear. We report here that the kinesin KIF14 associates with the PDZ domain of Radil and negatively regulates Rap1-mediated inside-out integrin activation by tethering Radil on microtubules. The depletion of KIF14 led to increased cell spreading, altered focal adhesion dynamics, and inhibition of cell migration and invasion. We also show that Radil is important for breast cancer cell proliferation and for metastasis in mice. Our findings provide evidence that the concurrent up-regulation of Rap1 activity and increased KIF14 levels in several cancers is needed to reach optimal levels of Rap1–Radil signaling, integrin activation, and cell–matrix adhesiveness required for tumor progression.     

Proline-rich tyrosine kinase 2 and Rac activation by chemokine and integrin receptors controls NK cell transendothelial migration

Protein tyrosine kinase activation is an important requisite for leukocyte migration. Herein we demonstrate that NK cell binding to endothelium activates proline-rich tyrosine kinase 2 (Pyk-2) and the small GTP binding protein Rac that are coupled to integrin and chemokine receptors. Chemokine-mediated, but not integrin-mediated, Pyk-2 and Rac activation was sensitive to pretreatment of NK cells with pertussis toxin, a pharmacological inhibitor of G(i) protein-coupled receptors. Both Pyk-2 and Rac are functionally involved in chemokine-induced NK cell migration through endothelium or ICAM-1 or VCAM-1 adhesive proteins, as shown by the use of recombinant vaccinia viruses encoding dominant negative mutants of Pyk-2 and Rac. Moreover, we found that Pyk-2 is associated with the Rac guanine nucleotide exchange factor Vav, which undergoes tyrosine phosphorylation upon integrin triggering. Finally, we provide direct evidence for the involvement of Pyk-2 in the control of both chemokine- and integrin-mediated Rac activation. Collectively, our results indicate that Pyk-2 acts as a receptor-proximal link between integrin and chemokine receptor signaling, and the Pyk-2/Rac pathway plays a pivotal role in the control of NK cell transendothelial migration.     

RSK2 Protein Suppresses Integrin Activation and Fibronectin Matrix Assembly and Promotes Cell Migration

Modulation of integrin activation is important in many cellular functions including adhesion, migration, and assembly of the extracellular matrix. RSK2 functions downstream of Ras/Raf and promotes tumor cell motility and metastasis. We therefore investigated whether RSK2 affects integrin function. We report that RSK2 mediates Ras/Raf inactivation of integrins. As a result, we find that RSK2 impairs cell adhesion and integrin-mediated matrix assembly and promotes cell motility. Active RSK2 appears to affect integrins by reducing actin stress fibers and disrupting focal adhesions. Moreover, RSK2 co-localizes with the integrin activator talin and is present at integrin cytoplasmic tails. It is thereby in a position to modulate integrin activation and integrin-mediated migration. Activation of RSK2 promotes filamin phosphorylation and binding to integrins. We also find that RSK2 is activated in response to integrin ligation to fibronectin. Thus, RSK2 could participate in a feedback loop controlling integrin function. These results reveal RSK2 as a key regulator of integrin activity and provide a novel mechanism by which it may promote cell migration and cancer metastasis.     

Protein tyrosine kinases in neutrophil activation and recruitment

Migration of leukocytes into tissue is a key element of innate and adaptive immunity. The first contact of leukocytes with endothelial cells is mediated by engagement of selectins with their counter-receptors which results in leukocyte rolling. During rolling, leukocytes collect different inflammatory signals that activate intracellular signaling pathways. Integration of these signals induces leukocyte activation, firm arrest, post-adhesion strengthening, intravascular crawling, and transmigration. In neutrophils, like in T-cells and platelets, both G-protein-coupled receptor-dependent and -independent activation pathways exist that lead to integrin activation. Accumulating evidence suggests that different protein tyrosine kinases play key roles in signal transduction pathways regulating neutrophil activation and recruitment to inflammatory sites. This review focuses on the role of protein tyrosine kinases of the Src, Syk, and Tec families for neutrophil activation and recruitment.     

Role of integrin-linked kinase in leukocyte recruitment

Chemokines modulate leukocyte integrin avidity to coordinate adhesion and subsequent transendothelial migration, although the sequential signaling pathways involved remain poorly characterized. Here we show that integrin-linked kinase (ILK), a 59-kDa serine-threonine protein kinase that interacts principally with beta(1) integrins, is highly expressed in human mononuclear cells and is activated by exposure of leukocytes to the chemokine monocyte chemoattractant protein-1. Biochemical inhibitor studies show that chemokine-triggered activation of ILK is downstream of phosphoinositide 3-kinase. In functional assays under physiologically relevant flow conditions, overexpression of wild-type ILK in human monocytic cells diminishes beta(1) integrin/vascular cell adhesion molecule-1-dependent firm adhesion to human endothelial cells. These data implicate ILK in the dynamic signaling events involved in the regulation of leukocyte integrin avidity for endothelial substrates.     

S100A9 mediates neutrophil adhesion to fibronectin through activation of beta2 integrins

Neutrophil migration from the blood to inflammatory sites follows a cascade of events, in which adhesion to endothelial cells and extracellular matrix proteins is essential. S100A8, S100A9, and S100A12 are small abundant proteins found in human neutrophil cytosol and presumed to be involved in leukocyte migration. Here we investigated the S100 proteins' activities in neutrophil tissue migration by evaluating their effects on neutrophil adhesion to certain extracellular matrix proteins. S100A9 induced adhesion only to fibronectin and was the only S100 protein that stimulated neutrophil adhesion to this extracellular matrix protein. Experiments with blocking antibodies revealed that neither beta1 nor beta3 integrins were strongly involved in neutrophil adhesion to fibronectin, contrary to what the literature predicted. In contrast, neutrophil adhesion to fibronectin was completely inhibited by anti-beta2 integrins, suggesting that S100A9-induced specific activation of beta2 integrin is essential to neutrophil adhesion.     

Leukocyte rolling velocities and migration are optimized by cooperative L-selectin and intercellular adhesion molecule-1 functions

Selectin family members largely mediate initial tethering and rolling of leukocytes on vascular endothelium, whereas integrin and Ig family members are essential for leukocyte firm adhesion. To quantify functional synergy between L-selectin and Ig family members during leukocyte rolling, the EA.hy926 human vascular endothelial line was transfected with either fucosyltransferase VII (926-FtVII) cDNA to generate L-selectin ligands alone or together with ICAM-1 cDNA (926-FtVII/ICAM-1). The ability of transfected 926 cells to support human leukocyte interactions was assessed in vitro using parallel plate flow chamber assays. Lymphocyte rolling on 926-FtVII cells was increased by approximately 70% when ICAM-1 was expressed at physiological levels. Although initial tether formation was similar for both cell types, lymphocyte rolling was 26% slower on 926-FtVII/ICAM-1 cells. Pretreatment of lymphocytes with an anti-CD18 mAb eliminated the increase in rolling, and all rolling was blocked by anti-L-selectin mAb. In addition, rolling velocities of lymphocytes from CD18-hypomorphic mice were 48% faster on 926-FtVII/ICAM-1 cells, with a similar reduction in rolling frequency relative to wild-type lymphocytes. CD18-hypomorphic lymphocytes also showed an approximately 40% decrease in migration to peripheral and mesenteric lymph nodes during in vivo migration assays compared with wild-type lymphocytes. Likewise, wild-type lymphocyte migration to peripheral lymph nodes was reduced by approximately 50% in ICAM-1(-/-) recipient mice. Similar to human lymphocytes, human neutrophils showed enhanced rolling interactions on 926-FtVII/ICAM-1 cells, but also firmly adhered. Thus, in addition to mediating leukocyte firm adhesion, CD18 integrin/ICAM-1 interactions regulate leukocyte rolling velocities and thereby optimize L-selectin-mediated leukocyte rolling.     

Cyclin-dependent kinase inhibitors block leukocyte adhesion and migration

Leukocyte trafficking is a tightly regulated process essential for an appropriate inflammatory response. We now report a new adhesion pathway that allows unstimulated leukocytes to adhere to and migrate through exposed endothelial matrix or high-density ligand, a process we have termed ligand-induced adhesion. This ligand-induced adhesion is integrin mediated, but in contrast to phorbol ester-stimulated adhesion, it is not dependent on the small GTPase Rap-1 activity. Instead, we show a critical role for cyclin-dependent kinase (Cdk) 4 in ligand-induced adhesion by three independent lines of evidence: inhibition by pharmacological inhibitors of Cdk, inhibition by dominant-negative construct of Cdk4, and inhibition by Cdk4 small interfering RNA. The major substrate of Cdk4, Rb, is not required for ligand-induced adhesion, suggesting the involvement of a novel Cdk4 substrate. We also demonstrate that Cdk4(-/-) mice have impaired recruitment of lymphocytes to the lung following injury. The finding that Cdk inhibitors can block leukocyte adhesion and migration may expand the clinical indications for this emerging class of therapeutics.     

The talin-tail interaction places integrin activation on FERM ground

Integrins are essential receptors for the development and functioning of multicellular animals because they mediate cell migration and cell adhesion, and regulate cell proliferation and apoptosis. Cellular regulation of the affinity of integrins for ligands - so-called 'integrin activation' - is a central property of these receptors. Integrin activation controls cell adhesion, migration and extracellular matrix assembly, thereby contributing to processes such as angiogenesis, tumor cell metastasis, inflammation, the immune response and hemostasis. Recent studies indicate that a crucial, final step in integrin activation is the binding of talin, a cytoskeletal protein, to the cytoplasmic domain of the integrin beta subunit. These results provide a focus for unraveling the many biochemical pathways implicated in integrin activation and suggest a general structural model for the connections between integrins and diverse cellular signal transduction pathways.     

JNK2 promotes endothelial cell alignment under flow

Endothelial cells in straight, unbranched segments of arteries elongate and align in the direction of flow, a feature which is highly correlated with reduced atherosclerosis in these regions. The mitogen-activated protein kinase c-Jun N-terminal kinase (JNK) is activated by flow and is linked to inflammatory gene expression and apoptosis. We previously showed that JNK activation by flow is mediated by integrins and is observed in cells plated on fibronectin but not on collagen or basement membrane proteins. We now show thatJNK2 activation in response to laminar shear stress is biphasic, with an early peak and a later peak. Activated JNK localizes to focal adhesions at the ends of actin stress fibers, correlates with integrin activation and requires integrin binding to the extracellular matrix. Reducing JNK2 activation by siRNA inhibits alignment in response to shear stress. Cells on collagen, where JNK activity is low, align slowly. These data show that an inflammatory pathway facilitates adaptation to laminar flow, thereby revealing an unexpected connection between adaptation and inflammatory pathways.