Publications

Leukocyte microvilli are elastic actin-rich projections implicated in rapid sensing and penetration across glycocalyx barriers. Microvilli are critical for the capture and arrest of flowing lymphocytes by high endothelial venules, the main lymph node portal vessels. T lymphocyte arrest involves subsecond activation of the integrin LFA-1 by the G-protein-coupled receptor CCR7 and its endothelial-displayed ligands, the chemokines CCL21 and CCL19. The topographical distribution of CCR7 and of LFA-1 in relation to lymphocyte microvilli has never been elucidated. We applied the recently developed microvillar cartography imaging technique to determine the topographical distribution of CCR7 and LFA-1 with respect to microvilli on peripheral blood T lymphocytes. We found that CCR7 is clustered on the tips of T cell microvilli. The vast majority of LFA-1 molecules were found on the cell body, likely assembled in macroclusters, but a subset of LFA-1, 5% of the total, were found scattered within 20 nm from the CCR7 clusters, implicating these LFA-1 molecules as targets for inside-out activation signals transmitted within a fraction of a second by chemokine-bound CCR7. Indeed, RhoA, the key GTPase involved in rapid LFA-1 affinity triggering by CCR7, was also found to be clustered near CCR7. In addition, we observed that the tyrosine kinase JAK2 controls CCR7-mediated LFA-1 affinity triggering and is also highly enriched on tips of microvilli. We propose that tips of lymphocyte microvilli are novel signalosomes for subsecond CCR7-mediated inside-out signaling to neighboring LFA-1 molecules, a critical checkpoint in LFA-1-mediated lymphocyte arrest on high endothelial venules.

Microtubules (MTs) control cell shape and intracellular cargo transport. The role of MT turnover in the migration of slow-moving cells through endothelial barriers remains unclear. To irreversibly interfere with MT disassembly, we have used the MT-stabilizing agent zampanolide (ZMP) in Β16F10 melanoma as amodel of slow-moving cells. ZMP-treated B16 cells failed to follow chemotactic gradients across rigid confinements and could not generate stable sub-endothelial pseudopodia under endothelial monolayers. In vivo, ZMP-treated Β16 cells failed to extravasate though lung capillaries. In contrast to melanoma cells, the chemotaxis and transendothelial migration of ZMP-treated Tcells were largely conserved. This is afirst demonstration that MT disassembly is akey checkpoint in the directional migration of cancer cells but not of lymphocytes.

The mechanisms by which the nuclear lamina of tumor cells influences tumor growth and migration are highly disputed. Lamin A and its variant lamin C are key lamina proteins that control nucleus stiffness and chromatin conformation. Downregulation of lamin A/C in two prototypic metastatic lines, B16F10 melanoma and E0771 breast carcinoma, facilitated cell squeezing through rigid pores, and reduced heterochromatin content. Surprisingly, both lamin A/C knockdown cells grew poorly in 3D spheroids within soft agar, and lamin A/C deficient cells derived from spheroids transcribed lower levels of the growth regulator Yap1. Unexpectedly, the transendothelial migration of both cancer cells in vitro and in vivo, through lung capillaries, was not elevated by lamin A/C knockdown and their metastasis in lungs was even dramatically reduced. Our results are the first indication that reduced lamin A/C content in distinct types of highly metastatic cancer cells does not elevate their transendothelial migration (TEM) capacity and diapedesis through lung vessels but can compromise lung metastasis at a post extravasation level.

Neutrophils provide first line of host defense against bacterial infections utilizing glycolysis for their effector functions. How glycolysis and its major byproduct lactate are triggered in bone marrow (BM) neutrophils and their contribution to neutrophil mobilization in acute inflammation is not clear. Here we report that bacterial lipopolysaccharides (LPS) or Salmonella Typhimurium triggers lactate release by increasing glycolysis, NADPH-oxidase-mediated reactive oxygen species and HIF-1α levels in BM neutrophils. Increased release of BM lactate preferentially promotes neutrophil mobilization by reducing endothelial VE-Cadherin expression, increasing BM vascular permeability via endothelial lactate-receptor GPR81 signaling. GPR81-/- mice mobilize reduced levels of neutrophils in response to LPS, unless rescued by VE-Cadherin disrupting antibodies. Lactate administration also induces release of the BM neutrophil mobilizers G-CSF, CXCL1 and CXCL2, indicating that this metabolite drives neutrophil mobilization via multiple pathways. Our study reveals a metabolic crosstalk between lactate-producing neutrophils and BM endothelium, which controls neutrophil mobilization under bacterial infection.

Cell migration is indispensable for various biological processes including angiogenesis, wound healing, and immunity. In general, there are two different migration modes described, the mesenchymal migration mode and the amoeboid migration mode. Neutrophils rapidly migrate toward the sites of injury, infection, and inflammation using the amoeboid migration mode which is characterized by cell polarization and a high migration velocity. During site-directed trafficking of neutrophils from the blood stream into the inflamed tissue, neutrophils must first withstand shear stress while migrating on the 2-dimensional endothelial surface. Subsequently, they have to cross different physical barriers during the extravasation process including the squeezing through the compact endothelial monolayer that comprises the blood vessel, the underlining basement membrane and then the 3-dimensional meshwork of extracellular matrix (ECM) proteins in the tissue. Therefore, neutrophils have to rapidly switch between distinct migration modes such as intraluminal crawling, transmigration, and interstitial migration to pass these different confinements and mechanical barriers. The nucleus is the largest and stiffest organelle in every cell and is therefore the key cellular element involved in cellular migration through variable confinements. This review highlights the importance of nuclear deformation during neutrophil crossing of such confinements, with a focus on transendothelial migration and interstitial migration. We discuss the key molecular components involved in the nuclear shape changes that underlie neutrophil motility and squeezing through cellular and ECM barriers. Understanding the precise molecular mechanisms that orchestrate these distinct neutrophil migration modes introduces an opportunity to develop new therapeutic concepts for controlling pathological neutrophil-driven inflammation.

Transendothelial migration (TEM) of lymphocytes and neutrophils is associated with the ability of their deformable nuclei to displace endothelial cytoskeletal barriers. Lamin A is a key intermediate filament component of the nuclear lamina that is downregulated during granulopoiesis. When elevated, lamin A restricts nuclear squeezing through rigid confinements. To determine if the low lamin A expression by leukocyte nuclei is critical for their exceptional squeezing ability through endothelial barriers, we overexpressed this protein in granulocyte-like differentiated HL-60 cells. A 10-fold higher lamin A expression did not interfere with chemokinetic motility of these granulocytes on immobilized CXCL1. Furthermore, these lamin A high leukocytes exhibited normal chemotaxis toward CXCL1 determined in large pore transwell barriers, but poorly squeezed through 3 μm pores toward identical CXCL1 gradients. Strikingly, however, these leukocytes successfully completed paracellular TEM across inflamed endothelial monolayers under shear flow, albeit with a small delay in nuclear squeezing into their sub-endothelial pseudopodia. In contrast, CXCR2 mediated granulocyte motility through collagen I barriers was dramatically delayed by lamin A overexpression due to a failure of lamin A high nuclei to translocate into the pseudopodia of the granulocytes. Collectively, our data predict that leukocytes maintain a low lamin A content in their nuclear lamina in order to optimize squeezing through extracellular collagen barriers but can tolerate high lamin A content when crossing the highly adaptable barriers presented by the endothelial cytoskeleton.

The tumor suppressor p53 limits tumorigenesis by inducing apoptosis, cell cycle arrest, and senescence. Although p53 is known to limit inflammation during tumor development, its role in regulating chronic lung inflammation is less well understood. To elucidate the function of airway epithelial p53 in such inflammation, we subjected genetically modified mice, whose bronchial epithelial club cells lack p53, to repetitive inhalations of lipopolysaccharide (LPS), an exposure that leads to severe chronic bronchitis and airway senescence in wild-type mice. Surprisingly, the club cell p53 knockout mice exhibited reduced airway senescence and bronchitis in response to chronic LPS exposure and were significantly protected from global lung destruction. Furthermore, pharmacological elimination of senescent cells also protected wild-type mice from chronic LPS-induced bronchitis. Our results implicate p53 in induction of club-cell senescence and correlate epithelial cell senescence of chronic airway inflammation and lung destruction. Sagiv et al. find that senescence and p53 in bronchial epithelial cells promote chronic lung inflammation and COPD-like disease. Genetic or pharmacological reduction in senescent cell number blunts chronic inflammation and limits disease progression.

Activation of endothelial cells by IL-1p triggers the expression of multiple inflammatory cytokines and leukocyte-attracting chemokines. The machineries involved in the secretion of these inducible proteins are poorly understood. With the use of genome-wide transcriptional analysis of inflamed human dermal micro vascular endothelial cells, we identified several IL-1 beta induced candidate regulators of these machineries and chose to focus our study on TNF-alpha-induced protein 2 (myeloid-secretory). The silencing of myeloid secretory did not affect the ability of inflamed endothelial cells to support the adhesion and crawling of effector T lymphocytes. However, the ability of these lymphocytes to complete transendothelial migration across myeloid-secretory-silenced human dermal microvascular endothelial cells was inhibited significantly. These observed effects on lymphocyte trans endothelial migration were recovered completely when exogenous promigratory chemokine CXCL12 was overlaid on the endothelial barrier. A polarized secretion assay suggested that the silencing of endothelial myeloid-secretory impairs T effector transendothelial migration by reducing the preferential secretion of endothelial-produced CCL2, a key transendothelial migration-promoting chemokine for these lymphocytes, into the basolateral endothelial compartment. Myeloid secretory silencing also impaired the preferential secretion of other endothelial-produced inflammatory chemokines, as well as cytokines, such as IL-6 and GMCSF, into the basolateral endothelial compartment. This is the first evidence of a novel inflammation -inducible machinery that regulates polarized secretion of endothelial CCL2 and other inflammatory chemokines and cytokines into basolateral endothelial compartments and facilitates the ability of endothelial CCL2 to promote T cell transendothelial migration.

Results of a study of the substructure of the highest transverse momentum (p(T)) jets observed by the CDF Collaboration are presented. Events containing at least one jet with p(T) > 400 GeV/c in a sample corresponding to an integrated luminosity of 5.95 fb(-1), collected in 1.96 TeV proton-antiproton collisions at the Fermilab Tevatron collider, are selected. A study of the jet mass, angularity, and planar-flow distributions is presented, and the measurements are compared with predictions of perturbative quantum chromodynamics. A search for boosted top-quark production is also described, leading to a 95% confidence level upper limit of 38 fb on the production cross section of top quarks with p(T) > 400 GeV/c.

Amplified HER2, which encodes a member of the epidermal growth factor receptor (EGFR) family, is a target of effective therapies against breast cancer. In search for similarly targetable genomic aberrations, we identified copy number gains in SYNJ2, which encodes the 5'-inositol lipid phosphatase synaptojanin 2, as well as overexpression in a small fraction of human breast tumors. Copy gain and overexpression correlated with shorter patient survival and a low abundance of the tumor suppressor microRNA miR-31. SYNJ2 promoted cell migration and invasion in culture and lung metastasis of breast tumor xenografts in mice. Knocking down SYNJ2 impaired the endocytic recycling of EGFR and the formation of cellular lamellipodia and invadopodia. Screening compound libraries identified SYNJ2-specific inhibitors that prevented cell migration but did not affect the related neural protein SYNJ1, suggesting that SYNJ2 is a potentially druggable target to block cancer cell migration.

A single G protein-coupled receptor (GPCR) can activate multiple signaling cascades based on the binding of different ligands. The biological relevance of this feature in immune regulation has not been evaluated. The chemoldne-binding GPCR CXCR3 is preferentially expressed on CD4+ T cells, and canonically binds 3 structurally related chemokines: CXCL9, CXCL10, and CXCL11. Here we have shown that CXCL10/CXCR3 interactions drive effector Th1 polarization via STAT1, STAT4, and STAT5 phosphorylation, while CXCL11/CXCR3 binding induces an im.munotolerizing state that is characterized by IL-10(hi) (Tr) and IL-4(hi) (Th2) cells, mediated via p70 ldnase/mTOR in STAT3- and STAT6-dependent pathways. CXCL11 binds CXCR3 with a higher affinity than CXCL10, suggesting that CXCL11 has the potential to restrain inflammatory autoimmunity. We generated a CXCL11-Ig fusion molecule and evaluated its use in the EAE model of inflammatory autoimmune disease. Administration of CXCL11-Ig during the first episode of relapsing EAE in SJL/J mice not only led to rapid remission, but also prevented subsequent relapse. Using GFP-expressing effector CD4(+) T cells, we observed that successful therapy was associated with reduced accumulation of these cells at the autoimmune site. Finally, we showed that very low doses of CXCL11 rapidly suppress signs of EAE in C57BL/6 mice lacking functional CXCL11.

During sterile inflammation, emigrating leukocytes sequentially engage subsets of pericytes associated with blood vessels and acquire adhesive, migratory and survival signals.

Recent evidence suggests that kindlin-3 is a major coactivator, required for most, if not all, integrin activities. Here we studied the function of kindlin-3 in regulating NK cell activation by studying a patient with kindlin-3 deficiency (leukocyte adhesion deficiency-III). We found that kindlin-3 is required for NK cell migration and adhesion under shear force. Surprisingly, we also found that kindlin-3 lowers the threshold for NK cell activation. Loss of kindlin-3 has a pronounced effect on NK cell-mediated cytotoxicity triggered by single activating receptors. In contrast, for activation through multiple receptors, kindlin-3 deficiency is overcome and target cells killed. The realization that NK cell activity is impaired, but not absent in leukocyte adhesion deficiency, may lead to the development of more efficient therapy for this rare disease. (Blood. 2012;120(19):3915-3924)

Death-associated protein kinase (DAPk) is a tumor suppressor thought to inhibit cancer by promoting apoptosis and autophagy. Because cancer progression is linked to inflammation, we investigated the in vivo functions of DAPk in lung responses to various acute and chronic inflammatory stimuli. Lungs of DAPk knockout (KO) mice secreted higher concentrations of IL-6 and keratinocyte chemoattractant (or chemokine [C-X-C motif] ligand 1) in response to transient intranasal administrations of the Toll-like receptor-4 (TLR4) agonist LPS. In addition, DAPk-null macrophages and neutrophils were hyperresponsive to ex vivo stimulation with LPS. DAPk-null neutrophils were also hyperresponsive to activation via Fc receptor and Toll-like receptor-3, indicating that the suppressive functions of this kinase are not restricted to TLR4 pathways. Even after the reconstitution of DAPk-null lungs with DAPk-expressing leukocytes by transplanting wild-type (WT) bone marrow into lethally irradiated DAPk KO mice, the chimeric mice remained hypersensitive to both acute and chronic LPS challenges, as well as to tobacco smoke exposure. DAPk-null lungs reconstituted with WT leukocytes exhibited elevated neutrophil content and augmented cytokine secretion in the bronchoalveolar space, as well as enhanced epithelial cell injury in response to both acute and chronic inflammatory conditions. These results suggest that DAPk attenuates a variety of inflammatory responses, both in lung leukocytes and in lung epithelial cells. The DAPk-mediated suppression of lung inflammation and airway injury may contribute to the tumor-suppressor functions of this kinase in epithelial carcinogenesis.

The role of integrins in leukocyte migration across endothelial barriers is widely accepted. In contrast, the contribution of integrins to interstitial motility of leukocytes is still elusive. Chemokine binding to G-protein-coupled receptors expressed on the surface of leukocytes plays key roles in both of these processes by directly activating integrin conformations favorable for ligand binding and integrin micro-clustering. Chemokines can also serve as weak adhesive ligands and potent inducers of actin cytoskeleton remodeling. Real-time assays utilizing live imaging microscopy have been implemented to dissect these versatile roles of chemokines in different leukocyte migration processes. Here, we review several in vitro assays useful for exploring the contribution of chemokine signals and shear forces to integrin activation and function during various stages of leukocyte transendothelial migration. In addition, we describe a new assay that assesses the contribution of chemokines to integrin-independent interstitial leukocyte motility. These assays can also follow the outcome of specific genetic or biochemical manipulations of either the leukocyte or the endothelial barrier on distinct migratory steps. Following fixation, subcellular changes in the distribution of integrin subsets and of specific integrin-associated adaptors can be further dissected by immunofluorescence tools and by ultrastructural electron microscopic analysis.

Lymphocyte arrest and spreading on ICAM-1-expressing APCs require activation of lymphocyte LFA-1 by TCR signals, but the conformational switches of this integrin during these critical processes are still elusive. Using Ab probes that distinguish between different LFA-1 conformations, we found that, unlike strong chemokine signals, potent TCR stimuli were insufficient to trigger LFA-1 extension or headpiece opening in primary human lymphocytes. Nevertheless, LFA-1 in these TCR-stimulated T cells became highly adhesive to both anchored and mobile surface-bound ICAM-1, although it failed to bind soluble ICAM-1 with measurable affinity. Rapid rearrangement of LFA-1 by immobilized ICAM-1 switched the integrin to an open headpiece conformation within numerous scattered submicron focal dots that did not readily collapse into a peripheral LFA-1 ring. Headpiece-activated LFA-1 microclusters were enriched with talin but were devoid of TCR and CD45. Notably, LFA-1 activation by TCR signals as well as subsequent T cell spreading on ICAM-1 took place independently of cytosolic Ca(2+). In contrast to LFA-1-activating chemokine signals, TCR activation of LFA-1 readily took place in the absence of external shear forces. LFA-1 activation by TCR signals also did not require internal myosin II forces but depended on intact actin cytoskeleton. Our results suggest that potent TCR signals fail to trigger LFA-1 headpiece activation unless the integrin first gets stabilized by surface-bound ICAM-1 within evenly scattered actin-dependent LFA-1 focal dots, the quantal units of TCR-stimulated T cell arrest and spreading on ICAM-1. The Journal of Immunology, 2010, 185: 7394-7404.

We investigated the effect of laquinimod on inflammatory demyelination, axonal damage, cytokine profiles and migratory capacities of lymphocytes in C57BL/6 mice with active EAE induced with MOG(35-55) peptide. The mice were treated at disease induction and after disease onset. Spinal cords were assessed histologically. Cytokines and adhesive properties were analyzed in splenocytes. Preventive and therapeutic laquinimod treatment reduced clinical signs, inflammation, and demyelination. VLA-4-mediated adhesiveness and pro-inflammatory cytokines such as IL-17 were down-regulated in treated animals. Within lesions, treated mice showed similar axonal densities, but less acute axonal damage than controls. Laquinimod might thus protect myelin and axons by decreasing pro-inflammatory cytokines and impairing the migratory capacity of lymphocytes. (c) 2010 Elsevier B.V. All rights reserved.

Adhesion and motility of mammalian leukocytes are essential requirements for innate and adaptive immune defense mechanisms. We show here that the guanine nucleotide exchange factor cytohesin-1, which had previously been demonstrated to be an important component of beta-2 integrin activation in lymphocytes, regulates the activation of the small GTPase RhoA in primary dendritic cells (DCs). Cytohesin-1 and RhoA are both required for the induction of chemokine-dependent conformational changes of the integrin beta-2 subunit of DCs during adhesion under physiological flow conditions. Furthermore, use of RNAi in murine bone marrow DCs (BM-DCs) revealed that interference with cytohesin-1 signaling impairs migration of wild-type dendritic cells in complex 3D environments and in vivo. This phenotype was not observed in the complete absence of integrins. We thus demonstrate an essential role of cytohesin-1/RhoA during ameboid migration in the presence of integrins and further suggest that DCs without integrins switch to a different migration mode. (Blood. 2009; 113: 5801-5810)

Homing to secondary lymphoid organs and bone marrow (BM) is a central aspect of leukemic pathophysiology. We investigated the roles of the two major lymphocyte integrins LFA-1 and VLA-4 on B-cell chronic lymphocytic leukemia (CLL) cells in these processes. We found that the majority of CLL cells expressed significantly reduced LFA-1 due to low beta 2 integrin transcripts. VLA-4 expression was heterogenous but underwent rapid activation by the BM chemokine CXCL12. CLL cells failed to transmigrate across VCAM-1-expressing, ICAM-1-expressing, and CXCL12-expressing endothelium, whereas when LFA-1 expression was regained in subsets of CLL cells, these lymphocytes rapidly transmigrated the endothelium. Furthermore, when injected into tail veins of immunodeficient mice, normal B cells rapidly homed to lymph nodes (LN) in a LFA-1-dependent manner, whereas CLL cells did not. Nevertheless, only residual CLL subsets could reenter BM, whereas both normal and CLL cells homed to the mice spleen in an LFA-1-independent and VLA-4-independent manner. Our results suggest that CLL cells have a reduced capacity to adhere and transmigrate through multiple vascular endothelial beds and poorly home to lymphoid organs other than spleen. Integrin blocking could thus be an efficient strategy to prevent circulating CLL cells from reaching prosurvival niches in LNs and BM but not in spleen. [Cancer Res 2009;69(7):3121-30]

Cell adhesion is a complex event dictated by the properties of individual adhesion molecules. It is desirable to link their individual properties to the adhesive behavior of a whole cell. Here, we examine with atomic force microscopic (AFM) and flow chamber experiments how the exogenous activation of the major T cell integrin VLA-4 affects the adhesiveness of T-lymphocytes to the endothelial VLA-4 ligand, VCAM-1. The atomic force measurements are performed on substrates with different VCAM-1 densities to compare the properties of single adhesive bonds on low-density ligands with the effect of multiple bonds on higher ligand densities. We determine the basal off-rate and the potential width of the single adhesive VLA-4 -VCAM-1 bond by Monte-Carlo simulations. We show that activating VLA-4 with magnesium increases the potential width of the bond from 0.18 nm to 0.44 nm, but hardly affects the basal off-rate of single VLA-4 -VCAM-1 bonds measured at low VCAM-1 density, lowering it only from 1.2 s(-1) to 0.9 s(-1). At higher VCAM-1 densities, the apparent off-rate of high avidity VLA-4 -VCAM-1 interactions is significantly reduced by magnesium to

The arrest of rolling leukocytes at target vascular sites depends on rapid activation of their vascular integrins at endothelial contacts enriched with specific Ig superfamily (IgSF) ligands. Accumulating data suggest that these integrins acquire high affinity to these endothelial ligands in a process tightly regulated by a variety of cytoskeletal conformational changes in their α- and β-subunits. These alterations are controlled by inside-out signals that are induced primarily by chemokines displayed on the endothelium and transduced by G protein-coupled receptors (GPCRs) on the responding leukocytes. The GPCR signals can propagate within milliseconds at submicron ranges. Upon binding their ligands, GPCR-stimulated integrins also undergo outside-in conformational activation, which is accelerated by applied forces. The integrin activator, talin—a universal cytoskeletal adaptor in integrinmediated focal adhesions—plays an instrumental role in this bidirectional activation process, which is critical for leukocyte arrest on endothelial IgSF ligands. Multiple effectors in the immediate vicinity to integrin–talin complexes can determine the extent to which talin may translate a given chemokine signal into a fully productive integrin activation. Substantial heterogeneity exists in the pathways used by different cell types to translate a given combination of chemokines and integrin ligands into leukocyte arrest and subsequent adhesion strengthening on various target endothelia. This chapter highlights the main molecular players in the earliest events of chemokine signaling to lymphocyte integrins and proposes modalities used by rapid chemokine signals to trigger cytoskeletal rearrangements in integrin tails, leading to rapid integrin activation and leukocyte arrest on vascular endothelium.

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.

Lymphocyte motility in lymph nodes is regulated by chemokines, but the contribution of integrins to this motility remains obscure. Here we examined lymphocyte migration over CCR7-binding chemokines that 'decorate' lymph node stroma. In a shear-free environment, surface-bound lymph node chemokines but not their soluble counterparts promoted robust and sustained T lymphocyte motility. The chemokine CCL21 induced compartmentalized clustering of the integrins LFA-1 and VLA-4 in motile lymphocytes, but both integrins remained nonadhesive to ligands on lymphocytes, dendritic cells and stroma. The application of shear stress to lymphocytes interacting with CCL21 and integrin ligands promoted robust integrin-mediated adhesion. Thus, lymph node chemokines that promote motility and strongly activate lymphocyte integrins under shear forces fail to stimulate stable integrin adhesiveness in extravascular shear-free environments.

The movement of leukocytes from the blood into peripheral tissues plays a key role in immunity as well as chronic inflammatory and autoimmune diseases. The shear force of blood flow presents special challenges to leukocytes as they establish adhesion on the vascular endothelium and migrate into the underlying tissues. Integrins are a family of cell adhesion and signaling molecules, whose function can be regulated to meet these challenges. The affinity of integrins for their vascular ligands can be stimulated in subseconds by chemoattractant signaling. This aids in inducing leukocyte adhesion under flow conditions. Further, linkage of these integrins to the actin cytoskeleton also helps to establish adhesion to the endothelium under flow conditions. In the case of alpha 4 beta 1 integrins, this linkage of the integrin to the cytoskeleton is mediated in part by the binding of paxillin to the alpha 4 integrin subunit and the subsequent binding of paxillin to the cytoskeleton molecule talin. The movement of leukocytes along the vascular endothelium and in between endothelial cells requires the temporal and spatial regulation of small guanosine triphosphatases, such as Rac1. We describe mechanisms through which alpha 4 beta 1 integrin signaling regulates appropriate Rac activation to drive leukocyte migration.

Leukocyte and platelet integrins rapidly alter their affinity and adhesiveness in response to various activation (inside-out) signals. A rare leukocyte adhesion deficiency (LAD), LAD-III, is associated with severe defects in leukocyte and platelet integrin activation. We report two new LAD cases in which lymphocytes, neutrophils, and platelets share severe defects in beta(1), beta(2), and beta(3) integrin activation. Patients were both homozygous for a splice junction mutation in their CaIDAG-GEFI gene, which is a key Rap-1/2 guanine exchange factor (GEF). Both mRNA and protein levels of the GEF were diminished in LAD lymphocytes, neutrophils, and platelets. Consequently, LAD-II platelets failed to aggregate because of an impaired alpha(IIb)beta(3) activation by key agonists. beta(2) integrins on LAD-III neutrophils were unable to mediate leukocyte arrest on TNF alpha-stimulated endothelium, despite normal selectin-mediated rolling. In situ subsecond activation of neutrophil beta(2) integrin adhesiveness by surface-bound chemoattractants and of primary T lymphocyte LFA-1 by the CXCL12 chemokine was abolished. Chemokine inside-out signals also failed to stimulate lymphocyte LFA-1 extension and high affinity epitopes. Chemokine-triggered VLA-4 adhesiveness in T lymphocytes was partially defective as well. These studies identify CaIDAG-GEFI as a critical regulator of inside-out integrin activation in human T lymphocytes, neutrophils, and platelets.

Recruitment of lymphocytes to the lymph nodes requires L-selectin-mediated recognition of carbohydrate determinants presented by O-glycans. New work shows that N-glycans 'decorated' with the same determinants also contribute to lymphocyte homing.

The chemokine receptor CXCR3 is predominantly expressed on activated T and natural killer (NK) cells. CXCR3 and its ligands, CXCL11, CXCL10, and CXCL9, play a major role in T-helper 1 (Th1)-dependent inflammatory responses. CXCL11 is the most dominant physiological inducer of adhesion, migration, and internalization of CXCR3. To study the role of CXCR3 carboxyl-terminus and the third intracellular (3i) loop in chemokine-mediated migration, adhesion, and CXCR3 internalization, we generated CXCR3 receptors mutated in their distal (Ser-Thr domain) or proximal (trileucine domain) membrane carboxyl terminus, and/or the third intracellular loop. We found that migration of CXCR3-expressing HEK 293 cells toward CXCL11 was pertussis toxin-dependent and required the membrane proximal carboxyl terminus of CXCR3. Internalization induced by CXCL11 and protein kinase C (PKC) activation was also regulated by the membrane proximal carboxyl terminus; however, only CXCL11 induced internalization required the LLL motif of this region. Internalization and Ca2+ flux induced by CXCL11 were independent of the 3i loop S245, whereas migration at high CXCL11 concentrations, integrin-dependent adhesion, and actin polymerization were S245 dependent. Our findings indicate that CXCL11-dependent CXCR3 internalization and cell migration are regulated by the CXCR3 membrane proximal carboxyl terminus, whereas adhesion is regulated by the 3i loop S245. Thus, distinct conformational changes induced by a given CXCR3 ligand trigger different downstream effectors of adhesion, motility, and CXCR3 desensitization.

The capacity of integrins to mediate adhesiveness is modulated by their cytoplasmic associations. In this study, we describe a novel mechanism by which alpha(4)-integrin adhesiveness is regulated by the cytoskeletal adaptor paxillin. A mutation of the alpha(4) tail that disrupts paxillin binding, alpha(4)(Y991A), reduced talin association to the alpha(4)beta(1) heterodimer, impaired integrin anchorage to the cytoskeleton, and suppressed alpha(4)beta(1)-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 alpha(4)-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.

It is widely believed that rolling lymphocytes require successive chemokine-induced signaling for lymphocyte function associated antigen 1 (LFA-1) to achieve a threshold avidity that will mediate lymphocyte arrest. Using an in vivo model of lymphocyte arrest, we show here that LFA-1-mediated arrest of lymphocytes rolling on high endothelial venules bearing LFA-1 ligands and chemokines was abrupt. In vitro flow chamber models showed that endothelium-presented but not soluble chemokines triggered instantaneous extension of bent LFA-1 in the absence of LFA-1 ligand engagement. To support lymphocyte adhesion, this extended LFA-1 conformation required immediate activation by its ligand, intercellular adhesion molecule 1. These data show that chemokine-triggered lymphocyte adhesiveness involves a previously unrecognized extension step that primes LFA-1 for ligand binding and firm adhesion.

Purpose of review: In the last 2-3 years our understanding of leukocyte adhesion cascades has increased, mainly in defining new pathways by which integrin activation occurs on circulating leukocytes recruited to sites of inflammation. While defects in the integrin structure (leukocyte adhesion deficiency (LAD) I) and in the selectin glycoprotein ligand biosynthesis (LAD II) have been described in the past few decades, a newly recognized defect in the activation of integrins (LAD III) was only recently delineated. The clinical manifestations and molecular basis of this syndrome and related cases will be reviewed. Recent findings: While in LAD I and II the defect in the adhesion cascade is restricted to leukocytes, all four cases of LAD III described to date also had defects in platelet aggregation. These patients suffered from recurrent bacterial infections and a severe bleeding tendency. All cases were reported to have activation defects in all major integrin subfamily members expressed in circulating leukocytes and platelets. In one case there was a defect in Rap1, which is a crucial protein in the inside-out and outside-in (ligand-induced) signaling underlying integrin activation mainly by cytokines. In this case, both chemokines and cytokines were unable to activate Rap1 leading to severe adhesive defects analyzed in vitro. Summary: While in LAD I and II the primary genetic defect is known, in the newly described LAD III the primary event leading to the defect is still unknown, despite a clear biochemical defect in Rap1 activation. The molecular basis or the defect in integrin activation may be different in the various cases described so far. It seems logical, however, to assume that in all reported cases, a key component of inside-out signaling to integrins activation is involved.

Intercellular adhesion molecule (ICAM)-2 is highly expressed on platelets and endothelium and is a counter-receptor for the leukocyte integrin, lymphocyte function-associated antigen-1 (LFA-1) and for the dendritic cell-specific, ICAM-grabbing non-integrin (DC-SIGN) protein. In this study, we investigated structural and functional differences between ICAM-2 from platelets and that from endothelial cells. The isoelectric point (pI) of ICAM-2 from HUVEC was pH 3.5 - 4.3, whereas that of platelet ICAM-2 was more acidic at pH 3.0 - 3.7. This charge difference was abolished by treatment with N-glycanase or neuraminidase, thus it was due to cell-specific N-linked glycosylation. Purified, immobilized platelet ICAM-2 supported 50% less adhesion of LFA-1-bearing T cells than did purified HUVEC ICAM-2 and no adhesion was observed of monocyte-derived immature dendritic cells via DC-SIGN to platelet ICAM-2. Treatment of platelet ICAM-2 with neuraminidase abolished these functional differences. These findings demonstrated that physiologic sialylation of platelet ICAM-2 renders it less able than endothelial ICAM-2 to support adherence of leukocytes.

Leukocyte recruitment to inflamed and lymphoid tissues is mediated by sequential adhesive interactions between specialized vascular receptors and their endothelial counterligands (1, 2, 3). Following rolling and arrest on the endothelium, circulating immune cells locomote on and extravasate through the endothelial cell (EC) barrier (4, 5, 6). These steps must be delicately coordinated to allow leukocyte motility while maintaining leukocyte resistance to detachment by the high shear forces constantly exerted at the vessel wall. The key signals, which trigger leukocyte arrest on endothelial integrin ligands, are elicited by chemokines, presented on the apical surface of the endothelial lining at sites of leukocyte diapedesis (7, 8, 9). Recent observations from our lab (10) as well as from other labs (11, 12, 13) have pointed out that shear forces may transduce, together with endothelial chemokines, potent promigratory signals to adherent leukocytes at endothelial interfaces. These studies suggest that in vitro transendothelial migration (TEM) model systems should incorporate both the physiological blood flow conditions and chemokine presentation profiles that exist at sites of leukocyte emigration in the vasculature.

L-selectin is a leukocyte cell-surface protein that facilitates the rolling of leukocytes along the endothelium, a process that leads to leukocyte migration to a site of infection. Preventing L-selectin-mediated rolling minimizes leukocyte adhesion and extravasation; therefore, compounds that inhibit rolling may act as anti-inflammatory agents. To investigate the potential role of multivalent ligands as rolling inhibitors, compounds termed neoglycopolymers were synthesized that possess key structural features of physiological L-selectin ligands. Sulfated neoglycopolymers substituted with sialyl Lewis x derivatives (3',6-disulfo Lewis x or 6-sulfo sialyl Lewis x) or a sulfatide analog (3,6-disulfo galactose) inhibited L-selectin-mediated roiling of lymphoid cells. Functional analysis of the inhibitory ligands indicates that they also induce proteolytic release of L-selectin. Thus, their inhibitory potency may arise from their ability to induce shedding. Our data indicate that screening for compounds that promote L-selectin release can identify ligands that inhibit rolling.

L-selectin-mediated tethers result in leukocyte rolling only above a threshold in shear. Here we present biophysical modeling based on recently published data from flow chamber experiments, which supports the interpretation that L-selectin-mediated tethers below the shear threshold correspond to single L-selectin carbohydrate bonds dissociating on the time scale of milliseconds, whereas L-selectin-mediated tethers above the shear threshold are stabilized by multiple bonds and fast rebinding of broken bonds, resulting in tether lifetimes on the time scale of 10(-1) seconds. Our calculations for cluster dissociation suggest that the single molecule rebinding rate is of the order of 10(4) Hz. A similar estimate results if increased tether dissociation for tail-truncated L-selectin mutants above the shear threshold is modeled as diffusive escape of single receptors from the rebinding region due to increased mobility. Using computer simulations, we show that our model yields first-order dissociation kinetics and exponential dependence of tether dissociation rates on shear stress. Our results suggest that multiple contacts, cytoskeletal anchorage of L-selectin, and local rebinding of ligand play important roles in L-selectin tether stabilization and progression of tethers into persistent rolling on endothelial surfaces.

Trafficking of human CD34(+) stem/progenitor cells (HSCs/HPCs) is regulated by chemokines, cytokines, proteolytic enzymes, and adhesion molecules. We report that the adhesion receptor CD44 and its major ligand, hyaluronic acid (HA), are essential for homing into the bone marrow (BM) and spleen of nonobese diabetic/ severe combined immunodeficient (NOD/ SCID) mice and engraftment by human HSCs. Homing was blocked by anti-CD44 monoclonal antibodies (mAbs) or by soluble HA, and it was significantly impaired after intravenous injection of hyaluronidase. Furthermore, stromal cell-derived factor-1 (SDF-1) was found to be a rapid and potent stimulator of progenitor, adhesion to immobilized HA, leading to formation of actin-containing protrusions with CD44 located at their tips. HPCs migrating on HA toward a gradient of SDF-1 acquired spread and polarized morphology with CD44 concentrating at the pseudopodia at the leading edge. These morphologic alterations were not observed when the progenitors were first exposed to anti-CD44 mAbs, demonstrating a crosstalk between CD44 and CXCR4 signaling. Unexpectedly, we found that HA is expressed on human BM sinusoidal endothelium and endosteum, the regions where SDF-1 is also abundant. Taken. together, our data suggest a key role for CD44 and HA in SDF-1-dependent transendothelial migration of HSCs/HPCs and their final anchorage within specific niches of the BM.

L-selectin is a key lectin essential for leukocyte capture and rolling on vessel walls. Functional adhesion of L-selectin requires a minimal threshold of hydrodynamic shear. Using high temporal resolution videomicroscopy, we now report that L-selectin engages its ligands through exceptionally labile adhesive bonds (tethers) even below this shear threshold. These tethers share a lifetime of 4 ms on distinct physiological ligands, two orders of magnitude shorter than the lifetime of the P-selectin-PSGL-1 bond. Below threshold shear, tether duration is not shortened by elevated shear stresses. However, above the shear threshold, selectin tethers undergo 1 4-fold stabilization by shear-driven leukocyte transport. Notably, the cytoplasmic tail of L-selectin contributes to this stabilization only above the shear threshold. These properties are not shared by P-selectin- or VLA-4-mediated tethers. L-selectin tethers appear adapted to undergo rapid avidity enhancement by cellular transport, a specialized mechanism not used by any other known adhesion receptor.

To extravasate the bloodstream at specific targets, circulating immune cells must activate their integrins to undergo rapid in situ modulation of affinity or avidity for their endothelial ligands. This activation involves specialized sub-second G-protein signal transduction triggered by endothelium-displayed chemo-attractants-primarily chemokines-and their cognate leukocyte-expressed G-protein-coupled receptors (GPCRs). Recently, we reported a rare autosomal-recessive leukocyte adhesion deficiency (LAD) syndrome associated with a defective ability of integrins to undergo GPCR-mediated stimulation at endothelial contacts. This LAD shows significant similarities to a group of integrin-activation syndromes reported in leukocytes and platelets. Here, the mechanisms by which GPCRs might regulate leukocyte and platelet integrins are outlined with respect to this new family of LAD cases. We propose to term this the LAD-III family.

Leukocyte arrest on vascular endothelium under disruptive shear flow is a multistep process that requires in situ integrin activation on the leukocyte surface by endothelium-displayed chemoattractants, primarily chemokines. A genetic deficiency of leukocyte adhesion to endothelium associated with defective beta2 integrin expression or function (LAD-1) has been described. We now report a novel severe genetic disorder in this multistep process associated with functional defects in multiple leukocyte integrins, reflected in recurrent infections, profound leukocytosis, and a bleeding tendency. This syndrome is associated with an impaired ability of neutrophil and lymphocyte beta1 and beta2 integrins to generate high avidity to their endothelial ligands and arrest cells on vascular endothelium in response to endothelial chemoattractant signals. Patient leukocytes roll normally on endothelial selectins, express intact integrins and G protein-coupled chemokine receptors (GPCR), spread on integrin ligands, and migrate normally along a chemotactic gradient. Activation of beta2 integrins in response to GPCR signals and intrinsic soluble ligand binding properties of the very late activation antigen-4 (VLA-4) integrin are also retained in patient leukocytes. Nevertheless, all integrins fall to generate firm adhesion to immobilized ligands in response to in situ GPCR-mediated activation by chemokines or chemoattractants, a result of a primary defect in integrin rearrangement at ligand-bearing contacts. This syndrome is the first example of a human integrin-activation deficiency associated with defective GPCR stimulation of integrin avidity at subsecond contacts, a key step in leukocyte arrest on vascular endothelium under shear flow. (C) 2003 by The American Society of Hematology.

Chemokines presented on specialized endothelial surfaces rapidly up-regulate leukocyte integrin avidity and firm arrest through G(i)-protein signaling. Here we describe a novel, G-protein-independent, down-regulatory activity of apical endothelial chemokines in destabilizing L-selectin-mediated leukocyte rolling. Unexpectedly, this anti-adhesive chemokine suppression of rolling does not involve L-selectin shedding. Destabilization of rolling is induced only by immobilized chemokines juxtaposed to L-selectin ligands and is an energy-dependent process. Chemokines are found to interfere with a subsecond stabilization of selectin tethers necessary for persistent rolling. This is a first indication that endothelial chemokines can attenuate in situ L-selectin adhesion to endothelial ligands at subsecond contacts. This negative feedback mechanism may underlie the jerky nature of rolling mediated by L-selectin in vivo.

Chronic myelogenous leukemia (CML), a malignant myeloproliferative disorder originating from a pluripotent stem cell expressing the bcr-abl oncogene, is characterized by abnormal release of the expanded, malignant stem cell clone from the bone marrow (BM) into the circulation. Moreover, immature CD34(+) CML cells have lower adhesion to stromal cells and fibronectin as well as lower engraftment potential in severe combined immune-deficient (SCID) and nonobese diabetic (NOD)/SCID mice than normal CD34(+) cells. We report in this study that leukemic Philadelphia chromosome-positive (Ph+)CD34(+) cells from newly diagnosed CML patients that express the chemokine receptor CXCR4 migrate in response to stromal-derived factor-1 (SDF-1). However, normal Ph-CD34(+)CXCR4(+) cells derived from the same patient have significantly higher migration levels toward SDF-1. In contrast to their transwell migration potential, the SDF-1-mediated integrin-dependent polarization and migration of the Ph(+)CD34(+)CXCR4(+) cells through extracellular matrix-like gels were significantly lower than for normal cells. Concomitantly, binding of these cells to vascular cell adhesion molecule-1 or fibronectin, in the presence of SDF-1, was also substantially lower. These findings suggest a major role for SDF-1-mediated, integrin-dependent BM retention of Ph(+)CD34(+) cells.

Cell motility is an essential element of tumor dissemination, allowing organ infiltration by cancer cells. Using mouse LB lymphoma cells transfected with standard CD44 (CD44s) cDNA (LB-TRs cells) or with the alternatively spliced CD44 variant CD44v4-v10 (CD44v) cDNA (LB-TRv cells), we explored their CD44-dependent cell migration. LB-TRv cells, but not LB-TRs or parental LB cells, bound soluble hyaluronic acid (HA) and other glycosaminoglycans (GAGS), and exclusively formed, under physiological shear force, rolling attachments on HA substrate. Furthermore, LB-TRv cells, but not LB-TRs cells or their parental LB cells, displayed accelerated local tumor formation and enhanced accumulation in the peripheral lymph nodes after s.c. inoculation. The aggressive metastatic behavior of i.v.-injected LB-TRV cells, when compared with that of other LB-transfectants, is attributed to more efficient migration to the lymph nodes, rather than to local growth in the lymph node. Injection of anti-CD44 monoclonal antibody or of the enzyme hyaluronidase also prevented tumor growth in lymph nodes of BALB/c mice inoculated with LB-TRv cells. The enhanced in vitro rolling and enhanced in vivo local tumor growth and lymph node invasion disappeared in LB cells transfected with CD44v cDNA bearing a point mutation at the HA binding site, located at the distal end of the molecule constant region. These findings show that the interaction of cell surface CD44v with HA promotes cell migration both in vitro and in vivo, and they contribute to our understanding of the mechanism of cell trafficking, including tumor spread.

The interaction of integrin alpha (4)beta (1) with endothelial VCAM-1 controls the trafficking of lymphocytes from blood into peripheral tissues. Cells actively regulate the affinity of alpha (4)beta (1) for VCAM-1 (activation). To investigate the biological function of alpha (4)beta (1) activation, we isolated Jurkat T cell lines with defective alpha (4)beta (1) activation. Using these cells, we found that alpha (4)beta (1)-stimulated alpha (L)beta (2)-dependent cell migration was dramatically reduced in cells with defects in alpha (4)beta (1) activation. These cells required 20 times more VCAM-1 to promote alpha (L)beta (2)-dependent cell migration. This defect was at the level of alpha (4)beta (1) affinity as an activating alpha (4)beta (1) Ab rescued alpha (4)beta (1)-stimulated alpha (L)beta (2)-dependent migration. In contrast, migration of alpha (4)beta (1) activation-defective cells on VCAM-1 alone was enhanced at higher VCAM-1 densities. Thus, alpha (4)beta (1) activation determines a set point or threshold at which VCAM-1 can regulate alpha (L)beta (2)-dependent as well as alpha (4)beta (1)-dependent cell migration. Changes in this set point may specify preferred anatomical sites of integrin-dependent leukocyte emigration from the bloodstream.

Leukocyte transendothelial migration (TEM) is thought to be a chemotactic process controlled by chemokine gradients across the endothelium. Using cytokine-activated human umbilical vascular endothelial cells (HUVECs) as a model of inflamed endothelium, we have shown that apical endothelial chemokines can trigger robust peripheral blood lymphocyte (PBL) migration across endothelial cells, Lymphocyte TEM was promoted by physiological shear stress applied continuously to migrating lymphocytes. Lymphocyte integrins, intact actin cytoskeleton and G(i) protein-mediated chemokine signaling, but not a chemotactic gradient, were mandatory for TEM, PBL TEM did not require intracellular free calcium or intact phosphatidyl inositol kinase activity in migrating lymphocytes. Thus, lymphocyte TEM is promoted by fluid shear-induced mechanical signals coupled to G(i) protein signals at apical endothelial zones.

In circulating lymphocytes, the VLA-4 integrin preexists in multiple affinity states that mediate spontaneous tethering, rolling, and arrest on its endothelial ligand, vascular cell adhesion molecule-1 (VCAM-1), The regulation and function of VLA-4 affinity in lymphocytes has never been elucidated. We show here that p56(lck), the major Src kinase in T cells, is a key regulator of high affinity VLA-4, This high affinity is essential for the rapid development of firm adhesion of resting T cells to VCAM-1 and to their extracellular matrix ligand, fibronectin, Lck-regulated VLA-4 function does not require intact TCR nor several key components of the TCR signaling pathway, including ZAP-70 and SLP-76, Furthermore, stimulation of p56(lck) by the phosphatase inhibitor, pervanadate, triggers firm VLA-4-dependent adhesion to VCAM-1, Although Lck is not required for chemokine receptor signaling to mitogen-activated protein kinase, the presence of Lck-regulated high affinity VLA-4 also facilitates firm adhesion triggered by the chemokine, SDF-1, at short-lived contacts. Surprisingly, bond formation rates, ability to tether cells to VLA-4 ligand, and VLA-4 tether bond stability under shear flow are not affected by VLA-4 affinity or Lck activity. Thus, the ability of high affinity VLA-4 to arrest cells on VCAM-1 under flow arises from instantaneous post-ligand strengthening rather than from increased kinetic stability of individual VLA-4 bonds. These results suggest that p56(lck) maintains high affinity VLA-4 on circulating lymphocytes, which determines their ability to strengthen VLA-4 adhesion and rapidly respond to proadhesive chemokine signals at endothelial sites.

The mechanism by which immature B cells are sequestered from encountering foreign antigens present in lymph nodes or sites of inflammation, before their final maturation in the spleen, has not been elucidated. We show here that immature B cells fail to home to the lymph nodes. These cells can actively exclude themselves from antigen-enriched sites by downregulating their integrin-mediated adhesion to the extracellular matrix protein, Fibronectin. This inhibition is mediated by interferon gamma secretion. Perturbation of interferon gamma activity in vivo leads to the homing of immature B cells to the lymph nodes. This is the first example of autocrine regulation of immune cell migration to sites of foreign antigen presentation.

The migration of T cells into extravascular sites of inflammation is regulated by information derived from the molecular structure of the invaded tissue and from chemokine and cytokine gradients in the context of the extracellular matrix (ECM). Although recent studies have highlighted the role of particular chemoattractants in leukocyte migration, to date little Is known about how specific combinations of contextual signals control the migration of leukocytes and their localization at sites of inflammation. Here me studied the interplay between a pleiotropic cytokine, TNF-alpha, and two prototypic chemoattractants, RANTES and stromal cell-derived factor-1 alpha (SDF-1 alpha), on human CD45RO(+) T cells migrating within an ECM-like contest, For this purpose, we used a newly constructed three-dimensional gel system designed to follow, in real time, the migration of individual leukocytes along chemotactic gradients in vitro. We found that TNF-alpha, which binds the ECM protein fibronectin and lacks adhesion- and migration-promoting effects of its own, can act as a proadhesive cytokine on T cells exposed to RANTES and SDF-1 alpha, Furthermore, fibronectin-complexed TNF-alpha provided anchorage signals to the T cells as they moved directionally along chemoattractive gradients. This effect of TNF-alpha required an intact TNF-alpha receptor II subtype on the migrating T cells. The anchoring effect of TNF-alpha appears to be specific; IL-2, an integrin-activating proadhesive cytokine, does not transmit stoppage signals to T cell migration induced by RANTES, Thus, TNF-alpha present in the ECM at sites of inflammation may function to anchor T cells recruited to these sites by chemotactic signals.

The interaction of cells with the extracellular matrix regulates cell adhesion, motility, growth, survival and differentiation through integrin-mediated signal transduction, Here we demonstrate that galectin-8, a secreted mammalian beta-galactoside binding protein, inhibits adhesion of human carcinoma (1299) cells to plates coated with integrin ligands, and induces cell apoptosis, Pretreatment of the cells with Mn2+, which increases the affinity of integrins for their ligands, abolished the inhibitory effects of galectin-8, The inhibitory effects of galectin-8 were specific and were not mimicked by plant lectins or other galectins (galectin-1 and galectin-3). In accordance with its anti-adhesive effects, transfection of galectin-8 cDNA into 1299 cells significantly reduced (by 75%) colony formation, when compared to the number of colonies formed by cells transfected With an empty vector. Affinity chromatography over immobilized galectin-8 indicated that few membrane proteins interacted with galectin-8 in a sugar-dependent manner. Microsequencing and western immunoblotting revealed that alpha(3)beta(1) integrin derived from 1299 as well as other cells (e.g. HeLa and human endothelial cells) is a major galectin-8 binding-protein. Furthermore, immunoprecipitation and immunohistochemical studies suggested that endogenous galectin-8, secreted from 1299 cells, forms complexes with alpha(3)beta(1) integrins expressed on the surface of 1299 cells. Galectin-8 also interacts with other members of the integrin family, like alpha(6)beta(1) integrins, In contrast, galectin-8 only minimally interacts with alpha(4) or beta(3) integrins, We propose that galectin-8 is an integrin binding-protein that interacts to a different extent with several, but not all members of the integrin family. Binding of galectin-8 modulates integrin interactions with the extracellular matrix and thus regulates cell adhesion and cell survival.

Selectins mediate the initial tethering and rolling of leukocytes on vessel walls. Adhesion by selectins is a function of both ligand recognition at equilibrium and mechanical properties of the selectin-ligand bond under applied force. We describe an EGF domain mutant of L-selectin with profoundly augmented adhesiveness over that of native L-selectin but conserved ligand specificity. This mutant, termed LPL, was derived by a substitution of the EGF-like domain of L selectin with the homologous domain from P-selectin. The mutant bound soluble carbohydrate L-selectin ligand with affinity comparable with that of native L-selectin but interacted with all surface-bound ligands much more readily than native L-selectin, in particular under elevated shear flow. Tethers mediated by both native and mutant L-selectin exhibited similar lifetimes under a range of shear stresses, but the rate of bond formation by the mutant was at least 10-fold higher than that of native L-selectin toward distinct L-selectin ligands. Enhanced rate of bond formation by the mutant was associated with profoundly stronger rolling interactions and reduced dependence of rolling on a threshold of shear stress. This is the first demonstration that the EGF domain can modulate the binding of the lectin domain of a selectin to surface-immobilized ligands under shear flow without affecting the equilibrium properties of the selectin toward soluble ligands.

We have developed a novel 3-D gel reconstituted with major extracellular matrix (ECM) glycoproteins to follow the dynamics of migration of human T cells locomoting, in real-time, on gradients formed by representative chemoattractants: the C-C chemokine RANTES, and the pro-inflammatory cytokine IL-2. In the absence of chemoattractants, none of the T cells migrated directionally and the levels of random migration or cell polarization were low. However, major fractions of T cells placed in IL-2 and RANTES gradients in the gels polarized immediately after exposure to the chemoattractants. Shortly after polarization, 25% of the T cells migrated, in either a random or directional fashion, towards the sources of the chemoattractants; additional 5-10% of the cells remained polarized but stationary. The number of T cells migrating directionally towards RANTES or IL-2 peaked along with the formation of the chemotactic gradients. The directional migration of T cells was increased by a short pre-exposure to low doses of IL-2, which did not alter the level of expression of the beta(1) integrins. The directional migration of T cells towards IL-2 and RANTES was mediated by IL-2R and pertussis toxin-sensitive receptors, respectively, and the directional, and to a lesser degree, the random locomotion of T cells induced by both chemoattractants required intact tyrosine kinase signaling and activities of the alpha(4) alpha(5) and, to a lesser degree, the alpha(2) and alpha(6) members the beta(1) integrins. Our system enables the real-time tracking of individual locomoting lymphocytes and the analysis of their dynamic interactions with ECM components and cytokines. (C) 1999 Elsevier Science B.V. All rights reserved.

Synthetic carbohydrate and glycoprotein mimics displaying sulfated saccharide residues have been assayed for their L-selectin inhibitory properties under static and flow conditions. Polymers displaying the L-selectin recognition epitopes 3',6-disulfo Lewis x(Glc) (3-O-SO3-Gal beta 1 alpha 4(Fuc alpha 1 alpha 3)-6-O-SO3-Glc beta-OR) and 3',6'-disulfo Lewis x(Glc) (3,6-di-O-SO3-Gal beta 1 alpha 4(Fuc alpha 1 alpha 3)Glc beta-OR) both inhibit L-selectin binding to heparin under static, cell-free binding conditions with similar efficacies. Under conditions of shear flow, however, only the polymer displaying 3',6-disulfo Lewis x(Glc) inhibits the rolling of L-selectin-transfected cells on the glycoprotein ligand GlyCAM-1, Although it has been shown to more effective than sialyl Lewis x at blocking the L-selectin-GlyCAM-1 interaction in static binding studies, the corresponding monomer had no effect in the dynamic assay. These data indicate that multivalent ligands are far more effective inhibitors of L-selectin-mediated rolling than their monovalent counterparts and that the inhibitory activities are dependent on the specific sulfation pattern of the recognition epitope, Importantly, our results indicate the L-selectin specificity for one ligand over another found in static, cell-free binding assays is not necessarily retained under the conditions of shear flow. The results suggest that monovalent or polyvalent carbohydrate or glycoprotein mimetics that inhibit selectin binding in static assays may not block the more physiologically relevant process of selectin-mediated rolling.

The putative effects of interleukin (IL)-7, operating in the context of extracellular matrix (ECM), on the adhesion of human T cells were examined. Recombinant human IL-7 was found to bind ECM or fibronectin (FN) with IC50 values of 10-100 nM. Nanogram amounts of both soluble and, especially, FN- or ECM-bound IL-7, which differentially affected the morphologies of FN-adherent T cells, induced the adhesion of resting CD4(+) and CD8(+) T cells in dose-dependent and beta 1 integrin-dependent manners. Under static and flow conditions, soluble IL-7 also induced the binding of unstimulated T cells to vascular cell adhesion molecule-1, suggesting that this cytokine can also modulate integrin binding to endothelial cell ligands. The effects of affinity modulation by IL-7 of FN-specific beta 1 integrins depend on the presence of soluble FN, which inhibited T cell adhesion to FN induced by FN-bound IL-7 or by an integrin-specific affinity-modulating monoclonal antibody, but not by soluble IL-7 or phorbol 12-myristate 13-acetate. These findings provide an example of a major ECM integrin ligand, FN, which is capable of modulating its adhesive interactions with specific immune cells by associating with and presenting a cytokine in a bio-active state.

The selectins, a family of Ca2+-dependent lectins, are expressed on inflamed vascular endothelium and some leukocyte subsets, and mediate adhesive contacts between blood cells and vessel walls. These interactions are loose and reversible, operate under conditions of shear flow, and result in leukocyte rolling along the vessel wall. The structure of the selectins and their ligands makes them uniquely suited for supporting the type of bond formation and dissociation that must prevail in order for a cell to be able to roll under conditions of flow. Because rolling precedes (and appears to be essential for) the integrin-mediated firm arrest before extravasation in response to inflammatory or infectious stimuli, inhibition of selectin function has potential for anti-inflammatory therapy, but also presents some significant challenges because of the complexity of the processes involved.

Interaction of leukocytes in flow with adherent Ieukocytes may contribute to their accumulation at sites of inflammation. Using L-selectin immobilized in a flow chamber, a model system that mimics presentation of L-selectin by adherent leukocytes, we characterize ligands for L-selectin on leukocytes and show that they mediate tethering and rolling in shear now. We demonstrate the presence of L-selectin ligands on granulocytes, monocytes, and myeloid and lymphoid cell lines, and not on peripheral blood T lymphocytes. These ligands are calcium dependent, sensitive to protease and neuraminidase, and structurally distinct from previously described ligands for L-selectin on high endothelial venules (HEV). Differential sensitivity to O-sialoglycoprotease provides evidence for ligand activity on both mucin-like and nonmucin-like structures. Transfection with fucosyltransferase induces expression of functional L-selectin ligands on both a lymphoid cell line and a nonhematopoietic cell line. L-selectin presented on adherent cells is also capable of supporting tethering and rolling interactions in physiologic shear flow. L-selectin ligands on leukocytes may be important in promoting leukocyte-leukocyte and subsequent leukocyte-endothelial interactions in vivo, thereby enhancing leukocyte localization at sites of inflammation.

Little is known about how lymphocytes migrate within secondary lymphoid organs, Stromal cells and their associated reticular fibers form a network of fibers that radiate from high endothelial venules to all areas of the lymph node and may provide a scaffold for lymphocyte migration, We studied interactions of lymphocytes with cultured human tonsillar stromal cells and their extracellular matrix using shear stress to distinguish transient interactions from firm adhesion. Tonsillar lymphocytes and SKW3 T lymphoma cells tethered and rolled on monolayers of cultured tonsillar stromal cells and their matrix. A significant proportion of these rolling interactions were independent of divalent cations and were mediated by CD44 binding to hyaluronan, as shown by inhibition with mAb to CD44, soluble hyaluronan, hyaluronidase treatment of the substrate, and O-glycoprotease treatment of the rolling cells. O-glycoprotease treatment of the substrate also blocked binding completely to stromal matrix and partially to stromal monolayers. SKW3 cells tethered and rolled on plastic;immobilized hyaluronan, confirming the specificity of this interaction. By contrast? monolayers of resting or stimulated human umbilical vein endothelial cells failed to support CD44- and hyaluronan-dependent rolling. SKW3 cells added under flow conditions to frozen sections of human tonsil bound and rolled along reticular fibers in the presence of EDTA. Rolling was blocked by either CD44 mAb or hyaluronan. We propose that lymphocytes migrating through secondary lymphoid organs may use CD44 to bind to hyaluronan immobilized on stromal cells and reticular fibers.

Keywords: STRAIN

Previous studies have demonstrated that the leukocyte integrin Mac-1 adheres to several cell surface and soluble ligands including intercellular adhesion molecule-1, fibrinogen, iC3b, and factor X. However, experiments with Mac-1-expressing transfectants, purified Mac-1, and mAbs to Mac-1 indicate the existence of additional ligands. In this paper, we demonstrate a direct interaction between Mac-1 and heparan sulfate glycans. Heparin affinity resins immunoprecipitate Mac-1, and neutrophils and transfectant cells that express Mac-1 bind to heparin and heparan sulfate, but not to other sulfated glycosaminoglycans. Inhibition studies with mAbs and chemically modified forms of heparin suggest the I domain as a recognition site on Mac-1 for heparin, and suggest that either N- or O-sulfation is sufficient for heparin to bind efficiently to Mac-1. Under conditions of continuous flow in which heparins and E-selectin are cosubstrates, neutrophils tether to E-selectin and form firm adhesions through a Mac-1-heparin interaction.

Selectin interactions with glycolipids have been examined previously under static conditions, whereas physiologic interactions mediated by selectins take place under flow. We find that under physiologic flow conditions, sialyl Lewis(x) (sLe(x)) glycolipid and sialyl Lewis(a) (sLe(a)) neoglycolipid support tethering and rolling adhesions of Chinese hamster ovary (CHO) cells expressing E-selectin and lymphoid and myeloid cells expressing L-selectin. These selectin-mediated adhesions persist at the highest shear stresses that occur in postcapillary venules in vivo and occur at lower site densities than found for sLe(x) on neutrophils. The interactions are Ca2+-dependent and can be specifically and completely blocked with anti-selectin mAbs. Asialo nonfucosylated glycolipids are inactive, and sulfatide supports weak tethering, but not rolling, of L-selectin-expressing cells. Rolling velocities and resistance to detachment are related to the glycolipid site density and fall within the range measured for neutrophil and myeloid cell rolling on substrates containing purified selectins. These observations are the first indication that glycolipids can interact with selectins in physiologic flow conditions, and can contribute to rolling adhesions.

The effects of cytokines on immune cells may be influenced by their milieu, such as the extracellular matrix (ECM), in the vicinities of which cytokines and inflammatory cells interact and function. Previously, we demonstrated that TNF-alpha bound to fibronectin (FN) and augments the level of adhesion of activated CD4(+) cells. Herein, we examined the mechanisms of this pro-adhesive activity of TNF-alpha and its putative physiologic consequences using human or rat CD4(+) cells. A brief exposure of CD4(+) cells to low dosages of soluble TNF-alpha or of FN- or laminin-bound TNF-alpha synergized with PMA to enhance the integrin-mediated binding of CD4(+) cells to these immobilized ECM moieties. TNF-alpha-enhanced adhesion of CD4(+) cells did not delay or inhibit the subsequent detachment of the cells from the substrate, and adhesion was increased provided the cells were treated with TNF-alpha immediately after their exposure to PMA. Th is indicates that the enhancing effect of TNF-alpha requires a previous activation of the cells. When TNF-alpha was immobilized on FN, less TNF-alpha was required to induce CD4(+) cell binding to FN. Soluble, and to a greater extent FN-bound, TNF-alpha synergizes with PMA to intensify protein tyrosine phosphorylation in FN-bound CD4(+) cells, and this effect of TNF-alpha was inhibited by inhibitors of tyrosine kinase. That FN-bound or soluble TNF-alpha also amplified the binding of an Ag-specific autoimmune rat T cell line to immobilized FN, emphasizes the physiologic relevance of our findings. Thus, the signal transduction and cell adhesive properties of ECM glycoproteins may be modulated upon their association with TNF-alpha, and matrix-linked TNF-alpha may recruit and direct immune cells to inflammatory sites.

The Arg-Gly-Asp (RGD) cell adhesion motif has been demonstrated in various studies to play a pivotal role in leucocyte and platelet interactions with plasma and extracellular matrix (ECM) glycoproteins. The recognition of the RGD sequence is mediated by heterodimeric receptors designated integrins of the beta(1) subfamily, expressed on distinct cell types, including T lymphocytes. We have recently shown that flexible non-peptidic mimetics of RGD, in which the two ionic side groups were separated by a linear spacer of 11 atoms, bound specifically to the platelet integrin alpha(IIb)beta(3), and inhibited T cell-mediated immune responses. The present study was designed to (i) further characterize the structural requirements for RGD interactions with CD4(+) T cells, and (ii) examine the mechanisms by which the RGD mimetics interfere with immune cell reactivity in vivo. We now report that freezing the conformational degrees of freedom in the spacer chain, which fixes the relative orientation of the guanidinium and carboxylate side groups in a favourable manner, results in a higher level of inhibition of T cell binding to immobilized fibronectin, an RGD-containing ECM glycoprotein. In vivo, treatment of mice with relatively low doses of the RGD mimetics, but not the RGD peptide, inhibited the elicitation of an adoptively transferred DTH reaction. This inhibition was achieved by direct impairment of the ability of antigen-primed lymph node cells to migrate and accumulate in inflammatory sites. Hence, we suggest that the design and production of non-peptidic mimetics of RGD offers a novel approach to study defined parameters related to the structure-function requirements of small adhesion epitopes. Furthermore, this approach could be used therapeutically to inhibit pathological processes which depend on RGD recognition.

The interaction of the activated platelet integrin, glycoprotein IIb-IIIa (GPIIb-IIIa) with fibrinogen and von-Willebrand factor (vWF) is essential for platelet aggregation. The minimal structure required for this integrin's binding to fibrinogen is the Arg-Gly-Asp (RGD) sequence. Inasmuch as normal level of GPIIb-IIIa-RGD interactions are required for maintaining hemostasis, elevated platelet aggregation can cause adverse pathological effects. We have previously reported that nonpeptidic mimetics of RGD, consisting of carboxylate and guanidinium groups of Asp and Arg divided by a linear 11-atom spacer, acquired a significant affinity for the GPIIb-IIIa integrin and inhibited platelet aggregation. The structural requirements for the interactions df the RGD sequence with GPIIb-IIIa and the inhibitory potential of a newly designed series of mimetics on platelet aggregation and interactions with extracellular matrix (ECM) were assayed herein. Adenosine-diphosphate (ADP)-induced platelet aggregation was inhibited in a dose-dependent manner by various RGD mimetics, with a maximal inhibition of 80-100% with an IC50 of 3 mu M for the most potent inhibitor, NS-11, in which a six-membered ring was introduced into the spacer chain, which exceeded the IC50 attained with the original RGDS peptide. The inhibitory effect of the RGD mimetics was attributed to their specific interaction with the GPIIb-IIIa integrin, since these mimetics inhibited the binding of the PAC-1 mAb to GPIIb-IIIA. Furthermore, the binding of I-125-labeled fibrinogen to platelets was inhibited by the RGD surrogates in a dose-dependent and saturable manner. The RGD-mimetics also inhibited up to 70% the adhesion, aggregation, and deposition of platelets onto ECM. Thus, we suggest that the novel nonpeptidic mimetics of RGD described herein, which were shown to be resistant to proteolytic digestion, would be valuable in novel therapeutic approaches to treat in RGD-dependent pathological disorders involving p

Although cells of the immune system often function under feverish conditions, the effects of elevated temperatures on T cells have not been fully elucidated. Herein, the effects of a 1-hr exposure to 41-degrees of CD4+ human T cell were studied. Heat-shock treatment of activated CD4+ T cells reduced their adhesion to fibronectin and laminin, the major adhesive glycoproteins of the extracellular matrix (ECM) by 25-40%. This decrease was partially due to a minor decrease in the surface expression of beta1 integrins, which specifically interact with fibronectin and laminin. In contrast, the capacities of heat-stressed T cells to proliferate and to secrete tumour necrosis factor-alpha (TNF-alpha) were increased upon cell activation. In vivo, heat-treated antigen-primed murine T cells, injected directly into the antigen challenging site, induced a more severe delayed-type hypersensitivity (DTH) response than those not exposed to elevated temperatures. In contrast, the same heat-treated cells inoculated intravenously did not induce DTH, suggesting that these cells were impaired with respect to penetration of blood vessel walls. Thus, the effects of heat shock on key cellular functions are expressed in different manners: T-cell-ECM adhesiveness and subsequent extravasation are impaired, whereas their abilities to proliferate and to secrete TNF-alpha are augmented.

The pivotal role of the Arg-Gly-Asp (RGD) peptide motif in integrin-mediated cell adhesive interactions with extracellular matrix and plasma proteins stimulated the present design of nonpeptidic mimetics of this sequence. To probe the structural requirements for RGD recognition by integrins, we designed various structural mimetics of the tripeptide sequence, which consist of differentially spaced guanidinium and carboxylic groups. We now report that structures which contain guanidinium and carboxylic groups separated by an 11-carbon atom backbone mimic the distal configuration of functional RGD sequence. These compounds acquire a considerable affinity for the RGD-dependent platelet alpha(IIb)beta3 integrin. As a result, these mimetics specifically inhibited platelet aggregation with an IC50 at the submillimolar range and interfered with RGD-dependent adhesion of CD4+ T-lymphocytes and metastatic tumor cells to immobilized fibronectin and vitronectin. A structural mimetic of the Arg-Gly-Glu (RGE) sequence, and structures with incorrect spacing between the functional groups, failed to inhibit these adhesive interactions. Furthermore, substitution of the guanidinium group by a primary amine abrogated the RGD-mediated biological effects. In vivo, an RGD surrogate effectively inhibited the elicitation of a delayed-type hypersensitivity reaction mediated by CD4+ T-cells, while the RGE mimetic did not. This interference suggests for a central role for RGD recognition in the regulation of immune responses. These proteolytically stable RGD mimetics may thus serve as useful therapeutic agents in versatile pathologic processes which depend on RGD recognition.