Development of Novel Promiscuous Anti-Chemokine Peptibodies for Treating Autoimmunity and Inflammation.

Chemokines and their receptors play critical roles in the progression of autoimmunity and inflammation. Typically, multiple chemokines are involved in the development of these pathologies. Indeed, targeting single chemokines or chemokine receptors has failed to achieve significant clinical benefits in treating autoimmunity and inflammation. Moreover, the binding of host atypical chemokine receptors to multiple chemokines as well as the binding of chemokine-binding proteins secreted by various pathogens can serve as a strategy for controlling inflammation. In this work, promiscuous chemokine-binding peptides that could bind and inhibit multiple inflammatory chemokines, such as CCL2, CCL5, and CXCL9/10/11, were selected from phage display libraries. These peptides were cloned into human mutated immunoglobulin Fc-protein fusions (peptibodies). The peptibodies BKT120Fc and BKT130Fc inhibited the ability of inflammatory chemokines to induce the adhesion and migration of immune cells. Furthermore, BKT120Fc and BKT130Fc also showed a significant inhibition of disease progression in a variety of animal models for autoimmunity and inflammation. Developing a novel class of antagonists that can control the courses of diseases by selectively blocking multiple chemokines could be a novel way of generating effective therapeutics.

CD151 Regulates T-Cell Migration in Health and Inflammatory Bowel Disease.

The continuous recirculation of mature lymphocytes and their entry into the peripheral lymph nodes are crucial for the development of an immune response to foreign antigens. Occasionally, the entry and the subsequent response of T lymphocytes in these sites lead to severe inflammation and pathological conditions. Here, we characterized the tetraspanin molecule, CD151, as a regulator of T cell motility in health and in models of inflammatory bowel disease. CD151 formed a cell surface complex with VLA-4 and LFA-1 integrins, and its activation led to enhanced migration of T cells. Picomolar levels of CCL2 that were previously shown to inhibit T-cell migration to lymph nodes suppressed CD151 expression and dissociated CD151-integrin complexes in T lymphocytes, resulting in attenuated migration toward T-cell attractant chemokines. To directly inhibit CD151 function, a truncated CD151 peptide fragment mimicking of the CD151 extracellular loop was designed. CD151 extracellular loop inhibited T-cell migration in vitro and in vivo and attenuated the development of dextrane sulfate sodium-induced colitis. Thus, CD151 is a key orchestrator of T cell motility; interference with its proper function results in attenuated progression of inflammatory bowel disease.

Loss of kindlin-3 alters the threshold for NK cell activation in human leukocyte adhesion deficiency-III.

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.

Transendothelial migration of lymphocytes mediated by intraendothelial vesicle stores rather than by extracellular chemokine depots.

Chemokines presented by the endothelium are critical for integrin-dependent adhesion and transendothelial migration of naive and memory lymphocytes. Here we found that effector lymphocytes of the type 1 helper T cell (T(H)1 cell) and type 1 cytotoxic T cell (T(C)1 cell) subtypes expressed adhesive integrins that bypassed chemokine signals and established firm arrests on variably inflamed endothelial barriers. Nevertheless, the transendothelial migration of these lymphocytes strictly depended on signals from guanine nucleotide-binding proteins of the G(i) type and was promoted by multiple endothelium-derived inflammatory chemokines, even without outer endothelial surface exposure. Instead, transendothelial migration-promoting endothelial chemokines were stored in vesicles docked on actin fibers beneath the plasma membranes and were locally released within tight lymphocyte-endothelial synapses. Thus, effector T lymphocytes can cross inflamed barriers through contact-guided consumption of intraendothelial chemokines without surface-deposited chemokines or extraendothelial chemokine gradients.

Kindlin-3 is required for the stabilization of TCR-stimulated LFA-1:ICAM-1 bonds critical for lymphocyte arrest and spreading on dendritic cells.

Kindlin-3 is a key lymphocyte function-associated antigen-1 (LFA-1) coactivator deleted in leukocyte adhesion deficiency-III (LAD-III). In the present study, we investigated the involvement of this adaptor in lymphocyte motility and TCR-triggered arrest on ICAM-1 or on dendritic cells (DCs). Kindlin-3-null primary T cells from a LAD-III patient migrated normally on the major lymph node chemokine CCL21 and engaged in normal TCR signaling. However, TCR activation of Kindlin-3-null T lymphocytes failed to trigger the robust LFA-1-mediated T-cell spreading on ICAM-1 and ICAM-1-expressing DCs that is observed in normal lymphocytes. Kindlin-3 was also essential for cytoskeletal anchorage of the LFA-1 heterodimer and for microclustering of LFA-1 within ventral focal dots of TCR-stimulated lymphocytes spread on ICAM-1. Surprisingly, LFA-1 on Kindlin-3-null lymphocytes migrating over CCL21 acquired normal expression of an epitope associated with the conformational activation of the key headpiece domain, ß I. This activated LFA-1 was highly responsive to TCR-triggered ICAM-1-driven stop signals in normal T cells locomoting on CCL21, but not in their Kindlin-3-null T-cell counterparts. We suggest that Kindlin-3 selectively contributes to a final TCR-triggered outside-in stabilization of bonds generated between chemokine-primed LFA-1 molecules and cell-surface ICAM-1.

Occupancy of lymphocyte LFA-1 by surface-immobilized ICAM-1 is critical for TCR- but not for chemokine-triggered LFA-1 conversion to an open headpiece high-affinity state.

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.

Talin1 is required for integrin-dependent B lymphocyte homing to lymph nodes and the bone marrow but not for follicular B-cell maturation in the spleen.

Talin1 is a key integrin coactivator. We investigated the roles of this cytoskeletal adaptor and its target integrins in B-cell lymphogenesis, differentiation, migration, and function. Using CD19 Cre-mediated depletion of talin1 selectively in B cells, we found that talin1 was not required for B-cell generation in the bone marrow or for the entry of immature B cells to the white pulp of the spleen. Loss of talin1 also did not affect B-cell maturation into follicular B cells but compromised differentiation of marginal zone B cells. Nevertheless, serum IgM and IgG levels remained normal. Ex vivo analysis of talin1-deficient spleen B cells indicated a necessary role for talin1 in LFA-1 and VLA-4 activation stimulated by canonical agonists, but not in B-cell chemotaxis. Consequently, talin1 null B splenocytes could not enter lymph nodes nor return to the bone marrow. Talin1 deficiency in B cells was also impaired in the humoral response to a T cell-dependent antigen. Collectively, these results indicate that talin1 is not required for follicular B-cell maturation in the spleen or homeostatic humoral immunity but is critical for integrin-dependent B lymphocyte emigration to lymph nodes and optimal immunity against T-dependent antigens.

TAp63 regulates VLA-4 expression and chronic lymphocytic leukemia cell migration to the bone marrow in a CD74-dependent manner.

The hallmark of chronic lymphocytic leukemia (CLL) is the relentless accumulation of mature lymphocytes, mostly due to their decreased apoptosis. CD74 was recently shown to serve as a survival receptor on CLL cells. In this study, we show that stimulation of CD74 with its natural ligand, migration inhibitory factor, initiates a signaling cascade that results in upregulation of TAp63, which directly regulates CLL survival. In addition, TAp63 expression elevates the expression of the integrin VLA-4, particularly during the advanced stage of the disease. Blocking of CD74, TAp63, or VLA-4 inhibits the in vivo homing of CLL cells to the bone marrow (BM). Thus, CD74 and its target genes TAp63 and VLA-4 facilitate migration of CLL cells back to the BM, where they interact with the supportive BM environment that rescues them from apoptosis. These results could form the basis of novel therapeutic strategies aimed at blocking homing of CLL cells in their return to the BM and attenuating their survival.

Loss of Kindlin-3 in LAD-III eliminates LFA-1 but not VLA-4 adhesiveness developed under shear flow conditions.

Leukocyte adhesion deficiency (LAD)-III is associated with homozygous stop codon mutations in Kindlin-3, the hematopoietic member of the Kindlin family of integrin coactivators. In addition, a subgroup of LAD-III patients has a homozygous splice junction mutation in and reduced expression of the Rap-1 guanine nucleotide exchange factor, CalDAG-GEFI (CDGI). In this study, we compared the adhesive properties of the leukocyte function-associated antigen-1 (LFA-1) and very late activation antigen-4 (VLA-4) integrins in both primary and activated leukocytes derived from these 2 LAD-III subgroups. Primary lymphocytes lacking both Kindlin-3 and CDGI lost all firm T-cell receptor-stimulated LFA-1 adhesiveness, in contrast to LAD-III lymphocytes deficient in Kindlin-3 alone. Effector T cells expanded from all tested LAD-III variants expressed normal CDGI, but lacked Kindlin-3. These Kindlin-3-null effector T cells exhibited total loss of inside-out LFA-1 activation by chemokine signals as well as abrogated intrinsic LFA-1 adhesiveness. Surprisingly, VLA-4 in Kindlin-3-null resting or effector lymphocytes retained intrinsic rolling adhesions to vascular cell adhesion molecule-1 and exhibited only partial defects in chemokine-stimulated adhesiveness to vascular cell adhesion molecule-1. Deletion of the putative beta(1) Kindlin-3 binding site also retained VLA-4 adhesiveness. Thus, our study provides the first evidence that Kindlin-3 is more critical to LFA-1 than to VLA-4-adhesive functions in human lymphocytes.

Beta-arrestin 2 is required for the induction and strengthening of integrin-mediated leukocyte adhesion during CXCR2-driven extravasation.

Leukocyte extravasation involves interdependent signaling pathways underlying the complex dynamics of firm adhesion, crawling, and diapedesis. While signal transduction by agonist-bound chemokine receptors plays a central role in the above responses, it is unclear how it contributes to the sustained and concurrent nature of such responses, given the rapid kinetics of chemokine-induced trimeric G protein coupling and homologous desensitization. Our findings unveil a novel role of beta-arrestins in regulating the activation of signaling pathways underlying discrete integrin-mediated steps in CXCR2-driven leukocyte extravasation. By combining in vivo approaches in beta-arrestin knockout mice with in vitro studies in engineered cellular models, we show that membrane-recruited beta-arrestin 2 is required for the onset and maintenance of shear stress-resistant leukocyte adhesion mediated by both beta(1) and beta(2) integrins. While both beta-arrestin isoforms are required for rapid keratinocyte-derived chemokine (KC)-induced arrest onto limiting amounts of vascular cell adhesion molecule-1 (VCAM-1), adhesion strengthening under shear is selectively dependent on beta-arrestin 2. The latter synergizes with phospholipase C in promoting activation of Rap1A and B, both of which co-operatively control subsecond adhesion as well as postarrest adhesion stabilization. Thus, receptor-induced Galpha(i) and beta-arrestins act sequentially and in spatially distinct compartments to promote optimal KC-induced integrin-dependent adhesion during leukocyte extravasation.

Lymphocyte crawling and transendothelial migration require chemokine triggering of high-affinity LFA-1 integrin.

Endothelial chemokines are instrumental for integrin-mediated lymphocyte adhesion and transendothelial migration (TEM). By dissecting how chemokines trigger lymphocyte integrins to support shear-resistant motility on and across cytokine-stimulated endothelial barriers, we found a critical role for high-affinity (HA) LFA-1 integrin in lymphocyte crawling on activated endothelium. Endothelial-presented chemokines triggered HA-LFA-1 and adhesive filopodia at numerous submicron dots scattered underneath crawling lymphocytes. Shear forces applied to endothelial-bound lymphocytes dramatically enhanced filopodia density underneath crawling lymphocytes. A fraction of the adhesive filopodia invaded the endothelial cells prior to and during TEM and extended large subluminal leading edge containing dots of HA-LFA-1 occupied by subluminal ICAM-1. Memory T cells generated more frequent invasive filopodia and transmigrated more rapidly than their naive counterparts. We propose that shear forces exerted on HA-LFA-1 trigger adhesive and invasive filopodia at apical endothelial surfaces and thereby promote lymphocyte crawling and probing for TEM sites.

Circulating B-cell chronic lymphocytic leukemia cells display impaired migration to lymph nodes and bone marrow.

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 beta2 integrin transcripts. VLA-4 expression was heterogeneous 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.

Anti-inflammatory effects of an inflammatory chemokine: CCL2 inhibits lymphocyte homing by modulation of CCL21-triggered integrin-mediated adhesions.

Our studies focus on the pathways that restrict homing of specific subsets of immune cells, and thereby fine-tune the immune response at specific lymphoid and peripheral tissues. Here, we report that CCL2 (at picomolar [pM] levels) renders both murine and human T cells defective in their ability to develop CCR7-triggered activation of LFA-1- and LFA-1-mediated adhesion strengthening to endothelial ICAM-1 both in vitro and in vivo. CCL2 also attenuated lymphocyte chemotaxis toward lymph node chemokines. Consequently, low-dose CCL2 inhibited lymphocyte homing to peripheral lymph nodes but did not affect lymphocyte trafficking through the spleen. Impaired homing of lymphocytes to peripheral lymph nodes resulted in attenuated progression of both asthma and adjuvant arthritis. Thus, pM levels of circulating CCL2 can exert global suppressive effects on T-cell trafficking and differentiation within peripheral lymph nodes, and may be clinically beneficial as an anti-inflammatory agent.

A crosstalk between intracellular CXCR7 and CXCR4 involved in rapid CXCL12-triggered integrin activation but not in chemokine-triggered motility of human T lymphocytes and CD34+ cells.

The chemokine CXCL12 promotes migration of human leukocytes, hematopoietic progenitors, and tumor cells. The binding of CXCL12 to its receptor CXCR4 triggers Gi protein signals for motility and integrin activation in many cell types. CXCR7 is a second, recently identified receptor for CXCL12, but its role as an intrinsic G-protein-coupled receptor (GPCR) has been debated. We report that CXCR7 fails to support on its own any CXCL12-triggered integrin activation or motility in human T lymphocytes or CD34(+) progenitors. CXCR7 is also scarcely expressed on the surface of both cell types and concentrates right underneath the plasma membrane with partial colocalization in early endosomes. Nevertheless, various specific CXCR7 blockers get access to this pool and attenuate the ability of CXCR4 to properly rearrange by surface-bound CXCL12, a critical step in the ability of the GPCR to trigger optimal CXCL12-mediated stimulation of integrin activation in T lymphocytes as well as in CD34(+) cells. In contrast, CXCL12-triggered CXCR4 signaling to early targets, such as Akt as well as CXCR4-mediated chemotaxis, is insensitive to identical CXCR7 blocking. Our findings suggest that although CXCR7 is not an intrinsic signaling receptor for CXCL12 on lymphocytes or CD34(+) cells, its blocking can be useful for therapeutic interference with CXCR4-mediated activation of integrins.

RhoA is involved in LFA-1 extension triggered by CXCL12 but not in a novel outside-in LFA-1 activation facilitated by CXCL9.

Chemokines presented on endothelial tissues instantaneously trigger LFA-1-mediated arrest on ICAM-1 via rapid inside-out and outside-in (ligand-driven) LFA-1 activation. The GTPase RhoA was previously implicated in CCL21-triggered LFA-1 affinity triggering in murine T lymphocytes and in LFA-1-dependent adhesion strengthening to ICAM-1 on Peyer's patch high endothelial venules stabilized over periods of at least 10 s. In this study, we show that a specific RhoA 23/40 effector region is vital for the initial LFA-1-dependent adhesions of lymphocytes on high endothelial venules lasting 1-3 s. Blocking the RhoA 23/40 region in human T lymphocytes in vitro also impaired the subsecond CXCL12-triggered LFA-1-mediated T cell arrest on ICAM-1 by eliminating the rapid induction of an extended LFA-1 conformational state. However, the inflammatory chemokine CXCL9 triggered robust LFA-1-mediated T lymphocyte adhesion to ICAM-1 at subsecond contacts independently of the RhoA 23/40 region. CXCL9 did not induce conformational changes in the LFA-1 ectodomain, suggesting that particular chemokines can activate LFA-1 through outside-in post ligand binding stabilization changes. Like CXCL9, the potent diacylglycerol-dependent protein kinase C agonist PMA was found to trigger LFA-1 adhesiveness to ICAM-1 also without inducing integrin extension or an a priori clustering and independently of the RhoA 23/40 region. Our results collectively suggest that the 23/40 region of RhoA regulates chemokine-induced inside-out LFA-1 extension before ligand binding, but is not required for a variety of chemokine and non-chemokine signals that rapidly strengthen LFA-1-ICAM-1 bonds without an a priori induction of high-affinity extended LFA-1 conformations.

Lymph node chemokines promote sustained T lymphocyte motility without triggering stable integrin adhesiveness in the absence of shear forces.

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.

Membranal cholesterol is not required for L-selectin adhesiveness in primary lymphocytes but controls a chemokine-induced destabilization of L-selectin rolling adhesions.

Cholesterol-enriched lipid microdomains regulate L-selectin signaling, but the role of membrane cholesterol in L-selectin adhesion is unclear. Arrest chemokines are a subset of endothelial chemokines that rapidly activate leukocyte integrin adhesiveness under shear flow. In the absence of integrin ligands, these chemokines destabilize L-selectin-mediated leukocyte rolling. In the present study, we investigated how cholesterol extraction from the plasma membrane of peripheral blood T or B cells affects L-selectin adhesions and their destabilization by arrest chemokines. Unlike the Jurkat T cell line, whose L-selectin-mediated adhesion is cholesterol dependent, in primary human PBLs and in murine B cells and B cell lines, cholesterol depletion did not impair any intrinsic adhesiveness of L-selectin, consistent with low selectin partitioning into lipid rafts in these cells. However, cholesterol raft disruption impaired the ability of two arrest chemokines, CXCL12 and CXCL13, but not of a third arrest chemokine, CCL21, to destabilize L-selectin-mediated rolling of T lymphocytes. Actin capping by brief incubation with cytochalasin D impaired the ability of all three chemokines to destabilize L-selectin rolling. Blocking of the actin regulatory phosphatidylinositol lipid, phosphatidylinositol 4,5-bisphosphate, did not affect chemokine-mediated destabilization of L-selectin adhesions. Collectively, our results suggest that L-selectin adhesions are inhibited by actin-associated, cholesterol-stabilized assemblies of CXCL12- and CXCL13-binding receptors on both T and B lymphocytes. Thus, the regulation of L-selectin by cholesterol-enriched microdomains varies with the cell type as well as with the identity of the destabilizing chemokine.

Talin 1 and paxillin facilitate distinct steps in rapid VLA-4-mediated adhesion strengthening to vascular cell adhesion molecule 1.

VLA-4 (alpha4beta1) is a key integrin in lymphocytes, interacting with endothelial vascular cell adhesion molecule 1 (VCAM-1) on blood vessels and stroma. To dissect the contribution of the two cytoskeletal VLA-4 adaptor partners paxillin and talin to VLA-4 adhesiveness, we transiently knocked them down in Jurkat T cells and primary resting human T cells by small interfering RNA silencing. Paxillin was required for VLA-4 adhesiveness to low density VCAM-1 under shear stress conditions and was found to control mechanical stability of bonds mediated by the alpha4 subunit but did not affect the integrin affinity or avidity to VCAM-1 in shear-free conditions. Talin 1 maintained VLA-4 in a high affinity conformation, thereby promoting rapid VLA-4 adhesion strengthening to VCAM-1 under both shear stress and shear-free conditions. Talin 1, but not paxillin, was required for VLA-4 to undergo optimal stimulation by the prototypic chemokine, CXCL12, under shear stress conditions. Interestingly, talin 1 and paxillin played the same distinct roles in VLA-4 adhesions of primary T lymphocytes, although VLA-4 affinity to VCAM-1 was at least 200-fold lower in these cells than in Jurkat cells. Collectively, our results suggest that whereas paxillin is a mechanical regulator of VLA-4 bonds generated in the absence of chemokine signals and low VCAM-1 occupancy, talin 1 is a versatile VLA-4 affinity regulator implicated in both spontaneous and chemokine-triggered rapid adhesions to VCAM-1.

A LAD-III syndrome is associated with defective expression of the Rap-1 activator CalDAG-GEFI in lymphocytes, neutrophils, and platelets.

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 CalDAG-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-III 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 TNFalpha-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 CalDAG-GEFI as a critical regulator of inside-out integrin activation in human T lymphocytes, neutrophils, and platelets.

DOCK2 regulates chemokine-triggered lateral lymphocyte motility but not transendothelial migration.

Rac GTPases are key regulators of leukocyte motility. In lymphocytes, chemokine-mediated Rac activation depends on the CDM adaptor DOCK2. The present studies addressed the role of DOCK2 in chemokine-triggered lymphocyte adhesion and motility. Rapid chemokine-triggered activation of both LFA-1 and VLA-4 integrins took place normally in DOCK2-/- T lymphocytes under various shear flow conditions. Consequently, DOCK2-/- T cells arrested normally on TNFalpha-activated endothelial cells in response to integrin stimulatory chemokine signals, and their resistance to detachment was similar to that of wild-type (wt) T lymphocytes. Nevertheless, DOCK2-/- T lymphocytes exhibited reduced microvillar collapse and lamellipodium extension in response to chemokine signals, ruling out a role for these events in integrin-mediated adhesion strengthening. Strikingly, arrested DOCK2-/- lymphocytes transmigrated through a CCL21-presenting endothelial barrier with similar efficiency and rate as wt lymphocytes but, unlike wt lymphocytes, could not locomote away from the transmigration site of the basal endothelial side. DOCK2-/- lymphocytes also failed to laterally migrate over multiple integrin ligands coimmobilized with chemokines. This is a first indication that T lymphocytes use 2 different chemokine-triggered actin remodeling programs: the first, DOCK2 dependent, to locomote laterally along apical and basal endothelial surfaces; the second, DOCK2 independent, to cross through a chemokine-bearing endothelial barrier.