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.

The three-dimensional organization of chromatin contributes to transcriptional control, but information about native chromatin distribution is limited. Imaging chromatin in live Drosophila larvae, with preserved nuclear volume, revealed that active and repressed chromatin separates from the nuclear interior and forms a peripheral layer underneath the nuclear lamina. This is in contrast to the current view that chromatin distributes throughout the nucleus. Furthermore, peripheral chromatin organization was observed in distinct Drosophila tissues, as well as in live human effector T lymphocytes and neutrophils. Lamin A/C up-regulation resulted in chromatin collapse toward the nuclear center and correlated with a significant reduction in the levels of active chromatin. Physical modeling suggests that binding of lamina-associated domains combined with chromatin self-attractive interactions recapitulate the experimental chromatin distribution profiles. Together, our findings reveal a novel mode of mesoscale organization of peripheral chromatin sensitive to lamina composition, which is evolutionary conserved.

Neutrophils are the most prevalent leukocytes in the human body. They have a pivotal role in the innate immune response against invading bacterial and fungal pathogens, while recent emerging evidence also demonstrates their role in cancer progression and anti-tumor responses. The efficient execution of many neutrophil effector responses requires the presence of β2 integrins, in particular CD11a/CD18 or CD11b/CD18 heterodimers. Although extensively studied at the molecular level, the exact signaling cascades downstream of β2 integrins still remain to be fully elucidated. In this review, we focus mainly on inside-out and outside-in signaling of these two β2 integrin members expressed on neutrophils and describe differences between various neutrophil stimuli with respect to integrin activation, integrin ligand binding, and the pertinent differences between mouse and human studies. Last, we discuss how integrin signaling studies could be used to explore the therapeutic potential of targeting β2 integrins and the intracellular signaling cascade in neutrophils in several, among other, inflammatory conditions in which neutrophil activity should be dampened to mitigate disease.

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.

Chronic lymphocytic leukemia (CLL) outgrowth depends on signals from the microenvironment. We have previously found that in vitro reconstitution of this microenvironment induces specific variant isoforms of the adhesion molecule CD44, which confer human CLL with high affinity to hyaluronan (HA). Here, we determined the in vivo contribution of standard CD44 and its variants to leukemic B-cell homing and proliferation in Tcl1 transgenic mice with a B-cell-specific CD44 deficiency. In these mice, leukemia onset was delayed and leukemic infiltration of spleen, liver, and lungs, but not of bone marrow, was decreased. Competitive transplantation revealed that CLL homing to spleen and bone marrow required functional CD44. Notably, enrichment of CD44v6 variants particularly in spleen enhanced CLL engraftment and proliferation, along with increased HA binding. We recapitulated CD44v6 induction in the human disease and revealed the involvement of MAPK and NF-κB signaling upon CD40 ligand and B-cell receptor stimulation by in vitro inhibition experiments and chromatin immunoprecipitation assays. The investigation of downstream signaling after CD44v6-HA engagement uncovered the activation of extracellular signal-regulated kinase and p65. Consequently, anti-CD44v6 treatment reduced leukemic cell proliferation in vitro in human and mouse, confirming the general nature of the findings. In summary, we propose a CD44-NF-κB-CD44v6 circuit in CLL, allowing tumor cells to gain HA binding capacity and supporting their proliferation.

Activation of endothelial cells by IL-1β 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 microvascular endothelial cells, we identified several IL-1β2 induced candidate regulators of these machineries and chose to focus our study on TNF-α-induced protein 2 (myeloid-secretory). The silencing of myeloidsecretory 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 transendothelial 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. Myeloidsecretory 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.

Heparanase, the exclusive mammalian heparan sulfate-degrading enzyme, has been suggested to be utilized by leukocytes to penetrate through the dense basement membranes surrounding blood venules. Despite its established role in tumor cell invasion, heparanase function in leukocyte extravasation has never been demonstrated. We found that T_H1/T_C1-type effector T cells are highly enriched for this enzyme, with a 3.6-fold higher heparanase mRNA expression compared with naive lymphocytes. Using adoptive transfer of wild-type and heparanase-deficient effector T cells into inflamed mice, we show that T-cell heparanase was not required for extravasation inside inflamed lymph nodes or skin. Leukocyte extravasation through acute inflamed skin vessels was also heparanase independent Furthermore, neutrophils emigrated to the inflamed peritoneal cavity independently of heparanase expression on either the leukocytes or on the endothelial and mesothelial barriers, and overexpression of the enzyme on neutrophils did not facilitate their emigration. However, heparanase absence significantly reduced monocyte emigration into the inflamed peritoneal cavity. These results collectively suggest that neither leukocyte nor endothelial heparanase is required for T-cell and neutrophil extravasation through inflamed vascular barriers, whereas this enzyme is required for optimal monocyte recruitment to inflamed peritoneum.

Background: The existing shortage of animal models that properly mimic the progression of early-stage human lung cancer from a solitary confined tumor to an invasive metastatic disease hinders accurate characterization of key interactions between lung cancer cells and their stroma. We herein describe a novel orthotopic animal model that addresses these concerns and consequently serves as an attractive platform to study tumor-stromal cell interactions under conditions that reflect early-stage lung cancer. Methods: Unlike previous methodologies, we directly injected small numbers of human or murine lung cancer cells into murine's left lung and longitudinally monitored disease progression. Next, we used green fluorescent protein-tagged tumor cells and immuno-fluorescent staining to determine the tumor's microanatomic distribution and to look for tumor-infiltrating immune cells and stromal cells. Finally, we compared chemokine gene expression patterns in the tumor and lung microenvironment. Results: We successfully generated a solitary pulmonary nodule surrounded by normal lung parenchyma that grew locally and spread distally over time. Notably, we found that both fibroblasts and leukocytes are recruited to the tumor's margins and that distinct myeloid cell attracting and CCR2-binding chemokines are specifically induced in the tumor microenvironment. Conclusion: Our orthotopic lung cancer model closely mimics the pathologic sequence of events that characterizes early-stage human lung cancer propagation. It further introduces new means to monitor tumor-stromal cell interactions and offers unique opportunities to test therapeutic targets under conditions that reflect early-stage lung cancer. We argue that for such purposes our model is superior to lung cancer models that are based either on genetic induction of epithelial transformation or on ectopic transplantation of malignant cells.

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 chemokine-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 immunotolerizing state that is characterized by IL-10^hi (Tr1) and IL-4^hi (Th2) cells, mediated via p70 kinase/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.

The ATLAS detector at the Large Hadron Collider is used to search for highmass resonances decaying to an electron-positron pair or a muon-antimuon pair. The search is sensitive to heavy neutral Z gauge bosons, Randall-Sundrum gravitons, Z* bosons, techni-mesons, Kaluza-Klein Z/γ bosons, and bosons predicted by Torsion models. Results are presented based on an analysis of pp collisions at a center-of-mass energy of 7 TeV corresponding to an integrated luminosity of 4.9 fb^-1 in the e ⁺e^- channel and 5.0 fb^-1 in the μ⁺μ^- channel. A Z boson with Standard Model-like couplings is excluded at 95% confidence level for masses below 2.22 TeV. A Randall-Sundrum graviton with coupling k /M̄_Pl = 0.1 is excluded at 95% confidence level for masses below 2.16 TeV. Limits on the other models are also presented, including Technicolor and Minimal Z Models. Copyright CERN.

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 secretedhigher concentrations of IL-6 andkeratinocyte 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.

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, β I. This activated LFA-1 was highly responsive to TCRtriggered 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.

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.

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.

We have previously demonstrated that the chemokine IFN-γ inducible protein 10 (IP-10) and its receptor CXCR3, are overexpressed in myasthenia gravis (MG) and its animal model experimental autoimmune MG (EAMG). We now studied the potential of modulating rat EAMG by interference in CXCR3/IP-10 signaling. Two different approaches were used: 1) blocking IP-10 by IP-10-specific antibodies and 2) inhibiting the CXCR3 chemokine receptor by a CXCR3 antagonist. Treatment by either of these reagents led to suppression of EAMG suggesting that inhibition of CXCR3/IP-10 signaling can be considered as a potential treatment modality for MG.

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.

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.

The mechanisms underlying leukocyte migration across endothelial barriers are still largely elusive. Integrin activation by chemokine signals is a key checkpoint in this process. Most of the current knowledge on transendothelial migration (TEM) of leukocytes has been derived from in vitro modified Boyden-chamber transfilter migration assays. In these assays, leukocyte migration toward chemokine gradients established across an endothelial barrier is measured under shear-free conditions. Consequently, these assays do not address the critical contribution of shear forces to dynamic integrin activation and redistribution at focal lymphocyte-endothelial contacts. Endothelial chemokines are displayed at high levels on blood vessel walls in vivo and play critical roles in both integrin activation and polarization of leukocytes on blood vessels, yet transwell assays do not assess the role of these chemokines in leukocyte TEM. To overcome these two drawbacks, several laboratories, including our group, developed assays based on in vitro live imaging microscopy to follow leukocyte migration across endothelial barriers that display defined compositions of integrin-stimulatory chemokines. These assays not only successfully simulate physiologic TEM processes but also enable the tracking and dissection of leukocyte adhesion, motility, and crossing of endothelial barriers in real time and under physiologic flow conditions. In addition, fluorescent tagging of membranes, adhesion molecules, and cytoskeletal regulatory elements on the endothelial barrier or the leukocyte can provide key spatial and temporal information on the mode of activity of these elements during distinct stages of leukocyte TEM. After fixation, subcellular changes in the redistribution of these key molecules can be further dissected by immunofluorescence tools and by ultrastructural analysis based on scanning and transmission electron microscopy.

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 β-arrestins in regulating the activation of signaling pathways underlying discrete integrin-mediated steps in CXCR2-driven leukocyte extravasation. By combining in vivo approaches in β-arrestin knockout mice with in vitro studies in engineered cellular models, we show that membrane-recruited β-arrestin 2 is required for the onset and maintenance of shear stress-resistant leukocyte adhesion mediated by both β₁ and β₂ integrins. While both β-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 β-arrestin 2. The latter synergizes with phospholipase C in promoting activation of Rap1A and B, both of which cooperatively control subsecond adhesion as well as postarrest adhesion stabilization. Thus, receptor-induced Gα_i and β-arrestins act sequentially and in spatially distinct compartments to promote optimal KC-induced integrin-dependent adhesion during leukocyte extravasation.

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.

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.

VLA-4 (α₄β₁) 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 α₄ 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.

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.

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 burgeoning field of leukocyte trafficking has created new and exciting opportunities in the clinic. Trafficking signals are being defined that finely control the movement of distinct subsets of immune cells into and out of specific tissues. Because the accumulation of leukocytes in tissues contributes to a wide variety of diseases, these 'molecular codes' have provided new targets for inhibiting tissue-specific inflammation, which have been confirmed in the clinic. However, immune cell migration is also critically important for the delivery of protective immune responses to tissues. Thus, the challenge for the future will be to identify the trafficking molecules that will most specifically inhibit the key subsets of cells that drive disease processes without affecting the migration and function of leukocytes required for protective immunity.

The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor, CXCR4, play a major role in migration, retention, and development of hematopoietic progenitors in the bone marrow. We report the direct involvement of atypical PKC-ζ in SDF-1 signaling in immature human CD34 ⁺-enriched cells and in leukemic pre-B acute lymphocytic leukemia (ALL) G2 cells. Chemotaxis, cell polarization, and adhesion of CD34⁺ cells to bone marrow stromal cells were found to be PKC-ζ dependent. Overexpression of PKC-ζ in G2 and U937 cells led to increased directional motility to SDF-1. Interestingly, impaired SDF-1-induced migration of the pre-B ALL cell line B1 correlated with reduced PKC-ζ expression. SDF-1 triggered PKC-ζ phosphorylation, translocation to the plasma membrane, and kinase activity. Furthermore we identified PI3K as an activator of PKC-ζ, and Pyk-2 and ERK1/2 as downstream targets of PKC-ζ. SDF-1-induced proliferation and MMP-9 secretion also required PKC-ζ activation. Finally, we showed that in vivo engraftment, but not homing, of human CD34 ⁺-enriched cells to the bone marrow of NOD/SCID mice was PKC-ζ dependent and that injection of mice with inhibitory PKC-ζ pseudosubstrate peptides resulted in mobilization of murine progenitors. Our results demonstrate a central role for PKC-ζ in SDF-1-dependent regulation of hematopoietic stem and progenitor cell motility and development.

Lymphocyte transendothelial migration (TEM) is promoted by fluid shear signals and apical endothelial chemokines. Studying the role of these signals in neutrophil migration across differently activated HUVEC in a flow chamber apparatus, we gained new insights into how neutrophils integrate multiple endothelial signals to promote TEM. Neutrophils crossed highly activated HUVEC in a β₂ integrin-dependent manner but independently of shear. In contrast, neutrophil migration across resting or moderately activated endothelium with low-level β₂ integrin ligand activity was dramatically augmented by endothelial-presented chemoattractants, conditional to application of physiological shear stresses and intact β₂ integrals. Shear stress signals were found to stimulate extensive neutrophil invaginations into the apical endothelial interface both before and during TEM. A subset of invaginating neutrophils completed transcellular diapedesis through individual endothelial cells within

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.

The mechanism that regulates the preferential accumulation of NKT cells in the BM is unknown. The BM endothelium constitutively expresses selectins, the integrin ligands VCAM-1 and ICAM-1, and the chemokine CXCL12. Both NK and NKT subsets of cells exhibited similar tethering and rolling interactions on both P-selectin and E-selectin and expressed similar levels of the integrins, VLA-4 and LFA-1. Although NKT cells express higher levels of CXCR4 than NK cells, CXCL12 (the ligand for CXCR4) rapidly stimulates similar levels of adhesion of NK and NKT cells to VCAM-1 and ICAM-1. In both subsets, the arrest on VCAM-1 was dependent on high affinity VLA-4 and the homing of these cells to the BM of NOD/SCID was VLA-4-dependent. However, as opposed to the situation for NK cells, CXCL12 preferentially triggers, under shear flow, the rolling on VCAM-1 and transendothelial migration of NKT cells. Moreover, over-expression of high levels of CXCR4 on the YT NK cell line enables them to migrate in response to CXCL12. This study therefore suggests an important role for CXCR4 levels of expression and for VLA-4 in regulating the accumulation of NKT cells in the BM.

During their migration into inflammatory sites, immune cells, such as T cells, secrete extracellular matrix (ECM)-degrading enzymes, such as heparanase, which, under mildly acidic conditions, degrade heparan sulfate proteoglycans (HSPG). We have previously shown that at pH 7.2, human placental heparanase loses its enzymatic activity, while retaining its ability to bind HSPG and promote T cell adhesion to unfractionated ECM. We now demonstrate that the 65-kDa recombinant human heparanase, which is devoid of enzymatic activity, but can still bind HSPG, captures T cells under shear flow conditions and mediates their rolling and arrest, in the absence or presence of stromal cell-derived factor 1α (SDF-1α; CXCL12), in an α₄β ₁-VCAM-1-dependent manner. Furthermore, heparanase binds to and induces T cell adhesion to key ECM components, like fibronectin and hyaluronic acid, in β₁ integrin- and CD44-specific manners, respectively, via the activation of the protein kinase C and phosphatidylinositol 3-kinase intracellular signaling machineries. Although the nature of the putative T cell heparanase-binding moiety is unknown, it appears that heparanase exerts its proadhesive activity by interacting with the T cells' surface HSPG, because pretreatment of the cells with heparinase abolished their subsequent response to heparanase. Also, heparanase augmented the SDF-1α-triggered phosphorylation of Pyk-2 and extracellular signal-regulated kinase-2 implicated in integrin functioning. Moreover, heparanase, which had no chemotactic effect on T cells on its own, augmented the SDF-1α-induced T cell chemotaxis across fibronectin. These findings add another dimension to the known versatility of heparanase as a key regulator of T cell activities during inflammation, both in the context of the vasculature and at extravascular sites.

Leukocytes are arrested on endothelial cells before moving through vessel intercellular junctions. The identification of another beta(2) integrin-dependent step may provide insights into the events and signals required for transmigration.

Recently, we reported a rare leukocyte adhesion deficiency (LAD) associated with severe defects in integrin activation by chemokine signals, despite normal ligand binding of leukocyte integrins. We now report that the small GTPase, Rap1, a key regulator of inside-out integrin activation is abnormally regulated in LAD Epstein-Barr virus (EBV) lymphocyte cells. Both constitutive and chemokine-triggered activation of Rap1 were abolished in LAD lymphocytes despite normal chemokine signaling. Nevertheless, Rap1 expression and activation by phorbol esters were intact, ruling out an LAD defect in Rap1 guanosine triphosphate (GTP) loading. The very late antigen 4 (VLA-4) integrin abnormally tethered LAD EBV lymphocytes to its ligand vascular cell adhesion molecule 1 (VCAM-1) under shear flow due to impaired generation of high-avidity contacts despite normal ligand binding and intact avidity to surface-bound anti-VLA-4 monoclonal antibody (mAb). Thus, a defect in constitutive Rap1 activation results in an inability of ligand-occupied integrins to generate high-avidity binding to ligand under shear flow. This is a first report of an inherited Rap1 activation defect associated with a pathologic disorder in leukocyte integrin function, we herein term it "LAD-III."

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 14-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 chemoattractants - 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.

The arrest of rolling leukocytes on target endothelium is predominantly mediated by integrins, which pre-exist in largely inactive states on circulating immune cells and need to be activated in situ. These adhesion receptors acquire high avidity upon encounter with endothelial-displayed chemokines or chemoattractants, which are ligands to specific G protein-coupled receptors (GPCRs) on the leukocyte surface. In order to arrest, the leukocyte must constantly integrate endothelial-based signals as it moves along the vessel wall. It is unclear whether the chemokine signal is locally transmitted at the endothelial contact zone or whether the rolling leukocyte accumulates successive chemokine signals to reach a threshold global activation. Recent in vitro and in vivo data suggest that the induction of high integrin avidity by endothelial chemokine-transduced G_i-signals is a general mechanism that has evolved to locally enhance integrin avidity to ligand within subseconds at restricted leukocyte-endothelial contacts. In addition, a second specialized mechanism, involving stepwise signals integrated by selectin ligands on rolling cells, seems to activate integrins on the entire leukocyte surface. This GPCR-independent and much slower pathway (10¹-10² seconds) is transmitted through rolling engagements of neutrophils, primarily on E-selectin. We propose that these two mechanisms are differentially used by distinct leukocyte subsets at various vascular beds, providing much larger combinatorial diversity of integrin activation on rolling leukocytes than previously predicted.

Selectin counterreceptors are glycoprotein scaffolds bearing multiple carbohydrate ligands with exceptional ability to tether flowing cells under disruptive shear forces. Bond clusters may facilitate formation and stabilization of selectin tethers. L-selectin ligation has been shown to enhance L-selectin rolling on endothelial surfaces. We now report that monoclonal antibodies-induced L-selectin dimerization enhances L-selectin leukocyte tethering to purified physiological L-selectin ligands and glycopeptides. Microkinetic analysis of individual tethers suggests that leukocyte rolling is enhanced through the dimerization-induced increase in tether formation, rather than by tether stabilization. Notably, L-selectin dimerization failed to augment L-selectin-mediated adhesion below a threshold ligand density, suggesting that L-selectin dimerization enhanced adhesiveness only to properly clustered ligand. In contrast, an epidermal growth factor domain substitution of L-selectin enhanced tether formation to L-selectin ligands irrespective of ligand density, suggesting that this domain controls intrinsic ligand binding properties of L-selectin without inducing L-selectin dimerization. Strikingly, at low ligand densities, where L-selectin tethering was not responsive to dimerization, elevated shear stress restored sensitivity of tethering to selectin dimerization. This is the first indication that shear stress augments effective selectin ligand density at local contact sites by promoting L-selectin encounter of immobilized ligand.

In order to extravasate the bloodstream at specific sites of inflammation or antigen presentation, circulating leukocytes must rapidly translate specific adhesive and stimulatory signals into firm adhesion. Leukocyte arrest is nearly exclusively mediated by integrin receptors. Recent in vitro and in vivo evidence suggests that specialized integrins support reversible tethers that slow down selectin-initiated rolling of leukocytes prior to their arrest. In situ activation of integrin avidity by ligand and chemokine signaling can take place within fractions of seconds, resulting either in augmented reversible adhesions or immediate arrest on the vascular endothelium. The ability of leukocyte integrins to rapidly respond to these in situ avidity modulators appears to depend on preformed affinity and clustering states, which are internally regulated by cytoskeletal constraints on integrin conformation and mobility. We discuss potential regulatory mechanisms by which a given set of chemokine receptors and integrins may interact to rapidly generate high avidity, shear-resistant integrin-mediated leukocyte arrest on vascular endothelium.

L-selectin is a leukocyte lectin that mediates leukocyte capture and rolling in the vasculature. The cytoplasmic domain of L-selectin has been shown to regulate leukocyte rolling. In this study, the regulatory mechanisms by which this domain controls L-selectin adhesiveness were investigated. We report that an L-selectin mutant generated by truncation of the COOH-terminal 11 residues of L-selectin tail, which impairs association with the cytoskeletal protein α-actinin, could capture leukocytes to glycoprotein L-selectin ligands under physiological shear flow. However, the conversion of initial tethers into rolling was impaired by this partial tail truncation, and was completely abolished by a further four-residue truncation of the L-selectin tail. Physical anchorage of both cell-free tail-truncated mutants within a substrate fully rescued their adhesive deficiencies. Microkinetic analysis of full-length and truncated L-selectin-mediated rolling at millisecond temporal resolution suggests that the lifetime of unstressed L-selectin tethers is unaffected by cytoplasmic tail truncation. However, cytoskeletal anchorage of L-selectin stabilizes the selectin tether by reducing the sensitivity of its dissociation rate to increasing shear forces. Low force sensitivity (reactive compliance) of tether lifetime is crucial for selectins to mediate leukocyte rolling under physiological shear stresses. This is the first demonstration that reduced reactive compliance of L-selectin tethers is regulated by cytoskeletal anchorage, in addition to intrinsic mechanical properties of the selectin-carbohydrate bond.

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.

The recruitment of circulating leukocytes at vascular sites in target tissue has been linked to activation of Gi-protein signaling in leukocytes by endothelial chemokines. The mechanisms by which apical and subendothelial chemokines regulate leukocyte adhesion to and migration across endothelial barriers have been elusive. We recently found that endothelial chemokines not only stimulate integrin-mediated arrest on vascular endothelial ligands but also trigger earlier very late antigen (VLA)-4 integrin-mediated capture (tethering) of lymphocytes to vascular cell adhesion molecule 1 (VCAM-1)-bearing surfaces by extremely rapid modulation of integrin clustering at adhesive contact zones. This rapid modulation of integrin avidity requires chemokine immobilization in juxtaposition with the VLA-4 ligand VCAM-1. We also observed that endothelial-bound chemokines promote massive lymphocyte transendothelial migration (TEM). It is interesting that chemokine-promoted lymphocyte TEM requires continuous exposure of lymphocytes but not of the endothelial barrier to fluid shear. It is noteworthy that lymphocyte stimulation by soluble chemokines did not promote lymphocyte TEM. Our results suggest new roles for apical endothelial chemokines both in triggering lymphocyte capture to the endothelial surface and in driving post-arrest events that promote lymphocyte transmigration across endothelial barriers under shear flow.

Dendritic cells (DCs) are recruited from blood into tissues to patrol for foreign antigens. After antigen uptake and processing, DCs migrate to the secondary lymphoid organs to initiate immune responses. We now show that DC-SIGN, a DC-specific C-type lectin, supports tethering and rolling of DC-SIGN-positive cells on the vascular ligand 1CAM-2 under shear flow, a prerequisite for emigration from blood. The DC-SIGN-1CAM-2 interaction regulates chemokine-induced transmigration of DCs across both resting and activated endothelium. Thus, DC-SIGN is central to the unusual trafficking capacity of DCs, further supported by the expression of DC-SIGN on precursors in blood and on immature and mature DCs in both peripheral and lymphoid tissues.

Leukocyte recruitment to target tissue is initiated by weak rolling attachments to vessel wall ligands followed by firm integrin-dependent arrest triggered by endothelial chemokines. We show here that immobilized chemokines can augment not only arrest but also earlier integrin-mediated capture (tethering) of lymphocytes on inflamed endothelium. Furthermore, when presented in juxtaposition to vascular cell adhesion molecule 1 (VCAM-1), the endothelial ligand for the integrin very late antigen 4 (VLA-4, α4β1), chemokines rapidly augment reversible lymphocyte tethering and rolling adhesions on VCAM-1. Chemokines potentiate VLA-4 tethering within

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.

By differential display we demonstrated that antibody-mediated ligation of the GPI-linked protein product of E48, a newly discovered human Ly-6 gene, upregulates the expression of the FX enzyme in 3 lines of head and neck squamous carcinoma cells. FX is responsible for the last step in the synthesis of GDP-L-fucose. The up-regulation of FX was E48 ligand-specific. 22AWT head and neck squamous carcinoma cells expressing high levels of E48 expressed significantly higher levels of FX than the E48 antisense transfected 22AWT cells (8-3 cells). The former cells also expressed higher levels of two major fucosylated glycans (the selectin ligand, Sialyl Lewis a, and VIM-2) than the E48 antisense transfectants. Conversely, transfection of cells from the 14CWT line expressing very low levels of E48 with E48 cDNA caused an up-regulated expression of FX and of the two fucosylated glycans in the 14C-CMV16 transfectants. Moreover, the expression levels of Sialyl Lewis a was significantly up-regulated on HNSCC upon ligation of E48 by anti-E48 antibodies. The functional significance of the E48-mediated up-regulation of Sialyl Lewis a was demonstrated in rolling experiments on E-selectin bearing surfaces under physiological conditions of shear flow and on tumor necrosis factor α-activated human umbilical venous endothelial cells. Only high E48/FX/Sialyl Lewis a expressing 14C-CMV16 cells could roll on purified E- selectin or establish E-selectin dependent rolling on the activated human umbilical venous endothelial cells. Low E48/FX/Sialyl Lewis a expressing 14CWT cells did not roll. These results show that E48 controls the expression of the FX enzyme and of certain fucosylated E-selectin ligands by HNSCC. E48 may thus function as a key regulator of the adhesiveness of these tumor cells to inflamed vessel walls expressing E-selectin.

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 β₁ 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 α₄, α₅, and, to a lesser degree, the α₂ and α₆ members the β₁ integrins. Our system enables the real-time tracking of individual locomoting lymphocytes and the analysis of their dynamic interactions with ECM components and cytokines.

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- SO₃Galβ1α4(Fuca1α3)-6-O-SO₃-GIcβ-OR) and 3',6'-disulfo Lewis x(Glc) (3,6-di-O-SO₃-Galβ1α4(Fucα1α3)Glcβ-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.

Two mechanisms have been proposed for regulating rolling velocities on selectins, These are (a) the intrinsic kinetics of bond dissociation, and (b) the reactive compliance, i.e,, the susceptibility of the bond dissociation reaction to applied force. To determine which of these mechanisms explains the 7.5-11.5-fold faster rolling of leukocytes on L-selectin than on E-and P-selectins, we have compared the three selectins by examining the dissociation of transient tethers. We find that the intrinsic kinetics for tether bond dissociation are 7-10-fold more rapid for L-selectin than for E-and P-selectins, and are proportional to the rolling velocities through these selectins. The durations of pauses during rolling correspond to the duration of transient tethers on low density substrates. Moreover, applied force increases dissociation kinetics less for L-selectin than for E-and P-selectins, demonstrating that reactive compliance is not responsible for the faster rolling through L-selectin, Further measurements provide a biochemical and biophysical framework for understanding the molecular basis of rolling. Displacements of tethered cells during flow reversal, and measurements of the distance between successive pauses during rolling provide estimates of the length of a tether and the length of the adhesive contact zone, and suggest that rolling occurs with as few as two tethers per contact zone. Tether bond lifetime is an exponential function of the force on the bond, and the upper limit for the tether bond spring constant is of the same order of magnitude as the estimated elastic spring constant of the lectin-EGF unit. Shear uniquely enhances the rate of L-selectin transient tether formation, and conversion of tethers to rolling adhesions, providing further understanding of the shear threshold requirement for rolling through L-selectin.

Antibodies immobilized on the wall of a flow chamber can support leukocyte rolling in shear flow, IgM mAb to Lewis(x) (CD15) and sialyl Lewis(x) (CD15s). which are carbohydrate antigens related to selectin ligands, plus mAb to CD48 and CD59, could mediate rolling, IgM and IgG mAb to L-selectin (CD62L), lymphocyte function-associated antigen 1 (CD11a), CD43, intercellular adhesion molecule 3 (CD50), and CD45 mediated only firm adhesion, In contrast to selectins, antibodies supported rolling only within a restricted range of site densities and wall shear stresses, outside of which firm adhesion or detachment occurred, When wall shear stress was increased, rolling velocity increased rapidly far antibodies but not for selectins, The kinetics of dissociation from the substrate of transiently tethered cells also increased more rapidly as a function of shear stress for antibodies than for selectins, These comparisons emphasize a number of remarkable features of selectins, including the lack of development of firm adhesion, and suggest that specialized molecular or cellular mechanisms must be required to explain their ability to support rolling over a wide range of environmental variables.

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.

SELECTINS are cell adhesion molecules that bind carbohydrate ligands and promote interaction between leukocytes and the vessel wall in vascular shear flow^1,2. Selectinligand bonds have high mechanical strength, allowing initial tethering to the vessel wall through one or few bonds, and have fast on and off rates, permitting rolling in response to hydrodynamic drag³. The L-selectin molecule on leukocytes binds to peripheral node addressin on high endothelial venules of lymph nodes to mediate leukocyte rolling^4,5 and binds to a ligand on neutrophils to mediate rolling of leukocytes over one another⁶. Here we describe a surprising mechanism for regulation of these interactions, both in vitro and in vivo. Shear above a critical threshold is required to promote and maintain rolling interactions through L-selectin, but not through E-selectin, P-selectin or VCAM-1. The shear threshold requirement for L-selectin may be physiologically important in low shear to prevent inappropriate aggregation of leukocytes and interaction with the vessel wall.

For functional studies of the integrin alpha(4) cytoplasmic domain, we have expressed the following in K562 and Chinese hamster ovary (CHO) cells: 1) wild-type alpha(4) (called X4C4), 2) two chimeric forms of alpha(4) (called X4C2 and X4C5) that contain the cytoplasmic domains of alpha(2) and alpha(5), respectively, and 3) alpha(4) with no cytoplasmic domain (X4CO). Cytoplasmic domain exchange had no effect on VLA-4-dependent static cell adhesion or tethering to VCAM-1 in conditions of shear flow. However, the presence of the alpha(2) alpha(5) tails markedly or a enhanced VLA-4-dependent K562 cells spreading (X4C2 > X4C5 > X4C4 > X4CO), increased localization of VLA-4 into focal adhesion-like complexes in CHO cells (X4C2 > X4C5 > X4C4), and strengthened CHO and K562 cell resistance to detachment from VCAM-1 in conditions of shear flow (X4C2 > X4C5 > X4C4 > X4CO). Conversely, the alpha(4) tail supported greater VLA-4-dependent haptotactic and chemotactic cell migration. In the absence of any alpha tail (i.e., X4CO), robust focal adhesions were observed, even though cell spreading and adhesion strengthening were minimal. Thus, such focal adhesions may have relatively little functional importance, and should not be compared with focal adhesions formed when alpha tails are present. Together, these results indicate that all three alpha-chain tails exert defined positive effects (compared with no tail at all), but suggest that the alpha(4) cytoplasmic domain may be specialized to engage in weaker cytoskeletal interactions, leading to diminished focal adhesion formation, cell spreading, and adhesion strengthening, while augmenting cell migration and facilitating rolling under shear flow. These properties of the alpha(4) tail are consistent with the role of alpha 4 lymphocytes, monocytes and eosinophils.

Selectins have previously been shown to tether a flowing leukocyte to a vessel wall and mediate rolling. Here, we report that an integrin, VLA-4, can also support tethering and rolling. Blood T lymphocytes and alpha 4 integrin-transfected cells can tether in shear flow, and then roll, through binding of the integrin VLA-4 to purified VCAM-1 on the wall of a flow chamber. VLA-4 transfectants showed similar tethering and rolling on TNF-stimulated endothelium. Tethering efficiency, rolling velocity, and resistance to detachment are related to VCAM-1 density. Tethering and rolling did not occur on ICAM-1, fibronectin, or fibronectin fragments, and tethering did not require integrin activation or the presence of an alpha 4 cytoplasmic domain. Arrest of rolling cells on VCAM-1 occurred spontaneously, and/or was triggered by integrin activating agents Mn2+, phorbol ester, and mAb TS2/16. These agents, and the alpha 4 cytoplasmic domain, promoted increased resistance to detachment. Together the results show that VLA-4 is a versatile integrin that can mediate tethering, rolling, and firm arrest on VCAM-1.

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-α 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-α and its putative physiologic consequences using human or rat CD4⁺ cells. A brief exposure of CD4⁺ cells to low dosages of soluble TNF-α or of FN- or laminin-bound TNF-α synergized with PMA to enhance the integrin-mediated binding of CD4⁺ cells to these immobilized ECM moieties. TNF-α-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-α immediately after their exposure to PMA. This indicates that the enhancing effect of TNF-α requires a previous activation of the cells. When TNF-α was immobilized on FN, less TNF-α was required to induce CD4⁺ cell binding to FN. Soluble, and to a greater extent FN-bound, TNF-α synergizes with PMA to intensify protein tyrosine phosphorylation in FN-bound CD4⁺ cells, and this effect of TNF-α was inhibited by inhibitors of tyrosine kinase. That FN-bound or soluble TNF-α 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-α, and matrix-linked TNF-α may recruit and direct immune cells to inflammatory sites.

Certain inflammatory cytokines and growth factors have been previously shown to interact with glycosaminoglycan moieties of the extracellular matrix (ECM). We have examined the association of the pleiotropic cytokine TNF-α with glycoprotein constituents of ECM. TNF-α interacted with fibronectin (FN) and laminin, and to a lesser degree with collagen. The major binding site for TNF-α on FN was localized to its 30-kDa N-terminal fragment (FN- N') with a K(i) in the sub-nM range. The binding of ¹²⁵I-labeled TNF-α to immobilized FN or FN-N' persisted for at least 24 h, and was specifically inhibited by antibodies to FN, mAb directed against the FN-N' domain, unlabeled TNF-α, and by the truncated forms of TNF-α receptors. Once bound to immobilized FN or FN-N', the cytokine could not be released by the soluble TNF-α-receptors, although it could be released by anti-TNF-α Ab. TNF-α was also found to interact with soluble FN, although with a lower affinity. Similar to the soluble cytokine, the FN-bound TNF-α appears to be functional; it augmented the β₁-integrin-mediated adhesiveness of activated CD4⁺ human T cells to the glycoprotein. Hence, binding of TNF-α to immobilized FN, which modifies its functional accessibility to soluble TNF- α receptors, does not abolish but rather may locally restrict its activity. This study suggests that a major ECM glycoprotein can present, in a restricted manner, a functional adhesion-modulating cytokine to immune cells, and that ECM glycoproteins may regulate their intrinsic cell-adhesive properties by associating with cytokines.

In the present study, we have demonstrated the feasibility of targeting a proteolytic enzyme, via the high-affinity avidin-biotin system, to act in a highly selective manner upon a cell surface-associated antibody. As an example of this approach, a cell-bound biotinylated monoclonal antibody could be removed efficiently by means of biotinylated proteinase K, bridged to streptavidin. Only low levels of cell death were observed using this procedure. The approach may prove useful for a variety of applications, including the recovery of antibody-free positively selected cell populations.

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 II-atom spacer, acquired a significant affinity for the GPIIb-IIIa integrin and inhibited platelet aggregation. The structural requirements for the interactions of 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 μM 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 ¹²⁵I-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 platelet-ECM interactions.

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 α_IIbβ₃ 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.