Selectin avidity modulation by chemokines at subsecond endothelial contacts: a novel regulatory level of leukocyte trafficking.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
R Alon
Publications and source records attributed to R Alon.
Cytoplasmic anchorage of L-selectin controls leukocyte capture and rolling by increasing the mechanical stability of the selectin tether.
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 alpha-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.
Amino Acid Sequence↗
The affinity of integrin alpha(4)beta(1) governs lymphocyte migration.
The interaction of integrin alpha(4)beta(1) with endothelial VCAM-1 controls the trafficking of lymphocytes from blood into peripheral tissues. Cells actively regulate the affinity of alpha(4)beta(1) for VCAM-1 (activation). To investigate the biological function of alpha(4)beta(1) activation, we isolated Jurkat T cell lines with defective alpha(4)beta(1) activation. Using these cells, we found that alpha(4)beta(1)-stimulated alpha(L)beta(2)-dependent cell migration was dramatically reduced in cells with defects in alpha(4)beta(1) activation. These cells required 20 times more VCAM-1 to promote alpha(L)beta(2)-dependent cell migration. This defect was at the level of alpha(4)beta(1) affinity as an activating alpha(4)beta(1) Ab rescued alpha(4)beta(1)-stimulated alpha(L)beta(2)-dependent migration. In contrast, migration of alpha(4)beta(1) activation-defective cells on VCAM-1 alone was enhanced at higher VCAM-1 densities. Thus, alpha(4)beta(1) activation determines a set point or threshold at which VCAM-1 can regulate alpha(L)beta(2)-dependent as well as alpha(4)beta(1)-dependent cell migration. Changes in this set point may specify preferred anatomical sites of integrin-dependent leukocyte emigration from the bloodstream.
Cell Adhesion↗
Shear forces promote lymphocyte migration across vascular endothelium bearing apical chemokines.
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.
Animals↗
An altered peptide ligand inhibits the activities of matrix metalloproteinase-9 and phospholipase C, and inhibits T cell interactions with VCAM-1 induced in vivo by a myasthenogenic T cell epitope.
Myasthenia gravis (MG) is a T cell-regulated, antibody-mediated autoimmune disease. Immunization with two myasthenogenic peptides, p195-212 and p259-271, which are sequences of the human acetylcholine receptor, resulted in MG-associated immune responses. A dual altered peptide ligand (APL) composed of the two APLs of the myasthenogenic peptides inhibited, in vitro and in vivo, those responses. This study was aimed at understanding the mechanism(s) underlying the in vivo inhibitory properties of the dual APL. To this end, we analyzed T cells of mice that were immunized with p259-271 for their adhesiveness toward vascular cell adhesion molecule 1, for the activity of their secreted matrix metalloproteinases (MMPs), and for their intracellular phospholipase C (PLC) activity. Immunization with p259-271 triggered the above three activities and in vivo administration of the dual APL inhibited the latter. Thus, treatment of mice with the dual APL interferes with functions required for T cells to migrate and interact with the self-AChR. This is the first indication that very late antigen 4, MMP-9, and PLC are targets for immunomodulation of autoreactive T cells by altered peptide ligands.
Amino Acid Sequence↗
Encephalitogenic lymphoblast recruitment to resting CNS microvasculature: a natural immunosurveillance mechanism?
Explore the source record for details and available documents.
Adoptive Transfer↗
CD44-dependent lymphoma cell dissemination: a cell surface CD44 variant, rather than standard CD44, supports in vitro lymphoma cell rolling on hyaluronic acid substrate and its in vivo accumulation in the peripheral lymph nodes.
Cell motility is an essential element of tumor dissemination, allowing organ infiltration by cancer cells. Using mouse LB lymphoma cells transfected with standard CD44 (CD44s) cDNA (LB-TRs cells) or with the alternatively spliced CD44 variant CD44v4-v10 (CD44v) cDNA (LB-TRv cells), we explored their CD44-dependent cell migration. LB-TRv cells, but not LB-TRs or parental LB cells, bound soluble hyaluronic acid (HA) and other glycosaminoglycans (GAGs), and exclusively formed, under physiological shear force, rolling attachments on HA substrate. Furthermore, LB-TRv cells, but not LB-TRs cells or their parental LB cells, displayed accelerated local tumor formation and enhanced accumulation in the peripheral lymph nodes after s.c. inoculation. The aggressive metastatic behavior of i.v.-injected LB-TRV cells, when compared with that of other LB-transfectants, is attributed to more efficient migration to the lymph nodes, rather than to local growth in the lymph node. Injection of anti-CD44 monoclonal antibody or of the enzyme hyaluronidase also prevented tumor growth in lymph nodes of BALB/c mice inoculated with LB-TRv cells. The enhanced in vitro rolling and enhanced in vivo local tumor growth and lymph node invasion disappeared in LB cells transfected with CD44v cDNA bearing a point mutation at the HA binding site, located at the distal end of the molecule constant region. These findings show that the interaction of cell surface CD44v with HA promotes cell migration both in vitro and in vivo, and they contribute to our understanding of the mechanism of cell trafficking, including tumor spread.
Adjuvants, Immunologic↗
Novel chemokine functions in lymphocyte migration through vascular endothelium under shear flow.
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.
Animals↗
The Src kinase p56(lck) up-regulates VLA-4 integrin affinity. Implications for rapid spontaneous and chemokine-triggered T cell adhesion to VCAM-1 and fibronectin.
In circulating lymphocytes, the VLA-4 integrin preexists in multiple affinity states that mediate spontaneous tethering, rolling, and arrest on its endothelial ligand, vascular cell adhesion molecule-1 (VCAM-1). The regulation and function of VLA-4 affinity in lymphocytes has never been elucidated. We show here that p56(lck), the major Src kinase in T cells, is a key regulator of high affinity VLA-4. This high affinity is essential for the rapid development of firm adhesion of resting T cells to VCAM-1 and to their extracellular matrix ligand, fibronectin. Lck-regulated VLA-4 function does not require intact TCR nor several key components of the TCR signaling pathway, including ZAP-70 and SLP-76. Furthermore, stimulation of p56(lck) by the phosphatase inhibitor, pervanadate, triggers firm VLA-4-dependent adhesion to VCAM-1. Although Lck is not required for chemokine receptor signaling to mitogen-activated protein kinase, the presence of Lck-regulated high affinity VLA-4 also facilitates firm adhesion triggered by the chemokine, SDF-1, at short-lived contacts. Surprisingly, bond formation rates, ability to tether cells to VLA-4 ligand, and VLA-4 tether bond stability under shear flow are not affected by VLA-4 affinity or Lck activity. Thus, the ability of high affinity VLA-4 to arrest cells on VCAM-1 under flow arises from instantaneous post-ligand strengthening rather than from increased kinetic stability of individual VLA-4 bonds. These results suggest that p56(lck) maintains high affinity VLA-4 on circulating lymphocytes, which determines their ability to strengthen VLA-4 adhesion and rapidly respond to proadhesive chemokine signals at endothelial sites.
Blotting, Western↗
Autocrine secretion of interferon gamma negatively regulates homing of immature B cells.
The mechanism by which immature B cells are sequestered from encountering foreign antigens present in lymph nodes or sites of inflammation, before their final maturation in the spleen, has not been elucidated. We show here that immature B cells fail to home to the lymph nodes. These cells can actively exclude themselves from antigen-enriched sites by downregulating their integrin-mediated adhesion to the extracellular matrix protein, fibronectin. This inhibition is mediated by interferon gamma secretion. Perturbation of interferon gamma activity in vivo leads to the homing of immature B cells to the lymph nodes. This is the first example of autocrine regulation of immune cell migration to sites of foreign antigen presentation.
Animals↗
TNF-alpha associated with extracellular matrix fibronectin provides a stop signal for chemotactically migrating T cells.
The migration of T cells into extravascular sites of inflammation is regulated by information derived from the molecular structure of the invaded tissue and from chemokine and cytokine gradients in the context of the extracellular matrix (ECM). Although recent studies have highlighted the role of particular chemoattractants in leukocyte migration, to date little is known about how specific combinations of contextual signals control the migration of leukocytes and their localization at sites of inflammation. Here we studied the interplay between a pleiotropic cytokine, TNF-alpha, and two prototypic chemoattractants, RANTES and stromal cell-derived factor-1alpha (SDF-1alpha), on human CD45RO+ T cells migrating within an ECM-like context. For this purpose, we used a newly constructed three-dimensional gel system designed to follow, in real time, the migration of individual leukocytes along chemotactic gradients in vitro. We found that TNF-alpha, which binds the ECM protein fibronectin and lacks adhesion- and migration-promoting effects of its own, can act as a proadhesive cytokine on T cells exposed to RANTES and SDF-1alpha. Furthermore, fibronectin-complexed TNF-alpha provided anchorage signals to the T cells as they moved directionally along chemoattractive gradients. This effect of TNF-alpha required an intact TNF-alpha receptor II subtype on the migrating T cells. The anchoring effect of TNF-alpha appears to be specific; IL-2, an integrin-activating proadhesive cytokine, does not transmit stoppage signals to T cell migration induced by RANTES. Thus, TNF-alpha present in the ECM at sites of inflammation may function to anchor T cells recruited to these sites by chemotactic signals.
Adjuvants, Immunologic↗
Subsecond induction of alpha4 integrin clustering by immobilized chemokines stimulates leukocyte tethering and rolling on endothelial vascular cell adhesion molecule 1 under flow conditions.
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, alpha4beta1), chemokines rapidly augment reversible lymphocyte tethering and rolling adhesions on VCAM-1. Chemokines potentiate VLA-4 tethering within <0.1 s of contact through Gi protein signaling, the fastest inside-out integrin signaling events reported to date. Although VLA-4 affinity is not altered upon chemokine signaling, subsecond VLA-4 clustering at the leukocyte-substrate contact zone results in enhanced leukocyte avidity to VCAM-1. Endothelial chemokines thus regulate all steps in adhesive cascades that control leukocyte recruitment at specific vascular beds.
Antibodies, Monoclonal↗
The LFA-1 integrin supports rolling adhesions on ICAM-1 under physiological shear flow in a permissive cellular environment.
The LFA-1 integrin is crucial for the firm adhesion of circulating leukocytes to ICAM-1-expressing endothelial cells. In the present study, we demonstrate that LFA-1 can arrest unstimulated PBL subsets and lymphoblastoid Jurkat cells on immobilized ICAM-1 under subphysiological shear flow and mediate firm adhesion to ICAM-1 after short static contact. However, LFA-1 expressed in K562 cells failed to support firm adhesion to ICAM-1 but instead mediated K562 cell rolling on the endothelial ligand under physiological shear stress. LFA-1-mediated rolling required an intact LFA-1 I-domain, was enhanced by Mg2+, and was sharply dependent on ICAM-1 density. This is the first indication that LFA-1 can engage in rolling adhesions with ICAM-1 under physiological shear flow. The ability of LFA-1 to support rolling correlates with decreased avidity and impaired time-dependent adhesion strengthening. A beta2 cytoplasmic domain-deletion mutant of LFA-1, with high avidity to immobilized ICAM-1, mediated firm arrests of K562 cells interacting with ICAM-1 under shear flow. Our results suggest that restrictions in LFA-1 clustering mediated by cytoskeletal attachments may lock the integrin into low-avidity states in particular cellular environments. Although low-avidity LFA-1 states fail to undergo adhesion strengthening upon contact with ICAM-1 at stasis, these states are permissive for leukocyte rolling on ICAM-1 under physiological shear flow. Rolling mediated by low-avidity LFA-1 interactions with ICAM-1 may stabilize rolling initiated by specialized vascular rolling receptors and allow the leukocyte to arrest on vascular endothelium upon exposure to stimulatory endothelial signals.
Cations, Divalent↗
An activated L-selectin mutant with conserved equilibrium binding properties but enhanced ligand recognition under shear flow.
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.
Animals↗
The chemokine SDF-1 activates the integrins LFA-1, VLA-4, and VLA-5 on immature human CD34(+) cells: role in transendothelial/stromal migration and engraftment of NOD/SCID mice.
Hematopoietic stem cell homing and engraftment require several adhesion interactions, which are not fully understood. Engraftment of nonobese/severe combined immunodeficiency (NOD/SCID) mice by human stem cells is dependent on the major integrins very late activation antigen-4 (VLA-4); VLA-5; and to a lesser degree, lymphocyte function associated antigen-1 (LFA-1). Treatment of human CD34(+) cells with antibodies to either VLA-4 or VLA-5 prevented engraftment, and treatment with anti-LFA-1 antibodies significantly reduced the levels of engraftment. Activation of CD34(+) cells, which bear the chemokine receptor CXCR4, with stromal derived factor 1 (SDF-1) led to firm adhesion and transendothelial migration, which was dependent on LFA-1/ICAM-1 (intracellular adhesion molecule-1) and VLA-4/VCAM-1 (vascular adhesion molecule-1). Furthermore, SDF-1-induced polarization and extravasation of CD34(+)/CXCR4(+) cells through the extracellular matrix underlining the endothelium was dependent on both VLA-4 and VLA-5. Our results demonstrate that repopulating human stem cells functionally express LFA-1, VLA-4, and VLA-5. Furthermore, this study implies a novel approach to further advance clinical transplantation.
Animals↗
The GPI-linked Ly-6 antigen E48 regulates expression levels of the FX enzyme and of E-selectin ligands on head and neck squamous carcinoma cells.
By differential display we demonstrated that antibody-mediated ligation of the GPI-linked protein product of E48, a newly discovered human Ly-6 gene, up-regulates 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 alpha-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.
Antigens, Ly↗
DC-SIGN-ICAM-2 interaction mediates dendritic cell trafficking.
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 ICAM-2 under shear flow, a prerequisite for emigration from blood. The DC-SIGN-ICAM-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.
Antigens, CD↗
Cytokine profile and T cell adhesiveness to endothelial selectins: in vivo induction by a myasthenogenic T cell epitope and immunomodulation by a dual altered peptide ligand.
Myasthenia gravis (MG) is a T cell-regulated antibody-mediated autoimmune disease. Immunization with two myasthenogenic peptides, p195-212 and p259-271, that are sequences of the human acetylcholine receptor alpha subunit was shown to induce experimental autoimmune MG (EAMG)-associated immune responses. A peptide composed of the two altered peptide ligands (APL) of the myasthenogenic peptides (designated as dual APL) inhibited, in vitro and in vivo, those responses. The objectives of this study were to examine (i) whether in vivo T cell activation by p259-271 affects the cytokine profile and the T cell migration ability, and (ii) whether the latter are immunomodulated by in vivo administration of the dual APL. Our results showed that immunization of mice with p259-271 enriched the population of lymph node and spleen cells with subsets of T cells with strong adhesiveness towards E- and P-selectins. This enrichment was associated with an acquisition of a T(h)1-type cytokine profile. Treatment of the immunized mice with the dual APL interfered with both the migratory potential of the autoreactive T cells, and the production of the T(h)1-type cytokines IL-2 and IFN-gamma (known to play a pathogenic role in MG and EAMG). T cells derived from APL-treated mice acquired a T(h)3-type cytokine profile, characterized by the secretion of the immunosuppresive cytokine transforming growth factor-ss. Thus, our results suggest that T cell selectin ligands and T cell-derived cytokines are involved in the induction and immunomodulation of EAMG- and MG-associated T cell responses.
Animals↗