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Y van Kooyk

Publications and source records attributed to Y van Kooyk.

At least 55 records · Page 3Linked to original sources
PubMed1997

Dual role of the actin cytoskeleton in regulating cell adhesion mediated by the integrin lymphocyte function-associated molecule-1.

Intracellular signals are required to activate the leukocyte-specific adhesion receptor lymphocyte function-associated molecule-1 (LFA-1; CD11a/CD18) to bind its ligand, intracellular adhesion molecule-1 (ICAM-1). In this study, we investigated the role of the cytoskeleton in LFA-1 activation and demonstrate that filamentous actin (F-actin) can both enhance and inhibit LFA-1-mediated adhesion, depending on the distribution of LFA-1 on the cell surface. We observed that LFA-1 is already clustered on the cell surface of interleukin-2/phytohemagglutinin-activated lymphocytes. These cells bind strongly ICAM-1 and disruption of the actin cytoskeleton inhibits adhesion. In contrast to interleukin-2/phytohemagglutinin-activated peripheral blood lymphocytes, resting lymphocytes, which display a homogenous cell surface distribution of LFA-1, respond poorly to intracellular signals to bind ICAM-1, unless the actin cytoskeleton is disrupted. On resting peripheral blood lymphocytes, uncoupling of LFA-1 from the actin cytoskeleton induces clustering of LFA-1 and this, along with induction of a high-affinity form of LFA-1, via "inside-out" signaling, results in enhanced binding to ICAM-1, which is dependent on intact intermediate filaments, microtubules, and metabolic energy. We hypothesize that linkage of LFA-1 to cytoskeletal elements prevents movement of LFA-1 over the cell surface, thus inhibiting clustering and strong ligand binding. Release from these cytoskeletal elements allows lateral movement and activation of LFA-1, resulting in ligand binding and "outside-in" signaling, that subsequently stimulates actin polymerization and stabilizes cell adhesion.

Actin Cytoskeleton↗
PubMed1997

Cytoplasmic tails of beta 1, beta 2, and beta 7 integrins differentially regulate LFA-1 function in K562 cells.

The beta 2 integrin lymphocyte function-associated antigen 1 (LFA-1) mediates activation-dependent adhesion of lymphocytes. To investigate whether lymphocyte-specific elements are essential for LFA-1 function, we expressed LFA-1 in the erythroleukemic cell line K562, which expresses only the integrin very late antigen 5. We observed that LFA-1-expressing K562 cannot bind to intercellular adhesion molecule 1-coated surfaces when stimulated by phorbol 12-myristate 13-acetate (PMA), whereas the LFA-1-activating antibody KIM185 markedly enhanced adhesion. Because the endogenously expressed beta 1 integrin very late antigen 5 is readily activated by PMA, we investigated the role of the cytoplasmic domain of distinct beta subunits in regulating LFA-1 function. Transfection of chimeric LFA-1 receptors in K562 cells reveals that replacement of the beta 2 cytoplasmic tail with the beta 1 but not the beta 7 cytoplasmic tail completely restores PMA responsiveness of LFA-1, whereas a beta 2 cytoplasmic deletion mutant of LFA-1 is constitutively active. Both deletion of the beta 2 cytoplasmic tail or replacement by the beta 1 cytoplasmic tail alters the localization of LFA-1 into clusters, thereby regulating LFA-1 activation and LFA-1-mediated adhesion to intercellular adhesion molecule 1. These data demonstrate that distinct signaling routes activate beta 1 and beta 2 integrins through the beta-chain and hint at the involvement of lymphocyte-specific signal transduction elements in beta 2 and beta 7 integrin activation that are absent in the nonlymphocytic cell line K562.

Amino Acid Sequence↗
PubMed1996

Induction of LFA-1 on pluripotent CD34+ bone marrow cells does not affect lineage commitment.

Leukocyte function associated antigen 1 (LFA-1) is an adhesion molecule indispensable in immune and inflammatory reactions, but its role in hematopoiesis remains obscure. Since LFA-1 is predominantly expressed by leukocytes, it is considered as a marker of late stage stem cell maturation when expressed on CD34+ bone marrow cells, and represents more mature hematopoietic progenitor cells. We observed that freshly isolated CD34+ bone marrow cells express LFA-1, and that the level of expression is highly variable. Interestingly, the expression of LFA-1 specific activation epitope L16 on these cells is low, even after culture. This demonstrates the LFA-1 is not activated, as was confirmed by low adhesion to ICAM-1. Culturing sorted CD34+ LFA-1+ cells in single cell per well assays in medium supplemented with SCF, Epo, IL-3, Il-6, GM-CSF, and G-CSF revealed that they gave rise to dispersed macrophage-like colonies, supporting the notion that CD34+LFA-1+ cells indeed consist of a mature committed cell population. In contrast, sorted CD34+LFA-1- cells had high proliferative potential and developed into large multilineage colonies within 14 days of culture. Unanticipated, in time course experiments we observed that these CD34+LFA-1- cells expressed LFA-1 within 24 hours upon culture. This induction was neither caused by the monoclonal antibody used to tag CD34 cells, nor dependent on growth factors present in the medium. These findings demonstrate that two populations of CD34+LFA-1+ cells can be discriminated: leukocyte lineage committed CD34+ cells in freshly isolated bone marrow cells, and multipotent CD34+ cells that acquired LFA-1 upon in vitro culture. These in vitro findings support the hypothesis that once contacts with bone marrow stroma are lost, LFA-1 is upregulated by default, due to the lack of negative regulating signals from stromal cells. This might also explain the widely variable expression of LFA-1 as a result of crowding of cells in the bone marrow with subsequent loss of contact with stroma and upregulation of LFA-1, providing those cells with adhesion receptors enabling migration in the periphery.

Antigens, CD34↗
PubMed1996

Antibodies that selectively inhibit leukocyte function-associated antigen 1 binding to intercellular adhesion molecule-3 recognize a unique epitope within the CD11a I domain.

Several studies indicate that the I domain located in the alpha chain (CD11a) of leukocyte function-associated antigen-1 (LFA-1; CD11a/CD18) plays an essential role in ligand recognition. We recently identified three distinct epitopes (IdeA, IdeB, and IdeC) within the CD11a I domain, recognized by antibodies that block binding of LFA-1 to intercellular adhesion molecules (ICAM) 1, 2, and 3. In the present study, we used a series of human/murine CD11a I domain chimeras, to localize a fourth I domain epitope (IdeD), recognized by three independently derived anti-CD11a antibodies that selectively block the binding of LFA-1 to ICAM-3, but not to ICAM-1. The IdeD epitope depended on human CD11a residues Asp182 and Ser184 and was not present in CD11b or CD11c. Although mutation of Asp182 and Ser184 failed to abolish ICAM-3 adhesion of LFA-1 transfectants, alignment of these residues with the crystal structure of the CD11a I domain suggested that the IdeD epitope is located in close proximity to residues (Ile126 and Asn129) recently implicated in the ICAM-3 binding site. Interestingly, the IdeB and IdeC epitopes appeared to be in close proximity of a divalent cation binding pocket within the CD11a I domain that regulates both ICAM-1 and ICAM-3 adhesion. Taken together, these data indicate that distinct regions of the CD11a I domain contain epitopes for antibodies that either selectively inhibit binding of LFA-1 to ICAM-3, or interfere with both ICAM-1 and ICAM-3 binding of LFA-1.

Amino Acid Sequence↗
PubMed1996

Critical amino acids in the lymphocyte function-associated antigen-1 I domain mediate intercellular adhesion molecule 3 binding and immune function.

We have identified amino acid residues within the evolutionarily conserved I domain of the alpha-chain (CD11a) of the leukocyte integrin leukocyte function-associated antigen (LFA) 1 that are critical for intercellular adhesion molecule (ICAM) 3 (CD50) binding. ICAM-3, a ligand of LFA-1, is thought to mediate intercellular adhesion essential for the initiation of immune responses. Using a panel of human/murine I domain chimeras and point mutants, we observed that the Ile-Lys-Gly-Asn motif, located in the NH2-terminal part of the CD11a I domain, is required for ICAM-3 but not ICAM-1 binding. These findings demonstrate that the I domain of CD11a contains distinct functional subdomains for ligand specific binding. An aspartic acid located at position 137, which is essential to ICAM-1/LFA-1 interactions (Edwards, C.P., M. Champe, T. Gonzalez, M.E. Wessinger, S.A. Spencer, L.G. Presta, P.W. Berman, and S.C. Bodary. 1995. J. Biol. Chem. 270:12635-12640), was also critical for ICAM-3 binding, whereas Ser at position 139 did not effect ICAM-1 or ICAM-3 binding. A synthetic peptide containing the Ile-Lys-Gly-Asn motif inhibited ICAM-3-dependent adhesion and proliferation of T cells at micromolar concentrations, suggesting that this peptide interferes with immune recognition. These observations underscore the importance of ICAM-3 in leukocyte function, and may lead to development of a new category of immunosuppressive agents.

Amino Acid Sequence↗
PubMed1996

Activation and increased expression of adhesion molecules on peripheral blood lymphocytes is a mechanism for the immediate lymphocytopenia after administration of OKT3.

We investigated the mechanism by which antihuman CD3 monoclonal antibodies of the isotypes IgG2a (eg, OKT3) and IgA (eg, IXA) can induce the rapid disappearance of virtually all circulating T lymphocytes. We hypothesize that upregulation of adhesion molecules on the lymphocyte membrane contributes to this effect. However, this hypothesis is difficult to test, because of the inherent lymphocytopenia and/or shifts in lymphocyte populations between intra and extra-vascular compartments. Therefore, studies in vitro were performed, as well. Analysis of peripheral blood lymphocytes isolated at several times after addition of OKT3 or IXA to whole blood of healthy individuals showed an immediate increase in the proportion of T cells expressing NKI-L16, an activation epitope on CD11a/CD18. Likewise, an increase in CD11b/CD18 expression occurred. In parallel experiments, a transiently increased adhesion of T cells to endothelial cell monolayers was observed. This adhesion could be completely blocked by anti-CD18 or anti-CD11a monoclonal antibodies and only partly by an anti-CD11b antibody. Our data indicate that upregulation of activation epitopes of CD11a/CD18, as well as increased expression of CD11b/CD18 on T lymphocytes, may result in increased adhesion of these cells to intercellular adhesion molecule-1 (ICAM-1) and ICAM-2 on vascular endothelium. This phenomenon may, at least, partly explain the rapidly occurring peripheral lymphocytopenia observed in vivo.

Adult↗
PubMed1996

Competition between lymphocyte function-associated antigen 1 (CD11a/CD18) and Mac-1 (CD11b/CD18) for binding to intercellular adhesion molecule-1 (CD54).

Both human integrin receptors Mac-1 (CD11b/CD18,CR3)and lymphocyte function-associated antigen (LFA)-1 (CD11a/CD18) have been demonstrated to bind human intercellular adhesion molecule-1 (ICAM-1) (CD54). Here we show that LFA-1 and Mac-1 can bind to ICAM-1 in the mouse as well. Interestingly, we observed that binding of murine LFA-1 dominates over Mac-1 for binding to ICAM-1. Using three different murine macrophage cell lines that express distinct levels of LFA-1 and Mac-1 on their cell surface, we could only detect Mac-1-dependent adhesion to ICAM-1 when little or no LFA-1 is expressed on the cell surface. When LFA-1 and Mac-1 are expressed at similar levels, the LFA-1/ICAM-1 interaction dominates over Mac-1/ICAM-1 interaction, indicating that there is a competition of LFA-1 and Mac-1 for ICAM-1 binding.

Animals↗
PubMed1996

Fibrinogen binding to ICAM-1 on EA.hy 926 endothelial cells is dependent on an intact cytoskeleton.

Fibrinogen is a ligand for Intercellular Adhesion Molecule-1 (ICAM-1), and enhances monocyte-endothelial cell interaction by coupling Mac-1 on monocytes to ICAM-1 on endothelial cells. We investigated the role of the cytoskeleton in fibrinogen binding to the human endothelial cell line EA.hy 926 using immunofluorescence techniques. In this cell line TNF alpha induced the simultaneous appearance of stress fibers and of ICAM-1, which was clustered predominantly on endothelial cell projections. Incubation of TNF alpha-stimulated endothelial cells with fibrinogen resulted in binding of fibrinogen to ICAM-1 on these cell projections. Disruption of the cytoskeleton by cytocholasin B abolished fibrinogen binding. Activation of protein kinase C with 12-O-tetradecanoyl phorbol-13-acetate resulted in simultaneous loss of both stress fibers and fibrinogen binding. These results suggest that a connection between ICAM-1 and the cytoskeleton results in clustering of ICAM-1 on cell projections, which is required for fibrinogen binding.

Cell Line↗
PubMed1995

Ins and outs of LFA-1.

Leukocyte function-associated molecule 1 (LFA-1) is an integrin that plays a major role in the immune system. Recent findings demonstrate that LFA-1 has a two-way signaling function, mediating cell adhesion and stimulating intracellular processes at the same time. Here, Marijke Lub, Yvette van Kooyk and Carl Figdor discuss the 'inside-out' and 'outside-in' signaling properties of LFA-1, as a prototype leukocyte integrin, in normal and malignant T cells. They integrate data into a model that highlights the role of the cytoskeleton in the regulation of LFA-1.

Animals↗
PubMed1994

Distinct binding of T lymphocytes to ICAM-1, -2 or -3 upon activation of LFA-1.

LFA-1 (CD11a/CD18) mediates leukocyte adhesion by binding to one of its ligands: ICAM-1, ICAM-2 or ICAM-3. Here, we investigated whether stimuli known to induce adhesion to ICAM-1 were also capable of inducing LFA-1-mediated adhesion of T lymphocytes to ICAM-2 and -3 transfectants. We observed that phorbol 12-myristate 13-acetate, Mn2+, cross-linking of CD3 or activating antibodies against LFA-1 enhanced LFA-1-mediated T cell adhesion to ICAM-2 and -3, although to a lesser extent than to ICAM-1. These results indicate that, similar to what has been reported for adhesion to ICAM-1, activation of LFA-1 is also required for adhesion to ICAM-2 and -3. Furthermore, the results suggest that ICAM-1 is the major ligand for LFA-1 on activated T lymphocytes. Interestingly, we observed that in contrast to activating antibodies against CD18, activating antibodies against CD11a were incapable of inducing adhesion of LFA-1 to all three ligands. The antibody MEM-83 stimulated binding to ICAM-1, while at the same time inhibiting the interaction of LFA-1 with ICA8M-2 and -3. The antibody NKI-L16 selectively induced adhesion to ICAM-1 and -2, but not to ICAM-3. Our results suggest that different conformations of LFA-1 are required to support adhesion to ICAM-1, -2 or -3, and that ligands may bind on different sites of the LFA-1 molecule.

Animals↗
PubMed1994

Extracellular Ca2+ modulates leukocyte function-associated antigen-1 cell surface distribution on T lymphocytes and consequently affects cell adhesion.

Transition of leukocyte function-associated antigen-1 (LFA-1), from an inactive into an activate state depends on the presence of extracellular Mg2+ and/or Ca2+ ions. Although Mg2+ is directly involved in ligand binding, the role of Ca2+ in LFA-1 mediated adhesion remained obscure. We now demonstrate that binding of Ca2+, but not Mg2+, directly correlates with clustering of LFA-1 molecules at the cell surface of T cells, thereby facilitating LFA-1-ligand interaction. Using a reporter antibody (NKI-L16) that recognizes a Ca(2+)-dependent epitope on LFA-1, we found that Ca2+ can be bound by LFA-1 with different strength. We noticed that weak binding of Ca2+ is associated with a dispersed LFA-1 surface distribution on T cells and with non-responsiveness of these cells to stimuli known to activate LFA-1. In contrast, stable binding of Ca2+ by LFA-1 correlates with a patch-like surface distribution and vivid ligand binding after activation of LFA-1. Mg(2+)-dependent ligand binding does not affect binding of Ca2+ by LFA-1 as measured by NKI-L16 expression, suggesting that Mg2+ binds to a distinct site, and that both cations are important to mediate adhesion. Only Sr2+ ions can replace Ca2+ to express the L16 epitope, and to induce clustering of LFA-1 at the cell surface. We conclude that Ca2+ is involved in avidity regulation of LFA-1 by clustering of LFA-1 molecules at the cell surface, whereas Mg2+ is important in regulation of the affinity of LFA-1 for its ligands.

Calcium↗
PubMed1993

Lymphocyte function-associated antigen 1 dominates very late antigen 4 in binding of activated T cells to endothelium.

Lymphocyte function-associated antigen 1/intercellular adhesion molecule 1 (LFA-1/ICAM-1)-and very late antigen 4/vascular cell adhesion molecule 1 (VLA-4/VCAM-1)-mediated adhesion of T lymphocytes to endothelial cells (EC) can be regulated by increased expression of ICAM-1 and VCAM-1 upon cytokine treatment of EC, or by activation of the integrin molecules LFA-1 and VLA-4 on T cells. Here, we provide evidence that preferential usage of LFA-1 over VLA-4 is yet another mechanism to control T cell adhesion. We observed that binding of activated T lymphocytes, as opposed to resting T cells, to EC is essentially mediated through LFA-1 and not through VLA-4. VLA-4-mediated adhesion of T cells to EC is only found when LFA-1 is not expressed or not functional, as observed for several T cell leukemia cell lines. These results suggest that LFA-1-mediated adhesion dominates and may downregulate VLA-4-mediated adhesion through an unidentified mechanism.

Animals↗
PubMed1993

Role of LFA-1/ICAM-1 in interleukin-2-stimulated lymphocyte proliferation.

Major adhesion routes between lymphoid cells involve the receptor/ligand pairs LFA-1/ICAM-1 and CD2/LFA-3, in addition to VLA or CD44 molecules. In this study we evaluated the role of these adhesion receptors in the proliferative response of lymphoid cells to interleukin-2 (IL-2). Blocking studies were performed with a panel of monoclonal antibodies (mAb) directed against these adhesion molecules. Selective inhibition of recombinant (r)IL-2-induced cell proliferation was observed with mAb directed against the alpha or beta subunit of LFA-1 or to its ligand ICAM-1. Interestingly, rIL-2-induced proliferation was also inhibited by NKI-L16, and anti-1 alpha antibody known to enhance cell-cell interaction. Resting lymphocytes were preferentially susceptible to the inhibition, particularly in an early phase of culture and when stimulated with a relatively low dose of rIL-2. By using mAb that specifically could block distinct rIL-2 activation pathways, LFA-1/ICAM-1 interaction was found to be required for p55 IL-2 receptor (IL-2R)-mediated interaction of rIL-2 with its high-affinity receptor, but not for p75 IL-2R-mediated responses. Furthermore, it was shown that the rIL-2 response of T lymphocytes, but not of natural killer cells, was dependent on LFA-1/ICAM-1 interaction. This suggests that LFA-1/ICAM-1 interaction is required for an optimal rIL-2 response of cells capable of IL-2 secretion. Our data provide evidence for the hypothesis that adhesion receptor-directed release of IL-2 may result in a locally high concentration of IL-2 that triggers high-affinity IL-2R signaling and up-regulates p55 IL-2R to enhance cytokine responsiveness.

Antibodies, Monoclonal↗
PubMed1993

Role of intracellular Ca2+ levels in the regulation of CD11a/CD18 mediated cell adhesion.

CD2, CD3, and MHC class II have been demonstrated to stimulate lymphocyte function-associated antigen (LFA)-1 (CD11a/CD18) mediated adhesion (Van Kooyk et al., 1989, Dustin and Springer, 1989; Mourad et al., 1990). Activation of LFA-1 may be mediated by different intracellular signals generated from these stimuli, since previous findings suggest that triggering of LFA-1 through CD2 or CD3 leads to sustained and transient cell adhesion respectively (Van Kooyk et al., 1989). We investigated the role of intracellular signalling pathways in more detail. The results demonstrate that, in addition to protein tyrosine kinase (PTK) and protein kinase C (PKC) mediated signalling, increase in cytosolic-free calcium ([Ca2+]i) levels play a major role in the activation of LFA-1. The calcium ionophore Ionomycin, which increases [Ca2+]i is capable of directly activating LFA-1. Furthermore, activation of LFA-1 by triggering through CD2, CD3 or MHC class II is associated with an increase in [Ca2+]i levels, with kinetics that directly correlate with cell adhesiveness. Moreover, entry of extracellular Ca2+ via Ca-channels is involved in both the CD3- and MHC class II, as well as part of the CD2 induced LFA-1 activation. Depletion of intracellular calcium results in unresponsiveness of LFA-1 to these stimuli, further demonstrating a regulatory role for [Ca2+]i in LFA-1 mediated adhesion.

Animals↗
PubMed1992

Adhesion of T and B lymphocytes to extracellular matrix and endothelial cells can be regulated through the beta subunit of VLA.

Investigating the regulation of very late antigen (VLA)-mediated functions, we found that TS2/16, a mAb directed against the beta chain of the VLA group of integrins, can induce binding of resting peripheral blood lymphocytes, cloned T lymphocytes, and Epstein Barr virus-transformed B cells to extracellular matrix components, fibronectin, laminin, and collagen, but not to fibrinogen. The antibody stimulates VLA-4-, VLA-5-, and VLA-6-mediated binding. Furthermore, it induces VLA-4-mediated binding to vascular cell adhesion molecule-1 expressed by rTNF-alpha-stimulated endothelial cells, but it does not stimulate homotypic aggregation of cells as described for a number of anti-VLA-4 alpha antibodies (Bednarczyk, J.L., and B. W. McIntyre. 1990. J. Immunol. 144: 777-784; Campanero, M. R., R. Pulido, M. A. Ursa, M. Rodríguez-Moya, M. O. de Landázuri, and F. Sánchez-Madrid. 1990. J. Cell Biol. 110:2157-2165). Therefore, the stimulating activity of this anti-beta 1 antibody clearly contrasts with that of the anti-VLA-4 alpha antibodies, which induce homotypic cell aggregation, but not binding of cells to extracellular matrix components or endothelial cells, indicating that TS2/16 may generate different signals. The observation that also F(ab')2 or Fab fragments of this anti-beta 1 antibody stimulate binding to extracellular matrix components and endothelial cells excludes the possibility that binding requires receptor crosslinking, or is Fc receptor mediated. Induction of this adhesion is cation and energy dependent and requires an intact cytoskeleton. Although changes in the conformation of VLA integrins induced by this antibody may regulate their functional activity, the dependence on metabolic energy indicates that intracellular processes may also play a role.

Antibodies, Monoclonal↗