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1.
Abstract

Leukocyte function associated antigen 1 (LFA-1) and intercellular adhesion molecule 1 (ICAM-1) have been shown to be critical for adhesion process and immune response. Modulation or inhibition of the interaction between LFA-1/ICAM-1 interactions can result in therapeutic effects. Our group and others have shown that peptides derived from ICAM- 1 or LFA-1 inhibit adhesion in a homotypic T-cell adhesion assay. It is likely that the peptides derived from ICAM-1 bind to LFA-1 and peptides derived from LFA-1 bind to ICAM- 1 and inhibit the adhesion interaction. However, there are no concrete experimental evidence to show that peptides bind to either LFA-1 or ICAM-1 and inhibit the adhesion. Using NMR, CD and docking studies we have shown that an LFA-1 derived peptide binds to soluble ICAM-1. Docking studies using “autodock” resulted in LFA-1 peptide interacting with the ICAM-1 protein near Glu34. The proposed model based on our experimental data indicated that the LFA-1 peptide interacts with the protein via three intermolecular hydrogen bonds. Hydrophobic interactions also play a role in stabilizing the complex.  相似文献   

2.
ICAM-1 (CD54): a counter-receptor for Mac-1 (CD11b/CD18)   总被引:13,自引:0,他引:13       下载免费PDF全文
《The Journal of cell biology》1990,111(6):3129-3139
While the leukocyte integrin lymphocyte function-associated antigen (LFA)-1 has been demonstrated to bind intercellular adhesion molecule (ICAM)-1, results with the related Mac-1 molecule have been controversial. We have used multiple cell binding assays, purified Mac- 1 and ICAM-1, and cell lines transfected with Mac-1 and ICAM-1 cDNAs to examine the interaction of ICAM-1 with Mac-1. Stimulated human umbilical vein endothelial cells (HUVECs), which express a high surface density of ICAM-1, bind to immunoaffinity-purified Mac-1 adsorbed to artificial substrates in a manner that is inhibited by mAbs to Mac-1 and ICAM-1. Transfected murine L cells or monkey COS cells expressing human ICAM-1 bind to purified Mac-1 in a specific and dose-dependent manner; the attachment to Mac-1 is more temperature sensitive, lower in avidity, and blocked by a different series of ICAM-1 mAbs when compared to LFA-1. In a reciprocal assay, COS cells cotransfected with the alpha and beta chain cDNAs of Mac-1 or LFA-1 attach to immunoaffinity- purified ICAM-1 substrates; this adhesion is blocked by mAbs to ICAM-1 and Mac-1 or LFA-1. Two color fluorescence cell conjugate experiments show that neutrophils stimulated with fMLP bind to HUVEC stimulated with lipopolysaccharide for 24 h in an ICAM-1-, Mac-1-, and LFA-1- dependent fashion. Because cellular and purified Mac-1 interact with cellular and purified ICAM-1, we conclude that ICAM-1 is a counter receptor for Mac-1 and that this receptor pair is responsible, in part, for the adhesion between stimulated neutrophils and stimulated endothelial cells.  相似文献   

3.
Leukocyte function associated antigen 1 (LFA-1) and intercellular adhesion molecule 1 (ICAM-1) have been shown to be critical for adhesion process and immune response. Modulation or inhibition of the interaction between LFA-1/ICAM-1 interactions can result in therapeutic effects. Our group and others have shown that peptides derived from ICAM-1 or LFA-1 inhibit adhesion in a homotypic T-cell adhesion assay. It is likely that the peptides derived from ICAM-1 bind to LFA-1 and peptides derived from LFA-1 bind to ICAM-1 and inhibit the adhesion interaction. However, there are no concrete experimental evidence to show that peptides bind to either LFA-1 or ICAM-1 and inhibit the adhesion. Using NMR, CD and docking studies we have shown that an LFA-1 derived peptide binds to soluble ICAM-1. Docking studies using "autodock" resulted in LFA-1 peptide interacting with the ICAM-1 protein near Glu34. The proposed model based on our experimental data indicated that the LFA-1 peptide interacts with the protein via three intermolecular hydrogen bonds. Hydrophobic interactions also play a role in stabilizing the complex.  相似文献   

4.
Intercellular adhesion molecule 1 (ICAM-1, CD54) is a member of the Ig superfamily and is a counterreceptor for the beta 2 integrins: lymphocyte function-associated antigen 1 (LFA-1, CD11a/CD18), complement receptor 1 (MAC-1, CD11b/CD18), and p150,95 (CD11c/CD18). Binding of ICAM-1 to these receptors mediates leukocyte-adhesive functions in immune and inflammatory responses. In this report, we describe a cell-free assay using purified recombinant extracellular domains of LFA-1 and a dimeric immunoadhesin of ICAM-1. The binding of recombinant secreted LFA-1 to ICAM-1 is divalent cation dependent (Mg2+ and Mn2+ promote binding) and sensitive to inhibition by antibodies that block LFA-1-mediated cell adhesion, indicating that its conformation mimics that of LFA-1 on activated lymphocytes. We describe six novel anti-ICAM-1 monoclonal antibodies, two of which are function blocking. Thirty-five point mutants of the ICAM-1 immunoadhesin were generated and residues important for binding of monoclonal antibodies and purified LFA-1 were identified. Nineteen of these mutants bind recombinant LFA-1 equivalently to wild type. Sixteen mutants show a 66-2500-fold decrease in LFA-1 binding yet, with few exceptions, retain binding to the monoclonal antibodies. These mutants, along with modeling studies, define the LFA-1 binding site on ICAM-1 as residues E34, K39, M64, Y66, N68, and Q73, that are predicted to lie on the CDFG beta-sheet of the Ig fold. The mutant G32A also abrogates binding to LFA-1 while retaining binding to all of the antibodies, possibly indicating a direct interaction of this residue with LFA-1. These data have allowed the generation of a highly refined model of the LFA-1 binding site of ICAM-1.  相似文献   

5.
The red cell ICAM-4/LW blood group glycoprotein, which belongs to the family of intercellular adhesion molecules (ICAMs), has been reported to interact with CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1) beta(2) integrins. To better define the basis of the ICAM-4/beta(2) integrin interaction, we have generated wild-type, domain-deleted and mutated recombinant chimeric ICAM-4-Fc proteins and analyzed their interaction in a cellular adhesion assay with LFA-1 and Mac-1 L-cell stable transfectants. We found that monoclonal antibodies against CD11a, CD11b, CD18, or LW(ab) block adhesion of transfectant L-cells to immobilized ICAM-4-Fc protein and that the ICAM-4/beta(2) integrin interaction was highly sensitive to the presence of the divalent cations Ca(2+) and Mg(2+). Deletion of individual Ig-domains D1 or D2 of the extracellular part of ICAM-4 showed that LFA-1 binds to the first Ig-like domain, whereas the Mac-1 binding site encompassed both the first and the second Ig-like domains. Based on the crystal structure of ICAM-2, we propose a model for the Ig-like domains D1 and D2 of ICAM-4. Accordingly, by site-directed mutagenesis of 22 amino acid positions spread out on all faces of the ICAM-4 molecule, we identified four exposed residues, Leu(80), Trp(93), and Arg(97) on the CFG face and Trp(77) on the E-F loop of domain D1 that may contact LFA-1 as part of the binding site. However, the single and double mutants R52E and T91Q on the CFG face of domain D1, which correspond to the key residues Glu(34) and Gln(73) for ICAM-1 binding to LFA-1, had no effect on LFA-1 binding. In contrast, all mutants on the CFG face of domain D1 and residues Glu(151) and Thr(154) in the C'-E loop of the domain D2 seem to play a dominant role in Mac-1 binding. These data suggest that the binding site for LFA-1 on ICAM-4 overlaps but is distinct from the Mac-1 binding site.  相似文献   

6.
The interaction between leukocyte function-associated antigen-1(LFA-1) and intercellular adhesion molecule-1 (ICAM-1) plays a pivotal role in cellular adhesion including the extravasation and inflammatory response of leukocytes, and also in the formation of immunological synapse. However, irregular expressions of LFA-1 or ICAM-1 or both may lead to autoimmune diseases, metastasis cancer, etc. Thus, the LFA-1/ICAM-1 interaction may serve as a potential therapeutic target for the treatment of these diseases. Here, we developed one simple ‘in solution’ steady state fluorescence resonance energy transfer (FRET) technique to obtain the dissociation constant (Kd) of the interaction between LFA-1 and ICAM-1. Moreover, we developed the assay into a screening platform to identify peptides and small molecules that inhibit the LFA-1/ICAM-1 interaction. For the FRET pair, we used Alexa Fluor 488-LFA-1 conjugate as donor and Alexa Fluor 555-human recombinant ICAM-1 (D1-D2-Fc) as acceptor. From our quantitative FRET analysis, the Kd between LFA-1 and D1-D2-Fc was determined to be 17.93±1.34 nM. Both the Kd determination and screening assay were performed in a 96-well plate platform, providing the opportunity to develop it into a high-throughput assay. This is the first reported work which applies FRET based technique to determine Kd as well as classifying inhibitors of the LFA-1/ICAM-1 interaction.  相似文献   

7.
In their active state, beta(2)-integrins, such as LFA-1, mediate the firm arrest of leukocytes by binding intercellular adhesion molecules (ICAMs) expressed on endothelium. Although the primary function of LFA-1 is assumed to be the ability to mediate firm adhesion, recent work has shown that LFA-1 can contribute to cell tethering and rolling under hydrodynamic flow, a role previously largely attributed to the selectins. The inserted (I) domain of LFA-1 has recently been crystallized in the wild-type (wt) and locked-open conformations and has been shown to, respectively, support rolling and firm adhesion under flow when expressed in alpha(L)beta(2) heterodimers or as isolated domains on cells. Here, we report results from cell-free adhesion assays where wt I-domain-coated polystyrene particles were allowed to interact with ICAM-1-coated surfaces in shear flow. We show that wt I-domain can independently mediate the capture of particles from flow and support their rolling on ICAM-1 surfaces in a manner similar to how carbohydrate-selectin interactions mediate rolling. Adhesion is specific and blocked by appropriate antibodies. We also show that the rolling velocity of I-domain-coated particles depends on the wall shear stress in flow chamber, I-domain site density on microsphere surfaces, and ICAM-1 site density on substrate surfaces. Furthermore, we show that rolling is less sensitive to wall shear stress and ICAM-1 substrate density at high density of I-domain on the microsphere surface. Computer simulations using adhesive dynamics can recreate bead rolling dynamics and show that the mechanochemical properties of ICAM-1-I-domain interactions are similar to those of carbohydrate-selectin interactions. Understanding the biophysics of adhesion mediated by the I-domain of LFA-1 can elucidate the complex roles this integrin plays in leukocyte adhesion in inflammation.  相似文献   

8.
To analyze the binding requirements of LFA-1 for its two most homologous ligands, ICAM-1 and ICAM-3, we compared the effects of various LFA-1 activation regimes and a panel of anti-LFA-1 mAbs in T cell binding assays to ICAM-1 or ICAM-3 coated on plastic. These studies demonstrated that T cell binding to ICAM-3 was inducible both from the exterior of the cell by Mn2+ and from the interior by an agonist of the "inside-out" signaling pathway. T cells bound both ICAM ligands with comparable avidity. A screen of 29 anti-LFA-1 mAbs led to the identification of two mAbs specific for the alpha subunit of LFA-1 which selectively blocked adhesion of T cells to ICAM-3 but not ICAM-1. These two mAbs, YTH81.5 and 122.2A5, exhibited identical blocking properties in a more defined adhesion assay using LFA-1 transfected COS cells binding to immobilized ligand. Blocking was not due to a steric interference between anti-LFA-1 mAbs and N-linked carbohydrate residues present on ICAM-3 but not ICAM-1. The epitopes of mAbs YTH81.5 and 122.2A5 were shown to map to the I domain of the LFA-1 alpha subunit. A third I domain mAb, MEM-83, has been previously reported to uniquely activate LFA-1 to bind ICAM-1 (Landis, R. C., R. I. Bennett, and N. Hogg. 1993. J. Cell Biol. 120:1519-1527). We now show that mAb MEM-83 is not able to stimulate binding of T cells to ICAM-3 over a wide concentration range. Failure to induce ICAM-3 binding by mAb MEM-83 was not due to a blockade of the ICAM-3 binding site on LFA-1. This study has demonstrated that two sets of functionally distinct mAbs recognizing epitopes in the I domain of LFA-1 are able to exert differential effects on the binding of LFA-1 to its ligands ICAM-1, and ICAM-3. These results suggest for the first time that LFA-1 is capable of binding these two highly homologous ligands in a selective manner and that the I domain plays a role in this process.  相似文献   

9.
Intercellular adhesion molecule-1 (ICAM-1) is found on the surface of many hemopoietic and non-hemopoietic cells and can function as an adhesive ligand for the integrin, leukocyte function associated molecule-1 (LFA-1, CD11a/CD18). ICAM-1/LFA-1 interaction is thought to be of importance in many immune mediated cell-cell adhesion reactions. Recently, the major human rhinovirus (HRV) receptor has been identified as ICAM-1. HRV has been shown to bind specifically to ICAM-1 on transfected COS cells and to purified ICAM-1, which has been adsorbed to plastic microtiter wells. We have compared the ability of ICAM-1 expressed on the surface of human fibroblasts (FB) to function as a receptor for HRV as well as a receptor for LFA-1-bearing human T lymphocytes. We show that FB stimulation by the cytokines IFN-gamma or IL-1, both known inducers of ICAM-1 synthesis and expression in FB, induced an increase in HRV binding to treated cells, which could be inhibited by antibody to ICAM-1. In contrast, only IFN-gamma and not IL-1 treatment of FB resulted in an increased adhesion of T lymphocytes. Binding of HRV to IFN-gamma-treated FB inhibited the subsequent adhesion of T cells. We also show that prior stimulation of FB with IL-1 enhanced the adhesion of HRV to IFN-gamma-stimulated cells, although IL-1 pretreatment was inhibitory for T cell adhesion. As these two cytokines both up-regulate ICAM-1 on the surface of human FB, the contrasting effects of IFN-gamma and IL-1 on human FB ICAM-1 adhesion to HRV and to LFA-1 suggest that qualitative as well as quantitative alterations of the ICAM-1 molecule may contribute to its specificity of ligand recognition.  相似文献   

10.
Lymphocyte adhesion to target cells is mediated, in part, by the interaction of lymphocyte function-associated Ag-1 (LFA-1) with intercellular adhesion molecule-1 (ICAM-1). Cells of the B cell line, JY, express both coreceptors and have been used as a model for intercellular adhesion mediated by these molecules. Elevation of the intracellular cAMP concentration ([cAMP]i), by any of several reagents, for periods as brief as 30 min, led to enhanced intercellular adhesion in a concentration dependent manner 5 to 8 h later. Two protein kinase A inhibitors, KT5720 and H-89, but not the protein kinase C inhibitor calphostin C, blocked the effects of elevated [cAMP]i. These data suggest a role for protein kinase A in this response. The adhesion augmented by increased [cAMP]i was due to LFA-1/ICAM-1 interactions between cells because it was blocked by either anti-LFA-1 or anti-ICAM-1 mAb. Elevated [cAMP]i induced cell surface patching of LFA-1, but not ICAM-1, and this redistribution preceded intercellular adhesion. Finally, redistribution of LFA-1 was not mediated by the cytoskeleton. These results suggest a model in which transient activation of protein kinase A results in increased local concentration of LFA-1 at the cell surface and in augmented long term adhesion mediated by this integrin.  相似文献   

11.
Intercellular adhesion molecule-1 (ICAM-1) on the surface of cultured umbilical vein and saphenous vein endothelial cells was upregulated between 2.5- and 40-fold by rIL-1, rTNF, LPS and rIFN gamma corresponding to up to 5 X 10(6) sites/cell. Endothelial cell ICAM-1 was a single band of 90 kD in SDS-PAGE. Purified endothelial cell ICAM-1 reconstituted into liposomes and bound to plastic was an excellent substrate for both JY B lymphoblastoid cell and T lymphoblast adhesion. Adhesion to endothelial cell ICAM-1 in planar membranes was blocked completely by monoclonal antibodies to lymphocyte function associated antigen-1 (LFA-1) or ICAM-1. Adhesion to artificial membranes was most sensitive to ICAM-1 density within the physiological range found on resting and stimulated endothelial cells. Adhesion of JY B lymphoblastoid cells, normal and genetically LFA-1 deficient T lymphoblasts and resting peripheral blood lymphocytes to endothelial cell monolayers was also assayed. In summary, LFA-1 dependent (60-90% of total adhesion) and LFA-1-independent basal adhesion was observed and the use of both adhesion pathways by different interacting cell pairs was increased by monokine or lipopolysaccharide stimulation of endothelial cells. The LFA-1-dependent adhesion could be further subdivided into an LFA-1/ICAM-1-dependent component which was increased by cytokines and a basal LFA-1-dependent, ICAM-1-independent component which did not appear to be affected by cytokines. We conclude that ICAM-1 is a regulated ligand for lymphocyte-endothelial cell adhesion, but at least two other major adhesion pathways exist.  相似文献   

12.
Summary Immunohistological analysis of sections prepared from human palatine tonsils revealed marked differences in the distribution of the adhesion molecule, leucocyte function antigen-1 (LFA-1) and its counter receptor, intercellular adhesion molecule-1 (ICAM-1). Light microscopy showed that LFA-1 was restricted to the leucocytes, particularly the lymphocytes. In contrast, staining of ICAM-1 was predominantly confined to the vascular endothelium with the greatest expression seen on the morphologically distinct high endothelial venules in the parafollicular areas; these are the sites that appear to support lymphocyte migration. Electron microscopy revealed that ICAM-1 was present on the luminal and lateral surfaces of the high endothelium and absent from the abluminal surface supported by basal lamina. The ICAM-1 was also absent from those surfaces of the endothelium that were in close contact with intravascular lymphocytes. Other cells stained by the anti-ICM-1 antibody included dendritic cells, plasma cells and epithelial cells in the reticulated crypt epithelium and in the upper strata of the non-keratinised stratified squamous epithelium. The high expression of LFA-1 was most prominent on lymphocytes, low on antigen-presenting cells and activated lymphoid cells, and not detectable on plasma cells, epithelial and endothelial cells. We propose that LFA-1/ICAM-1 binding participates in mediating the transendothelial migration of lymphocytes across the high endothelial venules of palatine tonsil.  相似文献   

13.
Intercellular adhesion molecule 1 (ICAM-1, CD54) binds to the integrin LFA-1 (CD11a/CD18), promoting cell adhesion in immune and inflammatory reactions. ICAM-1 is also subverted as a receptor by the major group of rhinoviruses. Electron micrographs show that ICAM-1 is a bent rod, 18.7 nm long, suggesting a model in which the five immunoglobulin-like domains are oriented head to tail at a small angle to the rod axis. ICAM-1 sequences important to binding LFA-1, rhinovirus, and four monoclonal antibodies were identified through the characterization of chimeric ICAM-1 molecules and mutants. The amino-terminal two immunoglobulin-like domains of ICAM-1 appear to interact conformationally. Domain 1 of ICAM-1 contains the primary site of contact for both LFA-1 and rhinovirus; the presence of domains 3-5 markedly affects the accessibility of the binding site for rhinovirus and less so for LFA-1. The binding sites appear to be distinct but overlapping; rhinovirus binding also differs from LFA-1 binding in its lack of divalent cation dependence. Our analysis suggests that rhinoviruses mimic LFA-1 in binding to the most membrane-distal, and thus most accessible, site of ICAM-1.  相似文献   

14.
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.  相似文献   

15.
The intercellular adhesion molecule-3 (ICAM-3) is a counter receptor for the integrin LFA-1 that supports cell-cell adhesion dependent functions. ICAM-3 is a member of the immunoglobulin superfamily possessing five immunoglobulin-like domains. Here, we characterize the overall shape of ICAM-3 and the amino acid residues involved in binding LFA-1 and monoclonal antibodies (Mab). Electron microscopic observations show that ICAM-3 is predominantly a straight rod of 15 nm in length, suggesting a head to tail arrangement of the immunoglobulin-like domains. Six out of nine ICAM-3 Mab described blocked the interaction with LFA-1 to varying degrees. Domain assignment of blocking Mab epitopes and characterization of LFA-1-dependent cell adhesion to ICAM-3 mutants demonstrate that the amino-terminal domain of ICAM-3 interacts with LFA-1. A conserved amino acid motif including residues E37 and T38 form an integrin binding site (IBS) in ICAM-3. This motif has also been shown to function as an IBS in ICAM-1 and VCAM-1 and hence may form a common site of contact in all CAMs of this type. Other ICAM-3 residues critical to adhesive interactions, such as Q75, conserved in ICAM-1 and ICAM-2, but not VCAM-1, may confer specificity to LFA-1 binding. This residue, Q75, is predicted to locate in a model of ICAM-3 to the same site as RGD in the immunoglobulin-like domain of fibronectin that binds several integrins. This suggests an evolutionary relationship between ICAMs and fibronectin interactions with integrins.  相似文献   

16.
The intercellular adhesion molecule-3 (ICAM-3) is a counter receptor for the integrin LFA-1 that supports cell-cell adhesion dependent functions. ICAM-3 is a member of the immunoglobulin superfamily possessing five immunoglobulin-like domains. Here, we characterize the overall shape of ICAM-3 and the amino acid residues involved in binding LFA-1 and monoclonal antibodies (Mab). Electron microscopic observations show that ICAM-3 is predominantly a straight rod of 15 nm in length, suggesting a head to tail arrangement of the immunoglobulin-like domains. Six out of nine ICAM-3 Mab described blocked the interaction with LFA-1 to varying degrees. Domain assignment of blocking Mab epitopes and characterization of LFA-1-dependent cell adhesion to ICAM-3 mutants demonstrate that the amino-terminal domain of ICAM-3 interacts with LFA-1. A conserved amino acid motif including residues E37 and T38 form an integrin binding site (IBS) in ICAM-3. This motif has also been shown to function as an IBS in ICAM-1 and VCAM-1 and hence may form a common site of contact in all CAMs of this type. Other ICAM-3 residues critical to adhesive interactions, such as Q75, conserved in ICAM-1 and ICAM-2, but not VCAM-1, may confer specificity to LFA-1 binding. This residue, Q75, is predicted to locate in a model of ICAM-3 to the same site as RGD in the immunoglobulin-like domain of fibronectin that binds several integrins. This suggests an evolutionary relationship between ICAMs and fibronectin interactions with integrins.  相似文献   

17.
《The Journal of cell biology》1993,123(4):1007-1016
The interaction of lymphocyte function-associated antigen-1 (LFA-1) with its ligands mediates multiple cell adhesion processes of capital importance during immune responses. We have obtained three anti-ICAM-3 mAbs which recognize two different epitopes (A and B) on the intercellular adhesion molecule-3 (ICAM-3) as demonstrated by sequential immunoprecipitation and cross-competitive mAb-binding experiments. Immunoaffinity purified ICAM-3-coated surfaces were able to support T lymphoblast attachment upon cell stimulation with both phorbol esters and cross-linked CD3, as well as by mAb engagement of the LFA-1 molecule with the activating anti-LFA-1 NKI-L16 mAb. T cell adhesion to purified ICAM-3 was completely inhibited by cell pretreatment with mAbs to the LFA-1 alpha (CD11a) or the LFA-beta (CD18) integrin chains. Anti-ICAM-3 mAbs specific for epitope A, but not those specific for epitope B, were able to trigger T lymphoblast homotypic aggregation. ICAM-3-mediated cell aggregation was dependent on the LFA-1/ICAM-1 pathway as demonstrated by blocking experiments with mAbs specific for the LFA-1 and ICAM-1 molecules. Furthermore, immunofluorescence studies on ICAM-3-induced cell aggregates revealed that both LFA-1 and ICAM-1 were mainly located at intercellular boundaries. ICAM-3 was located at cellular uropods, which in small aggregates appeared to be implicated in cell-cell contacts, whereas in large aggregates it appeared to be excluded from cell-cell contact areas. Experiments of T cell adhesion to a chimeric ICAM-1-Fc molecule revealed that the proaggregatory anti-ICAM-3 HP2/19 mAb was able to increase T lymphoblast attachment to ICAM-1, suggesting that T cell aggregation induced by this mAb could be mediated by increasing the avidity of LFA-1 for ICAM-1. Moreover, the HP2/19 mAb was costimulatory with anti-CD3 mAb for T lymphocyte proliferation, indicating that enhancement of T cell activation could be involved in ICAM-3-mediated adhesive phenomena. Altogether, our results indicate that ICAM-3 has a regulatory role on the LFA-1/ICAM-1 pathway of intercellular adhesion.  相似文献   

18.
19.
To identify the intracellular signals which increase the adhesiveness of leukocyte function-associated antigen 1 (LFA-1), we established an assay system for activation-dependent adhesion through LFA-1/intercellular adhesion molecule 1 ICAM-1 using mouse lymphoid cells reconstituted with human LFA-1 and then introduced constitutively active forms of signaling molecules. We found that the phorbol myristate acetate (PMA)-responsive protein kinase C (PKC) isotypes (alpha, betaI, betaII, and delta) or phosphatidylinositol-3-OH kinase (PI 3-kinase) itself activated LFA-1 to bind ICAM-1. H-Ras and Rac activated LFA-1 in a PI 3-kinase-dependent manner, whereas Rho and R-Ras had little effect. Unexpectedly, Rap1 was demonstrated to function as the most potent activator of LFA-1. Distinct from H-Ras and Rac, Rap1 increased the adhesiveness independently of PI 3-kinase, indicating that Rap1 is a novel activation signal for the integrins. Rap1 induced changes in the conformation and affinity of LFA-1 and, interestingly, caused marked LFA-1/ICAM-1-mediated cell aggregation. Furthermore, a dominant negative form of Rap1 (Rap1N17) inhibited T-cell receptor-mediated LFA-1 activation in Jurkat T cells and LFA-1/ICAM-1-dependent cell aggregation upon differentiation of HL-60 cells into macrophages, suggesting that Rap1 is critically involved in physiological processes. These unique functions of Rap1 in controlling cellular adhesion through LFA-1 suggest a pivotal role as an immunological regulator.  相似文献   

20.
In order to identify a binding site for ligand intercellular adhesion molecule-1 (ICAM-1) on the beta 2 integrin lymphocyte function-associated antigen-1 (LFA-1), protein fragments of LFA-1 were made by in vitro translation of a series of constructs which featured domain-sized deletions starting from the N-terminus of the alpha subunit of LFA-1. Monoclonal antibodies and ICAM-1 were tested for their ability to bind to these protein fragments. Results show that the putative divalent cation binding domains V and VI contain an ICAM-1 binding site. A series of consecutive peptides covering these domains indicated two discontinuous areas as specific contact sites: residues 458-467 in domain V and residues 497-516 in domain VI. A three-dimensional model of these domains of LFA-1 was constructed based on the sequence similarity to known EF hands. The two regions critical for the interaction of LFA-1 with ICAM-1 lie adjacent to each other, the first next to the non-functional EF hand in domain V and the second coinciding with the potential divalent cation binding loop in domain VI. The binding of ICAM-1 with the domain V and VI region in solution was not sensitive to divalent cation chelation. In short, a critical motif for ICAM-1 binding to the alpha subunit of LFA-1 is shared between two regions of domains V and VI.  相似文献   

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