Lymphocyte function-associated antigen-1 (LFA-1) interaction with intercellular adhesion molecule-1 (ICAM-1) is one of at least three mechanisms for lymphocyte adhesion to cultured endothelial cells

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.     

Intercellular adhesion molecule-1 (ICAM-1) is involved in the cytolytic T lymphocyte interaction with a human synovial cell line

The cell surface molecules involved in the human cytolytic T lymphocyte (CTL)-synovial cell interaction may play an important role in T cell interactions with connective tissue mesenchymal cells. To examine the molecular basis for the CTL-synovial cell interaction, we immortalized synovial cell explants to establish the cell line SYN.SPP. The SYN.SPP cell line was compared to the established B lymphoblastoid cell line JY. Cell surface immunofluorescence demonstrated significantly different levels of the immunologically relevant cell surface molecules ICAM-1 and LFA-3. Both cell lines were used to stimulate CTL precursors. After several months in culture, CTL lines stimulated by the SYN.SPP and JY cell lines demonstrated HLA class I-directed cytolytic activity. The cell surface molecules utilized by the anti-SYN.SPP and anti-JY CTL lines were identified by monoclonal antibody (MAb) inhibition. MAb recognizing the CTL cell surface molecules CD3, CD8 and LFA-1 (CD11a) significantly inhibited CTL-mediated lysis of both target cells. An interesting observation was that the anti-SYN.SPP CTL line appeared to utilize the ICAM-1 and not the LFA-3 target cell molecule. In contrast, the anti-JY CTL line utilized the LFA-3 and not the ICAM-1 membrane molecule. These results indicate that CTL interactions with connective tissue mesenchymal cells may be regulated by a unique pattern of antigen nonspecific cell-cell interaction molecules.     

B cell-associated LFA-1 and T cell-associated ICAM-1 transiently cluster in the area of contact between interacting cells.

TNP-specific B cells interact with carrier-specific T hybridoma cells in an antigen-specific, MHC-restricted manner. The formation of T cell/B cell conjugates is time and temperature dependent and results in the formation of a broad area of close contact between the interacting cells. In order to determine which surface molecules on the two cells cluster at the interaction site. T cell/B cell conjugates were formalin-fixed at different times following conjugation and were stained with antibodies directed against cell surface molecules. Results of these studies indicate that the alpha- and beta-subunits of LFA-1 on B cells transiently cluster in the area of cell contact. Maximum clustering of LFA-1 occurs at 45 min, after which time LFA-1 redistributes on the surface of the B cells. Several other B cell-associated molecules (MHC Class II, ICAM-1, Ig, B220, J11D, or CD23) do not cluster at the interaction site at any time point. T cell-associated LFA-1 does not cluster with any specific pattern, but ICAM-1 does. Maximum clustering of ICAM-1 occurs 60 to 90 min after intercellular contact. After this time, ICAM-1 redistributes on the surface of the T cells. Although both the alpha- and beta-subunits of LFA-1 cluster at the interaction site on B cells, antibodies recognizing these subunits differ in their ability to affect conjugation. One antibody recognizing the alpha chain of LFA-1 (M17/4.2) inhibits T-cell/B cell conjugation, whereas another antibody that also recognizes the alpha chain-(G-48) enhances conjugation. In contrast, an antibody that recognizes LFA-1 beta (M18/2.a.8) has no effect. An antibody that recognizes ICAM-1 (YN/1.7), the ligand for LFA-1, inhibits conjugation. These data show that, during T cell/B cell interaction. LFA-1 on B cells and ICAM-1 on T cells transiently cluster with similar, albeit not identical, kinetics to the site of cell-cell contact.     

A human intercellular adhesion molecule (ICAM-1) distinct from LFA-1

Homotypic adhesion by phorbol ester-stimulated lymphocytes requires LFA-1 and Mg+2 and does not involve like-like interactions between LFA-1 molecules on adjacent cells. The latter finding suggested that a second molecule, distinct from LFA-1, also participates in LFA-1-dependent adhesion. The identification of such a molecule was the object of this investigation. After immunization with LFA-1-deficient EBV-transformed lymphoblastoid cells, a MAb was obtained that inhibits phorbol ester-stimulated aggregation of LFA-1+ EBV lines. This MAb defines a novel cell surface molecule, which is designated intercellular adhesion molecule 1 (ICAM-1). ICAM-1 is distinct from LFA-1 in both cell distribution and structure. In SDS-PAGE, ICAM-1 isolated from JY cells is a single chain of Mr = 90,000. As shown by MAb inhibition, ICAM-1 participates in phorbol ester-stimulated adhesion reactions of B lymphocyte and myeloid cell lines and T lymphocyte blasts. However, aggregation of one T lymphocyte cell line (SKW-3) was inhibited by LFA-1 but not ICAM-1 MAb. It is proposed that ICAM-1 may be a ligand in many, but not all, LFA-1-dependent adhesion reactions.     

Induction of tyrosine phosphorylation during ICAM-3 and LFA-1-mediated intercellular adhesion, and its regulation by the CD45 tyrosine phosphatase

Intercellular adhesion molecule (ICAM)-3, a recently described counter- receptor for the lymphocyte function-associated antigen (LFA)-1 integrin, appears to play an important role in the initial phase of immune response. We have previously described the involvement of ICAM-3 in the regulation of LFA-1/ICAM-1-dependent cell-cell interaction of T lymphoblasts. In this study, we further investigated the functional role of ICAM-3 in other leukocyte cell-cell interactions as well as the molecular mechanisms regulating these processes. We have found that ICAM-3 is also able to mediate LFA-1/ICAM-1-independent cell aggregation of the leukemic JM T cell line and the LFA-1/CD18-deficient HAFSA B cell line. The ICAM-3-induced cell aggregation of JM and HAFSA cells was not affected by the addition of blocking mAb specific for a number of cell adhesion molecules such as CD1 1a/CD18, ICAM-1 (CD54), CD2, LFA-3 (CD58), very late antigen alpha 4 (CD49d), and very late antigen beta 1 (CD29). Interestingly, some mAb against the leukocyte tyrosine phosphatase CD45 were able to inhibit this interaction. Moreover, they also prevented the aggregation induced on JM T cells by the proaggregatory anti-LFA-1 alpha NKI-L16 mAb. In addition, inhibitors of tyrosine kinase activity also abolished ICAM-3 and LFA-1- mediated cell aggregation. The induction of tyrosine phosphorylation through ICAM-3 and LFA-1 antigens was studied by immunofluorescence, and it was found that tyrosine-phosphorylated proteins were preferentially located at intercellular boundaries upon the induction of cell aggregation by either anti-ICAM-3 or anti-LFA-1 alpha mAb. Western blot analysis revealed that the engagement of ICAM-3 or LFA-1 with activating mAb enhanced tyrosine phosphorylation of polypeptides of 125, 70, and 38 kD on JM cells. This phenomenon was inhibited by preincubation of JM cells with those anti-CD45 mAb that prevented cell aggregation. Altogether these results indicate that CD45 tyrosine phosphatase plays a relevant role in the regulation of both intracellular signaling and cell adhesion induced through ICAM-3 and beta 2 integrins.     

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.     

Stimulation of B lymphocytes through surface Ig receptors induces LFA-1 and ICAM-1-dependent adhesion.

Engagement of the surface Ig receptor with anti-IgM antibodies stimulates murine B lymphocytes to markedly increase their expression of the cell adhesion molecules ICAM-1 and LFA-1. Stimulated B cells display increased homotypic adhesiveness and form spontaneous heterotypic conjugates with T lymphocytes. This latter T-B cell interaction is further enhanced if T cells have been previously activated with phorbol esters. In all cases, the formation of cell-cell conjugates is dependent on LFA-1-ICAM-1-mediated interactions as assessed in mAb blocking experiments. B lymphocytes stimulated with anti-IgM display a marked increase in binding to ICAM-1-transfected L cells. This cell-cell interaction is inhibited by anti-LFA-1 mAb binding to the B lymphocyte. Together, these results demonstrate that there is an induction of both ICAM-1 and LFA-1 on stimulated B cells and a corresponding increase in the adhesiveness of these cells. These findings suggest that Ag binding to the surface Ig receptor could prepare a B lymphocyte for subsequent interaction with a T lymphocyte. This provides insight into how efficient T-B collaboration may occur between very infrequent Ag-specific lymphocytes.     

The accessory function of murine intercellular adhesion molecule-1 in T lymphocyte activation. Contributions of adhesion and co-activation.

These studies demonstrate that the murine intercellular adhesion molecule-1 (ICAM-1) performs at least two roles in enhancing T cell activation. These two roles are evident in both of our experimental systems: with ICAM-1 expressed on the surface of transfected fibroblast cells, and with purified ICAM-1 immobilized on plastic. First, as has been documented by many investigators, ICAM-1 mediates adhesion between ICAM-1- and lymphocyte function-associated Ag-1 (LFA-1)-bearing cells. This adhesive interaction occurs even in the absence of T cell stimulation, although it is increased by addition of phorbol ester and calcium ionophore. Although ICAM-1 expression does markedly increase intercellular adhesion, the increase is significantly less than the improvement ICAM-1 expression makes in the Ag-presenting ability of MHC class II-transfected fibroblast cells. We have investigated whether this difference is due to LFA-1-mediated signaling, and we present data that demonstrates that although ICAM-1 does not deliver costimulatory signals required for T cell activation, the interaction of LFA-1 with ICAM-1 does synergize with TCR-transduced signals. This synergy is observed for ICAM-1 on live and on chemically fixed accessory cells, and for purified ICAM-1 molecules, but in all cases occurs only when the ICAM-1 and the TCR ligands are on the same surface. Finally, when the ICAM-1 is present on the surface of accessory cells, it enhances T cell activation by changing the Ag dose-dependence of the T cell, but when ICAM-1 and CD3 mAb are co-immobilized, ICAM-1 increases the peak response of the T cell without affecting the dose dependence of the response.     

Induction by IL 1 and interferon-gamma: tissue distribution, biochemistry, and function of a natural adherence molecule (ICAM-1)

ICAM-1 is a cell surface glycoprotein originally defined by a monoclonal antibody (MAb) that inhibits phorbol ester-stimulated leukocyte aggregation. Staining of frozen sections and immunofluorescence flow cytometry showed intercellular adhesion molecule-1 (ICAM-1) is expressed on non-hematopoietic cells such as vascular endothelial cells, thymic epithelial cells, certain other epithelial cells, and fibroblasts, and on hematopoietic cells such as tissue macrophages, mitogen-stimulated T lymphocyte blasts, and germinal center dendritic cells in tonsils, lymph nodes, and Peyer's patches. ICAM-1 staining on vascular endothelial cells is most intense in T cell areas in lymph nodes and tonsils showing reactive hyperplasia. ICAM-1 is expressed in low amounts on peripheral blood leukocytes. Phorbol ester-stimulated differentiation of myelomonocytic cell lines greatly increases ICAM-1 expression. ICAM-1 expression on dermal fibroblasts is increased threefold to fivefold by either interleukin 1 (IL 1) or interferon-gamma at 10 U/ml over a period of 4 or 10 hr, respectively. The induction is dependent on protein and mRNA synthesis and is reversible. ICAM-1 displays Mr heterogeneity in different cell types with a Mr of 97,000 on fibroblasts, 114,000 on the myelomonocytic cell line U937, and 90,000 on the B lymphoblastoid cell JY. ICAM-1 biosynthesis involves a Mr approximately 73,000 intracellular precursor. The non-N-glycosylated form resulting from tunicamycin treatment has a Mr of 55,000. ICAM-1 isolated from phorbol myristic acetate (PMA) stimulated U937 and from fibroblasts yields an identical major product of Mr = 60,000 after chemical deglycosylation. ICAM-1 MAb interferes with the adhesion of phytohemagglutinin blasts, and the adhesion of the cell line SKW3 to human dermal fibroblast cell layers. Pretreatment of fibroblasts but not lymphocytes with ICAM-1 MAb, and of lymphocytes but not fibroblasts with lymphocyte function-associated antigen 1 MAb inhibits adhesion. Intercellular adhesion is increased by prior exposure of fibroblasts to IL 1, and correlates with induction of ICAM-1.     

ICAM-3 interacts with LFA-1 and regulates the LFA-1/ICAM-1 cell adhesion pathway

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.     

The role of LFA-1/ICAM-1 interactions during murine T lymphocyte development.

We have examined the expression and function of the cell adhesion molecules LFA-1 (CD11a/CD18), ICAM-1 (CD54), and ICAM-2 in murine fetal thymic ontogeny and in the adult thymus. On fetal days 14 and 15, 40 to 50% of thymocytes coexpress high levels of LFA-1 and ICAM-1, as determined by flow cytometry. By day 16, more than 90% of fetal thymocytes are LFA-1+ ICAM-1hi, and all IL-2R+ cells are located in this population. Although LFA-1 expression remains unchanged thereafter, ICAM-1 expression appears to be differentially regulated in different thymocyte subpopulations, with CD4+8+ cells being ICAM-1lo and CD4-8- thymocytes remaining ICAM-1hi. ICAM-2 surface expression is dull on both fetal and adult thymocytes. Surprisingly, the expression of ICAM-1 is differentially up-regulated on T cells having a mature phenotype in thymus and in peripheral lymphoid organs, with CD8+ T cells bearing the highest amount of surface ICAM-1. Addition of anti-ICAM-1 or anti-LFA-1 antibodies to fetal thymic organ cultures results in the impaired generation of CD4+8+ cells. These results indicate that LFA-1/ICAM-1 interactions facilitate murine thymic development and suggest that cell adhesion molecules mediate important events in T cell differentiation.     

Level of ICAM-1 Surface Expression on Virus Producer Cells Influences both the Amount of Virion-Bound Host ICAM-1 and Human Immunodeficiency Virus Type 1 Infectivity

Using virions harvested from 293T cells stably expressing either low or high levels of surface ICAM-1, we determined that the number of virus-embedded host ICAM-1 proteins is positively influenced by the expression level of ICAM-1 on virus producer cells. Moreover, the increase in virion-bound host cell membrane ICAM-1 led to a concomitant enhancement of virus infectivity when a T-cell-tropic strain of human immunodeficiency virus type 1 (HIV-1) was used. The phenomenon was also seen when primary human cells were infected with virions pseudotyped with the envelope protein from a macrophage-tropic HIV-1 isolate, thus ruling out any envelope-specific effect. We also observed that target cells treated with NKI-L16, an anti-LFA-1 antibody known to increase the affinity of LFA-1 for ICAM-1, were markedly more susceptible to infection with HIV-1 particles bearing on their surfaces large numbers of host-derived ICAM-1 proteins. Given that cellular activation of leukocytes is known to modify the conformational state of LFA-1 and induce ICAM-1 surface expression, it is tempting to speculate that activation of virus-infected cells will lead to the production of HIV-1 particles bearing more host ICAM-1 on their surfaces and that such progeny virions will preferentially infect and replicate more efficiently in activated cells which are prevalent in lymphoid organs.     

Differential utilization of ICAM-1 and VCAM-1 during the adhesion and transendothelial migration of human T lymphocytes.

The comparative roles of the endothelial cell (EC) adhesion receptors VCAM-1 and ICAM-1 during the adhesion and transendothelial migration of T cells were examined. The adhesion of T cells to IL-1-activated EC was markedly, but not completely, inhibited by mAb to VCAM-1 as well as to its counter-receptor, VLA-4, whereas, T cell binding to IL-1-activated EC was not blocked by mAb to ICAM-1 or to its counter-receptor, LFA-1. In contrast, LFA-1/ICAM-1, but not VLA-4/VCAM-1, mediated much, but not all, of the binding of T cells to unstimulated EC. Activation of T cells with phorbol dibutyrate and ionomycin alter the receptor-counter-receptor pairs used for binding to EC. Regardless of the activation status of the EC, the binding of activated T cells was not blocked by mAb to VLA-4 or VCAM-1. Moreover, the binding of activated T cells to EC was blocked to a lesser degree by mAb to LFA-1 than that of resting T cells, and mAb to ICAM-1 blocked binding only modestly. The role of VCAM-1 and ICAM-1 during the transendothelial migration of T cells was also examined. Regardless of the activation status of the T cells or the EC, VCAM-1 was never found to function during transendothelial migration, even when it mediated the binding of resting T cells to IL-1-activated EC. In contrast, ICAM-1 played an important role in transendothelial migration under all of the conditions examined, including situations when T cell-EC binding was not mediated by ICAM-1. Immunoelectron microscopic analysis of transendothelial migration supported the conclusion that ICAM-1 but not VCAM-1 played a central role in this process. Thus, ICAM-1 was prominently and uniformly expressed at all EC membrane sites that were in contact with bound and migrating T cells, whereas VCAM-1 was localized to the luminal surface of IL-1-activated EC, but was often absent from the surface of the EC in contact with T cells undergoing transendothelial migration. These studies confirm that ICAM-1 and VCAM-1 play reciprocal roles in the binding of resting T cells to resting and IL-1-activated EC, respectively, but a less prominent role in the binding of activated T cells. Moreover, ICAM-1 but not VCAM-1 plays a role in transendothelial migration, regardless of the receptor-counter-receptor pairs used for initial binding.     

Role of ICAM-1 in antigen presentation demonstrated by ICAM-1 defective mutants

We report a methodology for selecting APC with mutations that have impaired their ability to present Ag to T cells. A20 B lymphoblastoid cells were mutagenized and then repeatedly cocultured with murine T-T hybridomas in the presence of specific Ag. During these cocultures, the T-T hybridomas kill the competent APC, allowing the outgrowth of inactive variants. Two variants, A20.M1 and A20.M2, were isolated and studied in detail. These variants are impaired in their ability to present multiple Ag to T cells. This defect is also observed for the presentation of processing independent peptides by fixed APC indicating that a lesion exists in a post-Ag processing step. The level of expression of MHC molecules is unaffected and the functional defect in the APC is not localized to a particular MHC molecule. In contrast, these mutants were found to have a selective decrease in the expression of the murine homolog of ICAM-1, and the residual ability of these cells to present Ag was not blocked by anti-ICAM-1 mAb. Conversely, Ag presentation by the wild-type A20 is inhibited by anti-ICAM-1 mAb. Similarly, anti-LFA-1 mAb inhibited the response of T cells to Ag presented by the wild-type A20 to a much greater degree than by the mutant cells, indicating that LFA-1 is involved in interaction of T cells with the former, but not latter, APC. In the apparent absence of a contribution of LFA-1 to the T cell-APC interaction, either as a result of mAb blocking or the disruption of the APC membrane, the mutant and wild-type APC have a similar level of Ag-presenting activity. Reconstitution of ICAM-1 expression in these mutants by transfection with murine ICAM-1 cDNA fully restores their ability to present Ag. Together these results demonstrate that a murine ICAM-1 homolog is expressed on A20 B cells, where it functions as a major cell interaction molecule. The degree of functional impairment in these mutant APC gives insight into the contribution of cell interaction molecules to efficient Ag presentation and T cell-B cell interaction. Finally, these results also demonstrate the feasibility of selecting APC with mutations affecting Ag presentation.     

Role of LFA-1 and VLA-4 in the adhesion of cloned normal and LFA-1 (CD11/CD18)-deficient T cells to cultured endothelial cells. Indication for a new adhesion pathway.

Patients with the leukocyte adhesion deficiency (LAD) syndrome have a genetic defect in the common beta 2-chain (CD18) of the leukocyte integrins. This defect can result in the absence of cell surface expression of all three members of the leukocyte integrins. We investigated the capacity of T cell clones obtained from the blood of an LAD patient and of normal T cell clones to adhere to human umbilical vein endothelial cells (EC). Adhesion of the number of LAD T cells to unstimulated EC was approximately half of that of leukocyte function-associated antigen (LFA)-1+ T cells. Stimulation of EC with human rTNF-alpha resulted in an average 2- and 2.5-fold increase in adhesion of LFA-1+ and LFA-1- cells, respectively. This effect was maximal after 24 h and lasted for 48 to 72 h. The involvement of surface structures known to participate in cell adhesion (integrins, CD44) was tested by blocking studies with mAb directed against these structures. Adhesion of LFA-1+ T cells to unstimulated EC was inhibited (average inhibition of 58%) with mAb to CD11a or CD18. Considerably less inhibition of adhesion occurred with mAb to CD11a or CD18 (average inhibition, 20%) when LFA-1+ T cells were incubated with rTNF-alpha-stimulated EC. The adhesion of LFA-1- T cells to EC stimulated with rTNF-alpha, but not to unstimulated EC, was inhibited (average inhibition, 56%) by incubation with a mAb directed to very late antigen (VLA)-4 (CDw49d). In contrast to LAD T cell clones and the LFA-1+ T cell line Jurkat, mAb to VLA-4 did not inhibit adhesion of normal LFA-1+ T cell clones to EC, whether or not the EC had been stimulated with rTNF-alpha. We conclude that the adhesion molecule pair LFA-1/intercellular adhesion molecule (ICAM)-1 plays a major role in the adhesion of LFA-1+ T cell clones derived from normal individuals to unstimulated EC. Adhesion of LFA-1-T cells to TNF-alpha-stimulated EC is mediated by VLA-4/vascular cell adhesion molecule (VCAM)-1 interactions. Since we were unable to reduce significantly the adhesion of cultured normal LFA-1+ T cells to 24 h with TNF-alpha-stimulated endothelium with antibodies that block LFA-1/ICAM-1 or VLA-4/VCAM-1 interactions, and lectin adhesion molecule-1 and endothelial leukocyte adhesion molecule-1 appeared not to be implicated, other as yet undefined cell surface structures are likely to participate in T cell/EC interactions.     

LFA-1- and ICAM-1-dependent homotypic aggregation of human thymocytes induced by JL1 engagement

Cell-cell adhesion is essential for the appropriate immune response, differentiation, and migration of lymphocytes. This important physiological event is reflected in vitro by homotypic cell aggregation. We have previously reported that a 120 kDa cell surface glycoprotein, JL1, is a unique protein specifically expressed by immature double positive (DP) human thymocytes which are in the process of positive and negative selections through the interaction between thymocyte and antigen-presenting cells (APCs). The function of the JL1 molecule, however, is yet to be identified. We show here that anti-JL1 monoclonal antibody (mAb) induced the homotypic aggregation of human thymocytes in a temperature- and Mg2+-dependent manner. It required an intact cytoskeleton and the interaction between leucocyte function associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) since it was blocked by cytochalasin B and D, and mAb against LFA-1 and ICAM-1 which are known to be involved in the aggregation of thymocytes. Translocation of phosphatidylserine (PtdSer) through the cell membrane was not detected, implying that the molecular mechanism of JL-1-induced homotypic aggregation is different from that of CD99-induced homotypic aggregation. In summary, JL1 is a cell surface molecule that induces homotypic adhesion mediated by the LFA-1 and ICAM-1 interaction and cytoskeletal reorganization. These findings suggest that JL1 may be an important regulator of thymocyte development and thymocyte-APC interaction.     

Regulation of LFA-1 avidity in human B cells. Requirements for dephosphorylation events for high avidity ICAM-1 binding.

Regulation of the avidity of LFA-1 (CD11a/CD18, alpha L beta 2) for its ligand ICAM-1 (CD54) was studied in human B cells by evaluating the effects of a phorbol ester, anti-IgM antibodies, staurosporine, and okadaic acid. We monitored changes in LFA-1 avidity by quantifying binding of cells to an immobilized rICAM-1 fusion protein. In this assay, the protein kinase C-activating phorbol ester PDB and anti-IgM antibodies, as well as the protein kinase inhibitor, staurosporine, were able to induce LFA-1-dependent binding to ICAM-1. This demonstrates that the high avidity state of LFA-1 can be induced by a protein kinase C-dependent and by a protein kinase C-independent pathway. Furthermore, treatment of the cells with the protein phosphatase inhibitor, okadaic acid, inhibited binding to ICAM-1. Treatment with staurosporine before addition of okadaic acid not only induced enhanced binding of cells to ICAM-1, but also dramatically reduced the ability of okadaic acid to inhibit binding. These results suggest a critical role for a protein phosphatase in inducing the high avidity state of LFA-1 as well as a role for a protein kinase in inducing the low avidity state of LFA-1.     

Binding of human rhinovirus and T cells to intercellular adhesion molecule-1 on human fibroblasts. Discordance between effects of IL-1 and IFN-gamma.

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.