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.     

Lymphatic endothelial murine chloride channel calcium-activated 1 is a ligand for leukocyte LFA-1 and Mac-1

The lymphatic circulation mediates drainage of fluid and cells from the periphery through lymph nodes, facilitating immune detection of lymph-borne foreign Ags. The 10.1.1 mAb recognizes a lymphatic endothelial Ag, in this study purified by Ab-affinity chromatography. SDS-PAGE and mass spectrometry identified murine chloride channel calcium-activated 1 (mCLCA1) as the 10.1.1 Ag, a 90-kDa cell-surface protein expressed in lymphatic endothelium and stromal cells of spleen and thymus. The 10.1.1 Ab-affinity chromatography also purified LFA-1, an integrin that mediates leukocyte adhesion to endothelium. This mCLCA1-LFA-1 interaction has functional consequences, as lymphocyte adhesion to lymphatic endothelium was blocked by 10.1.1 Ab bound to endotheliumor by LFA-1 Ab bound to lymphocytes. Lymphocyte adhesion was increased by cytokine treatment of lymphatic endothelium in association with increased expression of ICAM-1, an endothelial surface protein that is also a ligand for LFA-1. By contrast, mCLCA1 expression and the relative contribution of mCLCA1 to lymphocyte adhesion were unaffected by cytokine activation, demonstrating that mCLCA1 and ICAM-1 interactions with LFA-1 are differentially regulated. mCLCA1 also bound to the LFA-1-related Mac-1 integrin that is preferentially expressed on leukocytes. mCLCA1-mediated adhesion of Mac-1- or LFA-1-expressing leukocytes to lymphatic vessels and lymph node lymphatic sinuses provides a target for investigation of lymphatic involvement in leukocyte adhesion and trafficking during the immune response.     

I-domain of lymphocyte function-associated antigen-1 mediates rolling of polystyrene particles on ICAM-1 under flow

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.     

Down-regulation of cell adhesion molecules LFA-1 and ICAM-1 after in vitro treatment with the anti-TNF-alpha agent thalidomide.

The tumor necrosis factor-alpha (TNF-alpha) inhibitor thalidomide is known to be a potent modulator of host immunity, a potential treatment for autoimmune disorders such as rheumatoid arthritis (RA) and a treatment for complications of HIV-1 infection. RA is an autoimmune disease of the joints that has been associated with hyperactivity of lymphocytes and other leukocytes, over-expression of pro-inflammatory cytokines (TNF-alpha and IL-1) and chronic debilitating inflammation. Thalidomide may play a role in RA treatment by altering leukocyte function through down-modulation of cell adhesion molecules necessary for leukocyte migration to inflammatory sites. The present study investigates down-regulation of cell adhesion molecules (ICAM-1 and LFA-1) and decreases in cell-cell contacts between human T leukemic (CEM) cells and human umbilical vein endothelial cells (HUVEC) after thalidomide exposure. CEM cells were cultured in RPMI 1640 medium with 0, 10 or 50 microg/ml thalidomide, stained with fluorescent monoclonal antibodies specific to ICAM-1 and LFA-1 and expression was measured with flow cytometry. For cell-cell adhesion measurements, monolayers of HUVEC cultured in Kaign's F-12 medium were incubated with thalidomide treated CEM cells stained with calcein AM. Specific cell adhesion between the two cell types was visualized with fluorescence microscopy. Thalidomide treatment significantly reduced cell adhesion molecule expression in a dose-dependent fashion and inhibited HUVEC/CEM cell adhesion. These data support the hypothesis that thalidomide has modulatory actions on leukocyte functions through expression of cell adhesion molecules.     

Filamin B mediates ICAM-1-driven leukocyte transendothelial migration

During inflammation, the endothelium mediates rolling and firm adhesion of activated leukocytes. Integrin-mediated adhesion to endothelial ligands of the Ig-superfamily induces intracellular signaling in endothelial cells, which promotes leukocyte transendothelial migration. We identified the actin cross-linking molecule filamin B as a novel binding partner for intracellular adhesion molecule-1 (ICAM-1). Immune precipitation as well as laser scanning confocal microscopy confirmed the specific interaction and co-localization of endogenous filamin B with ICAM-1. Importantly, clustering of ICAM-1 promotes the ICAM-1-filamin B interaction. To investigate the functional consequences of filamin B binding to ICAM-1, we used small interfering RNA to reduce filamin B expression in ICAM-1-GFP expressing HeLa cells. We found that filamin B is required for the lateral mobility of ICAM-1 and for ICAM-1-induced transmigration of leukocytes. Reducing filamin B expression in primary human endothelial cells resulted in reduced recruitment of ICAM-1 to endothelial docking structures, reduced firm adhesion of the leukocytes to the endothelium, and inhibition of transendothelial migration. In conclusion, this study identifies filamin B as a molecular linker that mediates ICAM-1-driven transendothelial migration.     

Leukocyte rolling velocities and migration are optimized by cooperative L-selectin and intercellular adhesion molecule-1 functions

Selectin family members largely mediate initial tethering and rolling of leukocytes on vascular endothelium, whereas integrin and Ig family members are essential for leukocyte firm adhesion. To quantify functional synergy between L-selectin and Ig family members during leukocyte rolling, the EA.hy926 human vascular endothelial line was transfected with either fucosyltransferase VII (926-FtVII) cDNA to generate L-selectin ligands alone or together with ICAM-1 cDNA (926-FtVII/ICAM-1). The ability of transfected 926 cells to support human leukocyte interactions was assessed in vitro using parallel plate flow chamber assays. Lymphocyte rolling on 926-FtVII cells was increased by approximately 70% when ICAM-1 was expressed at physiological levels. Although initial tether formation was similar for both cell types, lymphocyte rolling was 26% slower on 926-FtVII/ICAM-1 cells. Pretreatment of lymphocytes with an anti-CD18 mAb eliminated the increase in rolling, and all rolling was blocked by anti-L-selectin mAb. In addition, rolling velocities of lymphocytes from CD18-hypomorphic mice were 48% faster on 926-FtVII/ICAM-1 cells, with a similar reduction in rolling frequency relative to wild-type lymphocytes. CD18-hypomorphic lymphocytes also showed an approximately 40% decrease in migration to peripheral and mesenteric lymph nodes during in vivo migration assays compared with wild-type lymphocytes. Likewise, wild-type lymphocyte migration to peripheral lymph nodes was reduced by approximately 50% in ICAM-1(-/-) recipient mice. Similar to human lymphocytes, human neutrophils showed enhanced rolling interactions on 926-FtVII/ICAM-1 cells, but also firmly adhered. Thus, in addition to mediating leukocyte firm adhesion, CD18 integrin/ICAM-1 interactions regulate leukocyte rolling velocities and thereby optimize L-selectin-mediated leukocyte rolling.     

Dynamic interaction of VCAM-1 and ICAM-1 with moesin and ezrin in a novel endothelial docking structure for adherent leukocytes

Ezrin, radixin, and moesin (ERM) regulate cortical morphogenesis and cell adhesion by connecting membrane adhesion receptors to the actin-based cytoskeleton. We have studied the interaction of moesin and ezrin with the vascular cell adhesion molecule (VCAM)-1 during leukocyte adhesion and transendothelial migration (TEM). VCAM-1 interacted directly with moesin and ezrin in vitro, and all of these molecules colocalized at the apical surface of endothelium. Dynamic assessment of this interaction in living cells showed that both VCAM-1 and moesin were involved in lymphoblast adhesion and spreading on the endothelium, whereas only moesin participated in TEM, following the same distribution pattern as ICAM-1. During leukocyte adhesion in static or under flow conditions, VCAM-1, ICAM-1, and activated moesin and ezrin clustered in an endothelial actin-rich docking structure that anchored and partially embraced the leukocyte containing other cytoskeletal components such as alpha-actinin, vinculin, and VASP. Phosphoinositides and the Rho/p160 ROCK pathway, which participate in the activation of ERM proteins, were involved in the generation and maintenance of the anchoring structure. These results provide the first characterization of an endothelial docking structure that plays a key role in the firm adhesion of leukocytes to the endothelium during inflammation.     

Interplay between rolling and firm adhesion elucidated with a cell-free system engineered with two distinct receptor-ligand pairs

The firm arrest of leukocytes to the endothelium during inflammation is known to be mediated by endothelial intercellular adhesion molecules (ICAMs) binding to activated integrins displayed on leukocyte surface. Selectin-ligand interactions, which mediate rolling, are believed to be important for facilitating firm adhesion, either by activating integrins or by facilitating the transition to firm adhesion by making it easier for integrins to bind. Although leukocytes employ two distinct adhesion molecules that mediate different states of adhesion, the fundamental biophysical mechanisms by which two pairs of adhesion molecules facilitate cell adhesion is not well understood. In this work, we attempt to understand the interaction between two molecular systems using a cell-free system in which polystyrene microspheres functionalized with the selectin ligand, sialyl Lewis(X) (sLe(X)), and an antibody against ICAM-1, aICAM-1, are perfused over P-selectin/ICAM-1 coated surfaces in a parallel plate flow chamber. Separately, sLe(X)/P-selectin interactions support rolling and aICAM-1/ICAM-1 interactions mediate firm adhesion. Our results show that sLe(X)/aICAM-1 microspheres will firmly adhere to P-selectin/ICAM-1 coated surfaces, and that the extent of firm adhesion of microspheres is dependent on wall shear stress within the flow chamber, sLe(X)/aICAM-1 microsphere site density, and P-selectin/ICAM-1 surface density ratio. We show that P-selectin's interaction with sLe(X) mechanistically facilitates firm adhesion mediated by antibody binding to ICAM-1: the extent of firm adhesion for the same concentration of aICAM-1/ICAM-1 interaction is greater when sLe(X)/P-selectin interactions are present. aICAM-1/ICAM-1 interactions also stabilize rolling by increasing pause times and decreasing average rolling velocities. Although aICAM-1 is a surrogate for beta(2)-integrin, the kinetics of association between aICAM-1 and ICAM-1 is within a factor of 1.5 of activated integrin binding ICAM-1, suggesting the findings from this model system may be insightful to the mechanism of leukocyte firm adhesion. In particular, these experimental results show how two molecule systems can interact to produce an effect not achievable by either system alone, a fundamental mechanism that may pervade leukocyte adhesion biology.     

Shear forces induce ICAM-1 nanoclustering on endothelial cells that impact on T-cell migration

The leukocyte-specific β₂-integrin LFA-1 and its ligand ICAM-1, expressed on endothelial cells (ECs), are involved in the arrest, adhesion, and transendothelial migration of leukocytes. Although the role of mechanical forces on LFA-1 activation is well established, the impact of forces on its major ligand ICAM-1 has received less attention. Using a parallel-plate flow chamber combined with confocal and super-resolution microscopy, we show that prolonged shear flow induces global translocation of ICAM-1 on ECs upstream of flow direction. Interestingly, shear forces caused actin rearrangements and promoted actin-dependent ICAM-1 nanoclustering before LFA-1 engagement. T cells adhered to mechanically prestimulated ECs or nanoclustered ICAM-1 substrates developed a promigratory phenotype, migrated faster, and exhibited shorter-lived interactions with ECs than when adhered to non mechanically stimulated ECs or to monomeric ICAM-1 substrates. Together, our results indicate that shear forces increase ICAM-1/LFA-1 bonds because of ICAM-1 nanoclustering, strengthening adhesion and allowing cells to exert higher traction forces required for faster migration. Our data also underscore the importance of mechanical forces regulating the nanoscale organization of membrane receptors and their contribution to cell adhesion regulation.     

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.     

Soluble intercellular adhesion molecule-1 (sICAM-1): an overview

Soluble intercellular adhesion molecule-1 (sICAM-1) represents a circulating form of ICAM-1 that is constitutively expressed or is inducible on the cell surface of different cell lines. It serves as a counter-receptor for the lymphocyte function-associated antigen (LFA-1). Interaction between ICAM-1, present on endothelial cells, and LFA-1 facilitates leukocyte adhesion and migration across the endothelium. ICAM-1 and its circulating form have been implicated in the development of any number of diseases.     

Mac-1, but not LFA-1, uses intercellular adhesion molecule-1 to mediate slow leukocyte rolling in TNF-alpha-induced inflammation

We have previously shown that Mac-1 and LFA-1 play a cooperative role in slow leukocyte rolling in inflamed vessels, and that, although both have a role in leukocyte adhesion, the contribution from LFA-1 exceeds that of Mac-1. In this study, we used mice deficient in ICAM-1 (ICAM-1(null)) to study the function of ICAM-1 as an endothelial ligand for Mac-1 and LFA-1. The cremaster muscles of these mice were treated with TNF-alpha and prepared for intravital microscopy. We found that the average rolling velocity in venules was not different in ICAM-1(null) mice (4.7 micro m/s) compared with wild-type mice (5.1 micro m/s). Similarly, leukocyte adhesion efficiency in ICAM-1(null) mice (0.11 +/- 0.01 mm) was similar to that in Mac-1(-/-) (0.12 +/- 0.03 mm) mice but significantly increased compared with that in LFA-1(-/-) (0.08 +/- 0.01 mm) mice and significantly reduced from that in wild type (0.26 +/- 0.04 mm). When both LFA-1 and ICAM-1 were blocked, rolling velocity increased, and adhesion efficiency and arrest decreased. However, blocking both Mac-1 and ICAM-1 had no greater effect than either blockade alone. We conclude that endothelial ICAM-1 is the main ligand responsible for slow leukocyte rolling mediated by Mac-1, but not 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.     

Rap1 is a potent activation signal for leukocyte function-associated antigen 1 distinct from protein kinase C and phosphatidylinositol-3-OH kinase

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.     

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.     

Force spectroscopy of LFA-1 and its ligands, ICAM-1 and ICAM-2

Single-molecule measurements of the interaction of leukocyte function-associated antigen-1 (LFA-1), expressed on Jurkat T cells, with intercellular adhesion molecules-1 and -2 (ICAM-1 and ICAM-2) were conducted using atomic force microscopy (AFM). The force spectra (i.e., unbinding force versus loading rate) of both the LFA-1/ICAM-1 and LFA-1/ICAM-2 interactions were acquired at a loading rate range covering 3 orders of magnitude (50-60,000 pN/s) and revealed a fast loading regime and a slow loading regime. This indicates that the dissociation of both complexes involves overcoming a steep inner and a wide outer activation barrier. LFA-1 binding to ICAM-1 and ICAM-2 was strengthened in the slow loading regime by the addition of Mg(2+). Differences in the dynamic strength of the LFA-1/ICAM-1 and LFA-1/ICAM-2 interactions can be attributed to the presence of wider barriers in the ICAM-2 complex, making it more responsive to a pulling force than the ICAM-1 complex.     

A role for ICAM-1 in maintenance of leukocyte-endothelial cell rolling interactions in inflamed arterioles

A key endothelial receptor in leukocyte-endothelial cell (EC) interactions is ICAM-1. ICAM-1 is constitutively expressed at low levels on vascular ECs, and its levels significantly increase following stimulation with many proinflammatory agents. This study provides evidence that in inflamed arterioles of anesthetized mice (65 mg/kg ip Nembutal), ICAM-1 mediates leukocyte rolling, in contrast to its expected role of mediating firm adhesion in venules. The number of leukocytes rolling on arteriolar ECs is decreased in ICAM-1 knockout (KO) compared with wild-type (WT) mice (KO, 6.0 +/- 0.9; WT, 12.0 +/- 1.0 leukocytes/40 s; P < 0.05), whereas the leukocyte-rolling number in venules remains unaffected (KO, 5.6 +/- 0.9; WT, 7.0 +/- 0.7 leukocytes/40 s; n = 13-15 sites). We also show that the fraction of leukocytes that is rolling on arteriolar ECs does so with a higher characteristic velocity (>70 microm/s), and, furthermore, that the distance over which rolling contacts with the arteriolar wall are maintained is ICAM-1 dependent. In ICAM-1 KO animals or in WT mice in the presence of ICAM-1-blocking antibody, leukocytes rolled significantly shorter distances over the sampled 200-microm vessel length compared with WT (68 +/- 6.7 and 55 +/- 9.4 vs. 85 +/- 12.9% total, respectively, n = 4 sites, P < 0.05). We also found evidence that in ICAM-1 KO mice, a significant fraction of leukocyte rolling and adhesive interactions with arteriolar ECs could be accounted for by upregulation of another adhesion molecule, VCAM-1, providing an important illustration of how expression of related proteins can be altered following genetic ablatement of a target molecule (in this case ICAM-1).     

ICAM-1 (CD54): a counter-receptor for Mac-1 (CD11b/CD18)

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.     

Regulation of locomotion and cell-cell contact area by the LFA-1 and ICAM-1 adhesion receptors.

We demonstrate complementary differences in the behavior of B lymphoblastoid cells adhering to LFA-1 or its counter-receptor ICAM-1. The interaction of B lymphoblastoid cells with glass-supported planar bilayers bearing LFA-1 or ICAM-1 was observed by time-lapse video microscopy, and the distribution of adhesion receptors on cells interacting with the planar bilayers was studied by immunofluorescence microscopy. B lymphoblasts formed a large contact area and crawled rapidly (up to 25 microns/min) on planar bilayers bearing ICAM-1. In contrast, these cells attached to planar bilayers bearing LFA-1 through a fixed point about which the cells actively pivoted, using a single stalk-like projection. Phorbol ester-stimulated lymphoblasts, which adhere more strongly to ICAM-1-bearing substrates than unstimulated lymphoblasts, were still capable of locomotion on ICAM-1. Phorbol ester stimulation of B lymphoblasts on planar bilayers bearing LFA-1 promoted a rapid conversion from "stalk" attachment to symmetrical spreading of the cell on the substrate. Cellular LFA-1 remained uniformly distributed on the cell surface during interaction with bilayers bearing purified ICAM-1 as determined by immunofluorescence. In contrast, ICAM-1 was concentrated in the stalk-like structure through which the unstimulated B lymphoblasts adhered to LFA-1 in planar bilayers, but ICAM-1 immunofluorescence became more uniformly distributed over the cell surface within minutes of phorbol ester addition. Neither LFA-1 or ICAM-1 colocalized with the prominent staining of filamentous actin in the ruffling membrane regions. Interaction through cell surface LFA-1 and ICAM-1, 2, or 3 promotes different cellular morphologies and behaviors, the correlation of which with previously observed patterns of lymphocyte interaction with different cell types is discussed.