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.     

Dynamic regulation of LFA-1 activation and neutrophil arrest on intercellular adhesion molecule 1 (ICAM-1) in shear flow

Neutrophil recruitment during acute inflammation is triggered by G-protein-linked chemotactic receptors that in turn activate beta(2) integrin (CD18), deemed a critical step in facilitating cell capture and arrest under the shear force of blood flow. A conformational switch in the I domain allosteric site (IDAS) and in CD18 regulates LFA-1 affinity for endothelial ligands including intercellular adhesion molecule 1 (ICAM-1). We examined the dynamics of CD18 activation in terms of the efficiency of neutrophil capture of ICAM-1, and we correlated this with the membrane topography of 327C, an antibody that recognizes the active conformation of CD18 I-like domain. Adhesion increased in direct proportion to chemotactic stimulus rising 7-fold over a log range of interleukin-8 (IL-8). A threshold dose of approximately 75 pm IL-8, corresponding to ligation of only approximately 10-100 receptors, was sufficient to activate approximately 20,000 CD18 and a rapid boost in the capture efficiency on ICAM-1. This was accompanied by a rapid redistribution of active LFA-1, but not Mac-1, into membrane patches, a necessary component for optimum adhesion efficiency. Shear-resistant arrest on a monolayer of ICAM-1 was reversed within minutes of chemotactic stimulation correlating with a shift from high to low affinity CD18 and dispersal of patches of active CD18. Mobility of active CD18 into high avidity patches was dependent on phosphatidylinositol 3-kinase activity and not F-actin polymerization. The data reveal that the number of chemotactic receptors bound and the topography and lifetime of high affinity LFA-1 tightly regulate the efficiency of neutrophil capture on ICAM-1.     

Cellular activation of leukocyte function-associated antigen-1 and its affinity are regulated at the I domain allosteric site

The I domain of the integrin LFA-1 possesses a ligand binding interface that includes the metal ion-dependent adhesion site. Binding of the LFA-1 ligand, ICAM-1 to the metal ion-dependent adhesion site is regulated by the I domain allosteric site (IDAS). We demonstrate here that intracellular signaling leading to activation of LFA-1 binding to ICAM-1 is regulated at the IDAS. Inhibitory mutations in or proximal to the IDAS are dominant to cytoplasmic signals that activate binding to ICAM-1. In addition, mutational activation at the IDAS greatly increases the binding of lymphocyte-expressed LFA-1 to ICAM-1 in response to PMA, but does not result in constitutive binding. Binding of a novel CD18 activation epitope mAb to LFA-1 in response to soluble ICAM-1 binding was also blocked by inhibitory and was enhanced by activating IDAS mutations. Surface plasmon resonance using soluble wild-type LFA-1 and an IDAS mutant of LFA-1 indicate that the IDAS can regulate a 6-fold change in the K(d) of ICAM-1 binding. The K(d) of wild-type LFA-1 (1.2 x 10(-1) s(-1)) differed with that of the activating IDAS mutant (1.9 x 10(-2) s(-1)), but their K(a) values were identical (2.2 x 10(5) M(-1)s(-1)). We propose that IDAS regulates the binding of LFA-1 to ICAM-1 activated by intracellular signals. IDAS can control the affinity state of LFA-1 with concomitant I domain and CD18 conformational changes.     

The role of CD11a/CD18-CD54 interactions in human T cell-dependent B cell activation.

The role of leukocyte function-associated Ag-1 (LFA-1, CD11a/CD18) and intercellular adhesion molecule 1 (ICAM-1, CD54) interactions in human T cell and B cell collaboration was examined by studying the effect of mAb to these determinants on B cell proliferation and differentiation stimulated by culturing resting B cells with CD4+ T cells activated with immobilized mAb to the CD3 molecular complex. In this model system, mAb to either the alpha (CD11a) or beta (CD18) chain of LFA-1 or ICAM-1 (CD54) inhibited B cell responses significantly. The mAb did not directly inhibit B cell function, inasmuch as T cell-independent activation induced by formalinized Staphylococcus aureus and IL-2 was not suppressed. Moreover, DNA synthesis and IL-2 production by immobilized anti-CD3-stimulated CD4+ T cells were not suppressed by the mAb to LFA-1 or ICAM-1. Although the mAb to LFA-1 inhibited enhancement of IL-2 production by co-culture of immobilized anti-CD3-stimulated CD4+ T cells with B cells, addition of exogenous IL-2 or supernatants of mitogen-activated T cells could not abrogate the inhibitory effects of the mAb to LFA-1 or ICAM-1 on B cell responses. Inhibition was most marked when the mAb were present during the initial 24 h in culture. Immobilized anti-CD3-stimulated LFA-1-negative CD4+ T cell clones from a child with leukocyte adhesion deficiency could induce B cell responses, which were inhibited by mAb to LFA-1 or ICAM-1. These results indicate that the interactions between LFA-1 and ICAM-1 play an important role in mediating the collaboration between activated CD4+ T cells and B cells necessary for the induction of B cell proliferation and differentiation, and for enhancement of IL-2 production by CD4+ T cells. Moreover, the data are consistent with a model of T cell-B cell collaboration in which interactions between LFA-1 on resting B cells and ICAM-1 on activated CD4+ T cells play a critical role in initial T cell-dependent B cell activation.     

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.     

F-actin flow drives affinity maturation and spatial organization of LFA-1 at the immunological synapse

Integrin-dependent interactions between T cells and antigen-presenting cells are vital for proper T cell activation, effector function, and memory. Regulation of integrin function occurs via conformational change, which modulates ligand affinity, and receptor clustering, which modulates valency. Here, we show that conformational intermediates of leukocyte functional antigen 1 (LFA-1) form a concentric array at the immunological synapse. Using an inhibitor cocktail to arrest F-actin dynamics, we show that organization of this array depends on F-actin flow and ligand mobility. Furthermore, F-actin flow is critical for maintaining the high affinity conformation of LFA-1, for increasing valency by recruiting LFA-1 to the immunological synapse, and ultimately for promoting intracellular cell adhesion molecule 1 (ICAM-1) binding. Finally, we show that F-actin forces are opposed by immobilized ICAM-1, which triggers LFA-1 activation through a combination of induced fit and tension-based mechanisms. Our data provide direct support for a model in which the T cell actin network generates mechanical forces that regulate LFA-1 activity at the immunological synapse.     

The actin cytoskeleton regulates LFA-1 ligand binding through avidity rather than affinity changes.

To elucidate the role of the cytoskeleton regulating avidity or affinity changes in the leukocyte adhesion receptor lymphocyte function-associated antigen-1 (LFA-1) (alpha(L)beta(2)), we generated mutant cytoplasmic LFA-1 receptors and expressed these into the erythroleukemic cell line K562. We determined whether intercellular adhesion molecule-1 (ICAM-1)-mediated adhesion of LFA-1, lacking parts of its cytoplasmic tails, is regulated through receptor diffusion/clustering and/or by altered ligand binding affinity. All cytoplasmic deletion mutants that lack the complete beta(2) cytoplasmic tail and/or the conserved KVGFFKR sequence in the alpha(L) cytoplasmic tail were constitutively active and expressed high levels of the activation epitopes NKI-L16 and M24. Surprisingly, whereas these mutants showed a clustered cell surface distribution of LFA-1, the ligand-binding affinity as measured by titration of soluble ligand ICAM-1 remained unaltered. The notion that redistribution of LFA-1 does not alter ligand-binding affinity is further supported by the finding that disruption of the cytoskeleton by cytochalasin D did not alter the binding affinity nor adhesion to ICAM-1 of these mutants. Most cytoplasmic deletion mutants that spontaneously bound ICAM-1 were not capable to spread on ICAM-1, demonstrating that on these mutants LFA-1 is not coupled to the actin cytoskeleton. From these data we conclude that LFA-1-mediated cell adhesion to ICAM-1 is predominantly regulated by receptor clustering and that affinity alterations do not necessarily coincide with strong ICAM-1 binding.     

Synoviocyte stimulation by the LFA-1-intercellular adhesion molecule-2-Ezrin-Akt pathway in rheumatoid arthritis

In rheumatoid arthritis (RA), the synovium is infiltrated by mononuclear cells that influence the proliferation and activation of fibroblast-like synoviocytes (FLS) through soluble mediators as well as cell-to-cell contact. To identify receptor-ligand pairs involved in this cross-talk, we cocultured T cells with FLS lines isolated from synovial tissues from RA patients. Coculture with T cells induced phosphorylation of Akt (Ser(473)) and its downstream mediators, GSK-3alpha/GSK-beta, FoxO1/3a, and mouse double minute-2, and enhanced FLS proliferation. T cell-mediated phospho-Akt up-regulation was unique for FLS as no such effect was observed upon interaction of T cells with dendritic cells and B cells. Akt activation was induced by all functional T cell subsets independent of MHC/Ag recognition and was also found with other leukocyte populations, suggesting the involvement of a common leukocyte cell surface molecule. Akt phosphorylation, enhanced in vitro FLS proliferation, and enhanced FLS IL-6 production was inhibited by blocking Abs to CD11a and ICAM-2 whereas Abs to ICAM-1 had a lesser effect. Selective involvement of the LFA-1-ICAM-2 pathway was confirmed by the finding of increased ezrin phosphorylation at Tyr(353) that is known to be downstream of ICAM-2 and supports cell survival through Akt activation. CD28(-) T cells, which are overrepresented in RA patients, have high CD11a cell surface expression and induce Akt phosphorylation in FLS more potently than their CD28(+) counterparts. These findings identify ICAM-2 as a potential therapeutic target to inhibit FLS activation in RA, allowing for a more selective intervention than broad LFA-1 inhibition.     

A novel LFA-1 activation epitope maps to the I domain

A panel of 21 alpha-subunit (CD11a) and 10 beta-subunit (CD18) anti-LFA- 1 mAbs was screened for ability to activate LFA-1. A single anti-CD11a mAb, MEM-83, was identified which was able to directly induce the binding of T cells to purified ICAM-1 immobilized on plastic. This ICAM- 1 binding could be achieved by monovalent Fab fragments of mAb MEM-83 at concentrations equivalent to whole antibody, was associated with appearance of the "activation reporter" epitope detected by mAb 24, and was completely inhibited by anti-ICAM-1 and LFA-1 blocking mAbs. The epitope recognized by mAb MEM-83 was distinct from that recognized by mAb NKI-L16, an anti-CD11a mAb previously reported to induce LFA-1 activation, in that it was constitutively present on freshly isolated peripheral blood mononuclear cells and was not divalent cation dependent for expression. The ICAM-1 binding activity induced by mAb MEM-83 was, however, dependent on the presence of Mg2+ divalent cations. Using an in vitro-translated CD11a cDNA deletion series, we have mapped the MEM-83 activation epitope to the "I" domain of the LFA- 1 alpha subunit. These studies have therefore identified a novel LFA-1 activation epitope mapping to the I domain of LFA-1, thereby implicating this domain in the regulation of LFA-1 binding to ICAM-1.     

Rolling adhesion of alphaL I domain mutants decorrelated from binding affinity

Activated lymphocyte function-associated antigen-1 (LFA-1, alphaLbeta2 integrin) found on leukocytes facilitates firm adhesion to endothelial cell layers by binding to intercellular adhesion molecule-1 (ICAM-1), which is up-regulated on endothelial cells at sites of inflammation. Recent work has shown that LFA-1 in a pre-activation, low-affinity state may also be involved in the initial tethering and rolling phase of the adhesion cascade. The inserted (I) domain of LFA-1 contains the ligand-binding epitope of the molecule, and a conformational change in this region during activation increases ligand affinity. We have displayed wild-type I domain on the surface of yeast and validated expression using I domain specific antibodies and flow cytometry. Surface display of I domain supports yeast rolling on ICAM-1-coated surfaces under shear flow. Expression of a locked open, high-affinity I domain mutant supports firm adhesion of yeast, while yeast displaying intermediate-affinity I domain mutants exhibit a range of rolling phenotypes. We find that rolling behavior for these mutants fails to correlate with ligand binding affinity. These results indicate that unstressed binding affinity is not the only molecular property that determines adhesive behavior under shear flow.     

Leukocyte function-associated antigen 1-mediated adhesion stability is dynamically regulated through affinity and valency during bond formation with intercellular adhesion molecule-1

Neutrophil rolling and transition to arrest on inflamed endothelium are dynamically regulated by the affinity of the beta(2) integrin CD11a/CD18 (leukocyte function associated antigen 1 (LFA-1)) for binding intercellular adhesion molecule (ICAM)-1. Conformational shifts are thought to regulate molecular affinity and adhesion stability. Also critical to adhesion efficiency is membrane redistribution of active LFA-1 into dense submicron clusters where multimeric interactions occur. We examined the influences of affinity and dimerization of LFA-1 on LFA-1/ICAM-1 binding by engineering a cell-free model in which two recombinant LFA-1 heterodimers are bound to respective Fab domains of an antibody attached to latex microspheres. Binding of monomeric and dimeric ICAM-1 to dimeric LFA-1 was measured in real time by fluorescence flow cytometry. ICAM-1 dissociation kinetics were measured while LFA-1 affinity was dynamically shifted by the addition of allosteric small molecules. High affinity LFA-1 dissociated 10-fold faster when bound to monomeric compared with dimeric ICAM-1, corresponding to bond lifetimes of 25 and 330 s, respectively. Downshifting LFA-1 into an intermediate affinity state with the small molecule I domain allosteric inhibitor IC487475 decreased the difference in dissociation rates between monomeric and dimeric ICAM-1 to 4-fold. When LFA-1 was shifted into the low affinity state by lovastatin, both monomeric and dimeric ICAM-1 dissociated in less than 1 s, and the dissociation rates were within 50% of each other. These data reveal the respective importance of LFA-1 affinity and proximity in tuning bond lifetime with ICAM-1 and demonstrate a nonlinear increase in the bond lifetime of the dimer versus the monomer at higher affinity.     

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.     

CD73 engagement promotes lymphocyte binding to endothelial cells via a lymphocyte function-associated antigen-1-dependent mechanism

CD73 is a GPI-anchored lymphocyte adhesion molecule possessing an ecto-5'-nucleotidase enzyme activity. In this work, we show that engagement of lymphocyte CD73 increases lymphocyte binding to cultured endothelial cells (EC) in an LFA-1-dependent fashion. Engagement of CD73 by an anti-CD73 mAb 4G4 increases the adhesion of lymphocytes to cultured EC by about 80% compared with that of lymphocytes treated with a negative control Ab, and the increased adhesion can be blocked by an anti-CD18 mAb. The CD73-regulated increase in lymphocyte adhesion is not due to a conformational change leading to high-affinity LFA-1 receptors as assayed using mAb 24 against an activation-induced epitope of the molecule. Instead, CD73 engagement induces clustering of LFA-1 that is inhibitable by calpeptin, indicating involvement of Ca(2+)-dependent activation of a calpain-like enzyme in this process. In conclusion, the results shown here demonstrate that CD73 regulates the avidity of LFA-1 by clustering. This indicates a previously undescribed role for CD73 in controlling the poorly characterized activation step in the multistep cascade of lymphocyte extravasation. Moreover, these results suggest that in physiological conditions the activation step may result in clustering of LFA-1 rather than in an affinity change of the molecule.     

The dendritic cell cytoskeleton promotes T cell adhesion and activation by constraining ICAM-1 mobility

Integrity of the dendritic cell (DC) actin cytoskeleton is essential for T cell priming, but the underlying mechanisms are poorly understood. We show that the DC F-actin network regulates the lateral mobility of intracellular cell adhesion molecule 1 (ICAM-1), but not MHCII. ICAM-1 mobility and clustering are regulated by maturation-induced changes in the expression and activation of moesin and α-actinin-1, which associate with actin filaments and the ICAM-1 cytoplasmic domain. Constrained ICAM-1 mobility is important for DC function, as DCs expressing a high-mobility ICAM-1 mutant lacking the cytoplasmic domain exhibit diminished antigen-dependent conjugate formation and T cell priming. These defects are associated with inefficient induction of leukocyte functional antigen 1 (LFA-1) affinity maturation, which is consistent with a model in which constrained ICAM-1 mobility opposes forces on LFA-1 exerted by the T cell cytoskeleton, whereas ICAM-1 clustering enhances valency and further promotes ligand-dependent LFA-1 activation. Our results reveal an important new mechanism through which the DC cytoskeleton regulates receptor activation at the immunological synapse.     

LFA-1 Affinity Regulation Is Necessary for the Activation and Proliferation of Naive T Cells

The activation of LFA-1 (lymphocyte function-associated antigen) is a critical event for T cell co-stimulation. The mechanism of LFA-1 activation involves both affinity and avidity regulation, but the role of each in T cell activation remains unclear. We have identified antibodies that recognize and block different affinity states of the mouse LFA-1 I-domain. Monoclonal antibody 2D7 preferentially binds to the low affinity conformation, and this specific binding is abolished when LFA-1 is locked in the high affinity conformation. In contrast, M17/4 can bind both the locked high and low affinity forms of LFA-1. Although both 2D7 and M17/4 are blocking antibodies, 2D7 is significantly less potent than M17/4 in blocking LFA-1-mediated adhesion; thus, blocking high affinity LFA-1 is critical for preventing LFA-1-mediated adhesion. Using these reagents, we investigated whether LFA-1 affinity regulation affects T cell activation. We found that blocking high affinity LFA-1 prevents interleukin-2 production and T cell proliferation, demonstrated by TCR cross-linking and antigen-specific stimulation. Furthermore, there is a differential requirement of high affinity LFA-1 in the activation of CD4⁺ and CD8⁺ T cells. Although CD4⁺ T cell activation depends on both high and low affinity LFA-1, only high affinity LFA-1 provides co-stimulation for CD8⁺ T cell activation. Together, our data demonstrated that the I-domain of LFA-1 changes to the high affinity state in primary T cells, and high affinity LFA-1 is critical for facilitating T cell activation. This implicates LFA-1 activation as a novel regulatory mechanism for the modulation of T cell activation and proliferation.LFA-1 (lymphocyte function-associated antigen), an integrin family member, is important in regulating leukocyte adhesion and T cell activation (1, 2). LFA-1 consists of the α_L (CD11a) and β₂ (CD18) heterodimer. The ligands for LFA-1, including intercellular adhesion molecule ICAM³-1, ICAM-2, and ICAM-3, are expressed on antigen-presenting cells (APCs), endothelial cells, and lymphocytes (1). Mice that are deficient in LFA-1 have defects in leukocyte adhesion, lymphocyte proliferation, and tumor rejection (3–5). Blocking LFA-1 with antibodies can prevent inflammation, autoimmunity, organ graft rejection, and graft versus host disease in human and murine models (6–10).LFA-1 is constitutively expressed on the surface of leukocytes in an inactive state. Activation of LFA-1 is mediated by inside-out signals from the cytoplasm (1, 11). Subsequently, activated LFA-1 binds to the ligands and transduces outside-in signals back into the cytoplasm that result in cell adhesion and activation (12, 13). The activation of LFA-1 is a critical event in the formation of the immunological synapse, which is important for T cell activation (2, 14, 15). The active state of LFA-1 is regulated by chemokines and the T cell receptor (TCR) through Rap1 signaling (16). LFA-1 ligation lowers the activation threshold and affects polarization in CD4⁺ T cells (17). Moreover, productive LFA-1 engagement facilitates efficient activation of cytotoxic T lymphocytes and initiates a distinct signal essential for the effector function (18–20). Thus, LFA-1 activation is essential for the optimal activation of T cells.The mechanism of LFA-1 activation involves both affinity (conformational changes within the molecule) and avidity (receptor clustering) regulation (21–23). The I-domain of the LFA-1 α_L subunit is the primary ligand-binding site and has been proposed to change conformation, leading to an increased affinity for ligands (24–26). The structural basis of the conformational changes in the I-domain of LFA-1 has been extensively characterized (27). Previously, we have demonstrated that the conformation of the LFA-1 I-domain changes from the low affinity to the high affinity state upon activation. By introducing disulfide bonds into the I-domain, LFA-1 can be locked in either the closed or open conformation, which represents the “low affinity” or “high affinity” state, respectively (28, 29). In addition, we identified antibodies that are sensitive to the affinity changes in the I-domain of human LFA-1 and showed that the activation-dependent epitopes are exposed upon activation (30). This study supports the presence of the high affinity conformation upon LFA-1 activation in cell lines. It has been demonstrated recently that therapeutic antagonists, such as statins, inhibit LFA-1 activation and immune responses by locking LFA-1 in the low affinity state (31–34). Furthermore, high affinity LFA-1 has been shown to be important for mediating the adhesion of human T cells (35, 36). Thus, the affinity regulation is a critical step in LFA-1 activation.LFA-1 is a molecule of great importance in the immune system, and its activation state influences the outcome of T cell activation. Our previous data using the activating LFA-1 I-domain-specific antibody MEM83 indicate that avidity and affinity of the integrin can be coupled during activation (37). However, whether affinity or avidity regulation of LFA-1 contributes to T cell activation remains controversial (23, 38, 39). Despite the recent progress suggesting that conformational changes represent a key step in the activation of LFA-1, there are considerable gaps to be filled. When LFA-1 is activated, the subsequent outside-in signaling contributes to T cell activation via immunological synapse and LFA-1-dependent signaling. It is critical to determine whether high affinity LFA-1 participates in the outside-in signaling and affects the cellular activation of T cells. Nevertheless, the rapid and dynamic process of LFA-1 activation has hampered further understanding of the role of high affinity LFA-1 in primary T cell activation. The affinity of LFA-1 for ICAM-1 increases up to 10,000-fold within seconds and involves multiple reversible steps (23). In addition, the activation of LFA-1 regulates both adhesion and activation of T cells, two separate yet closely associated cellular functions. When LFA-1 is constitutively expressed in the active state in mice, immune responses are broadly impaired rather than hyperactivated, suggesting the complexity of affinity regulation (40). Therefore, it is difficult to dissect the mechanisms by which high affinity LFA-1 regulates stepwise activation of T cells in the whole animal system.In the present study, we identified antibodies recognizing and blocking different affinity states of mouse LFA-1. These reagents allowed us to determine the role of affinity regulation in T cell activation. We found that blocking high affinity LFA-1 inhibited IL-2 production and proliferation in T cells. Furthermore, there is a differential requirement of high affinity LFA-1 in antigen-specific activation of CD4⁺ and CD8⁺ T cells. The activation of CD4⁺ T cells depends on both high and low affinity LFA-1. For CD8⁺ T cell activation, only high affinity LFA-1 provides co-stimulation. Thus, affinity regulation of LFA-1 is critical for the activation and proliferation of naive T cells.     

Effect of integrin beta 2 subunit truncations on LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) assembly, surface expression, and function

LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) are members of the beta2 integrins involved in leukocyte function during immune and inflammatory responses. We aimed to determine a minimized beta2 subunit that forms functional LFA-1 and Mac-1. Using a series of truncated beta2 variants, we showed that the subregion Q23-D300 of the beta2 subunit is sufficient to combine with the alphaL and alphaM subunits intracellularly. However, only the beta2 variants terminating after Q444 promote cell surface expression of LFA-1 and Mac-1. Thus, the major cysteine-rich region and the three highly conserved cysteine residues at positions 445, 447, and 449 of the beta2 subunit are not required for LFA-1 and Mac-1 surface expression. The surface-expressed LFA-1 variants are constitutively active with respect to ICAM-1 adhesion and these variants express the activation reporter epitope of the mAb 24. In contrast, surface-expressed Mac-1, both the wild type and variants, require 0. 5 mM MnCl2 for adhesion to denatured BSA. These results suggest that the role of the beta2 subunit in LFA-1- and Mac-1-mediated adhesion may be different.     

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.     

Divalent cation regulation of the function of the leukocyte integrin LFA-1

The integrin lymphocyte function-associated antigen-1 (LFA-1) expressed on T cells serves as a useful model for analysis of leukocyte integrin functional activity. We have assessed the role of divalent cations Mg2+, Ca2+, and Mn2+ in LFA-1 binding to ligand intercellular adhesion molecule-1 (ICAM-1) and induction of the divalent cation-dependent epitope recognized by mAb 24. Manganese strongly promoted both expression of the 24 epitope and T cell binding to ICAM-1 via LFA-1, suggesting that Mn2+ is able to directly alter the conformation of LFA-1 in a manner that favors ligand binding. Since Mn2+ also promotes functional activity of other integrins, parallels in mechanism of ligand binding may span the integrin family. In contrast, induction of 24 epitope expression by Mg2+ required removal of Ca2+ from T cell LFA-1 with EGTA. Furthermore, binding of mAb 24 to T cell LFA-1 in the presence of either Mn2+ or Mg2+ was found to be specifically inhibited by Ca2+, suggestive of a negative regulatory role for Ca2+ in the control of leukocyte integrin function. Analysis of T cell binding to ICAM-1 via LFA-1 in the presence of Mg2+ or Mn2+, confirmed that Ca2+ exerted inhibitory effects upon LFA-1 function. The implication of our findings is that Ca2+ bound with relatively high affinity to LFA-1 may serve to maintain an inactive state. Thus induction of function and 24 epitope expression may occur as a result of displacement of Ca2+ from leukocyte integrins or alternatively, such activators may be able to impose the required conformational change in the presence of bound Ca2+.     

Integrin leukocyte function-associated antigen-1-mediated cell binding can be activated by clustering of membrane rafts

The leukocyte function-associated antigen-1 (LFA-1) integrin (CD11a/CD18) is an important adhesion molecule for lymphocyte migration and the initiation of an immune response. At the cell surface, LFA-1 activity can be regulated by divalent cations that enhance receptor affinity but also by membrane clustering induced by treatment of cells with substances such as phorbol esters. Membrane clustering leads to increased LFA-1 avidity. We report here that LFA-1-mediated binding of mouse thymocytes or activated T lymphocytes to intercellular adhesion molecule 1 can be rapidly induced by clustering of membrane rafts using antibodies to the glycosylphophatidylinositol-anchored molecule CD24 or cholera toxin (CTx). CD24 and CD18 were found to co-localize in rafts and cross-linking with CTx lead to enhanced LFA-1 clustering. We observed that disruption of raft integrity by lowering the membrane cholesterol content abolished the CTx and the phorbol 12-myristate 13-acetate-induced LFA-1 binding but left the ability to activate LFA-1 with Mg(2+)/EGTA unimpaired. In contrast to activation with Mg(2+)/EGTA, activation via raft clustering was dependent on PI3-kinase, required cytoskeletal mobility, and was accompanied by Tyr phosphorylation of a 18-kDa protein. Our results support the notion that rafts as preformed adhesion platforms could be important for the rapid regulation of lymphocyte adhesion.     

2E8 Binds to the High Affinity I-domain in a Metal Ion-dependent Manner: A SECOND GENERATION MONOCLONAL ANTIBODY SELECTIVELY TARGETING ACTIVATED LFA-1*

The activation of leukocyte function-associated antigen-1 (LFA-1) plays a critical role in regulating immune responses. The metal ion-dependent adhesion site on the I-domain of LFA-1 α_L subunit is the key recognition site for ligand binding. Upon activation, conformation changes in the I-domain can lead LFA-1 from the low affinity state to the high affinity (HA) state. Using the purified HA I-domain locked by disulfide bonds for immunization, we developed an mAb, 2E8, that specifically binds to cells expressing the HA LFA-1. The surface plasmon resonance analysis has shown that 2E8 only binds to the HA I-domain and that the dissociation constant (K_D) for HA I-domain is 197 nm. The binding of 2E8 to the HA I-domain is metal ion-dependent, and the affinity decreased as Mn²⁺ was replaced sequentially by Mg²⁺ and Ca²⁺. Surface plasmon resonance analysis demonstrates that 2E8 inhibits the interaction of HA I-domain and ICAM-1. Furthermore, we found that 2E8 can detect activated LFA-1 on both JY and Jurkat cells using flow cytometry and parallel plate adhesion assay. In addition, 2E8 inhibits JY cell adhesion to human umbilical vein endothelial cells and homotypic aggregation. 2E8 treatment reduces the proliferation of both human CD4⁺ and CD8⁺ T cells upon OKT3 stimulation without the impairment of their cytolytic function. Taken together, these data demonstrate that 2E8 is specific for the high affinity form of LFA-1 and that 2E8 inhibits LFA-1/ICAM-1 interactions. As a novel activation-specific monoclonal antibody, 2E8 is a potentially useful reagent for blocking high affinity LFA-1 and modulating T cell activation in research and therapeutics.