Activation Of Leukocyte Function-associated Antigen-1 Research Articles

The Multiple Roles of the Cytosolic Adapter Proteins ADAP, SKAP1 and SKAP2 for TCR/CD3 -Mediated Signaling Events.

T cells are the key players of the adaptive immune response. They coordinate the activation of other immune cells and kill malignant and virus-infected cells. For full activation T cells require at least two signals. Signal 1 is induced after recognition of MHC/peptide complexes presented on antigen presenting cells (APCs) by the clonotypic TCR (T-cell receptor)/CD3 complex whereas Signal 2 is mediated via the co-stimulatory receptor CD28, which binds to CD80/CD86 molecules that are present on APCs. These signaling events control the activation, proliferation and differentiation of T cells. In addition, triggering of the TCR/CD3 complex induces the activation of the integrin LFA-1 (leukocyte function associated antigen 1) leading to increased ligand binding (affinity regulation) and LFA-1 clustering (avidity regulation). This process is termed “inside-out signaling”. Subsequently, ligand bound LFA-1 transmits a signal into the T cells (“outside-in signaling”) which enhances T-cell interaction with APCs (adhesion), T-cell activation and T-cell proliferation. After triggering of signal transducing receptors, adapter proteins organize the proper processing of membrane proximal and intracellular signals as well as the activation of downstream effector molecules. Adapter proteins are molecules that lack enzymatic or transcriptional activity and are composed of protein-protein and protein-lipid interacting domains/motifs. They organize and assemble macromolecular complexes (signalosomes) in space and time. Here, we review recent findings regarding three cytosolic adapter proteins, ADAP (Adhesion and Degranulation-promoting Adapter Protein), SKAP1 and SKAP2 (Src Kinase Associated Protein 1 and 2) with respect to their role in TCR/CD3-mediated activation, proliferation and integrin regulation.

Adhesive dynamics simulations quantitatively predict effects of kindlin-3 deficiency on T-cell homing.

Leukocyte adhesion is important for the proper functioning of the immune system. While leukocyte homing is mediated by adhesion receptors, the activation of these receptors is modulated by intracellular signaling molecules. In Leukocyte Adhesion Deficiency Type 3, the loss of the kindlin-3 prevents the activation of Leukocyte Function-associated Antigen-1 (LFA-1), which leads to a defect in adhesion, causing recurrent infections and bleeding disorders. Here, we use Integrated Signaling Adhesive Dynamics, a computer model of leukocyte rolling and adhesion combined with a simulated intracellular signaling cascade, to predict the response of T cells to depletion of kindlin-3. Our model predicts that cell adhesion is hypersensitive to the amount of kindlin-3 in the cell, while the rolling velocity is independent of kindlin-3 concentration. In addition, our simulation predicted that the time to stop, an important metric of adhesion, would increase with decreasing kindlin-3 expression. These predictions were confirmed experimentally in experiments using Jurkat cells with reduced expression of kindlin-3. These results suggest that Adhesive Dynamics is a versatile tool for quantifying adhesion in the immune response and predicting the effects of engineering cellular components.

Phosphorylation of the α-chain in the integrin LFA-1 enables β2-chain phosphorylation and α-actinin binding required for cell adhesion

The integrin leukocyte function-associated antigen-1 (LFA-1) plays a pivotal role in leukocyte adhesion and migration, but the mechanism(s) by which this integrin is regulated has remained incompletely understood. LFA-1 integrin activity requires phosphorylation of its β2-chain and interactions of its cytoplasmic tail with various cellular proteins. The α-chain is constitutively phosphorylated and necessary for cellular adhesion, but how the α-chain regulates adhesion has remained enigmatic. We now show that substitution of the α-chain phosphorylation site (S1140A) in T cells inhibits the phosphorylation of the functionally important Thr-758 in the β2-chain, binding of α-actinin and 14-3-3 protein, and expression of an integrin-activating epitope after treatment with the stromal cell-derived factor-1α. The presence of this substitution resulted in a loss of cell adhesion and directional cell migration. Moreover, LFA-1 activation through the T-cell receptor in cells expressing the S1140A LFA-1 variant resulted in less Thr-758 phosphorylation, α-actinin and talin binding, and cell adhesion. The finding that the LFA-1 α-chain regulates adhesion through the β-chain via specific phosphorylation at Ser-1140 in the α-chain has not been previously reported and emphasizes that both chains are involved in the regulation of LFA-1 integrin activity.

Dual functions of Rap1 are crucial for T-cell homeostasis and prevention of spontaneous colitis.

Rap1-GTP activates leukocyte function-associated antigen-1 (LFA-1) to induce arrest on the high endothelial venule (HEV). Here we show that Rap1-GDP restrains rolling behaviours of T cells on the peripheral lymph node addressin (PNAd), P-selectin and mucosal addressin cell adhesion molecule-1 (MadCAM-1) by inhibiting tether formation. Consequently, Rap1 deficiency impairs homing of naive T cells to peripheral lymph nodes, but accelerates homing of TH17 and TH1 cells to the colon, resulting in spontaneous colitis with tumours. Rap1-GDP associates with and activates lymphocyte-oriented kinase, which phosphorylates ERM (ezrin, radixin and moesin) in resting T cells. Phosphomimetic ezrin reduces the rolling of Rap1-deficient cells, and thereby decreases their homing into the colon. On the other hand, chemokines activate Rap1 at the plasma membrane within seconds, and Rap1-GTP binds to filamins, which diminishes its association with the β2 chain of LFA-1 and results in LFA-1 activation. This Rap1-dependent regulation of T-cell circulation prevents the onset of colitis.

TAGLN2 regulates T cell activation by stabilizing the actin cytoskeleton at the immunological synapse.

The formation of an immunological synapse (IS) requires tight regulation of actin dynamics by many actin polymerizing/depolymerizing proteins. However, the significance of actin stabilization at the IS remains largely unknown. In this paper, we identify a novel function of TAGLN2--an actin-binding protein predominantly expressed in T cells--in stabilizing cortical F-actin, thereby maintaining F-actin contents at the IS and acquiring LFA-1 (leukocyte function-associated antigen-1) activation after T cell receptor stimulation. TAGLN2 blocks actin depolymerization and competes with cofilin both in vitro and in vivo. Knockout of TAGLN2 (TAGLN2(-/-)) reduced F-actin content and destabilized F-actin ring formation, resulting in decreased cell adhesion and spreading. TAGLN2(-/-) T cells displayed weakened cytokine production and cytotoxic effector function. These findings reveal a novel function of TAGLN2 in enhancing T cell responses by controlling actin stability at the IS.

Regulation of integrin affinity on cell surfaces

Lymphocyte activation triggers adhesiveness of lymphocyte function-associated antigen-1 (LFA-1; integrin α(L)β(2)) for intercellular adhesion molecules (ICAMs) on endothelia or antigen-presenting cells. Whether the activation signal, after transmission through multiple domains to the ligand-binding αI domain, results in affinity changes for ligand has been hotly debated. Here, we present the first comprehensive measurements of LFA-1 affinities on T lymphocytes for ICAM-1 under a broad array of activating conditions. Only a modest increase in affinity for soluble ligand was detected after activation by chemokine or T-cell receptor ligation, conditions that primed LFA-1 and robustly induced lymphocyte adhesion to ICAM-1 substrates. By stabilizing well-defined LFA-1 conformations by Fab, we demonstrate the absolute requirement of the open LFA-1 headpiece for adhesiveness and high affinity. Interaction of primed LFA-1 with immobilized but not soluble ICAM-1 triggers energy-dependent affinity maturation of LFA-1 to an adhesive, high affinity state. Our results lend support to the traction or translational motion dependence of integrin activation.

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 Mn(2+) was replaced sequentially by Mg(2+) and Ca(2+). 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.

Blocking LFA-1 Activation with Lovastatin Prevents Graft-versus-Host Disease in Mouse Bone Marrow Transplantation

Graft-versus-host disease (GVHD) following bone marrow transplantation (BMT) is mediated by alloreactive donor T lymphocytes. Migration and activation of donor-derived T lymphocytes play critical roles in the development of GVHD. Leukocyte function-associated antigen-1 (LFA-1) regulates T cell adhesion and activation. We previously demonstrated that the I-domain, the ligand-binding site of LFA-1, changes from the low-affinity state to the high-affinity state on LFA-1 activation. Therapeutic antagonists, such as statins, inhibit LFA-1 activation and immune responses by modulating the affinity state of the LFA-1 I-domain. In the present study, we report that lovastatin blocked mouse T cell adhesion, proliferation, and cytokine production in vitro. Furthermore, blocking LFA-1 in the low-affinity state with lovastatin reduced the mortality and morbidity associated with GVHD in a murine BMT model. Specifically, lovastatin prevented T lymphocytes from homing to lymph nodes and Peyer's patches during the GVHD initiation phase and after donor lymphocyte infusion (DLI) after the establishment of GVHD. In addition, treatment with lovastatin impaired donor-derived T cell proliferation in vivo. Taken together, these results indicate the important role of lovastatin in the treatment of GVHD.

Loss of Kindlin-3 in LAD-III eliminates LFA-1 but not VLA-4 adhesiveness developed under shear flow conditions

AbstractLeukocyte adhesion deficiency (LAD)–III is associated with homozygous stop codon mutations in Kindlin-3, the hematopoietic member of the Kindlin family of integrin coactivators. In addition, a subgroup of LAD-III patients has a homozygous splice junction mutation in and reduced expression of the Rap-1 guanine nucleotide exchange factor, CalDAG-GEFI (CDGI). In this study, we compared the adhesive properties of the leukocyte function-associated antigen-1 (LFA-1) and very late activation antigen-4 (VLA-4) integrins in both primary and activated leukocytes derived from these 2 LAD-III subgroups. Primary lymphocytes lacking both Kindlin-3 and CDGI lost all firm T-cell receptor–stimulated LFA-1 adhesiveness, in contrast to LAD-III lymphocytes deficient in Kindlin-3 alone. Effector T cells expanded from all tested LAD-III variants expressed normal CDGI, but lacked Kindlin-3. These Kindlin-3–null effector T cells exhibited total loss of inside-out LFA-1 activation by chemokine signals as well as abrogated intrinsic LFA-1 adhesiveness. Surprisingly, VLA-4 in Kindlin-3–null resting or effector lymphocytes retained intrinsic rolling adhesions to vascular cell adhesion molecule-1 and exhibited only partial defects in chemokine-stimulated adhesiveness to vascular cell adhesion molecule-1. Deletion of the putative β1 Kindlin-3 binding site also retained VLA-4 adhesiveness. Thus, our study provides the first evidence that Kindlin-3 is more critical to LFA-1 than to VLA-4–adhesive functions in human lymphocytes.

Regulation of LFA-1–dependent inflammatory cell recruitment by Cbl-b and 14-3-3 proteins

AbstractInside-out signaling regulation of the β2-integrin leukocyte function–associated antigen-1 (LFA-1) by different cytoplasmic proteins, including 14-3-3 proteins, is essential for adhesion and migration of immune cells. Here, we identify a new pathway for the regulation of LFA-1 activity by Cbl-b, an adapter molecule and ubiquitin ligase that modulates several signaling pathways. Cbl-b−/− mice displayed increased macrophage recruitment in thioglycollate-induced peritonitis, which was attributed to Cbl-b deficiency in macrophages, as assessed by bone marrow chimera experiments. In vitro, Cbl-b−/− bone marrow–derived mononuclear phagocytes (BMDMs) displayed increased adhesion to endothelial cells. Activation of LFA-1 in Cbl-b–deficient cells was responsible for their increased endothelial adhesion in vitro and peritoneal recruitment in vivo, as the phenotype of Cbl-b deficiency was reversed in Cbl-b−/−LFA-1−/− mice. Consistently, LFA-1–mediated adhesion of BMDM to ICAM-1 but not VLA-4–mediated adhesion to VCAM-1 was enhanced by Cbl-b deficiency. Cbl-b deficiency resulted in increased phosphorylation of T758 in the β2-chain of LFA-1 and thereby in enhanced association of 14-3-3β protein with the β2-chain, leading to activation of LFA-1. Consistently, disruption of the 14-3-3/β2-integrin interaction abrogated the enhanced ICAM-1 adhesion of Cbl-b−/− BMDMs. In conclusion, Cbl-b deficiency activates LFA-1 and LFA-1–mediated inflammatory cell recruitment by stimulating the interaction between the LFA-1 β-chain and 14-3-3 proteins.

Activation of the Small GTPase Rac2 via the B Cell Receptor Regulates B Cell Adhesion and Immunological-Synapse Formation

The integrin leukocyte function-associated antigen-1 (LFA-1) is important in the promotion of B cell adhesion, thereby facilitating immunological synapse (IS) formation and B cell activation. Despite this significance, the associated signaling mechanisms regulating LFA-1 activation remain elusive. Here, we show that both isoforms of the small GTPase Rac expressed by primary B cells, Rac1 and Rac2, were activated rapidly downstream of Src-family kinases, guanine-nucleotide exchange factors Vav1 and Vav2, and phosphoinositide-3 kinase (PI3K) after BCR engagement. We identify Rac2, but not Rac1, as critical for B cell adhesion to intercellular adhesion molecule-1 (ICAM-1) and IS formation. Furthermore, B cells expressing constitutively active Rac2 are highly adhesive. We observe that Rac2-deficient B cells exhibit lower amounts of Rap1-GTP and severe actin polymerization defects, identifying a potential mechanism underlying their behavior. We postulate that this critical role for Rac2 in mediating B cell adhesion and IS formation might apply in all lymphocytes.

309: Blocking LFA-1 activation with lovastatin prevents graft-versus-host disease in mouse bone marrow transplantation

Leukocyte function associated antigen-1 (LFA-1) regulates T cell adhesion and activation. LFA-1 is constitutively expressed on cell surface in an inactive state. The control of LFA-1 activation is critical in inflammatory and immune responses. We demonstrated previously that the I-domain, the ligand binding site of LFA-1, changes from the low-affinity state to high-affinity state upon activation. Therapeutic antagonist, such as lovastatin, stabilizes the I-domain in the low-affinity state and inhibits the LFA-1 activation. Here, we report that lovastatin can block mouse T cell adhesion and proliferation in vitro. First, we demonstrated that lovastatin treatment reduced the mortality and morbidity in the mouse GVHD model. Lovastatin treatment significantly decreased GVHD mortality with 80% mice survived over 28 days, whereas more than 70% of the control mice died within the first 10 days, and the p values was 0.045. There were significantly reduced tissue damages in the skin, intestine and liver of lovastatin-treated mice. Second, we found lovastatin treatment reduced donor T cell homing to lymph nodes. There was a 65% reduction of CD4+ T cells homing to lymph nodes in lovastatin treatment group compared to control. The reduction of CD8+ T cells was greater with about 76% less cells homing to lymph nodes in the lovastatin treatment group. Third, we found lovastatin treatment reduced donor-derived T cell proliferation in vivo. There were 37% CD4+ and 31% CD8+ T cells remained undivided in the lymph nodes of the control mice at day 4 post-transplant. The lovastatin-treated mice had reduced proliferation kinetics of both CD4+ and CD8+ T cells with about 55% and 42% remained undivided. In the control lymph nodes, there were 42% CD4+ and 59% CD8+ T cells proliferated beyond 5th and 6th cell-divisions respectively, whereas the lovastatin treatment reduced the number to 31% and 48% respectively. In summary, we demonstrated here that lovastatin prevents both homing and proliferating of donor-derived T cells in the secondary lymphoid organs, which are crucial sites for alloreactive expansion. While most of the control mice died of acute GVHD within the first week of post-transplant when alloreactive T cells infiltrated the targeted organs, lovastatin treatment prevented the activation and expansion of donor-derived T cells, and thus reduced the GVHD mortality and morbidity. Our study provides rationale for a potential novel treatment for GVHD.

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.

Somatostatin down-regulates LFA-1 activation by modulating Rap1 expression in CD4 + and CD8 + T cells

Leukocyte function-associated antigen-1 (LFA-1) is one of the integrins that are expressed on the leukocytes, and has been shown to play an important role in leukocyte trafficking. The adhesive activity of LFA-1 is governed partially by the Rap1. This study examined that the relationship between LFA-1 and Rap1 mRNA expressions by anti-CD3 and anti-CD3+SOM treatment in the CD4 + and CD8 + T cells. The LFA-1 mRNA expression levels following the anti-CD3 and anti-CD3+SOM treatment for 30 min was greater on the CD8 + T cells, and the LFA-1 expression of the CD8 + T cells with anti-CD+SOM treatment was affected more severely than that of the CD4 + T cells. The Rap1 mRNA expression patterns following anti-CD3 and anti-CD3+SOM stimulation in the CD4 + and CD8 + T cells were similar to the LFA-1 expression patterns, and the expression level following anti-CD3+SOM treatment was suppressed more significantly in the CD8 + T cells. These results suggest that the difference in the Rap1 expression level after stimulation might explain the differences in the LFA-1 expression level on the T cell subsets, and that the down-regulation of Rap1 expression following SOM treatment is closely related to the diminished LFA-1 expression.

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.

Novel p-arylthio cinnamides as antagonists of leukocyte function-associated antigen-1/intracellular adhesion molecule-1 interaction. 2. Mechanism of inhibition and structure-based improvement of pharmaceutical properties.

The interaction between leukocyte function-associated antigen-1 (LFA-1) and intracellular adhesion molecule-1 (ICAM-1) has been implicated in inflammatory and immune diseases. Recently, a novel series of p-arylthio cinnamides has been described as potent antagonists of the LFA-1/ICAM-1 interaction. These compounds were found to bind to the I domain of LFA-1 using two-dimensional NMR spectroscopy of 15N-labeled LFA-1 I domain. On the basis of NOE studies between compound 1 and the I domain of LFA-1, a model of the complex was constructed. This model revealed that compound 1 does not directly inhibit ICAM-1 binding by interacting with the metal ion dependent adhesion site (MIDAS). Instead, it binds to the previously proposed I domain allosteric site (IDAS) of LFA-1 and likely modulates the activation of LFA-1 through its interaction with this regulatory site. A fragment-based NMR screening strategy was applied to identify small, more water-soluble ligands that bind to a specific region of the IDAS. When incorporated into the parent cinnamide template, the resulting analogues exhibited increased aqueous solubility and improved pharmacokinetic profiles in rats, demonstrating the power of this NMR-based screening approach for rapidly modifying high-affinity ligands.

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.

NMR solution structure of the inserted domain of human leukocyte function associated antigen-1

The interaction between the leukocyte function-associated antigen-1 (LFA-1) and the intercellular adhesion molecule is thought to be mediated primarily via the inserted domain (I-domain) in the α-subunit. The activation of LFA-1 is an early step in triggering the adhesion of leukocytes to target cells decorated with intercellular adhesion molecules. There is some disagreement in the literature over the respective roles of conformational changes in the I-domain and of divalent cations (Mg 2+, Mn 2+) in the activation of LFA-1 for intercellular adhesion molecule binding. X-ray crystallographic structures of the I-domains of LFA-1 and Mac-1 in the presence and absence of cations show structural differences in the C-terminal α-helix; this change was proposed to represent the active and inactive conformations of the I-domain. However, more recent X-ray results have called this proposal into question. The solution structure of the Mg 2+ complex of the I-domain of LFA-1 has been determined by NMR methods, using a model-based approach to nuclear Overhauser enhancement spectroscopy peak assignment. The protein adopts the same structure in solution as that of the published I-domain X-ray structures, but the C-terminal region, where the X-ray structures are most different from each other, is different again in the solution structures. The secondary structure of this helix is well formed, but NMR relaxation data indicate that there is considerable flexibility present, probably consisting of breathing or segmental motion of the helix. The conformational diversity seen in the various X-ray structures could be explained as a result of the inherent flexibility of this C-terminal region and as a result of crystal contacts. Our NMR data are consistent with a model where the C-terminal helix has the potential flexibility to take up alternative conformations, for example, in the presence and absence of the intercellular adhesion molecule ligand. The role of divalent cations appears from our results not to be as a direct mediator of a conformational change that alters affinity for the ligand. Rather, the presence of the cation appears to be involved in some other way in ligand binding, perhaps by acting as a bridge to the ligand and by modulation of the charge of the binding surface.

Constitutive Chemokine Production Results in Activation of Leukocyte Function-Associated Antigen-1 on Adult T-Cell Leukemia Cells

AbstractAdult T-cell leukemia (ATL) is characterized by massive infiltration of circulating ATL cells into a variety of tissues, a finding often associated with poor prognosis. Leukocyte migration from circulation into tissue depends on integrin-mediated adhesion to endothelium, and integrins are tightly regulated by several stimuli, such as inflammatory chemokines. However, the exact mechanisms that enhance adherence of leukemic cells to the endothelium and infiltration into tissues remain to be fully understood. We investigated the mechanisms of extravasation of leukemic cells using ATL cells and report the following novel features of endogenous chemokine-induced adhesion of ATL cells to the endothelium. ATL cells spontaneously adhered to endothelial cells without exogenous stimulation. Integrin leukocyte function-associated antigen-1 (LFA-1) on ATL cells was spontaneously activated. ATL cells produced high amounts of chemokines, macrophage inflammatory protein-1α (MIP-1α), and MIP-1β. Adhesion of ATL cells to endothelial cells and the expression of activated form of LFA-1 were reduced by pretreatment with pertussis toxin, wortmannin, or anti–MIP-1α and MIP-1β antibodies or transfection with antisense of MIP-1α or MIP-1β. Spontaneous polymerization of cytoskeletal F-actin was observed in ATL cells, which was also inhibited by pertussis toxin and wortmannin. We propose that ATL cells adhere to endothelial cells through an adhesion cascade similar to normal leukocytes and that the chemokines produced by ATL cells are involved in triggering integrin LFA-1 through cytoskeletal rearrangement induced by G-protein–dependent activation of phosphoinositide 3-kinases in an autocrine manner. These events result in a strong adhesion of ATL cells to the endothelium and spontaneous transendothelial migration.

Constitutive Chemokine Production Results in Activation of Leukocyte Function-Associated Antigen-1 on Adult T-Cell Leukemia Cells

Adult T-cell leukemia (ATL) is characterized by massive infiltration of circulating ATL cells into a variety of tissues, a finding often associated with poor prognosis. Leukocyte migration from circulation into tissue depends on integrin-mediated adhesion to endothelium, and integrins are tightly regulated by several stimuli, such as inflammatory chemokines. However, the exact mechanisms that enhance adherence of leukemic cells to the endothelium and infiltration into tissues remain to be fully understood. We investigated the mechanisms of extravasation of leukemic cells using ATL cells and report the following novel features of endogenous chemokine-induced adhesion of ATL cells to the endothelium. ATL cells spontaneously adhered to endothelial cells without exogenous stimulation. Integrin leukocyte function-associated antigen-1 (LFA-1) on ATL cells was spontaneously activated. ATL cells produced high amounts of chemokines, macrophage inflammatory protein-1α (MIP-1α), and MIP-1β. Adhesion of ATL cells to endothelial cells and the expression of activated form of LFA-1 were reduced by pretreatment with pertussis toxin, wortmannin, or anti–MIP-1α and MIP-1β antibodies or transfection with antisense of MIP-1α or MIP-1β. Spontaneous polymerization of cytoskeletal F-actin was observed in ATL cells, which was also inhibited by pertussis toxin and wortmannin. We propose that ATL cells adhere to endothelial cells through an adhesion cascade similar to normal leukocytes and that the chemokines produced by ATL cells are involved in triggering integrin LFA-1 through cytoskeletal rearrangement induced by G-protein–dependent activation of phosphoinositide 3-kinases in an autocrine manner. These events result in a strong adhesion of ATL cells to the endothelium and spontaneous transendothelial migration.