Lymphocyte Function-associated Antigen-1 Inhibition Research Articles

LFA-1/ICAM-1 Interaction as a Therapeutic Target in Dry Eye Disease.

Dry eye disease (DED) is a common ocular disorder associated with inflammation of the lacrimal gland and ocular surface. The interaction of the integrin lymphocyte function-associated antigen-1 (LFA-1) with its cognate ligand intercellular adhesion molecule-1 (ICAM-1) is known to have important roles in the interaction of a variety of cells involved in immune responses and inflammation, including those prominent in ocular surface inflammation. Lifitegrast, an LFA-1 antagonist that blocks binding of ICAM-1 to LFA-1, has recently been approved in the United States for the treatment of signs and symptoms of DED. In this review, we evaluate research findings to explore the potential role of LFA-1/ICAM-1 interaction in the pathophysiology of DED, and the evidence supporting LFA-1/ICAM-1 interaction as a rational therapeutic target in DED. The results of our review suggest that LFA-1/ICAM-1 interaction may play important roles in the cell-mediated immune response and inflammation associated with DED, including facilitating the homing of dendritic cells to the lymph nodes, interaction of dendritic cells with T cells and subsequent T cell activation/differentiation, migration of activated CD4+ T cells from the lymph nodes to the ocular surface, reactivation of T cells by resident antigen-presenting cells at the ocular surface, and recruitment and retention of LFA-1-expressing T cells in the conjunctival epithelium. Based on the available evidence, inhibition of LFA-1/ICAM-1 interaction represents a rational targeted approach in treating DED. Notably, inhibition of LFA-1/ICAM-1 binding with lifitegrast offers a novel approach to reducing ocular surface inflammation in this condition.

The Effects of LFA-1 Antagonism on Protein Sorting and T Cell Triggering

The large-scale spatial patterning of receptor-ligand pairs at intermembrane spaces are increasingly studied due to their contribution to cell signaling and downstream events. One example, the immunological synapse (IS), is a key event in the immune response. Crucial to the overall pattern at the T cell/antigen presenting cell (APC) interface are submicron clusters that become spatially organized to initiate T cell triggering. The T cell receptor (TCR) and its ligand, activating peptide-bound major histocompatibility complex (pMHC), form a large central cluster surrounded by a peripheral ring of lymphocyte function-associated antigen-1 (LFA-1) bound to its ligand intercellular adhesion molecule-1 (ICAM-1). These receptor-ligand interactions catalyze submicron clustering and subsequent centripetal transport by the actin cytoskeleton. In this work, we replace the APC with a biomimetic, fluid supported membrane with easily controlled protein composition. Using fluorescent microscopy visualization techniques, we have developed a reliable and robust assay to test the effects of LFA-1 inhibition. We hypothesize that LFA-1 antagonism through the use of a small-molecule inhibitor or an anti-LFA-1 antibody will alter protein sorting and T cell triggering. Unexpectedly, the small-molecule antagonist affects TCR spatial patterning in addition to the predicted disruption of LFA-1 ring formation. We further investigate this by studying the effects on actin morphology, calcium signaling, and IL-2 secretion. Our results provide a fundamental understanding of LFA-1 antagonism on the IS, TCR signaling, and actin behavior. In addition, our research can impact the development and analysis of drugs for immune-mediated disorders, which frequently target LFA-1 and inhibit ligand binding.

Sevoflurane Binds and Allosterically Blocks Integrin Lymphocyte Function-associated Antigen-1

Volatile anesthetics have been shown to modify immune cell functions via several mechanisms, some of which have been only partially elucidated. We demonstrated that isoflurane inhibits primary leukocyte integrin lymphocyte function-associated antigen-1 (LFA-1) by binding to the allosteric cavity critical for conformational activation to its high-affinity form. It remains to be determined whether the allosteric inhibition of LFA-1 by isoflurane can be generalized to other anesthetics such as sevoflurane. The effects of sevoflurane on the ability of LFA-1 to bind to its counter-ligand, intercellular adhesion molecule-1, was studied in leukocytes by flow cytometry. To examine whether sevoflurane acts directly on LFA-1, we measured ligand-binding using beads coated with purified LFA-1 protein. To distinguish between competitive versus allosteric inhibition, we analyzed the effects of sevoflurane on both wild-type and mutant-locked high-affinity LFA-1. One-way analysis of variance was employed for statistical analysis of the data. Nuclear magnetic resonance spectroscopy was used to identify sevoflurane binding site(s). Sevoflurane at clinically relevant concentrations inhibited the ligand-binding function of LFA-1 in leukocytes as well as in cell-free assays (P<0.05). Sevoflurane blocked wild-type but not locked high-affinity LFA-1, thereby demonstrating an allosteric mode of inhibition. Nuclear magnetic resonance spectroscopy revealed that sevoflurane bound to the allosteric cavity, to which LFA-1 allosteric antagonists and isoflurane also bind. This study suggests that sevoflurane also blocks the activation-dependent conformational changes of LFA-1 to the high-affinity form. The allosteric mode of action exemplified by sevoflurane and isoflurane via LFA-1 might represent one of the underlying mechanisms of anesthetic-mediated immunomodulation.

Efalizumab binding to the LFA-1 αLI domain blocks ICAM-1 binding via steric hindrance

Lymphocyte function-associated antigen 1 (LFA-1) plays important roles in immune cell adhesion, trafficking, and activation and is a therapeutic target for the treatment of multiple autoimmune diseases. Efalizumab is one of the most efficacious antibody drugs for treating psoriasis, a very common skin disease, through inhibition of the binding of LFA-1 to the ligand intercellular adhesion molecule 1 (ICAM-1). We report here the crystal structures of the Efalizumab Fab alone and in complex with the LFA-1 alpha(L) I domain, which reveal the molecular mechanism of inhibition of LFA-1 by Efalizumab. The Fab binds with an epitope on the inserted (I) domain that is distinct from the ligand-binding site. Efalizumab binding blocks the binding of LFA-1 to ICAM-1 via steric hindrance between its light chain and ICAM-1 domain 2 and thus inhibits the activities of LFA-1. These results have important implications for the development of improved antibodies and new therapeutic strategies for the treatment of autoimmune diseases.

Allosteric LFA-1 Inhibitors Modulate Natural Killer Cell Function

Natural killer (NK) cells are believed to play an important role in a variety of disease pathologies, including transplant rejection and autoimmunity. None of the therapeutic modalities currently available are known to potently interfere with NK cell activity. Here we demonstrate for the first time that low molecular weight inhibitors of the integrin lymphocyte function-associated antigen-1 (LFA-1) readily block NK cell adhesion, activation, and NK cell-mediated cytolysis in vitro, in contrast to other immunosuppressive agents. These effects were independent of the type of allosteric mechanism by which LFA-1 inhibition was achieved. In addition, we describe a simple, nonradioactive whole-blood assay that should be suitable to monitor NK cell activation in clinical practice. Taken together, our study underlines the importance of LFA-1 in NK cell effector functions and indicates that allosteric LFA-1 inhibitors may become important tools to further elucidate the therapeutic potential of NK cell modulation in immunological diseases.

Statins inhibit lymphocyte homing to peripheral lymph nodes

Lymphocyte homing to peripheral lymph nodes is governed by adhesion molecules, including lymphocyte function-associated antigen 1 (LFA-1). Statins are 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors and exert anti-inflammatory effects, e.g. inhibition of LFA-1. It is still not known whether statin compounds are capable of inhibiting lymphocyte homing in vivo. We used a cervical lymph node preparation to study the effects of simvastatin on lymphocyte adhesion to high endothelial venules (HEVs) by means of intravital fluorescence microscopy (IVM). IVM revealed that firm adhesion of lymphocytes to HEV endothelium critically depends on the adhesive function of LFA-1. The number of firmly adherent lymphocytes was reduced by 58% in LFA-1-deficient mice (P < 0.05 versus wild-type controls). As in mutant mice, acute treatment with simvastatin (i.p. injection at 2 hr prior to IVM) inhibited the firm adhesion of lymphocytes to HEV endothelium of wild-type animals by 63% (P < 0.05 versus vehicle-treated wild-type controls). In addition, acute treatment with the synthetic statin-derivate LFA878 also reduced firm lymphocyte adhesion in HEVs by 63% (P > 0.05 versus placebo-treated controls). Histological analysis after a 10-day treatment with simvastatin showed reduced cellularity of cervical lymph nodes, as indicated by a reduction of the relative area of haematoxylin-stained cell nuclei in cervical lymph node cross-sections from 94 +/- 0% in vehicle-treated controls to 77 +/- 3% in simvastatin-treated mice (P < 0.05). We conclude that statin compounds are capable of inhibiting lymphocyte homing to murine peripheral lymph nodes in vivo. This may have novel implications for the treatment of adaptive immune responses, e.g. transplant rejection.

Improved Lymphocyte Function-associated Antigen-1 (LFA-1) Inhibition by Statin Derivatives: MOLECULAR BASIS DETERMINED BY X-RAY ANALYSIS AND MONITORING OF LFA-1 CONFORMATIONAL CHANGES IN VITRO AND EX VIVO

The integrin lymphocyte function-associated antigen-1 (LFA-1) (alphaLbeta2; CD11a/CD18) plays an important role in leukocyte migration and T cell activation. LFA-1 is inhibited by the cholesterol-lowering drug lovastatin, which binds to an allosteric site of the alphaL I domain termed the lovastatin site (L-site). Here we report for the first time the x-ray structures of the LFA-1 I domain complexed with derivatives of lovastatin optimized for LFA-1 inhibition. This analysis identified two new subpockets within the L-site occupied by chemical groups of the statin derivatives but not by lovastatin itself. Occupancy of these L-site subpockets led to distinct conformational changes in LFA-1, which were detectable by an epitope-monitoring assay. We utilized this assay to demonstrate improved LFA-1 inhibition in human blood in vitro and in blood samples from treated animals ex vivo. Moreover, we demonstrate that the novel lovastatin-derived LFA-1 inhibitor LFA878 exhibits potent anti-inflammatory effects in carrageenan-induced rat paw edema. In summary, the findings reported here extend the understanding of LFA-1 inhibition at the molecular level, allow for the identification and design of LFA-1 inhibitors of further enhanced potency, and support the expectation that LFA-1 inhibitors binding to the L-site will be of therapeutic value in treating inflammatory diseases.

Selective in vivo growth of lymphocyte function- associated antigen-1-positive murine myeloma cells. Involvement of function-associated antigen-1-mediated homotypic cell-cell adhesion.

Lymphocyte function-associated antigen-1 (LFA-1) expression on multiple myeloma cells and its potential role in myeloma biology have been the subject of conflicting literature reports. In this study we used the 5T experimental mouse model to analyze the involvement of LFA-1 in myeloma cell bone marrow homing, survival, and growth. The 5T33MM vitro (5T33MMvt) myeloma line was used. LFA-1 and intercellular adhesion molecule-1 (ICAM-1) expression were analyzed by flow cytometry. A small molecule antagonist of LFA-1/ICAM interactions, BIRT 377, was used to block LFA-1 in vitro. Transendothelial migration was assessed by measuring migration through Transwells coated with bone marrow endothelial cells. Immediate in vivo homing was analyzed by tracing 51Cr-labeled cells. Invert microscopic cell counting was used to analyze homotypic cell adhesion. Cell cycle analysis was used to analyze apoptosis. S+G(2)/M phase analysis and 3H-thymidine incorporation were used to assess proliferation. Cells were separated into LFA-1(+) and LFA-1(-) fraction by magnetic activated cell sorting. 5T33MMvt cells had a heterogeneous LFA-1 expression and all cells were positive for the LFA-1 ligand ICAM-1. LFA-1 inhibition with BIRT 377 did not affect transendothelial migration of the 5T33MMvt cells; however, it did result in cell cluster scattering, indicating LFA-1 involvement in homotypic cell-cell adhesion. No effect was observed on apoptosis, but the percentage of cells in S+G(2)/M phase was decreased by 39%. 3H-thymidine incorporation confirmed this effect on 5T33MMvt cell proliferation (38% reduction). When 5T33MMvt cells were injected into animals, all myeloma cells isolated at the end stage of the disease were LFA-1(+) in contrast to the situation before injection. LFA-1(+) and LFA-1(-) MM cells had similar in vivo bone marrow homing capacities. Mice injected with LFA-1(+) 5T33MMvt cells developed myeloma (5/5) within 12 weeks after injection. In contrast, LFA-1(-) recipients did not develop the disease (0/5), even 1 year after tumor inoculation. Our data suggest that LFA-1-mediated homotypic cell-cell adhesion is involved in myeloma cell proliferation and raises the possibility that this interaction may have a crucial role in in vivo myeloma cell growth. LFA-1 does not appear to play a role in the bone marrow homing of these cells.

Lymphocyte function-associated antigen-1 blockade by statins: molecular basis and biological relevance.

Lymphocyte function-associated antigen-1 (LFA-1) belongs to the integrin family and plays an important role in leukocyte trafficking and in T-cell activation. Random screening of chemical libraries identified the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor lovastatin as an inhibitor of the LFA-1/intercellular adhesion molecule (ICAM)-1 interaction. The effect of lovastatin on LFA-1 was found to be unrelated to the inhibition of HMG-CoA reductase and to be mediated by lovastatin binding to a novel allosteric site within LFA-1. The biological relevance of LFA-1 inhibition by statins with respect to the overall benefit of this drug class is reviewed. The implications of the statin effect on LFA-1 for future drug design and therapy are discussed.

Small Molecule Inhibitors Induce Conformational Changes in the I Domain and the I-like Domain of Lymphocyte Function-associated Antigen-1: MOLECULAR INSIGHTS INTO INTEGRIN INHIBITION

The beta(2) integrin lymphocyte function-associated antigen-1 (LFA-1) is a conformationally flexible alpha/beta heterodimeric receptor, which is expressed on the surface of all leukocytes. LFA-1 mediates cell adhesion crucial for normal immune and inflammatory responses. Intracellular signals or cations are required to convert LFA-1 from a nonligand binding to a ligand binding state. Here we investigated the effect of small molecule inhibitors on LFA-1 by monitoring the binding of monoclonal antibodies mapped to different receptor domains. The inhibitors were found to not only induce epitope changes in the I domain of the alpha(L) chain but also in the I-like domain of the beta(2) chain depending on the individual chemical structure of the inhibitor and its binding site. For the first time, we provide strong evidence that the I-like domain represents a target for allosteric LFA-1 inhibition similar to the well established regulatory L-site on the I domain of LFA-1. Moreover, the antibody binding patterns observed in the presence of the various inhibitors establish a conformational interaction between the LFA-1 I domain and the I-like domain in the native receptor that is formed upon activation. Differentially targeting the binding sites of the inhibitors, the L-site and the I-like domain, may open new avenues for highly specific therapeutic intervention in diseases where integrins play a pathophysiological role.

Different roles for LFA-1 and VLA-4 integrins in T-B-cell interactions in vivo.

Adhesion molecules are critical in the cellular interactions involved in specific immune responses. They are used for homing, cell migration, cell-cell contact and, in some cases, for the delivery of costimulatory signals. Since the host-versus-graft (HVG) reaction represents a particular form of T-B-cell interaction, we have explored whether the inhibition of lymphocyte function-associated antigen-1/intracellular adhesion molecule-1 (LFA-1/ICAM-1) interactions and the signalling through very late activation antigen-4 (VLA-4) have any effect on the development of a lupus-like disease in BALB/c mice injected at birth with (BALB/cxC57BL/6)F1 spleen cells. In close association with the development of tolerance to donor allografts, these mice show a polyclonal activation of F1 donor B cells by alloreactive host CD4+ T cells, manifested by the production of autoantibodies (autoAbs) and the development of a mild glomerulonephritis. The dose of the monoclonal antibody (mAb) employed has been adjusted to block completely the molecule on the surface of peripheral lymphocytes without interfering with the induction of neonatal tolerance. Injection of saturating doses (100 microg/2 days) of either anti-LFA-1alpha or anti-ICAM-1 mAbs, but not anti-VLA-4alpha or anti-LFA-1beta mAbs, blocks the production of anti-ssDNA autoAbs and the thrombocytopenia characteristic of this HVG disease (HVGD). However, anti-VLA-4alpha treatment is only able to delay the production of autoAbs and the anti-LFA-1beta treatment, not to modify the evolution of the HVGD. These results point to the relevance of LFA-1/ICAM-1 interactions, but not of the VLA-4-mediated signal, in the polyclonal B-cell activation occurring during the allogeneic interactions between host T helper type 2 cells and donor B cells in HVGD.