LFA-1 Interaction Research Articles

CAR-mediated targeting of NK cells overcomes tumor immune escape caused by ICAM-1 downregulation

BackgroundThe antitumor activity of natural killer (NK) cells can be enhanced by specific targeting with therapeutic antibodies that trigger antibody-dependent cell-mediated cytotoxicity (ADCC) or by genetic engineering to express chimeric...

CAR T-cell Entry into Tumor Islets Is a Two-Step Process Dependent on IFNγ and ICAM-1.

Adoptive transfer of T cells expressing chimeric antigen receptors (CAR) has shown remarkable clinical efficacy against advanced B-cell malignancies but not yet against solid tumors. Here, we used fluorescent imaging microscopy and ex vivo assays to compare the early functional responses (migration, Ca2+, and cytotoxicity) of CD20 and EGFR CAR T cells upon contact with malignant B cells and carcinoma cells. Our results indicated that CD20 CAR T cells rapidly form productive ICAM-1-dependent conjugates with their targets. By comparison, EGFR CAR T cells only initially interacted with a subset of carcinoma cells located at the periphery of tumor islets. After this initial peripheral activation, EGFR CAR T cells progressively relocated to the center of tumor cell regions. The analysis of this two-step entry process showed that activated CAR T cells triggered the upregulation of ICAM-1 on tumor cells in an IFNγ-dependent pathway. The ICAM-1/LFA-1 interaction interference, through antibody or shRNA blockade, prevented CAR T-cell enrichment in tumor islets. The requirement for IFNγ and ICAM-1 to enable CAR T-cell entry into tumor islets is of significance for improving CAR T-cell therapy in solid tumors.

Retinoic acid exerts disease stage-dependent and tissue-specific effects on pristane-induced lupus

Abstract We previously showed that all-trans-retinoic acid (tRA) exacerbated pre-existing autoimmunity in lupus; however, its effects before disease onset are unknown. Here, using a pristane-induced model, we show that tRA exerts tissue-specific effects when given at the initiation vs. continuation phase of lupus. Pre-pristane treatment with tRA aggravated glomerulonephritis through increasing renal expression of pro-fibrotic protein laminin β1, activating bone marrow CD11b- conventional dendritic cells, increasing the splenic CD4:CD8 ratio, and upregulating the interaction of ICAM-1 and LFA-1 that led to the infiltration of inflammatory cells to the spleen. Transcriptomic analysis revealed that prior to lupus induction, tRA significantly upregulated genes associated with cell differentiation, activation, and migration. Moreover, compared to pristane alone, tRA pre-treatment potentiated, whereas tRA post-treatment significantly suppressed, the renal expression of proinflammatory TNF-α, IL-1β, CCL2, and CCL3. While it may benefit the kidney, post-pristane treatment with tRA worsened the onset and severity of pristane-induced arthritis through promoting neutrophil and mononuclear cell infiltration into the joints. Together, these findings suggest that tRA supplementation regardless of the time of administration promotes chemokine-driven migration and tissue-specific infiltration of inflammatory cells, thereby exacerbating lupus-associated kidney or joint inflammation. Interestingly, both pre- and post-treatments with tRA modulated the gut microbiota in a similar fashion, suggesting a gut microbiota-independent mechanism by which tRA affects the initiation vs. continuation phase of lupus.

A Multivalent ICAM-1 Binding Nanoparticle which Inhibits ICAM-1 and LFA-1 Interaction Represents a New Tool for the Investigation of Autoimmune-Mediated Dry Eye.

The autoimmune disorder, Sjögren’s syndrome (SS), is characterized by lymphocytic infiltration and loss of function of exocrine glands such as the lacrimal gland (LG) and salivary gland. SS-associated changes in the LG are associated with the development of autoimmune-mediated dry eye disease. We have previously reported the accumulation of intercellular adhesion molecule 1 (ICAM-1) in the LG of Non-Obese Diabetic (NOD) mice, a murine model of autoimmune-mediated dry eye in SS, in both LG acinar cells and infiltrating lymphocytes. ICAM-1 initiates T-cell activation and can trigger T-cell migration through binding to lymphocyte function-associated 1 antigen (LFA). To modulate this interaction, this study introduces a new tool, a multivalent biopolymeric nanoparticle assembled from a diblock elastin-like polypeptide (ELP) using the S48I48 (SI) ELP scaffold fused with a mouse ICAM-1 targeting peptide to form IBP-SI. IBP-SI forms a multivalent, monodisperse nanoparticle with a radius of 21.9 nm. Unlike the parent SI, IBP-SI binds mouse ICAM-1 and is internalized by endocytosis into transfected HeLa cells before it accumulates in lysosomes. In vitro assays measuring lymphocyte adhesion to Tumor Necrosis Factor TNF-α-treated bEnd.3 cells, which express high levels of ICAM-1, show that adhesion is inhibited by IBP-SI but not by SI, with IC50 values of 62.7 μM and 81.2 μM, respectively, in two different assay formats. IBP-SI, but not SI, also blocked T-cell proliferation in a mixed lymphocyte reaction by 74% relative to proliferation in an untreated mixed cell reaction. These data suggest that a biopolymeric nanoparticle with affinity for ICAM-1 can disrupt ICAM-1 and LFA interactions in vitro and may have further utility as an in vivo tool or potential therapeutic.

Cleaved endocan acts as a biologic competitor of endocan in the control of ICAM-1-dependent leukocyte diapedesis.

Dysregulated leukocyte diapedesis is a major contributor to acute severe inflammatory states like sepsis and acute respiratory distress syndrome, which are common conditions in critically ill subjects. Endocan is a circulating proteoglycan that binds to the leukocyte integrin LFA-1 and blocks its interaction with its endothelial ligand ICAM-1, subsequently leading to the inhibition of leukocyte recruitment. Recent data have highlighted the hypothetic role of p14, endocan's major catabolite found in the bloodstream of septic patients, as a potential antagonist of endocan, thus participating in the regulation of acute inflammation. We hereby characterize the role of p14 as a biologic competitor of endocan, through assessment of its molecular interactions with LFA-1, endocan, and ICAM-1, as well as its effects on human leukocyte trafficking. Using immunodetection assay, we report that p14 can bind to LFA-1, thus inhibiting the interaction between LFA-1 and endocan, which in turn leads to the restoration of the ICAM-1/LFA-1 interaction. In primary human T cells trafficking assays, we underline the absence of effect of p14 on ICAM-1-dependent adhesion and migration, as well as on transendothelial migration. However, in those models, p14 reverses the antimigratory effect of endocan. To conclude, our study supports the hypothesis of an antagonistic role of p14 versus endocan in its effect on the LFA-1/ICAM-1-dependent human leukocyte recruitment.

Retinoic Acid Exerts Disease Stage-Dependent Effects on Pristane-Induced Lupus.

We previously showed that all-trans-retinoic acid (tRA), an active metabolite of vitamin A, exacerbated pre-existing autoimmunity in lupus; however, its effects before the development of autoimmunity are unknown. Here, using a pristane-induced model, we show that tRA exerts differential effects when given at the initiation vs. continuation phase of lupus. Unlike tRA treatment during active disease, pre-pristane treatment with tRA aggravated glomerulonephritis through increasing renal expression of pro-fibrotic protein laminin β1, activating bone marrow conventional dendritic cells (cDCs), and upregulating the interaction of ICAM-1 and LFA-1 in the spleen, indicating an active process of leukocyte activation and trafficking. Transcriptomic analysis revealed that prior to lupus induction, tRA significantly upregulated the expression of genes associated with cDC activation and migration. Post-pristane tRA treatment, on the other hand, did not significantly alter the severity of glomerulonephritis; rather, it exerted immunosuppressive functions of decreasing circulatory and renal deposition of autoantibodies as well as suppressing the renal expression of proinflammatory cytokines and chemokines. Together, these findings suggest that tRA differentially modulate lupus-associated kidney inflammation depending on the time of administration. Interestingly, both pre- and post-pristane treatments with tRA reversed pristane-induced leaky gut and modulated the gut microbiota in a similar fashion, suggesting a gut microbiota-independent mechanism by which tRA affects the initiation vs. continuation phase of lupus.

Kupffer Cells Promote the Differentiation of Adult Liver Hematopoietic Stem and Progenitor Cells into Lymphocytes via ICAM-1 and LFA-1 Interaction.

It has been reported that the adult liver contains hematopoietic stem and progenitor cells (HSPCs), which are associated with long-term hematopoietic reconstitution activity. Hepatic hematopoiesis plays an important role in the generation of cells involved in liver diseases. However, how the progenitors differentiate into functional myeloid cells and lymphocytes in the liver microenvironment remains unknown. In the present study, HSPC transplantation experiments were used to confirm that adult murine liver HSPCs differentiate into both myeloid cells and lymphocytes (preferentially T cells) compared with bone marrow HSPCs. Using a coculture system comprised of kupffer cells and HSPCs, we found that kupffer cells promote adult liver HSPCs to primarily generate T cells and B cells. We then demonstrated that kupffer cells can also promote HSPC expansion. A blockade of intercellular cell adhesion molecule-1 (ICAM-1) in a liver HSPC and kupffer cell coculture system impaired the adhesion, expansion, and differentiation of HSPCs. These results suggest a critical role of kupffer cells in the maintenance and promotion of adult mouse liver hematopoiesis. These findings provide important insight into understanding liver extramedullary hematopoiesis and its significance, particularly under the state of some liver diseases, such as hepatitis, nonalcoholic fatty liver disease (NAFLD), and hepatocellular carcinoma (HCC).

44P - Role of ICAM-1/LFA-1 interaction in the angiogenic and desmoplasic response in liver metastasis

44P - Role of ICAM-1/LFA-1 interaction in the angiogenic and desmoplasic response in liver metastasis

T Cell Migration from Inflamed Skin to Draining Lymph Nodes Requires Intralymphatic Crawling Supported by ICAM-1/LFA-1 Interactions

T cells are the most abundant cell type found in afferent lymph, but their migration through lymphatic vessels (LVs) remains poorly understood. Performing intravital microscopy in the murine skin, we imaged Tcell migration through afferent LVs invivo. Tcells entered into and actively migrated within lymphatic capillaries but were passively transported in contractile collecting vessels. Intralymphatic Tcell number and motility were increased during contact-hypersensitivity-induced inflammation and dependent on ICAM-1/LFA-1 interactions. Invitro, blockade of endothelial cell-expressed ICAM-1 reduced Tcell adhesion, crawling, and transmigration across lymphatic endothelium and decreased Tcell advancement from capillaries into lymphatic collectors in skin explants. Invivo, Tcell migration to draining lymph nodes was significantly reduced upon ICAM-1 or LFA-1 blockade. Our findings indicate that Tcell migration through LVs occurs in distinct steps and reveal a key role for ICAM-1/LFA-1 interactions in this process.

Human CD56bright NK Cells Acquire Potent Anti-Leukemia Functionality Following IL-15 Priming

Natural killer (NK) cells are innate lymphoid cells that mediate anti-tumor responses via cytotoxicity and effector cytokine production. Human NK cells are divided into two subsets based on relative expression of CD56 (CD56bright and CD56dim) that classically participate in distinct functions. Cytotoxic CD56dim NK cells respond to tumor targets without prior stimulation, resulting in target cell death and transient secretion of effector cytokines (e.g. IFN-γ). In contrast, immunoregulatory CD56bright NK cells secrete abundant IFN-γ and other cytokines in response to cytokine receptor stimulation, but respond minimally to tumor target-based triggering. As a result of this dichotomy, translational strategies to enhance NK cell function for cancer immunotherapy have focused exclusively on the CD56dim subset.Based upon studies in mouse NK cells, we hypothesized IL-15 priming would enhance CD56bright anti-tumor functionality. Primary human NK cells from healthy donors were purified (>95% CD56+CD3-), cultured overnight in medium alone (control) or medium with 5 ng/mL rhIL-15 (primed), washed, and assayed for anti-tumor responses. IL-15 priming significantly enhanced multiple CD56bright NK cell functional responses to the prototypical AML target cell line K562 (CD107a+: control 20% vs. primed 59%, p<0.001; IFN-γ+: 3% vs. 27%, p<0.001; TNF: 3% vs. 20%, p<0.001), as well as primary AML blasts (N=3 unique AML sample: CD107a+,7% vs. 30%, p<0.001; IFN-γ 2% vs. 14%, p<0.001; TNF: 2% vs. 22%, p<0.001). IL-15-priming of CD56bright NK cells was evident after 1 hour, and peaked after only 6 hours. In addition, flow-sorted IL-15-primed CD56bright NK cells exhibited markedly increased killing of leukemia target cells. Similar results for functional comparisons were observed using primary NK cells from AML patients. Unexpectedly, IL-15-primed CD56bright NK cell anti-leukemia responses significantly exceeded those of IL-15-primed CD56dim NK cells. Multidimensional CyTOF analyses revealed that the maturity status of CD56dim NK cells did not determine the extent to which they could be primed by IL-15.In response to IL-15, we observed selective activation of the PI3K/Akt/mTOR (4.2 fold increase CD56bright NK cells, 1.2 CD56dim NK cells, p<0.001) and Ras/Raf/MEK/ERK (1.9 fold increase CD56bright NK cells, 1.2 CD56dim NK cells, p<0.001) pathways in CD56bright NK cells. The Jak/STAT pathway was strongly activated in both CD56bright and CD56dim subsets. These data suggested a signaling mechanism for preferential CD56bright NK cell priming by IL-15. Indeed, small molecule inhibitors of PI3K/Akt/mTOR and Ras/Raf/MEK/ERK pathways abrogated the anti-tumor responses of IL-15-primed CD56bright NK cells, supporting this idea. Several NK cell effector mechanisms were enhanced in IL-15-primed CD56bright NK cells, likely contributing to their augmented anti-tumor responsiveness. These included increased cytotoxic effector protein levels (granzyme B and perforin), as well as improved conjugate formation with tumor targets. Furthermore, blocking experiments demonstrated that IL-15-primed CD56bright NK cell anti-tumor responses depended on LFA-1, CD2, and NKG2D receptor interactions with target cells. Finally, since IL-15-based therapeutics are being investigated in clinical trials, we tested whether the IL-15 super-agonist complex ALT-803 primes CD56bright NK cells in vivo in cancer patients. There was an increase in leukemia target cell-triggered degranulation (CD107a+ unprimed 7% vs. primed 30%, p<0.001) and cytokine production (IFN-γ+ 6% vs. 31%, p<0.01; TNF+ 2% vs. 20%, p<0.05) by CD56bright NK cells 24 hours post-ALT-803 administration to multiple myeloma patients, compared to unprimed, pre-therapy values.Collectively, these results suggest that CD56bright NK cells play an under-appreciated anti-tumor role in settings of abundant IL-15, such as following lymphodepleting chemotherapy, during preparation for stem cell transplantation, at sites of inflammation, or after exogenous IL-15 administration. Since CD56bright NK cells have different in vivo tissue localization (secondary lymphoid organs), distinct inhibitory, activating, and chemokine receptor expression compared to CD56dim NK cells, and are thought to be the most abundant NK cell subset when considering all human tissues, this study identifies a promising NK cell subset to harness for cancer immunotherapy. DisclosuresNo relevant conflicts of interest to declare.

LFA-1 and Mac-1 integrins bind to the serine/threonine-rich domain of thrombomodulin

LFA-1 (αLβ2) and Mac-1 (αMβ2) integrins regulate leukocyte trafficking in health and disease by binding primarily to IgSF ligand ICAM-1 and ICAM-2 on endothelial cells. Here we have shown that the anti-coagulant molecule thrombomodulin (TM), found on the surface of endothelial cells, functions as a potentially new ligand for leukocyte integrins. We generated a recombinant extracellular domain of human TM and Fc fusion protein (TM-domains 123-Fc), and showed that pheripheral blood mononuclear cells (PBMCs) bind to TM-domains 123-Fc dependent upon integrin activation. We then demonstrated that αL integrin-blocking mAb, αM integrin-blocking mAb, and β2 integrin-blocking mAb inhibited the binding of PBMCs to TM-domains 123-Fc. Furthermore, we show that the serine/threonine-rich domain (domain 3) of TM is required for the interaction with the LFA-1 (αLβ2) and Mac-1 (αMβ2) integrins to occur on PBMCs. These results demonstrate that the LFA-1 and Mac-1 integrins on leukocytes bind to TM, thereby establishing the molecular and structural basis underlying LFA-1 and Mac-1 integrin interaction with TM on endothelial cells. In fact, integrin-TM interactions might be involved in the dynamic regulation of leukocyte adhesion with endothelial cells.

Timely blockade of ICAM-1.LFA-1 interaction prevents disease onset in a mouse model of emphysema.

It is becoming apparent that emphysema is partly driven by self-reactive T cells inducing inflammatory damage. Thus, T cells become targets for therapy similar to other autoimmune diseases. Costimulatory blockade therapy targets disease-specific T cells, rendering them ineffective by blocking a necessary costimulatory event on the T-cell surface. This therapy is tested here in mouse emphysema. Peptides representing contact domains of counter receptors LFA-1 and ICAM-1 were used as blockade therapy in elastase-induced emphysema. When administered during the first week after disease induction, blockade prevented lung destruction, reduced leukocyte infiltration and inhibited the decrease in T-cell CD4:CD8 ratio, also common in human emphysema. Costimulatory blockade therapy can affect the progress of emphysema.

Inhibition of LFA-1/ICAM-1 Interaction: A Therapeutic Strategy for Surmounting Inflammation

Leukocyte function associated antigen-1 (LFA-1, CD11a/CD18, αLβ2) is a cell surface adhesion protein expressed on leukocytes. Interaction of LFA-1 with its counter receptor intercellular adhesion molecule-1 (ICAM-1) promotes the migration of leukocytes to the site of inflammation and is responsible for a variety of cell adhesion events required for normal and pathological functions of the immune system. Thus, the LFA-1/ICAM-1 interaction is thought to play a role in the pathogenesis of inflammatory and auto-immune disease conditions such as psoriasis, multiple sclerosis, asthma, rheumatoid arthritis, inflammatory bowel disease and transplant rejection. LFA-1/ICAM-1 interaction inhibitors have been extensively studied as a therapeutic target for clinically important diseases; leading to the development of therapeutic antibodies, peptides, peptidomimetics, allosteric and competitive inhibitors. However, despite of promising preclinical results, the outcome of clinical trials with LFA-1/ICAM-1 interaction inhibitors has been inconsistent and met with limited clinical success. This article gives a brief account of rationale and efficacy of different LFA-1/ICAM-1 interaction inhibitors as promising therapeutic strategy for surmounting inflammatory disorders.

Cytoskeletal stiffness controls the threshold of T cell activation (IRM10P.747)

Abstract T cells require a dynamic cytoskeleton to effectively scan antigen-presenting cells and become fully activated. To study the function of cytoskeletal changes as they occur in real time during T cell activation, we modified an atomic force microscope to deliver antigen via the cantilever, simultaneously ligating T-cell receptors (TCRs) and measuring changes in cytoskeletal mechanics. We show that antigenically naïve T cells are mechanically stiff and their cytoskeletal structures undergo rapid relaxation at the site of TCR ligation. In contrast, antigen-experienced memory and effector T cells are already soft prior to TCR triggering. We found that cytoskeletal relaxation is due to depolymerization of actin, is proportional to antigen dose, and requires proximal TCR signaling pathways but not ICAM-1:LFA-1 interaction. We identified pathways controlling the stiffness of naïve T cells, and blocking these pathways resulted in naïve T cells become soft even prior to TCR triggering. Moreover, these soft yet antigen-inexperienced T cells showed a reduced threshold for allo- and antigen-specific activation compared to controls. Our results highlight the functional importance of cytoskeletal stuctures in tuning T cell responses to spatially localized signals from APCs.

An Anti-Human ICAM-1 Antibody Inhibits Rhinovirus-Induced Exacerbations of Lung Inflammation

Human rhinoviruses (HRV) cause the majority of common colds and acute exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Effective therapies are urgently needed, but no licensed treatments or vaccines currently exist. Of the 100 identified serotypes, ∼90% bind domain 1 of human intercellular adhesion molecule-1 (ICAM-1) as their cellular receptor, making this an attractive target for development of therapies; however, ICAM-1 domain 1 is also required for host defence and regulation of cell trafficking, principally via its major ligand LFA-1. Using a mouse anti-human ICAM-1 antibody (14C11) that specifically binds domain 1 of human ICAM-1, we show that 14C11 administered topically or systemically prevented entry of two major groups of rhinoviruses, HRV16 and HRV14, and reduced cellular inflammation, pro-inflammatory cytokine induction and virus load in vivo. 14C11 also reduced cellular inflammation and Th2 cytokine/chemokine production in a model of major group HRV-induced asthma exacerbation. Interestingly, 14C11 did not prevent cell adhesion via human ICAM-1/LFA-1 interactions in vitro, suggesting the epitope targeted by 14C11 was specific for viral entry. Thus a human ICAM-1 domain-1-specific antibody can prevent major group HRV entry and induction of airway inflammation in vivo.

Xiamenmycin Attenuates Hypertrophic Scars by Suppressing Local Inflammation and the Effects of Mechanical Stress

Hypertrophic scarring is a common disease affecting millions of people around the world, but there are currently no satisfactory drugs to treat the disease. Exaggerated inflammation and mechanical stress have been shown to be two main mechanisms of excessive fibrotic diseases. Here we found that a benzopyran natural product, xiamenmycin, could significantly attenuate hypertrophic scar formation in a mechanical stretch-induced mouse model. The compound suppressed local inflammation by reducing CD4+ lymphocyte and monocyte/macrophage retention in fibrotic foci and blocked fibroblast adhesion with monocytes. Both in vivo and in vitro studies found that the compound inhibited the mechanical stress-induced profibrotic effects by suppressing proliferation, activation, fibroblast contraction, and inactivating FAK, p38, and Rho guanosine triphosphatase signaling. Taken together, the compound could simultaneously suppress both the inflammatory and mechanical stress responses, which are the two pivotal pathological processes in hypertrophic scar formation, thus suggesting that xiamenmycin can serve as a potential agent for treating hypertrophic scar formation and other excessive fibrotic diseases.

Mechanical insights into the functional impacts of interactions between antigen-presenting cells and T cells (P5081)

Abstract Antigen recognition and discrimination by T lymphocyte are essential in initiating appropriate immune responses. The mechanisms underlying exquisite sensitivity and specificity of antigen discrimination are not fully elucidated but involved physical intercellular interactions between T cell and antigen-presenting cell (APC) including dendritic cells (DCs) and B cells. Using single cell force spectroscopy (SCFS), we have successfully probe mechanical interactions between DC and T cells in response to a panel of altered peptide ligands (APLs). In the presence of different type of APLs, DC:T conjugate displays different ranges of mechanical forces that match their corresponding T cell responsiveness. Strong cell-cell interaction forces are contributed not only by surface adhesion molecules including TCR:pMHC, CD28:B7 and ICAM-1:LFA-1 interaction pairs, but also dependent on the integrity of membrane cholesterol and cytoskeleton dynamics. As compared to B:T interactions, stronger DC:T cell-cell interactions are likely to provide a mechanically stable environment that induces potent functional T cell activation, suggesting that the measurement of mechanical forces could be a significant predictor of T cell functional activation efficacy. Our SCFS studies provide mechanical insights into the adhesive roles of cell surface proteins in regulating APC:T cell-cell interactions. Functional roles of TLR ligands in regulating APC:T interactions and T-cell responses will be discussed.

Recombinant production of human ICAM-1 chimeras by single step on column refolding and purification

The interaction of the adhesion molecule of the immunoglobulin family intercellular adhesion molecule 1 (ICAM-1) with its ligands such that the integrins LFA-1 and Mac-1 is crucial for the regulation of several physiological and pathophysiological processes like cell mediated-elimination of tumor or virus infected cells, cancer metastasis or inflammatory autoimmune processes. Thus, production of milligrams of protein is required to perform structural and functional studies as well as design novel approaches to find out new inhibitors of ICAM-1/LFA-1 interaction. Here we report on the production of a recombinant human ICAM-1 chimera comprising the first two extracellular domains of ICAM-1 linked to the Fc fraction of a human IgG1. To this aim we have used a cost-effective method based on the expression of a His-tagged protein in Escherichia coli followed by a single step of refolding and purification on immobilized metal affinity columns. This method is able to produce 3mg/l of bacterial culture in just 72h with purity greater than 95%. The identity and the native structure of refolded human ICAM-1 chimera were confirmed by biochemical and biophysical studies including SDS-electrophoresis, immunoblot, circular dichroism, isothermal titration calorimetry and fluorescence spectroscopy. Native folding and functional activity of the chimera were further confirmed by different cell biology studies, including B cell adhesion, T cell binding and inhibition of NK cell function. These studies indicate a high biological activity of the protein since it induces a 200-fold increase/mg of protein in B cell adhesion and the inhibitory dose 50 to block cell-mediated cytotoxicity is 10pg/effector cell. These analyses show that our protocol is able to produce a recombinant human ICAM-1 chimera fully active and useful to analyze the biological processes in which ICAM-1/LFA-1 interaction is critically involved.

KCa3.1 and TRPM7 channels at the uropod regulate migration of activated human T cells.

The migration of T lymphocytes is an essential part of the adaptive immune response as T cells circulate around the body to carry out immune surveillance. During the migration process T cells polarize, forming a leading edge at the cell front and a uropod at the cell rear. Our interest was in studying the involvement of ion channels in the migration of activated human T lymphocytes as they modulate intracellular Ca2+ levels. Ca2+ is a key regulator of cellular motility. To this purpose, we created protein surfaces made of the bio-polymer PNMP and coated with ICAM-1, ligand of LFA-1. The LFA-1 and ICAM-1 interaction facilitates T cell movement from blood into tissues and it is critical in immune surveillance and inflammation. Activated human T lymphocytes polarized and migrated on ICAM-1 surfaces by random walk with a mean velocity of ∼6 µm/min. Confocal microscopy indicated that Kv1.3, CRAC, and TRPM4 channels positioned in the leading-edge, whereas KCa3.1 and TRPM7 channels accumulated in the uropod. The localization of KCa3.1 and TRPM7 at the uropod was associated with oscillations in intracellular Ca2+ levels that we measured in this cell compartment. Further studies with blockers against Kv1.3 (ShK), KCa3.1 (TRAM-34), CRAC (SKF-96365), TRPM7 (2-APB), and TRPM4 (glibenclamide) indicated that blockade of KCa3.1 and TRPM7, and not Kv1.3, CRAC or TRPM4, inhibits the T cell migration. The involvement of TRPM7 in cell migration was confirmed with siRNAs against TRPM7. Downregulation of TRPM7 significantly reduced the number of migrating T cells and the mean velocity of the migrating T cells. These results indicate that KCa3.1 and TRPM7 selectively localize at the uropod of migrating T lymphocytes and are key components of the T cell migration machinery.

The expression of ICAM-1 on macrophages stimulated with Mycobacterium avium complex and its control by some regulatory cytokines

The interaction of LFA-1 on T lymphocytes with ICAM-1 on antigen presenting cells (APCs) is critical in determining conjugate formation between the APCs and T cells as well as activation of T cells. Recently, it was found that stimulation of THP-1 cells, a human monocyte M phi cell line, with Mycobacterium tuberculosis or its lipoarabinomannan, elicited the increase in the ICAM-1 expression. In addition, in cases of lepromatous leprosy patients with a serious defect in the M. leprae antigen-specific cellular immunity, keratinocytes in the leprosy lesions were lacking in the ICAM-1 expression. Therefore, ICAM-1 seems to participate in the host response to mycobacterial infections. Here, we studied the mode of the expression of ICAM-1 molecules on murine peritoneal M phis in response to stimulation with M. avium complex (MAC). In addition, the regulatory effect of some cytokines including TNF-alpha, IL-10, and transforming growth factor-beta (TGF-beta) on the ICAM-1 expression was studied. Monolayer cultures of peptone-starch induced murine peritoneal M phis were cultured in RPMI-1640 medium in the presence of MAC with or without test agents. At intervals, the M phis were stained with anti-ICAM-1 antibody (Ab) and then treated with alkaline phosphatase (Ap)-conjugated anti-1g Ab. After color development with NBT-BCIP substrate, percentage of the blue-stained (ICAM-1 positive) M phis was determined microscopically. The concentrations of TNF-alpha, IL-10, and TGF-beta in the M phi culture fluid was measured by ELISA using capture Ab, biotin-labelled capping Ab, and Ap-conjugated streptavidin. When M phis infected with MAC organisms were cultured in RPMI medium containing 10% fetal bovine serum or in serum-free GPI medium, a significant increase in their ICAM-1 expression was observed, reaching the peak at days 1 to 3, thereafter rapidly decreased and returned to the normal level by day 14. Further addition of TNF-alpha caused no significant change in the mode of the MAC-induced expression of ICAM-1. A transient increase in the IL-10 production of MAC-infected M phis was observed during the first 3-days cultivation, as in the case of TNF-alpha. On the other hand, TGF-beta production of the Mø population was initiated from day 3, and therafter increased gradually until day 14. ICAM-1 expression of the MAC-infected M phis was not influenced by the addition of IL-10, while anti-IL-10 Ab retarded the decline of ICAM-1 expression at day 7. On the other hand, the addition of TCF-beta attenuated the MAC infection-induced increase in the ICAM-1 expression significantly. In addition, anti-TGF-beta Ab significantly delayed the reduction of the ICAM-1 expression of MAC-infected M phis during days 3 to 7. These results indicate that, in the MAC-infected M phis. TGF-beta rather than IL-10 play important roles as a mediators for the late-phase down-regulation of ICAM-1 expression which had been transiently elevated by the action of TNF-alpha in the early phase of the Mø cultivation.