Extracellular ISG15 Signals Cytokine Secretion through the LFA-1 Integrin Receptor.

ISG15 is a ubiquitin-like protein that functions in innate immunity both as an intracellular protein modifier and as an extracellular signaling molecule that stimulates IFN-γ secretion. The extracellular function, important for resistance to mycobacterial disease, has remained biochemically uncharacterized. We have established an NK-92 cell-based assay for IFN-γ release, identified residues critical for ISG15 signaling, and identified the cell surface receptor as LFA-1 (CD11a/CD18; αLß2 integrin). LFA-1 inhibition blocked IFN-γ secretion, splenocytes from CD11a-/- mice did not respond to ISG15, and ISG15 bound directly to the αI domain of CD11a in vitro. ISG15 also enhanced secretion of IL-10, indicating a broader role for ISG15 in cytokine signaling. ISG15 engagement of LFA-1 led to the activation of SRC family kinases (SFKs) and SFK inhibition blocked cytokine secretion. These findings establish the molecular basis of the extracellular function of ISG15 and the initial outside-in signaling events that drive ISG15-dependent cytokine secretion.

Evaluation of cell adhesion molecules (LFA-1 and L-selectin) in ankylosing spondylitis patients after treatment with ß-D-mannuronic acid (M2000).

Background & objectives: To examine ß-D-mannuronic acid (M2000) effects on L-selectin shedding and leucocyte function-associated antigen-1 (LFA-1) expression as mechanisms of action of this drug in patients with ankylosing spondylitis (AS). Methods: To investigate the molecular consequences of ß-D-mannuronic acid on L-selectin shedding, flow cytometry method was used. Furthermore, the effect of it on LFA-1 gene expression was analyzed by using quantitative real time (qRT)-PCR technique. Results: The LFA-1 expression in patients with AS was higher than controls (P=0.046). The LFA-1 expression after 12 wk therapy with ß-D-mannuronic acid was meaningfully decreased (P=0.01). After 12 wk treatment with ß-D-mannuronic acid, the frequency of CD62L-expressing CD4+ T cells in patients with AS, was not considerably altered, compared to the patients before therapy (P=0.5). Furthermore, after 12 wk therapy with ß-D-mannuronic acid, L-selectin expression levels on CD4+ T-cells in patients with AS, were not remarkably changed, compared to the expression levels of these in patients before treatment (P=0.2). Interpretation & conclusions: The results of this study for the first time showed that ß-D-mannuronic acid can affect events of adhesion cascade in patients with AS. Moreover, ß-D-mannuronic acid presented as an acceptable benefit to AS patients and could aid in the process of disease management.

The Role of LFA-1 for the Differentiation and Function of Regulatory T Cells-Lessons Learned from Different Transgenic Mouse Models.

Regulatory T cells (Treg) are essential for the maintenance of peripheral tolerance. Treg dysfunction results in diverse inflammatory and autoimmune diseases with life-threatening consequences. ß2-integrins (CD11a-d/CD18) play important roles in the migration of leukocytes into inflamed tissues and cell signaling. Of all ß2-integrins, T cells, including Treg, only express CD11a/CD18, termed lymphocyte function-associated antigen 1 (LFA-1), on their surface. In humans, loss-of-function mutations in the common subunit CD18 result in leukocyte adhesion deficiency type-1 (LAD-1). Clinical symptoms vary depending on the extent of residual ß2-integrin function, and patients may experience leukocytosis and recurrent infections. Some patients can develop autoimmune diseases, but the immune processes underlying the paradoxical situation of immune deficiency and autoimmunity have been scarcely investigated. To understand this complex phenotype, different transgenic mouse strains with a constitutive knockout of ß2-integrins have been established. However, since a constitutive knockout affects all leukocytes and may limit the validity of studies focusing on their cell type-specific role, we established a Treg-specific CD18-floxed mouse strain. This mini-review aims to delineate the role of LFA-1 for the induction, maintenance, and regulatory function of Treg in vitro and in vivo as deduced from observations using the various ß2-integrin-deficient mouse models.

Understanding the Role of LFA-1 in Leukocyte Adhesion Deficiency Type I (LAD I): Moving towards Inflammation?

LFA-1 (Lymphocyte function-associated antigen-1) is a heterodimeric integrin (CD11a/CD18) present on the surface of all leukocytes; it is essential for leukocyte recruitment to the site of tissue inflammation, but also for other immunological processes such as T cell activation and formation of the immunological synapse. Absent or dysfunctional expression of LFA-1, caused by mutations in the ITGB2 (integrin subunit beta 2) gene, results in a rare immunodeficiency syndrome known as Leukocyte adhesion deficiency type I (LAD I). Patients suffering from severe LAD I present with recurrent infections of the skin and mucosa, as well as inflammatory symptoms complicating the clinical course of the disease before and after allogeneic hematopoietic stem cell transplantation (alloHSCT); alloHSCT is currently the only established curative treatment option. With this review, we aim to provide an overview of the intrinsic role of inflammation in LAD I.

Expression of TSLC1 in patients with HAM/TSP.

Human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is chronic myelopathy characterized by slowly progressive spastic paraparesis and urinary dysfunction. A few biomarkers in the cerebrospinal fluid are known to be related to disease activity, but no biomarker has been reported in peripheral blood. This study aims to explore the expression level of the adhesion molecule during the expression level of the adhesion molecule among HAM/TSP disease activity. In lymphocyte function-associated antigen 1 and DNAX accessory molecule 1, no variation in expression levels specific to HTLV-1 infection was observed in CD4-positive T cells; however, TSLC1 expression was higher in HAM patients than in asymptomatic carriers and non-infected persons. TSLC1 tended to be higher in patients whose symptoms were worsening. On the contrary, the expression level of TSLC1 in CD8-positive T cells was lower in HAM patients than in asymptomatic carriers, and this tendency was stronger in patients whose symptoms had deteriorated. No significant correlation was found between TSLC1 and either of the transcription factors Tax or HBZ in any T cell group. Therefore, TSLC1 expression in CD4-positive T cells might be a useful biomarker of HAM/TSP disease activity.

Assessment of Arf6 Deletion in PLB-985 Differentiated in Neutrophil-Like Cells and in Mouse Neutrophils: Impact on Adhesion and Migration.

Chemoattractant sensing, adhesiveness, and migration are critical events underlying the recruitment of neutrophils (PMNs) to sites of inflammation or infection. Defects in leukocyte adhesion or migration result in immunodeficiency disorders characterized by recurrent infections. In this study, we evaluated the role of Arf6 on PMN adhesion in vitro and on migration to inflammatory sites using PMN-Arf6 conditional knockout (cKO) mice. In PMN-like PLB-985 silenced for Arf6 fMLP-mediated adhesion to the ß2 integrin ligands, ICAM-1 and fibrinogen or the ß1/ß2 integrin ligand fibronectin was significantly reduced. Furthermore, overexpression of wild-type Arf6 promoted basal and fMLP-induced adhesion to immobilized integrin ligands, while overexpression of the dominant-negative Arf6 has the opposite effects. Using the Elane-Cre deleting mouse strains, we report that the level of Arf6 deletion in inflammatory PMNs isolated from the dorsal air pouches was stronger when compared to naïve cells isolated from the bone marrow. In PMN-Arf6 cKO mice, the recruitment of PMNs into the dorsal air pouch injected with LPS or the chemoattractant fMLP was significantly diminished. Impaired cell migration correlated with reduced cell surface expression of CD11a and CD11b in Arf6 cKO PMNs. Our results highlight that Arf6 regulates the activity and possibly the recycling of PMN integrins, and this compromises PMN migration to inflammatory sites.

Ligand- and cation-induced structural alterations of the leukocyte integrin LFA-1.

In αI integrins, including leukocyte function-associated antigen 1 (LFA-1), ligand-binding function is delegated to the αI domain, requiring extra steps in the relay of signals that activate ligand binding and coordinate it with cytoplasmic signals. Crystal structures reveal great variation in orientation between the αI domain and the remainder of the integrin head. Here, we investigated the mechanisms involved in signal relay to the αI domain, including whether binding of the ligand intercellular adhesion molecule-1 (ICAM-1) to the αI domain is linked to headpiece opening and engenders a preferred αI domain orientation. Using small-angle X-ray scattering and negative-stain EM, we define structures of ICAM-1, LFA-1, and their complex, and the effect of activation by Mn2+ Headpiece opening was substantially stabilized by substitution of Mg2+ with Mn2+ and became complete upon ICAM-1 addition. These agents stabilized αI-headpiece orientation, resulting in a well-defined orientation of ICAM-1 such that its tandem Ig-like domains pointed in the opposite direction from the ß-subunit leg of LFA-1. Mutations in the integrin ßI domain α1/α1' helix stabilizing either the open or the closed ßI-domain conformation indicated that α1/α1' helix movements are linked to ICAM-1 binding by the αI domain and to the extended-open conformation of the ectodomain. The LFA-1-ICAM-1 orientation described here with ICAM-1 pointing anti-parallel to the LFA-1 ß-subunit leg is the same orientation that would be stabilized by tensile force transmitted between the ligand and the actin cytoskeleton and is consistent with the cytoskeletal force model of integrin activation.

Pharmacological blockage of ICAM-1 improves angiotensin II-induced cardiac remodeling by inhibiting adhesion of LFA-1+ monocytes.

Intercellular adhesion molecule-1 (ICAM-1) is a member of an immunoglobulin-like superfamily of adhesion molecules that mediate leukocyte adhesion to vascular endothelium and are involved in several cardiovascular diseases, including ischemia-reperfusion injury, myocardial infarction, and atherosclerosis. However, the role of ICAM-1 in angiotensin II (ANG II)-induced cardiac remodeling in mice remains unclear. Wild-type mice were administered an IgG control or ICAM-1 neutralizing antibody (1 and 2 mg/mouse, respectively) and ANG II (1,000 ng·kg-1·min-1) for up to 14 days. Cardiac contractile function and structure were detected by echocardiography. Hypertrophy, fibrosis, and inflammation were assessed by histological examination. The infiltration of lymphocyte function-associated antigen-1 (LFA-1+) monocytes/macrophages was assessed by immunostaining. The mRNA expression of genes was evaluated by quantitative RT-PCR analysis. Protein levels were tested by immunoblotting. We found that ICAM-1 expression in ANG II-infused hearts and ICAM-1 levels in serum from human patients with heart failure were significantly increased. Moreover, ANG II infusion markedly enhanced ANG II-induced hypertension, caused cardiac contractile dysfunction, and promoted cardiac hypertrophy, fibrosis, and LFA-1+ macrophage infiltration. Conversely, blockage of ICAM-1 with a neutralizing antibody dose-dependently attenuated these effects. Moreover, our in vitro data further demonstrated that blocking ICAM-1 inhibited ANG II-induced LFA-1+ macrophage adhesion to endothelial cells and migration. In conclusion, these results provide novel evidence that blocking ICAM-1 exerts a protective effect in ANG II-induced cardiac remodeling at least in part through the modulation of adhesion and infiltration of LFA-1+ macrophages in the heart. Inhibition of ICAM-1 may represent a new therapeutic approach for hypertrophic heart diseases.NEW & NOTEWORTHY Leukocyte adhesion to vascular endothelium is a critical step in cardiovascular diseases. ICAM-1 is a member of immunoglobulin-like superfamily of adhesion molecules that binds LFA-1 to mediate leukocytes adhesion and migration. However, the significance of ICAM-1 in ANG II-induced cardiac remodeling remains unclear. This study reveals that blocking of ICAM-1 prevents ANG II-induced cardiac remodeling via modulating adhesion and migration of LFA-1+ monocytes, may serve as a novel therapeutic target for hypertensive cardiac diseases.

Not Just an Adhesion Molecule: LFA-1 Contact Tunes the T Lymphocyte Program.

The αLß2 integrin LFA-1 is known to play a key role in T lymphocyte migration, which is necessary to mount a local immune response, and is also the main driver of autoimmune diseases. This migration-triggering signaling process in T cells is tightly regulated to permit an immune response that is appropriate to the local trigger, as well as to prevent deleterious tissue-damaging bystander effects. Emerging evidence shows that, in addition to prompting a diverse range of downstream signaling cascades, LFA-1 stimulation in T lymphocytes modulates gene-transcription programs, including genetic signatures of TGF-ß and Notch pathways, with multifactorial biological outcomes. This review highlights recent findings and discusses molecular mechanisms by which LFA-1 signaling influence T lymphocyte differentiation into the effector subsets Th1, Th17, and induced regulatory T cells. We argue that LFA-1 contact with a cognate ligand, such as ICAM-1, independent of the immune synapse activates a late divergence in T cells' effector phenotypes, hence fine-tuning their functioning.

Adoptive Transfer of Phosphoantigen-Specific γδ T Cell Subset Attenuates Mycobacterium tuberculosis Infection in Nonhuman Primates.

The dominant Vγ2Vδ2 T cell subset recognizes phosphoantigen and exists only in humans and nonhuman primates. Despite the discovery of γδ T cells >30 y ago, a proof-of-concept study has not been done to prove the principle that the Vγ2Vδ2 T cell subset is protective against Mycobacterium tuberculosis and other infections. In this study, we used an adoptive cell-transfer strategy to define the protective role of Vγ2Vδ2 T cells in a primate tuberculosis (TB) model. Vγ2Vδ2 T cells for adoptive transfer displayed central/effector memory and mounted effector functions, including the production of anti-M. tuberculosis cytokines and inhibition of intracellular mycobacteria. They also expressed CXCR3/CCR5/LFA-1 trafficking/tissue-resident phenotypes and consistently trafficked to the airway, where they remained detectable from 6 h through 7 d after adoptive transfer. Interestingly, the test group of macaques receiving transfer of Vγ2Vδ2 T cells at weeks 1 and 3 after high-dose (500 CFU) M. tuberculosis infection exhibited significantly lower levels of M. tuberculosis infection burdens in lung lobes and extrapulmonary organs than did the control groups receiving PBLs or saline. Consistently, adoptive transfer of Vγ2Vδ2 T cells attenuated TB pathology and contained lesions primarily in the infection site of the right caudal lung lobe, with no or reduced TB dissemination to other lobes, spleen, or liver/kidney; in contrast, the controls showed widespread TB dissemination. The proof-of-concept finding supports the view that the dominant Vγ2Vδ2 T cell subset may be included in the rational design of a TB vaccine or host-directed therapy.

LFA-1 Activation in NK Cells and Their Subsets: Influence of Receptors, Maturation, and Cytokine Stimulation.

The integrin LFA-1 is essential for efficient activation and for cytotoxicity of NK cells because it initiates the assembly of the immunological synapse and mediates firm adhesion to the target. LFA-1 is also needed to polarize the cytotoxic machinery of the NK cell toward the target cell. The binding affinity and avidity of integrins can be regulated via inside-out signals from other receptors. In this article, we investigate the signals necessary to activate LFA-1 in human NK cells. Our data show that LFA-1 has a low ligand-binding activity in resting human NK cells, but it can be stimulated by triggering activating receptors, such as 2B4 or CD16, or by coactivation of different receptor combinations. Short-term stimulation of freshly isolated NK cells with cytokines, such as IL-15, IL-12, or IL-18, does not activate LFA-1 but increases the responsiveness of the cells to subsequent receptor stimulation. Different NK cell subsets vary in their ability to induce LFA-1 binding activity after activating receptor stimulation. Interestingly, the NK cell subsets that are more mature and possess higher cytotoxic potential also show the highest activation of LFA-1, which correlated with the expression of the small calcium-binding protein S100A4. Our data suggest that regulation of LFA-1 is one reason for the different activity of NK cells during differentiation.

Lentivirus-Mediated Gene Silencing of Lymphocyte Function-Associated Antigen 1 Inhibits Apoptosis of Hippocampal Neurons in Rats with Acute Cerebral Ischemia After Cerebral Lymphatic Blockage.

BACKGROUND/AIMS: Cerebral ischemia is considered to be the most common cause of stroke with high mortality. It occurs as a result of the damage of the hippocampal neurons with lymphocyte function-associated antigen (LFA)-1 being emphasized to play a role in the biological functions of hippocampal neurons. This study was conducted in order to investigate the effects of specific knockdown of LFA-1 expression by lentivirus had on the apoptosis of the hippocampal neurons, simulated by rat models of acute cerebral ischemia after cerebral lymphatic blockage. METHODS: A total of 60 Wistar rats were selected as subjects, among which 50 were used to establish models of the acute cerebral ischemia after cerebral lymphatic blockage, while the remaining 10 rats were treated with the sham operation. The underlying regulatory mechanisms regarding LFA-1 were analyzed with the treatment of si-LFA-1 and LFA-1 vector in the hippocampal CA1 area of brain tissues isolated from the rats with acute cerebral ischemia. The brain water content, electrolyte content, and blood-brain barrier permeability located in ischemic area of rats were measured. TUNEL staining and immunochemistry methods were employed in order to determine the apoptosis rate and positive levels of LFA-1, MMP-9, and Caspase-3. The mRNA and protein levels of related genes were also detected by means of RT-qPCR and western blot assay. RESULTS: The brain water content, Na+ and Ca+ contents, blood-brain barrier permeability, apoptosis rate, positive levels of LFA-1, MMP-9, and Caspase-3 were decreased, and the K+ content was increased in ischemic tissues treated with si-LFA-1. The mRNA and protein levels of LFA-1, MMP-9, Caspase-3, and Bax had all decreased, while the mRNA and protein levels of Bcl-2 were elevated in the hippocampal CA1 area of rat brain tissues treated with si-LFA-1. These situations could be reversed through the up-regulation of LFA-1. CONCLUSION: In conclusion, LFA-1 gene silencing could improve the acute cerebral ischemia after cerebral lymphatic blockage by inhibiting apoptosis of the hippocampal neurons in rats.

ICAM3 mediates tumor metastasis via a LFA-1-ICAM3-ERM dependent manner.

ICAM3 was reported to promote metastasis in tumors. However, the underlying mechanism remains elusive. Here, we disclosed that the expression of ICAM3 was closely correlated with the TNM stage of human breast and lung cancer, as well as the dominant overexpression in high aggressive tumor cell lines (231 and A549 cells). Moreover, the knockdown of ICAM3 inhibited tumor metastasis whereas the ectopic expression of ICAM3 promoted tumor metastasis both in vitro and in vivo. In addition, exploration of the underlying mechanism demonstrated that ICAM3 not only binds to LFA-1 with its extracellular domain and structure protein ERM but also to lamellipodia with its intracellular domain which causes a tension that pulls cells apart (metastasis). Furthermore, ICAM3 extracellular or intracellular mutants alternatively abolished ICAM3 mediated tumor metastasis in vitro and in vivo. As a therapy strategy, LFA-1 antibody or Lifitegrast restrained tumor metastasis via targeting ICAM3-LFA-1 interaction. In summary, the aforementioned findings suggest a model of ICAM3 in mediating tumor metastasis. This may provide a promising target or strategy for the prevention of tumor metastasis.

Protein tyrosine phosphatase PTPN22 regulates LFA-1 dependent Th1 responses.

A missense C1858T single nucleotide polymorphism within PTPN22 is a strong genetic risk factor for the development of multiple autoimmune diseases. PTPN22 encodes a protein tyrosine phosphatase that negatively regulates immuno-receptor proximal Src and Syk family kinases. Notably, PTPN22 negatively regulates kinases downstream of T-cell receptor (TCR) and LFA-1, thereby setting thresholds for T-cell activation. Alterations to the quality of TCR and LFA-1 engagement at the immune synapse and the regulation of downstream signals can have profound effects on the type of effector T-cell response induced. Here we describe how IFNγ+ Th1 responses are potentiated in Ptpn22-/- T-cells and in T-cells from mice expressing Ptpn22R619W (the mouse orthologue of the human genetic variant) as they age, or following repeated immune challenge, and explore the mechanisms contributing to the expansion of Th1 cells. Specifically, we uncover two LFA-1-ICAM dependent mechanisms; one T-cell intrinsic, and one T-cell extrinsic. Firstly, we found that in vitro anti-CD3/LFA-1 induced Th1 responses were enhanced in Ptpn22-/- T-cells compared to WT, whereas anti-CD3/anti-CD28 induced IFNy responses were similar. These data were associated with an enhanced ability of Ptpn22-/- T-cells to engage ICAM-1 at the immune synapse when incubated on planar lipid bilayers, and to form conjugates with dendritic cells. Secondly, we observed a T-cell extrinsic mechanism whereby repeated stimulation of WT OT-II T-cells with LPS and OVA323-339 pulsed Ptpn22-/- bone marrow derived dendritic cells (BMDCs) was sufficient to enhance Th1 cell development compared to WT BMDCs. Furthermore, this response could be reversed by LFA-1 blockade. Our data point to two related but distinct mechanisms by which PTPN22 regulates LFA-1 dependent signals to enhance Th1 development, highlighting how perturbations to PTPN22 function over time to regulate the balance of the immune response.

The interactive effects of genetic polymorphisms within LFA-1/ICAM-1/GSK-3ß pathway and environmental hazards on the development of Graves' opthalmopathy.

The purpose of this investigation was to explore the combined effects of single nucleotide polymorphisms (SNPs) within LFA-1/ICAM-1/GSK-3ß pathway and environmental hazards on susceptibility to Graves' opthalmopathy (GO) among a Chinese Han population. Altogether 305 GO patients and 283 Graves' disease (GD) subjects were recruited. Information relevant to the participants' age, gender, body mass index (BMI), regular physical activity, smoking history, alcohol intake, stressful work environment, stress at work, family history of thyroid disease and 131I treatment were summarized, and the participants' related SNPs of LFA-1/ICAM-1/GSK-3ß were also detected. Then the gene-gene and gene-environment interactions were evaluated by logistic regression model and multi-factor dimensionality reduction (MDR) modeling. The results exhibited that age, BMI, smoking history, stressful work, stress at home, family history of thyroid disease and 131I treatment appeared as potential indicators regulating GO risk, when either univariate or multivariate regression analysis was performed (all P < 0.05). Moreover, rs12716977 (T > C) and rs2230433 (G > C) of LFA-1, rs1799969 (G > A) and rs5498 (A > G) of ICAM-1, as well as rs6438552 (T > C) and rs334558 (T > C) of GSK-3ß were significantly associated with altered susceptibility to GO under the allelic models (all P < 0.05). Also haplotype TGAATC acted as a protective factor against GO risk (P < 0.05), whereas haplotype CGAACC largely elevated risk of GO (P < 0.05). Besides, logistic regression analysis demonstrated that rs12716927, rs5498 and rs6438552 all would affect the influences exerted by age, BMI, smoking history, stressful work, stress at home, family history of thyroid disease or 131I treatment on GO susceptibility (all P < 0.05). MDR modeling implied that the combined model of rs12716977, rs2230433 and rs1799969 was the supreme interactive model when BMI was co-assessed, and the interactive model of rs12716977, rs334558 and rs5491 was the most desirable among the smoking population. In conclusion, gene-gene and gene-environment interactions served as a crucial manner in affecting susceptibility to GO, providing solid evidences for screening effective GO-susceptible biomarkers and exploring potential GO treatment strategies.

A role for LFA-1 in delaying T-lymphocyte egress from lymph nodes.

Lymphocytes use the integrin leukocyte function-associated antigen-1 (LFA-1) to cross the vasculature into lymph nodes (LNs), but it has been uncertain whether their migration within LN is also LFA-1 dependent. We show that LFA-1 mediates prolonged LN residence as LFA-1(-/-) CD4 T cells have significantly decreased dwell times compared with LFA-1(+/+) T cells, a distinction lost in hosts lacking the major LFA-1 ligand ICAM-1. Intra-vital two-photon microscopy revealed that LFA-1(+/+) and LFA-1(-/-) T cells reacted differently when probing the ICAM-1-expressing lymphatic network. While LFA-1(+/+) T cells returned to the LN parenchyma with greater frequency, LFA-1(-/-) T cells egressed promptly. This difference in exit behaviour was a feature of egress through all assessed lymphatic exit sites. We show that use of LFA-1 as an adhesion receptor amplifies the number of T cells returning to the LN parenchyma that can lead to increased effectiveness of T-cell response to antigen. Thus, we identify a novel function for LFA-1 in guiding T cells at the critical point of LN egress when they either exit or return into the LN for further interactions.

SLAT promotes TCR-mediated, Rap1-dependent LFA-1 activation and adhesion through interaction of its PH domain with Rap1.

SLAT (also known as DEF6) promotes T cell activation and differentiation by regulating NFAT-Ca(2+) signaling. However, its role in TCR-mediated inside-out signaling, which induces integrin activation and T cell adhesion, a central process in T cell immunity and inflammation, has not been explored. Here, we show that SLAT is crucial for TCR-induced adhesion to ICAM-1 and affinity maturation of LFA-1 in CD4(+) T cells. Mechanistic studies revealed that SLAT interacts, through its PH domain, with a key component of inside-out signaling, namely the active form of the small GTPase Rap1 (which has two isoforms, Rap1A and Rap1B). This interaction has been further shown to facilitate the interdependent recruitment of Rap1 and SLAT to the T cell immunological synapse upon TCR engagement. Furthermore, a SLAT mutant lacking its PH domain drastically inhibited LFA-1 activation and CD4(+) T cell adhesion. Finally, we established that a constitutively active form of Rap1, which is present at the plasma membrane, rescues the defective LFA-1 activation and ICAM-1 adhesion in SLAT-deficient (Def6(-/-)) T cells. These findings ascribe a new function to SLAT, and identify Rap1 as a target of SLAT function in TCR-mediated inside-out signaling.

A mutation within the SH2 domain of slp-76 regulates the tissue distribution and cytokine production of iNKT cells in mice.

TCR ligation is critical for the selection, activation, and integrin expression of T lymphocytes. Here, we explored the role of the TCR adaptor protein slp-76 on iNKT-cell biology. Compared to B6 controls, slp-76(ace/ace) mice carrying a missense mutation (Thr428Ile) within the SH2-domain of slp-76 showed an increase in iNKT cells in the thymus and lymph nodes, but a decrease in iNKT cells in spleens and livers, along with reduced ADAP expression and cytokine response. A comparable reduction in iNKT cells was observed in the livers and spleens of ADAP-deficient mice. Like ADAP(-/-) iNKT cells, slp-76(ace/ace) iNKT cells were characterized by enhanced CD11b expression, correlating with an impaired induction of the TCR immediate-early gene Nur77 and a decreased adhesion to ICAM-1. Furthermore, CD11b-intrinsic effects inhibited cytokine release, concanavalin A-mediated inflammation, and iNKT-cell accumulation in the liver. Unlike B6 and ADAP(-/-) mice, the expression of the transcription factors Id3 and PLZF was reduced, whereas NP-1-expression was enhanced in slp-76(ace/ace) mice. Blockade of NP-1 decreased the recovery of iNKT cells from peripheral lymph nodes, identifying NP-1 as an iNKT-cell-specific adhesion factor. Thus, slp-76 contributes to the regulation of the tissue distribution, PLZF, and cytokine expression of iNKT cells via ADAP-dependent and -independent mechanisms.

Lung ICAM-1 and ICAM-2 support spontaneous intravascular effector lymphocyte entrapment but are not required for neutrophil entrapment or emigration inside endotoxin-inflamed lungs.

The pulmonary vasculature constitutively expresses the integrin lymphocyte function-associated antigen-1 ligands intercellular adhesion molecule (ICAM)-1 and -2. In this study, effector T cells were temporarily entrapped by the lung vasculature on their way to inflamed lymph nodes, and this entrapment was strongly reduced in ICAM-1 and -2 double-deficient mice (79 and 86% reduction for CD8(+) and CD4(+) effectors, respectively, compared with wild-type mice). Although the pulmonary vasculature has been suggested to be masked by the heparan sulfate-containing glycocalyx, which is susceptible to heparanase-mediated shedding, lung and lymphocyte heparanase have been found to be unnecessary for this entrapment. Systemic LPS induced rapid neutrophil entrapment in the lung vasculature, but in contrast to T-cell entrapment, this sequestration was ICAM-1, ICAM-2, and heparanase independent. Furthermore, neutrophil migration into the bronchoalveolar space induced by LPS inhalation and LPS-induced leakage of red blood cells into this space were not dependent on lung ICAMs or heparanase activity. Nevertheless, heparanase was critical for neutrophil accumulation in smoke-exposed lungs. Our results indicate that, whereas T cells use ICAM-1 and -2 for temporary pulmonary entrapment, neutrophils get sequestered and extravasate into inflamed lungs independent of ICAMs. This is the first demonstration that the pulmonary vasculature is differentially recognized by T cells and neutrophils.-Petrovich, E., Feigelson, S. W., Stoler-Barak, L., Hatzav, M., Solomon, A., Bar-Shai, A., Ilan, N., Li, J.-P., Engelhardt, B., Vlodavsky, I., Alon, R. Lung ICAM-1 and ICAM-2 support spontaneous intravascular effector lymphocyte entrapment but are not required for neutrophil entrapment or emigration inside endotoxin-inflamed lungs.

Coordinated expression of DNAM-1 and LFA-1 in educated NK cells.

The functional capacity of NK cells is dynamically tuned by integrated signals from inhibitory and activating cell surface receptors in a process termed NK cell education. However, the understanding of the cellular and molecular mechanisms behind this functional tuning is limited. In this study, we show that the expression of the adhesion molecule and activation receptor DNAX accessory molecule 1 (DNAM-1) correlates with the quantity and quality of the inhibitory input by HLA class I-specific killer cell Ig-like receptors and CD94/NKG2A as well as with the magnitude of functional responses. Upon target cell recognition, the conformational state of LFA-1 changed in educated NK cells, associated with rapid colocalization of both active LFA-1 and DNAM-1 at the immune synapse. Thus, the coordinated expression of LFA-1 and DNAM-1 is a central component of NK cell education and provides a potential mechanism for controlling cytotoxicity by functionally mature NK cells.