Ezrin/Radixin/Moesin proteins and flotillins cooperate to promote uropod formation in T cells

T cell uropods are enriched in specific proteins including adhesion receptors such as P-selectin glycoprotein ligand-1 (PSGL-1), lipid raft-associated proteins such as flotillins and ezrin/radixin/moesin (ERM) proteins which associate with cholesterol-rich raft domains and anchor adhesion receptors to the actin cytoskeleton. Using dominant mutants and siRNA technology we have tested the interactions among these proteins and their role in shaping the T cell uropod. Expression of wild type (WT) ezrin-EGFP failed to affect the morphology of human T cells or chemokine-induced uropod recruitment of PSGL-1 and flotillin-1 and -2. In contrast, expression of constitutively active T567D ezrin-EGFP induced a motile, polarized phenotype in some of the transfected T cells, even in the absence of chemokine. These cells featured F-actin-rich ruffles in the front and uropod enrichment of PSGL-1 and flotillins. T567D ezrin-EGFP was itself strongly enriched in the rear of the polarized T cells. Uropod formation induced by T567D ezrin-EGFP was actin-dependent as it was attenuated by inhibition of Rho-kinase or myosin II, and abolished by disruption of actin filaments. While expression of constitutively active ezrin enhanced cell polarity, expression of a dominant-negative deletion mutant of ezrin, 1-310 ezrin-EGFP, markedly reduced uropod formation induced by the chemokine SDF-1, T cell front-tail polarity, and capping of PSGL-1 and flotillins. Transfection of T cells with WT or T567D ezrin did not affect chemokine-mediated chemotaxis whereas 1-310 ezrin significantly impaired spontaneous 2D migration and chemotaxis. siRNA-mediated downregulation of flotillins in murine T cells attenuated moesin capping and uropod formation, indicating that ERM proteins and flotillins cooperate in uropod formation. In summary, our results indicate that activated ERM proteins function together with flotillins to promote efficient chemotaxis of T cells by structuring the uropod of migrating T cells.

Static and dynamic lengths of neutrophil microvilli

Containing most of the L-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) on their tips, microvilli are believed to promote the initial arrest of neutrophils on endothelium. At the rolling stage following arrest, the lifetimes of the involved molecular bonds depend on the pulling force imposed by the shear stress of blood flow. With two different methods, electron microscopy and micropipette manipulation, we have obtained two comparable neutrophil microvillus lengths, both ≈0.3 μm in average. We have found also that, under a pulling force, a microvillus can be extended (microvillus extension) or a long thin membrane cylinder (a tether) can be formed from it (tether formation). If the force is ≤34 pN (± 3 pN), the length of the microvillus will be extended; if the force is >61 pN (± 5 pN), a tether will be formed from the microvillus at a constant velocity, which depends linearly on the force. When the force is between 34 pN and 61 pN (transition zone), the degree of association between membrane and cytoskeleton in individual microvilli will dictate whether microvillus extension or tether formation occurs. When a microvillus is extended, it acts like a spring with a spring constant of ≈43 pN/μm. In contrast to a rigid or nonextendible microvillus, both microvillus extension and tether formation can decrease the pulling force imposed on the adhesive bonds, and thus prolonging the persistence of the bonds at high physiological shear stresses.

Quantifying the Effects of Contact Duration, Loading Rate, and Approach Velocity on P-Selectin-PSGL-1 Interactions Using AFM

Kinetics and its regulation by extrinsic physical factors govern selectin-ligand interactions that mediate tethering and rolling of circulating cells on the vessel wall under hemodynamic forces. While the force regulation of off-rate for dissociation of selectin-ligand bonds has been extensively studied, much less is known about how transport impacts the on-rate for association of these bonds and their stability. We used atomic force microscopy (AFM) to quantify how the contact duration, loading rate, and approach velocity affected kinetic rates and strength of bonds of P-selectin interacting with P-selectin glycoprotein ligand I (PSGL-1). We found a saturable relationship between the contact time and the rupture force, a biphasic relationship between the adhesion probability and the retraction velocity, a piece-wise linear relationship between the rupture force and the logarithm of the loading rate, and a threshold relationship between the approach velocity and the rupture force. These results provide new insights into how physical factors regulate receptor-ligand interactions.

Kinetic measurements of cell surface E-selectin/carbohydrate ligand interactions

Selectin/ligand interactions initiate the multistep adhesion and signaling cascades in the recruitment of leukocytes from circulation to inflamed tissues and may also play a role in tumor metastasis. Kinetic properties of these interactions are essential determinants governing blood-borne cells' tethering to and rolling on the vessel wall. Extending our recently developed micropipette method, we have measured the kinetic rates of E-selectin/ligand interactions. Red cells coated with an E-selectin construct were allowed to bind HL-60 or Colo-205 cells bearing carbohydrate ligands. Specific adhesions were observed to occur at isolated points, the frequency of which followed a Poisson distribution. These point attachments were formed at the same rate with both the HL-60 and Colo-205 cells (0.14 +/- 0.04 and 0.13 +/- 0.03 mum(2) s(-1) per unit density of E-selectin, respectively) but dissociated from the former at a rate twice as fast as did from the latter (0.92 +/- 0.23 and 0.44 +/- 0.10 s(-1), respectively). The reverse rates agree well with those measured by the flow chamber. The forward rates are orders of magnitude higher than those of Fc gamma receptors interacting with IgG measured under similar conditions, consistent with the rapid kinetics requirement for the function of E-selectin/ligand binding, which is to capture leukocytes on endothelial surfaces from flow.

PSGL-1 is dispensible for the development of active experimental autoimmune encephalomyelitis in SJL/J mice

The adhesion molecule P-selectin glycoprotein ligand (PSGL)-1 has been suggested to be involved in the immunopathogenesis of multiple sclerosis (MS). However, in C57BL/6 mice PSGL-1 was found to be dispensible for the development of MOG(aa35-55)-induced experimental autoimmune encephalomyelitis (EAE), an animal model for MS. To study, if involvement of PSGL-1 to EAE pathogenesis can be observed in another common mouse model, we backcrossed PSGL-1(-/-) mice for at least 12 generations into the SJL/J background and compared PLP(aa139-151) induced EAE in PSGL-1(-/-) SJL/J mice versus wild-type SJL/J mice. Here, we demonstrate that PSGL-1(-/-) SJL/J mice exhibited EAE pathogenesis indistinguishable from wild-type SJL/J mice. Our present study underscores and emphasizes previous observations that PSGL-1 is dispensible for EAE pathogenesis.

Mycobacteria-responsive sonic hedgehog signaling mediates programmed death-ligand 1-and prostaglandin E-2-induced regulatory T cell expansion

CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) are exploited by mycobacteria to subvert the protective host immune responses. The Treg expansion in the periphery requires signaling by professional antigen presenting cells and in particularly dendritic cells (DC). However, precise molecular mechanisms by which mycobacteria instruct Treg expansion via DCs are not established. Here we demonstrate that mycobacteria-responsive sonic hedgehog (SHH) signaling in human DCs leads to programmed death ligand-1 (PD-L1) expression and cyclooxygenase (COX)-2-catalyzed prostaglandin E-2 (PGE(2)) that orchestrate mycobacterial infection-induced expansion of Tregs. While SHH-responsive transcription factor GLI1 directly arbitrated COX-2 transcription, specific microRNAs, miR-324-5p and miR-338-5p, which target PD-L1 were downregulated by SHH signaling. Further, counter-regulatory roles of SHH and NOTCH1 signaling during mycobacterial-infection of human DCs was also evident. Together, our results establish that Mycobacterium directs a fine-balance of host signaling pathways and molecular regulators in human DCs to expand Tregs that favour immune evasion of the pathogen.

In vivo blockade of Ca(+2)-dependent nitric oxide synthases impairs expressions of L-selectin and PECAM-1

Interactions of leukocytes with endothelium play a role for the immune system modulated by endogenous agents, such as glucocorticoids and nitric oxide (NO). Glucocorticoids inhibit leukocyte-endothelial interactions whereas the role of NO is still controversial. In this study, the activity of Ca(+2)-dependent nitric oxide synthases was in vivo blocked in male Wistar rats by given L-NAME, 20 mg kg(-1) for 14 days dissolved in drinking water and expression of adhesion molecules involved in leukocyte-endothelial interactions was investigated. Expressions of L-selectin and PECAM-I in peripheral leukocytes and PECAM-1 in endothelial cells were reduced by L-NAME treatment. Only L-selectin expression was controlled at transcriptional levels. These effects were not dependent on endogenous glucocorticoids, as corticosterone levels were not altered in NAME-treated rats. Our results show that NO, produced at physiological levels, controls expression of constitutive adhesion molecules expressions in cell membranes by different mechanisms of action. Published by Elsevier Inc.

Immune cell entry into the central nervous system: involvement of adhesion molecules and chemokines

In multiple sclerosis and in its animal model experimental autoimmune encephalomyelitis (EAE), inflammatory cells migrate across the highly specialized endothelial blood-brain barrier (BBB) and gain access to the central nervous system (CNS). It is well established that leukocyte recruitment across this vascular bed is unique due to the predominant involvement of alpha4-integrins in mediating the initial contact to as well as firm adhesion with the endothelium. In contrast, the involvement of the selectins, L-selectin, E- and P-selectin and their respective carbohydrate ligands such as P-selectin glycoprotein (PSGL)-1 in this process has been controversially discussed. Intravital microscopic analysis of immune cell interaction with superficial brain vessels demonstrates a role for E- and P-selectin and their common ligand PSGL-1 in lymphocyte rolling. However, E- and P-selectin-deficient SJL- or C57Bl/6 mice or PSGL-1-deficient C57Bl/6 mice develop EAE indistinguishable from wild-type mice. Considering these apparently discrepant observations, it needs to be discussed whether the molecular mechanisms involved in leukocyte trafficking across superficial brain vessels are irrelevant for EAE pathogenesis or whether the therapeutic efficacy of targeting alpha4-integrins in EAE is truly dependent on the inhibition of leukocyte trafficking across the BBB.

Clinical impact of programmed cell death ligand 1 expression in colorectal cancer

BACKGROUND Programmed cell death 1 (PD-1) receptor triggering by PD ligand 1 (PD-L1) inhibits T cell activation. PD-L1 expression was detected in different malignancies and associated with poor prognosis. Therapeutic antibodies inhibiting PD-1/PD-L1 interaction have been developed. MATERIALS AND METHODS A tissue microarray (n=1491) including healthy colon mucosa and clinically annotated colorectal cancer (CRC) specimens was stained with two PD-L1 specific antibody preparations. Surgically excised CRC specimens were enzymatically digested and analysed for cluster of differentiation 8 (CD8) and PD-1 expression. RESULTS Strong PD-L1 expression was observed in 37% of mismatch repair (MMR)-proficient and in 29% of MMR-deficient CRC. In MMR-proficient CRC strong PD-L1 expression correlated with infiltration by CD8(+) lymphocytes (P=0.0001) which did not express PD-1. In univariate analysis, strong PD-L1 expression in MMR-proficient CRC was significantly associated with early T stage, absence of lymph node metastases, lower tumour grade, absence of vascular invasion and significantly improved survival in training (P=0.0001) and validation (P=0.03) sets. A similar trend (P=0.052) was also detectable in multivariate analysis including age, sex, T stage, N stage, tumour grade, vascular invasion, invasive margin and MMR status. Interestingly, programmed death receptor ligand 1 (PDL-1) and interferon (IFN)-γ gene expression, as detected by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) in fresh frozen CRC specimens (n=42) were found to be significantly associated (r=0.33, P=0.03). CONCLUSION PD-L1 expression is paradoxically associated with improved survival in MMR-proficient CRC.

A physiologic function for p-glycoprotein (MDR-1) during the migration of dendritic cells from skin via afferent lymphatic vessels

P-glycoprotein (MDR-1) is a well-known transporter that mediates efflux of chemotherapeutic agents from the intracellular milieu and thereby contributes to drug resistance. MDR-1 also is expressed by nonmalignant cells, including leukocytes, but physiologic functions for MDR-1 are poorly defined. Using an initial screening assay that included >100 mAbs, we observed that neutralizing mAbs MRK16, UIC2, and 4E3 against MDR-1 specifically and potently blocked basal-to-apical transendothelial migration of mononuclear phagocytes, a process that may mimic their migration into lymphatic vessels. Antagonists of MDR-1 then were used in a model of authentic lymphatic clearance. In this model, antigen-presenting dendritic cells (DC) migrate out of explants of cultured human skin and into the culture medium via dermal lymphatic vessels. DC and T cells derived from skin expressed MDR-1 on their surfaces. Addition of anti-MDR-1 mAbs MRK16, UIC2, or the MDR-1 antagonist verapamil to skin explants at the onset of culture inhibited the appearance of DC, and accompanying T cells, in the culture medium by approximately 70%. Isotype-matched control mAbs against other DC molecules including CD18, CD31, and major histocompatibility complex I did not block. In the presence of MDR-1 antagonists, epidermal DC were retained in the epidermis, in contrast to control conditions. In summary, this work identifies a physiologic function for MDR-1 during the mobilization of DC and begins to elucidate how these critical antigen-presenting cells migrate from the periphery to lymph nodes to initiate T lymphocyte-mediated immunity.

Association analysis of chromosome 1 migraine candidate genes

Migraine with aura (MA) is a subtype of typical migraine. Migraine with aura (MA) also encompasses a rare severe subtype Familial Hemiplegic Migraine (FHM) with several known genetic loci. The type 2 FHM (FHM-2) susceptibility locus maps to chromosome 1q23 and mutations in the ATP1A2 gene at this site have recently been implicated. We have previously provided evidence of linkage of typical migraine (predominantly MA) to microsatellite markers on chromosome 1, in the 1q31 and 1q23 regions. In this study, we have undertaken a large genomic investigation involving candidate genes that lie within the chromosome 1q23 and 1q31 regions using an association analysis approach. Methods We have genotyped a large population of case-controls (243 unrelated Caucasian migraineurs versus 243 controls) examining a set of 5 single nucleotide polymorphisms (SNPs) and the Fas Ligand dinucleotide repeat marker, located within the chromosome 1q23 and 1q31 regions. Results Several genes have been studied including membrane protein (ATP 1 subtype A4 and FasL), cytoplasmic glycoprotein (CASQ 1) genes and potassium (KCN J9 and KCN J10) and calcium (CACNA1E) channel genes in 243 migraineurs (including 85% MA and 15% of migraine without aura (MO)) and 243 matched controls. After correction for multiple testing, chi-square results showed non-significant P values (P > 0.008) across all SNPs (and a CA repeat) tested in these different genes, however results with the KCN J10 marker gave interesting results (P = 0.02) that may be worth exploring further in other populations. Conclusion These results do not show a significant role for the tested candidate gene variants and also do not support the hypothesis that a common chromosome 1 defective gene influences both FHM and the more common forms of migraine.

Effects of Molecular Length, Ortientation and Amino Acid Mutation on Kinetics of Selectin/Ligand interactions

Receptor/ligand interactions are basic issues to cell adhesion, which are important to many physiological and pathological processes such as lymphocyte-mediated cytotoxicity, tumor metastasis and inflammatory reactionl. Selectin/carbohydrate ligand bindings have been found to mediate the fast rolling of leukocytes on activated endothelial monolayer. Kinetic rate and binding affinity constants are essential determinants of cell adhesion...

Formation of Novel Di-Nuclear Lanthanide Luminescent Sm(III), Eu(III) and Tb(III) Triple Stranded Helicates from a C2 Symmetrically 2,6-Dicarboxylic Amide based 1,3-Xylene Ligand in CH3CN

The synthesis of the C2-symmetrical ligand 1 consisting of two naphthalene units connected to two pyridine-2,6-dicarboxamide moieties linked by a xylene spacer and the formation of LnIII-based (Ln1/4 Sm, Eu, Tb, and Lu) dimetallic helicates [Ln2 · 13] in MeCN by means of a metal-directed synthesis is described. By analyzing the metal-induced changes in the absorption and the fluorescence of 1, the formation of the helicates, and the presence of a second species [Ln2 · 12] was confirmed by nonlinear- regression analysis. While significant changes were observed in the photophysical properties of 1, the most dramatic changes were observed in the metal-centred lanthanide emissions, upon excitation of the naphthalene antennae. From the changes in the lanthanide emission, we were able to demonstrate that these helicates were formed in high yields (ca. 90% after the addition of 0.6 equiv. of LnIII), with high binding constants, which matched well with that determined from the changes in the absorption spectra. The formation of the LuIII helicate, [ Lu2 · 13 ] , was also investigated for comparison purposes, as we were unable to obtain accurate binding constants from the changes in the fluorescence emission upon formation of [Sm2 · 13], [Eu2 · 13], and [Tb2 · 13].