E-selectin Ligand Expression Research Articles

Differing contributions of CCR4, E-selectin and VLA-4 to the migration of CD4 memory and activated CD4+CD25+ T cells to dermal inflammation. (95.2)

Abstract CCR4 is on T cells in dermal inflammation and may mediate homing to skin. Our objective was to determine the expression and interaction of CCR4, E-selectin ligand (ESL) and a4β1 on memory and activated T cells in recruitment to dermal inflammation. A mAb to CCR4 (CR4.1) was developed. CCR4 was on ~10% of memory CD4 cells and 15% of these were ESL+. CCR4 and ESL were markedly increased on activated T cells. CCR4+ memory CD4 cells (memCCR4+) migrated 8-10 fold more to inflammation induced by cytokines, TLR agonists and DTH, than memCCR4- cells, and homed less to LNs. CCR4+ anti-TCR activated CD4 cells (actCCR4+) migrated only 50% better to skin than actCCR4- cells. E-selectin blockade inhibited 50-75% of actCCR4+, but not memCCR4 cell migration. a4β1 blockade had an inverse effect, i.e. inhibiting memCCR4+ more than actCCR4+ cells, while P-selectin blockade had no effect. Thus, CCR4 is on a subset of memCD4 cells with dermal tropism, but this selective homing is reduced on activated CD4+CD25+cells. The role of ESL and a4β1 also differs between activated and memory CCR4+ cells, with a decrease in the role of ESL and an increase in a4β1 with differentiation to long-term memory. (Supported by the CIHR).

Regulatory T Cells Sequentially Migrate from Inflamed Tissues to Draining Lymph Nodes to Suppress the Alloimmune Response

To determine the site and mechanism of suppression by regulatory T (Treg) cells, we investigated their migration and function in an islet allograft model. Treg cells first migrated from blood to the inflamed allograft where they were essential for the suppression of alloimmunity. This process was dependent on the chemokine receptors CCR2, CCR4, and CCR5 and P- and E-selectin ligands. In the allograft, Treg cells were activated and subsequently migrated to the draining lymph nodes (dLNs) in a CCR2, CCR5, and CCR7 fashion; this movement was essential for optimal suppression. Treg cells inhibited dendritic cell migration in a TGF-beta and IL-10 dependent fashion and suppressed antigen-specific T effector cell migration, accumulation, and proliferation in dLNs and allografts. These results showed that sequential migration from blood to the target tissue and to dLNs is required for Treg cells to differentiate and execute fully their suppressive function.

Skin-Homing Receptors on Effector Leukocytes Are Differentially Sensitive to Glyco-Metabolic Antagonism in Allergic Contact Dermatitis

T cell recruitment into inflamed skin is dependent on skin-homing receptor binding to endothelial (E)- and platelet (P)-selectin. These T cell receptors, or E- and P-selectin ligands, can be targeted by the metabolic fluorosugar inhibitor, 4-F-GlcNAc, to blunt cutaneous inflammation. Compelling new data indicate that, in addition to T cells, NK cells are also recruited to inflamed skin in allergic contact hypersensitivity (CHS) contingent on E- and P-selectin-binding. Using a model of allergic CHS, we evaluated the identity and impact of NK cell E-selectin ligand(s) on inflammatory responses and examined the oral efficacy of 4-F-GlcNAc. We demonstrated that the predominant E-selectin ligands on NK cells are P-selectin glycoprotein ligand-1 and protease-resistant glycolipids. We showed that, unlike the induced E-selectin ligand expression on activated T cells upon exposure to Ag, ligand expression on NK cells was constitutive. CHS responses were significantly lowered by orally administered 4-F-GlcNAc treatment. Although E-selectin ligand on activated T cells was suppressed, ligand expression on NK cells was insensitive to 4-F-GlcNAc treatment. These findings indicate that downregulating effector T cell E- and P-selectin ligand expression directly correlates with anti-inflammatory efficacy and provides new insight on metabolic discrepancies of E-selectin ligand biosynthesis in effector leukocytes in vivo.

Cutting Edge: Chemokine Receptor CCR4 Is Necessary for Antigen-Driven Cutaneous Accumulation of CD4 T Cells under Physiological Conditions

Dual expression of chemokine receptor CCR4 and E-selectin ligand is characteristic of skin-tropic CD4 T cells from blood, lymphoid organs, and the skin itself. A strong and specific correlation exists among CCR4, its ligand CCL17/TARC, and the cutaneous lymphocyte-homing process. Nevertheless, whether CCR4 function is required for skin-specific trafficking remains an open question, which we address in this study. We developed an Ag-specific, TCR-transgenic, murine CD4 T cell adoptive transfer model that induces a mixed Th1 and Th17 cutaneous response. Within the hosts, both CCR4(+/+) and CCR4(-/-) donor CD4 T cells contribute equally well to the circulating E-selectin ligand(+) pool in response to Ag. However, only CCR4(+/+) donor cells accumulate efficiently within the skin. CCR4(-/-) cells home normally to the peritoneum, showing that they do not have a general defect in lymphocyte trafficking. We conclude that under physiological conditions, CCR4 is a nonredundant, necessary component of skin-specific lymphocyte trafficking.

Varied levels of reactivity by different E-selectin/Fc constructs with cutaneous lymphocyte-associated antigen (CLA) + CD4 + T cells

T cells utilize the vascular adhesion molecule E-selectin to enter inflamed skin. T cells identified by the mAb HECA-452 [cutaneous lymphocyte-associated antigen (CLA) T cells] are enriched in E-selectin ligand expressing cells. However, the proportion of CLA + T cells reactive with an E-selectin/Fc chimeric construct, as determined by flow cytometry, can vary considerably between studies. One important variable in these studies has been the E-selectin/Fc chimera used to assess ligand expression. We therefore compared the reactivity of mouse, rat, and human E-selectin/Fc from the same widely used commercial source with peripheral blood CLA + CD4 + T cells, neutrophils, and the promyelocytic cell line HL-60 by flow cytometry and by shear flow assays. We observed that the binding activities of the different E-selectin/Fc chimeras were considerably different. Mouse E-selectin/Fc demonstrated the highest binding activity with human neutrophils and HL-60 cells by both assay approaches, whereas human E-selectin/Fc demonstrated the lowest binding activity. In addition, mouse E-selectin/Fc binding increased essentially in a linear manner with increasing expression of CLA by CD4 + T cells, whereas human and rat E-selectin/Fc binding occurred with only a subset of CLA + CD4 + T cells. We conclude that there is substantial variability in the reactivity of different E-selectin/Fc constructs, thus caution should be used when assessing E-selectin ligand expression with these reagents. For instance, the discordance in expression of CLA and E-selectin ligands by T cells may in part be due to the E-selectin/Fc construct being used.

G-CSF induces E-selectin ligand expression on human myeloid cells

Clinical use of G-CSF can result in vascular and inflammatory complications. To investigate the molecular basis of these effects, we analyzed the adherence of G-CSF-mobilized human peripheral blood leukocytes (ML) to inflamed (TNF-alpha-stimulated) vascular endothelium. Studies using parallel plate assays under physiologic flow conditions and intravital microscopy in a mouse inflammation model each showed that ML take part in heightened adhesive interactions with endothelium compared to unmobilized (native) blood leukocytes, mediated by markedly increased E-selectin receptor-ligand interactions. Biochemical studies showed that ML express the potent E-selectin ligand HCELL (ref. 8) and another, previously unrecognized approximately 65-kDa E-selectin ligand, and possess enhanced levels of transcripts encoding glycosyltransferases (ST3GalIV, FucT-IV and FucT-VII) conferring glycan modifications associated with E-selectin ligand activity. Enzymatic treatments and physiologic binding assays showed that HCELL and the approximately 65-kDa E-selectin ligand contribute prominently to the observed G-CSF-induced myeloid cell adhesion to inflamed endothelium. Treatment of normal human bone marrow cells with a pharmacokinetically relevant concentration of G-CSF in vitro resulted in increased expression of these two molecules, coincident with increased transcripts encoding pertinent glycosyltransferases and heightened E-selectin binding. These findings provide direct evidence for a role of G-CSF in the induction of E-selectin ligands on myeloid cells, thus providing mechanistic insight into the pathobiology of G-CSF complications.

Innate Immune Responses to Herpes Simplex Virus Type 2 Influence Skin Homing Molecule Expression by Memory CD4+Lymphocytes

Herpes simplex virus (HSV) infections of humans are characterized by intermittent, lytic replication in epithelia. Circulating HSV-specific CD4 T cells express lower levels of preformed cutaneous lymphocyte-associated antigen (CLA), a skin-homing receptor, than do circulating HSV-specific CD8 T cells but, paradoxically, move into infected skin earlier than CD8 cells. Memory CD4 T cells develop strong and selective expression of CLA and E-selectin ligand while responding to HSV antigen in vitro. We now show that interleukin-12, type I interferon, and transforming growth factor beta are each involved in CLA expression by memory HSV type 2 (HSV-2)-specific CD4 T cells in peripheral blood mononuclear cells (PBMC). A reduction of the number of monocytes and dendritic cells from PBMC reduces CLA expression by HSV-2-responsive CD4 lymphoblasts, while their reintroduction restores this phenotype, identifying these cells as possible sources of CLA-promoting cytokines. Plasmacytoid dendritic cells are particularly potent inducers of CLA on HSV-reactive CD4 T cells. These observations are consistent with cooperation between innate and acquired immunity to promote a pattern of homing receptor expression that is physiologically appropriate for trafficking to infected tissues.

Expression of Cutaneous Lymphocyte–Associated Antigen and E‐selectin Ligand by Circulating Human Memory CD4+T Lymphocytes Specific for Herpes Simplex Virus Type 2

Virus-specific memory T lymphocytes traffic to sites of viral infection. Herpes simplex virus (HSV) type 2-specific CD4(+) and CD8(+) T lymphocytes differ with regard to their homing kinetics to infected tissues. We studied the expression of cutaneous lymphocyte-associated antigen (CLA) and E-selectin ligand (ESL) by HSV-2-specific CD4(+) T lymphocytes. Virus-reactive T lymphocytes were identified ex vivo by CD154 or interferon-gamma up-regulation. We detected selective expression of CLA by HSV-2-reactive CD4(+) T lymphocytes, but at levels lower than those we previously observed for CD8(+) T lymphocytes. Short-term HSV-2-reactive CD4(+) lines generated from peripheral-blood mononuclear cells preferentially express CLA, compared with cytomegalovirus- or influenza-specific cells. CLA is expressed by HSV-2-reactive cells that are initially CLA negative before restimulation. Short-term culture-expanded HSV-2-specific CD4(+) T lymphocytes also selectively express ESL. These findings have implications for the optimization of vaccines for HSV and other cutaneous pathogens.

Dendritic Cell Immunization Route Determines CD8+ T Cell Trafficking to Inflamed Skin: Role for Tissue Microenvironment and Dendritic Cells in Establishment of T Cell-Homing Subsets

The effector/memory T cell pool branches in homing subsets selectively trafficking to organs such as gut or skin. Little is known about the critical factors in the generation of skin-homing CD8+ T cells, although they are crucial effectors in skin-restricted immune responses such as contact hypersensitivity and melanoma defense. In this study, we show that intracutaneous, but not i.v. injection of bone marrow-derived dendritic cells induced skin-homing CD8+ T cells with up-regulated E-selectin ligand expression and effector function in contact hypersensitivity. The skin-homing potential and E-selectin ligand expression remained stable in memory phase without further Ag contact. In contrast, i.p. injection induced T cells expressing the gut-homing integrin alpha(4)beta(7). Although differential expression of these adhesion molecules was strictly associated with the immunization route, the postulated skin-homing marker CCR4 was transiently up-regulated in all conditions. Interestingly, dendritic cells from different tissues effectively induced the corresponding homing markers on T cells in vitro. Our results suggest a crucial role for the tissue microenvironment and dendritic cells in the instruction of T cells for tissue-selective homing and demonstrate that Langerhans cells are specialized to target T cells to inflamed skin.

Prevention of leukocyte migration to inflamed skin with a novel fluorosugar modifier of cutaneous lymphocyte-associated antigen.

E-selectin and P-selectin on dermal postcapillary venules play critical roles in the migration of effector T cells into inflamed skin. P-selectin glycoprotein ligand-1 (PSGL-1) modified by alpha1,3-fucosyltransferase is the principal selectin ligand on skin-homing T cells and is required for effector T cell entry into inflamed skin. We have previously shown that a fluorinated analog of N-acetylglucosamine peracetylated-4-fluorinated-d-glucosamine (4-F-GlcNAc), inhibits selectin ligand expression on human T cell PSGL-1. To analyze 4-F-GlcNAc efficacy in dampening effector T cell migration to inflamed skin, we elicited allergic contact hypersensitivity (CHS) reactions in mice treated with 4-F-GlcNAc. We also investigated 4-F-GlcNAc efficacy on lymphocyte E-selectin ligand expression in LNs draining antigen-sensitized skin and on other immunological processes requisite for CHS responses. Our results showed that 4-F-GlcNAc treatment attenuated lymphocyte E-selectin ligand expression in skin-draining LNs and prevented CHS reactions. Significant reductions in inflammatory lymphocytic infiltrate were observed, while pathways related to antigenic processing and presentation and naive T cell recognition within skin-draining LNs were unaffected. These data indicate that 4-F-GlcNAc prevents CHS by inhibiting selectin ligand activity and the capacity of effector T cells to enter antigen-challenged skin without affecting the afferent phase of CHS.

Prevention of leukocyte migration to inflamed skin with a novel fluorosugar modifier of cutaneous lymphocyte-associated antigen

E-selectin and P-selectin on dermal postcapillary venules play critical roles in the migration of effector T cells into inflamed skin. P-selectin glycoprotein ligand-1 (PSGL-1) modified by α1,3-fucosyltransferase is the principal selectin ligand on skin-homing T cells and is required for effector T cell entry into inflamed skin. We have previously shown that a fluorinated analog of N-acetylglucosamine peracetylated-4-fluorinated-D-glucosamine (4-F-GlcNAc), inhibits selectin ligand expression on human T cell PSGL-1. To analyze 4-F-GlcNAc efficacy in dampening effector T cell migration to inflamed skin, we elicited allergic contact hypersensitivity (CHS) reactions in mice treated with 4-F-GlcNAc. We also investigated 4-F-GlcNAc efficacy on lymphocyte E-selectin ligand expression in LNs draining antigen-sensitized skin and on other immunological processes requisite for CHS responses. Our results showed that 4-F-GlcNAc treatment attenuated lymphocyte E-selectin ligand expression in skin-draining LNs and prevented CHS reactions. Significant reductions in inflammatory lymphocytic infiltrate were observed, while pathways related to antigenic processing and presentation and naive T cell recognition within skin-draining LNs were unaffected. These data indicate that 4-F-GlcNAc prevents CHS by inhibiting selectin ligand activity and the capacity of effector T cells to enter antigen-challenged skin without affecting the afferent phase of CHS.

Expression of α4β7 and E-selectin ligand by circulating memory B cells: implications for targeted trafficking to mucosal and systemic sites

We have examined the expression of homing receptors on circulating memory B cells subsets. Blood IgD+ (naive) B cells homogeneously express a high level of intestinal homing receptor, alpha4beta7, but IgD- (putative memory) B cells comprise distinct alpha4beta7+ and alpha4beta7- subsets. Naive and alpha4beta7+ memory B cells but not alpha4beta7- cells bind MAdCAM-1, suggesting that alpha4beta7 expression may predict B cell intestinal homing. In contrast, alpha4beta7+ and alpha4beta7- B cells bind well to VCAM-1, possibly allowing recruitment of both subsets to extra-intestinal sites, including those tissues of the "common mucosal immune system" characterized by vascular VCAM-1 expression. sIgA+ B cells, which are associated with mucosal immunity in the gut and elsewhere, are heterogeneous in homing receptor expression--with discrete subsets expressing alpha4beta7, L-selectin, and cutaneous lymphocyte antigen (CLA). sIgA+ CLA+ B cells are enriched by binding to E-selectin, suggesting that CLA may participate in B cell homing to nonintestinal mucosal tissues characterized by vascular E-selectin expression, such as chronically inflamed bronchial or oral mucosal. We conclude that circulating human peripheral blood memory B cells, like T cells, consist of discrete homing receptor-defined subsets. This diversity in homing phenotypes is apparent even among sIgA (presumptive mucosal) memory B cells, implying heterogeneity in trafficking mechanisms to different target mucosal surfaces.

Attenuated cardiac allograft vasculopathy in mice with targeted deletion of the transcription factor STAT4.

To study transcription factor signaling pathways that mediate cardiac allograft vasculopathy, we used mice with targeted gene deletion of signal transducer and activator of transcription (STAT)4 and STAT6 as recipients in our mouse cardiac transplant model of chronic rejection. At day 55 after transplantation, cardiac grafts placed into STAT4 -/- (n=10) had reduced frequency (24+/-2%) and severity (9+/-4%) of vascular occlusion compared with wild-type controls (n=7, frequency 70+/-12% [P<0.001], severity 25+/-6% [P<0.05]). This decrease was associated with reduced intragraft expression ((32)P RT-PCR and immunohistochemistry) of the Th1 signature cytokines interferon-gamma (P<0.001) and interleukin (IL)-2 (P<0.001). Furthermore, cardiac grafts in STAT4 -/- had fewer infiltrating CD45(+) mononuclear cells (99+/-27 cells/mm(3) compared with 551+/-168 cells/mm(3) in wild-type controls [P<0.05]) and reduced expression of P-selectin (P<0.001) and E-selectin (P<0.01) ligand, recently shown to regulate Th1 cell recruitment. In contrast, in grafts placed into STAT6 -/- (n=11), the development of cardiac allograft vasculopathy (frequency 62+/-8%, severity 28+/-6%) and Th2 cytokine profiles (IL-4, IL-10) were comparable to those in wild-type controls. Hence, we show that immune responses mediated by STAT4, but not STAT6, contribute to the development of cardiac allograft vasculopathy. We speculate that when present, STAT4-mediated signaling pathways may promote cardiac allograft vasculopathy by directing Th1-specific lymphocyte recruitment, activation, and effector functions.

Expression of E-selectin ligand in primary head and neck squamous cell carcinoma

Expression of E-selectin ligand in primary head and neck squamous cell carcinoma

A Circulating Bovine γδ T Cell Subset, Which Is Found in Large Numbers in the Spleen, Accumulates Inefficiently in an Artificial Site of Inflammation: Correlation with Lack of Expression of E-Selectin Ligands and L-Selectin

AbstractTissue-specific localization of TCR-defined subsets of γδ T cells has been widely reported; however, the mechanisms responsible for this phenomenon are poorly understood. We describe a bovine γδ T cell TCR-associated subset that preferentially localizes in the spleen. This subset was characterized by coexpression of CD8, and was found to lack surface expression of E-selectin ligands, GR Ag ligands, as well as low expression of L-selectin. The CD8-positive γδ T cell subset did not accumulate at sites of inflammation as efficiently as CD8-negative γδ T cells that, in contrast, express E-selectin and GR ligands and high levels of L-selectin. This is the first demonstration of a γδ T cell subset, which exhibits a defined tissue tropism, having a unique adhesion molecule expression profile. These results demonstrate that in some cases tissue-specific accumulation of γδ T cell subsets can be predicted by expression, or lack of expression, of defined homing molecules.

Expression of E-selectin and E-selectin ligands in human liver inflammation.

Lymphocyte recruitment to tissue is regulated by homing molecules expressed on subsets of leukocytes that bind to endothelial adhesion molecules termed addressins in target tissues. The addressins and homing molecules involved in recruiting lymphocytes to inflamed human liver are not known. E-selectin, which acts as an addressin for a subset of skin-infiltrating T cells that express a distinct E-selectin ligand called the cutaneous lymphocyte antigen (CLA), can be detected on endothelium in inflammatory liver diseases suggesting it might also act as an addressin for liver-infiltrating lymphocytes. To determine whether E-selectin does play such a role we looked for concomitant expression of E-selectin on hepatic endothelium and E-selectin ligands on infiltrating lymphocytes in liver sections from patients with primary biliary cirrhosis (PBC), acute allograft rejection, alcoholic liver disease, and normal liver. E-selectin was detected on vascular endothelium in association with lymphocytic infiltrates in all three liver diseases but not on normal endothelium. However, less that 10% of infiltrating lymphocytes expressed the only known lymphocyte E-selectin ligand, CLA. We used E-selectin chimeric fusion proteins to probe for novel E-selectin ligands on liver-infiltrating lymphocytes; however, less than 10% of lymphocytes had t he ability to bind the fusion proteins. Thus, E-selectin is unlikely to act as an addressin for liver-infiltrating lymphocytes, suggesting that other molecules are involved in lymphocyte recruitment to the liver. Despite the lack of binding to lymphocytes other structures in the liver expressed CLA and bound the E-selectin fusion proteins. Inflamed, but not normal, intrahepatic bile ducts expressed CLA and bound E-selectin, suggesting that they express activation dependent carbohydrate binding receptors. Furthermore, Kupffer cells did not express CLA but bound to both E-selectin fusion proteins, suggesting that they express potentially novel E-selectin ligands.

Expression of E-selectin and E-selectin ligands in human liver inflammation

Lymphocyte recruitment to tissue is regulated by homing molecules expressed on subsets of leukocytes that bind to endothelial adhesion molecules termed addressins in target tissues. The addressins and homing molecules involved in recruiting lymphocytes to inflamed human liver are not known. E-selectin, which acts as an addressin for a subset of skin-infiltrating T cells that express a distinct E-selectin ligand called the cutaneous lymphocyte antigen (CLA), can be detected on endothelium in inflammatory liver diseases suggesting it might also act as an addressin for liver-infiltrating lymphocytes. To determine whether E-selectin does play such a role we looked for concomitant expression of E-selectin on hepatic endothelium and E-selectin ligands on infiltrating lymphocytes in liver sections from patients with primary biliary cirrhosis (PBC), acute allograft rejection, alcoholic liver disease, and normal liver. E-selectin was detected on vascular endothelium in association with lymphocytic infiltrates in all three liver diseases but not on normal endothelium. However, less that 10% of infiltrating lymphocytes expressed the only known lymphocyte E-selectin ligand, CLA. We used E-selectin chimeric fusion proteins to probe for novel E-selectin ligands on liver-infiltrating lymphocytes; however, less than 10% of lymphocytes had t he ability to bind the fusion proteins. Thus, E-selectin is unlikely to act as an addressin for liver-infiltrating lymphocytes, suggesting that other molecules are involved in lymphocyte recruitment to the liver. Despite the lack of binding to lymphocytes other structures in the liver expressed CLA and bound the E-selectin fusion proteins. Inflamed, but not normal, intrahepatic bile ducts expressed CLA and bound E-selectin, suggesting that they express activation dependent carbohydrate binding receptors. Furthermore, Kupffer cells did not express CLA but bound to both E-selectin fusion proteins, suggesting that they express potentially novel E-selectin ligands. (Hepatology 1996 Sep;24(3):533-8)

Expression of E-selection and E-selectin ligands in human liver inflammation: [formula omitted]∗ [formula omitted] Liver Unit, Queen Elizabeth Hospital, Birmingham, UK and ∗Celltech Therapeutics, Slough, UK

Expression of E-selection and E-selectin ligands in human liver inflammation: [formula omitted]∗ [formula omitted] Liver Unit, Queen Elizabeth Hospital, Birmingham, UK and ∗Celltech Therapeutics, Slough, UK