IL-12, STAT4-dependent up-regulation of CD4(+) T cell core 2 beta-1,6-n-acetylglucosaminyltransferase, an enzyme essential for biosynthesis of P-selectin ligands

TCR activation of naive T cells in the presence of IL-12 drives polarization toward a Th1 phenotype and synthesis of P- and E-selectin ligands. Fucosyltransferase VII (Fuc-T VII) and core 2 beta-1,6-N-acetylglucosaminyltransferase (C2GnT) are critical for biosynthesis of selectin ligands. P-selectin glycoprotein ligand-1 is the best characterized ligand for P-selectin and also binds E-selectin. The contributions of TCR and cytokine signaling pathways to up-regulate Fuc-T VII and C2GnT during biosynthesis of E- and P-selectin ligands, such as P-selectin glycoprotein ligand 1, are unknown. IL-12 signals via the STAT4 pathway. Here, naive DO11.10 TCR transgenic and STAT4(-/-) TCR transgenic CD4(+) T cells were stimulated with Ag and IL-12 (Th1 condition), IL-4 (Th2), or neutralizing anti-IL-4 mAb only (Th0). The levels of Fuc-T VII and C2GnT mRNA in these cells were compared with their adhesive interactions with P- and E-selectin in vitro under flow. The data show IL-12/STAT4 signaling is necessary for induction of C2GnT, but not Fuc-TVII mRNA, and that STAT4(-/-) Th1 cells do not traffic normally to sites of inflammation in vivo, do not interact with P-selectin, and exhibit a partial reduction of E-selectin interactions under shear stress in vitro. Ag-specific TCR activation in CD4(+) T cells was sufficient to trigger induction of Fuc-TVII, but not C2GnT, mRNA and expression of E-selectin, but not P-selectin, ligands. Thus, Fuc-T VII and C2GnT are regulated by different signals during Th cell differentiation, and both cytokine and TCR signals are necessary for the expression of E- and P-selectin ligands.     

Expression of functional selectin ligands on Th cells is differentially regulated by IL-12 and IL-4

Immune responses may be qualitatively distinct depending on whether Th1 or Th2 cells predominate at the site of Ag exposure. T cell subset-specific expression of ligands for vascular selectins may underlie the distinct patterns of recruitment of Th1 or Th2 cells to peripheral inflammatory sites. Here we examine the regulation of selectin ligand expression during murine T helper cell differentiation. Large numbers of Th1 cells interacted with E- and P-selectin under defined flow conditions, while few Th2 and no naive T cells interacted. Th1 cells also expressed more fucosyltransferase VII mRNA than naive or Th2 cells. IL-12 induced expression of P-selectin ligands on Ag-activated naive T cells, even in the presence of IL-4, and on established Th2 cells restimulated in the presence of IL-12 and IFN-gamma. In contrast, Ag stimulation alone induced only E-selectin ligand. Interestingly, restimulation of established Th2 cells in the presence of IL-12 and IFN-gamma induced expression of P-selectin ligands but not E-selectin ligands; IFN-gamma alone did not enhance expression of either selectin ligand. In summary, functional P- and E-selectin ligands are expressed on most Th1 cells, few Th2 cells, but not naive T cells. Furthermore, selectin ligand expression is regulated by the cytokine milieu during T cell differentiation. IL-12 induces P-selectin ligand, while IL-4 plays a dominant role in down-regulating E-selectin ligand.     

Alpha(1,3)-fucosyltransferase VII and alpha(2,3)-sialyltransferase IV are up-regulated in activated CD4 T cells and maintained after their differentiation into Th1 and migration into inflammatory sites.

Activated Th1 CD4 T cells bind to P-selectin and migrate into inflamed tissue, whereas Th2 cells do not. We show that alpha(1, 3)-fucosyltransferase VII (FucT-VII) and alpha(2, 3)-sialyltransferase IV (ST3GalIV), which are crucial for the biosynthesis of functional P-selectin ligands, are absent in naive CD4 T cells, but are rapidly up-regulated upon activation. Th1 or Th2 differentiation in the presence of polarizing cytokines leads to down-regulation of FucT-VII mRNA selectively in Th2 but not in Th1 cells. Influencing the differentiation by varying the priming dose of antigenic peptide results in similar FucT-VII down-regulation only in Ag-specific Th2 cells. ST3GalIV levels remain elevated. FucT-VII and ST3GalIV mRNAs are also up-regulated by Th1 cells primed in vivo and recruited into the lymph nodes draining delayed-type hypersensitivity sites. We identify FucT-VII gene expression as a principal difference between Th1 and Th2 cells, and underscore the importance of FucT-VII and ST3GalIV expression for the biosynthesis of functional selectin ligands.     

TNF-alpha, IL-4, and IFN-gamma regulate differential expression of P- and E-selectin expression by porcine aortic endothelial cells

P- and E-selectin are surface glycoproteins that mediate leukocyte rolling on the surface of endothelium in inflammation. We have cloned porcine P-selectin cDNA and generated a mAb, 12C5, with which to examine P-selectin expression by porcine aortic endothelial cells (PAEC) in comparison with that of E-selectin. Basal expression by PAEC of P-selectin was greater than that of E-selectin, whereas E-selectin expression was more prominently enhanced than that of P-selectin by stimulation with TNF-alpha or IL-1alpha. Both human or porcine IL-4 led to an increase in P-selectin expression, with kinetics that were delayed compared with those seen following stimulation with TNF-alpha or IL-1alpha, but IL-4 did not stimulate expression of E-selectin. When cells were stimulated with TNF-alpha in the presence of IL-4, we observed enhanced P-selectin expression with a parallel reduction in E-selectin expression. Finally, the increase in P-selectin expression due to human IL-4 was reduced in the presence of porcine but not human IFN-gamma. These observations show that E-selectin and P-selectin expression are differentially regulated in PAEC, and that IL-4 leads to a shift in the relative surface density of the two molecules toward P-selectin. The ability of porcine IFN-gamma to inhibit IL-4-induced P-selectin expression suggests that the balance between Th1 and Th2 cytokine production may determine the relative densities of the two selectins in chronic immune-mediated inflammation. Because the increased expression of P-selectin induced by human IL-4 was not inhibited by human IFN-gamma, this balance may be shifted toward P-selectin expression in porcine xenografts infiltrated by human lymphocytes.     

Potent induction of alpha(1,3)-fucosyltransferase VII in activated CD4+ T cells by TGF-beta 1 through a p38 mitogen-activated protein kinase-dependent pathway

Homing of effector T cells to sites of inflammation, particularly in the skin, is dependent on T cell expression of ligands for the endothelial selectins. Underlying expression of these ligands is the expression of alpha(1,3)-fucosyltransferase VII (FucT-VII), a FucT essential for biosynthesis of selectin ligands. FucT-VII is sharply induced in activated T cells by IL-12, but cytokines other than IL-12 that induce FucT-VII and functional selectin ligands have not been identified, and are likely to be important in homing of T cells to other selectin-dependent sites. Screening of a number of cytokines known to be active on T cells identified only TGF-beta1 as able to up-regulate FucT-VII mRNA levels and selectin ligands on activated CD4 T cells. The sharp increase in FucT-VII induced by TGF-beta1 in activated T cells was completely blocked by pharmacologic inhibition of p38 mitogen-activated protein kinase, but was unaffected by mitogen-activated protein/extracellular signal-related kinase kinase inhibitors. The selective ability of TGF-beta1 to induce selectin ligands on activated T cells is likely important for T cell homing to the gut, which is a strongly selectin-dependent site, and correlates with the ability of TGF-beta1 to coordinately induce other gut-associated homing pathways.     

Regulation and Migratory Role of P-Selectin Ligands during Intestinal Inflammation

Dendritic cells from mesenteric lymph nodes (MLN) can convert retinal to retinoic acid (RA), which promotes induction of the gut-specific homing receptor α4β7. In contrast, priming within peripheral lymph nodes leads to upregulation of E- and P-selectin ligands (E- and P-lig). Apart from its α4β7 promoting effect, RA was shown to suppress E- and P-lig induction in vitro. However, enhanced frequencies of P-lig⁺ CD4⁺ T cells were reported during intestinal inflammation. To understand this contradiction, we first determined whether location of intestinal inflammation, that is, ileitis or colitis, affects P-lig induction. Both conditions promoted P-lig expression on CD4⁺ T cells; however, P-lig expressed on T cells facilitated Th1 cell recruitment only into the inflamed colon but not into inflamed small intestine induced by oral Toxoplasma gondii infection. A majority of P-lig⁺CD4⁺ T cells found within MLN during intestinal inflammation co-expressed α4β7 confirming their activation in the presence of RA. Mesenteric P-lig⁺CD4⁺ cells co-expressed the 130 kDa isoform of CD43 which requires activity of core 2 (beta)1,6-N-acetyl-glycosaminyltransferase-I (C2GlcNAcT-I) suggesting that C2GlcNAcT-I contributes to P-lig expression under these conditions. To test whether inflammatory mediators can indeed overrule the inhibitory effect of RA on P-lig expression we stimulated CD4⁺ T cells either polyclonal in the presence of IL-12 and IFNγ or by LPS-activated MLN-derived dendritic cells. Both conditions promoted P-lig induction even in the presence of RA. While RA impeded the induction of fucosyltransferase-VII it did not affect IL-12-dependent C2GlcNAcT-I induction suggesting that C2GlcNAcT-I can support P-lig expression even if fucosyltransferase-VII mRNA upregulation is dampened.     

Inhibition of Th1 immune response by glucocorticoids: dexamethasone selectively inhibits IL-12-induced Stat4 phosphorylation in T lymphocytes

Glucocorticoids are widely used in the therapy of inflammatory, autoimmune, and allergic diseases. As the end-effectors of the hypothalamic-pituitary-adrenal axis, endogenous glucocorticoids also play an important role in suppressing innate and cellular immune responses. Previous studies have indicated that glucocorticoids inhibit Th1 and enhance Th2 cytokine secretion. IL-12 promotes Th1 cell-mediated immunity, while IL-4 stimulates Th2 humoral-mediated immunity. Here, we examined the regulatory effect of glucocorticoids on key elements of IL-12 and IL-4 signaling. We first investigated the effect of dexamethasone on IL-12-inducible genes and showed that dexamethasone inhibited IL-12-induced IFN-gamma secretion and IFN regulatory factor-1 expression in both NK and T cells. This occurred even though the level of expression of IL-12 receptors and IL-12-induced Janus kinase phosphorylation remained unaltered. However, dexamethasone markedly inhibited IL-12-induced phosphorylation of Stat4 without altering its expression. This was specific, as IL-4-induced Stat6 phosphorylation was not affected, and mediated by the glucocorticoid receptor, as it was antagonized by the glucocorticoid receptor antagonist RU486. Moreover, transfection experiments showed that dexamethasone reduced responsiveness to IL-12 through the inhibition of Stat4-dependent IFN regulatory factor-1 promoter activity. We conclude that blocking IL-12-induced Stat4 phosphorylation, without altering IL-4-induced Stat6 phosphorylation, appears to be a new suppressive action of glucocorticoids on the Th1 cellular immune response and may help explain the glucocorticoid-induced shift toward the Th2 humoral immune response.     

Functional analysis of the combined role of the O-linked branching enzyme core 2 beta1-6-N-glucosaminyltransferase and dimerization of P-selectin glycoprotein ligand-1 in rolling on P-selectin

Leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) is expressed as a homodimer and mediates leukocyte rolling through interactions with endothelial P-selectin. Previous studies have shown that PSGL-1 must be properly modified by specific glycosyltransferases including alpha1,3-fucosyltransferase-VII, core 2 beta1-6-N-glucosaminyltransferase (C2GlcNAcT-I), one or more alpha2,3-sialytransferases, and a tyrosulfotransferase. In addition, dimerization of PSGL-1 through its sole extracellular cysteine (Cys(320)) is essential for rolling on P-selectin under shear conditions. In this report, we measured the contributions of both C2GlcNAcT-I glycosylation and dimerization of PSGL-1 to adhesive bonds formed during tethering and rolling of transfected cell lines on purified P-selectin. Tethering to P-selectin under flow increased with dimerization compared with cells expressing monomeric PSGL-1 (referred to as C320A). The rolling defects (decreased cellular accumulation, PSGL-1/P-selectin bond strengths and tethering rates, and increased velocities and skip distance) demonstrated by transfectants expressing monomeric PSGL-1 could be overcome by increasing the substrate P-selectin site density and by overexpressing C2GlcNAcT-I in C320A transfectants. Two molecular weight variants of PSGL-1 were isolated from cell lines transfected with PSGL-1, C320A, and/or C2GlcNAcT-I cDNAs, and these differences in electrophoretic mobility appeared to correlate with C2GlcNAcT-I expression. C320A transfectants expressing low molecular weight PSGL-1 had lower C2GlcNAcT-I levels (measured by reactivity to core 2 specific linkage antibody, CHO-131) and compromised rolling on P-selectin (regardless of site density) compared with C320A cells with high levels of C2GlcNAcT-I and high molecular weight PSGL-1. Both C2GlcNAcT-I glycosylation and PSGL-1 dimerization increased the rate of tethering to P-selectin under flow, whereas C2GlcNAcT-I levels primarily influenced tether bond strength.     

In vitro differentiation from naive to mature E-selectin binding CD4 T cells: acquisition of skin-homing properties occurs independently of cutaneous lymphocyte antigen expression

We previously showed that skin-homing CD4 T cells in peripheral blood can be subdivided into three populations on the basis of the expression pattern of the cutaneous lymphocyte Ag (CLA) and fucosyltransferase VII (FucT-VII): FucT-VII(+)CLA(-), FucT-VII(+)CLA(+), and FucT-VII(-)CLA(+). In view of the known late appearance of CLA during T cell differentiation, T cells programmed to attain skin-homing properties may start to generate E-selectin-binding epitopes at early stages of differentiation before induction of CLA expression. To this end, the in vitro differentiation from naive to CLA(+) memory T cells was followed after activation with anti-CD3 mAb. Here we demonstrate that naive skin-homing CD4 T cell precursors undergo a linear differentiation process from the FucT-VII(+)CLA(-) phenotype to the FucT-VII(+)CLA(+) phenotype and eventually to the FucT-VII(-)CLA(+) phenotype. The appearance of the FucT-VII(+)CLA(-) subset coincided with or could be immediately followed by the generation of E-selectin binding epitopes, and even after E-selectin-binding epitopes were no longer detectable, CLA remained expressed for prolonged periods of time, suggesting that induction of functional E-selectin ligands depends primarily on the expression of FucT-VII, but not CLA. Immunofluorescence and confocal microscopy studies of these T cells confirm that most E-selectin ligands were found independently of CLA expression.     

Stat5a inhibits IL-12-induced Th1 cell differentiation through the induction of suppressor of cytokine signaling 3 expression

In previous studies, we have shown that Th2 cell differentiation is diminished but Th1 cell differentiation is increased in Stat5a-deficient (Stat5a(-/-)) CD4(+) T cells. In the present study, we clarified the molecular mechanisms of Stat5a-mediated Th cell differentiation. We found that enhanced Th1 cell differentiation and the resultant IFN-gamma production played a dominant inhibitory role in the down-regulation of IL-4-induced Th2 cell differentiation of Stat5a(-/-) CD4(+) T cells. We also found that IL-12-induced Stat4 phosphorylation and Th1 cell differentiation were augmented in Stat5a(-/-) CD4(+) T cells. Importantly, the expression of suppressor of cytokine signaling (SOCS)3, a potent inhibitor of IL-12-induced Stat4 activation, was decreased in Stat5a(-/-) CD4(+) T cells. Moreover, a reporter assay showed that a constitutively active form of Stat5a but not Stat6 activated the SOCS3 promoter. Furthermore, chromatin immunoprecipitation assays revealed that Stat5a binds to the SOCS3 promoter in CD4(+) T cells. Finally, the retrovirus-mediated expression of SOCS3 restored the impaired Th cell differentiation of Stat5a(-/-) CD4(+) T cells. These results suggest that Stat5a forces the Th1/Th2 balance toward a Th2-type by preventing IL-12-induced Th1 cell differentiation through the induction of SOCS3.     

An alternate core 2 beta1,6-N-acetylglucosaminyltransferase selectively contributes to P-selectin ligand formation in activated CD8 T cells

Core 2 beta1,6-N-acetylglucosaminyltransferase (C2GlcNAcT) synthesizes essential core 2 O-glycans on selectin ligands, which mediate cell-cell adhesion required for lymphocyte trafficking. Although gene-deletion studies have implicated C2GlcNAcT-I in controlling selectin ligand-mediated cell trafficking, little is known about the role of the two other core 2 isoenzymes, C2GlcNAcT-II and C2GlcNAcT-III. We show that C2GlcNAcT-I-independent P-selectin ligand formation occurs in activated C2GlcNAcT-I(null) CD8 T cells. These CD8 T cells were capable of rolling under shear flow on immobilized P-selectin in a P-selectin glycoprotein ligand 1-dependent manner. RT-PCR analysis identified significant levels of C2GlcNAcT-III RNA, identifying this enzyme as a possible source of core 2 enzyme activity. Up-regulation of P-selectin ligand correlated with altered cell surface binding of the core 2-sensitive mAb 1B11, indicating that CD43 and CD45 are also physiological targets for this alternate C2GlcNAcT enzyme. Furthermore, C2GlcNAcT-I-independent P-selectin ligand induction was observed in an in vivo model. HY(tg) CD8 T cells from C2GlcNAcT-I(null) donors transferred into male recipients expressed P-selectin ligand in response to male Ag, although at reduced levels compared with wild-type HY(tg) CD8 T cells. Our data demonstrate that multiple C2GlcNAcT enzymes can contribute to P-selectin ligand formation and may cooperate with C2GlcNAcT-I in the control of CD8 T cell trafficking.     

Role of IFN-gamma in Th1 differentiation: IFN-gamma regulates IL-18R alpha expression by preventing the negative effects of IL-4 and by inducing/maintaining IL-12 receptor beta 2 expression

Two key events occur during the differentiation of IFN-gamma-secreting Th1 cells: up-regulation of IL-12Rbeta2 and IL-12-driven up-regulation of IL-18Ralpha. We previously demonstrated that IL-12-driven up-regulation of IL-18Ralpha expression is severely impaired in IFN-gamma(-/-) mice. However, it was unclear from these studies how IFN-gamma influenced IL-18Ralpha since IFN-gamma alone had no direct effect on IL-18Ralpha expression. In the absence of IL-4, IL-12-dependent up-regulation of IL-18Ralpha/IL-12Rbeta2 was independent of IFN-gamma. However, in the presence of IL-4, IFN-gamma functions to limit the negative effects of IL-4 on both IL-18Ralpha and IL-12Rbeta2. Neutralization of IL-4 restored IL-12-driven up-regulation of IL-18Ralpha/IL-12Rbeta2 in an IFN-gamma-independent fashion. In the absence of both IL-12 and IL-4, IFN-gamma up-regulates IL-12beta2 expression and primes IFN-gamma-producing Th1 cells. When T cells were primed in the presence of IL-4, no correlation was found between the levels of expression of the IL-18Ralpha or the IL-12Rbeta2 and the capacity of these cells to produce IFN-gamma, suggesting that IL-4 may also negatively affect IL-12-mediated signal transduction and thus Th1 differentiation. These data clarify the role of IFN-gamma in regulation of IL-18Ralpha/IL-12Rbeta2 during both IL-12-dependent and IL-12-independent Th1 differentiation.     

IL-12, but not IFN-alpha, promotes STAT4 activation and Th1 development in murine CD4+ T cells expressing a chimeric murine/human Stat2 gene

Humans and mice have evolved distinct pathways for Th1 cell development. Although IL-12 promotes CD4(+) Th1 development in both murine and human T cells, IFN-alphabeta drives Th1 development only in human cells. This IFN-alphabeta-dependent pathway is not conserved in the mouse species due in part to a specific mutation within murine Stat2. Restoration of this pathway in murine T cells would provide the opportunity to more closely model specific human disease states that rely on CD4(+) T cell responses to IFN-alphabeta. To this end, the C terminus of murine Stat2, harboring the mutation, was replaced with the corresponding human Stat2 sequence by a knockin targeting strategy within murine embryonic stem cells. Chimeric m/h Stat2 knockin mice were healthy, bred normally, and exhibited a normal lymphoid compartment. Furthermore, the murine/human STAT2 protein was expressed in murine CD4(+) T cells and was activated by murine IFN-alpha signaling. However, the murine/human STAT2 protein was insufficient to restore full IFN-alpha-driven Th1 development as defined by IFN-gamma expression. Furthermore, IL-12, but not IFN-alpha, promoted acute IFN-gamma secretion in collaboration with IL-18 stimulation in both CD4(+) and CD8(+) T cells. The inability of T cells to commit to Th1 development correlated with the lack of STAT4 phosphorylation in response to IFN-alpha. This finding suggests that, although the C terminus of human STAT2 is required for STAT4 recruitment and activation by the human type I IFNAR (IFN-alphabetaR), it is not sufficient to restore this process through the murine IFNAR complex.     

Regulation of PSGL-1 interactions with L-selectin, P-selectin, and E-selectin: role of human fucosyltransferase-IV and -VII

P-selectin glycoprotein ligand-1 (PSGL-1) interactions with selectins regulate leukocyte migration in inflammatory lesions. In mice, selectin ligand activity regulating leukocyte recruitment and lymphocyte homing into lymph nodes results from the sum of unequal contributions of fucosyltransferase (FucT)-IV and FucT-VII, with FucT-VII playing a predominant role. Here we have examined the role of human FucT-IV and -VII in conferring L-selectin, P-selectin, and E-selectin binding activities to PSGL-1. Lewis x (Le(x)) carbohydrate was generated at the CHO(dhfr)(-) cell surface by FucT-IV expression, whereas sialyl Le(x) (sLe(x)) was synthesized by FucT-VII. Both human FucT-IV and -VII had the ability to generate carbohydrate ligands that support L-selectin-, P-selectin-, and E-selectin-dependent rolling on PSGL-1, with FucT-VII playing a major role. Cooperation was observed between FucT-IV and -VII in recruiting L-, P-, or E-selectin-expressing cells on PSGL-1 and in regulating cell rolling velocity and stability. Additional rolling adhesion assays were performed to assess the role of Thr-57-linked core-2 O-glycans in supporting L-selectin-, P-selectin-, and E-selectin-dependent rolling on PSGL-1. These studies confirmed that core-2 O-glycans attached to Thr-57 play a critical role in supporting L- and P-selectin-dependent rolling and revealed that additional binding sites support >75% of E-selectin-mediated rolling. The observations presented here indicate that human FucT-IV and -VII both contribute and cooperate in regulating L-selectin-, P-selectin-, and E-selectin-dependent rolling on PSGL-1, with FucT-VII playing a predominant role in conferring selectin binding activity to PSGL-1.     

Synergistic effect of IL-2, IL-12, and IL-18 on thymocyte apoptosis and Th1/Th2 cytokine expression

In the periphery, IL-18 synergistically induces the expression of the Th1 cytokine IFN-gamma in the presence of IL-12 and the Th2 cytokines IL-5 and IL-13 in the presence of IL-2. Although the expression of these cytokines has been described in the thymus, their role in thymic development and function remains uncertain. We report here that freshly isolated thymocytes from C57BL/6 and BALB/c mice stimulated in vitro with IL-2-plus-IL-18 or IL-12-plus-IL-18 produce large amounts of IFN-gamma and IL-13. Analysis of the thymic subsets, CD4(-)CD8(-) (DN), CD4(+)CD8(+), CD4(+)CD8(-), and CD4(-)CD8(+) revealed that IL-18 in combination with IL-2 or IL-12 induces IFN-gamma and IL-13 preferentially from DN cells. Moreover, DN2 and DN3 thymocytes contained more IFN-gamma(+) cells than cells in the later stage of maturation. Additionally, IL-18 in combination with IL-2 induces CCR4 (Th2-associated) and CCR5 (Th1-associated) gene expression. In contrast, IL-18-plus-IL-12 specifically induced CCR5 expression. The IL-2-plus-IL-18 or IL-12-plus-IL-18 effect on IFN-gamma and IL-13 expression is dependent on Stat4 and NF-kappaB but independent of Stat6, T-bet, or NFAT. Furthermore, IL-12-plus-IL-18 induces significant thymocyte apoptosis when expressed in vivo or in vitro, and this effect is exacerbated in the absence of IFN-gamma. IL-12-plus-IL-18-stimulated thymocytes can also induce IA-IE expression on cortical and medullary thymic epithelial cells in an IFN-gamma-dependent manner. Thus, the combination of IL-2, IL-12, and IL-18 can induce phenotypic and functional changes in thymocytes that may alter migration, differentiation, and cell death of immature T cells inside the thymus and potentially affect the Th1/Th2 bias in peripheral immune compartments.     

The fucosyltransferase FucT-VII regulates E-selectin ligand synthesis in human T cells

Selectin-ligands on T cells contribute to the recruitment of circulating cells into chronic inflammatory lesions in the skin and elsewhere. This report provides the first evidence that a single fucosyltransferase, termed FucT-VII, controls the synthesis of E- selectin ligands in human T-lymphoblasts. The FucT-IV transferase (the ELFT enzyme), in contrast constructs lower avidity E-selectin ligands and requires enzyme levels found only in myeloid cells. Treatment of Jurkat cells with phorbol myristate acetate increased the expression of sialylated Lewis(x)-related sLe(x)related epitopes and induced the synthesis of E-selectin ligands functional at physiologic levels of linear shear-stress. Northern analysis revealed a parallel increase in the steady-state levels FucT-VII mRNA, but there were no increases in the two other leukocyte-associated fucosyltransferases (FucT-IV and VI). The stable transfection of the FucT-VII gene into Jurkat cells induced high levels of the sLe(x)-related epitopes and the synthesis of E-selectin ligands which equal or exceeded the avidity of those on circulating lymphocytes. The growth of T-lymphoblasts under conditions which induced expression of the sLe(x,a) epitopes increased the level of FucT-VII mRNA, the synthesis of sialylated-Lewis(x) structures by cell-free extracts and the synthesis of E-selectin ligands equal in avidity to those on FucT-VII transfectants. In contrast, neither the mRNA levels nor activities of the FucT-IV and VI enzymes increased in association with E-selectin ligand synthesis in T-lymphoblasts. Myeloid cell lines, unlike lymphoblasts, expressed high levels of both the FucT- VII and IV enzymes in conjunction with E-selectin ligands raising the possibility that both enzymes contributed to ligand synthesis. FucT-IV transfected Jurkat cells synthesized low avidity ligands for E-selectin but only in association with CDw65 (VIM-2) carbohydrate epitope. Only blood neutrophils and myeloid cell lines expressed this epitope at the levels associated with E-ligand synthesis in the transfectants. In contrast, native Jurkat cells, blood monocytes, blood lymphocytes, and cultured T-lymphoblasts expressed low levels or none. We conclude that FucT-VII is a principal regulator of E-selectin ligand synthesis in human T-lymphoblasts while both FucT-VII and FucT-IV may direct ligand synthesis in some myeloid cells.     

Helicobacter pylori infection interferes with epithelial Stat6-mediated interleukin-4 signal transduction independent of cagA,cagE, or VacA

Helicobacter pylori is a bacterial pathogen evolved to chronically colonize the gastric epithelium, evade immune clearance by the host, and cause gastritis, peptic ulcers, and even gastric malignancies in some infected humans. In view of the known ability of this bacterium to manipulate gastric epithelial cell signal transduction cascades, we determined the effects of H. pylori infection on epithelial IL-4-Stat6 signal transduction. HEp-2 and MKN45 epithelial cells were infected with H. pylori strains LC11 or 8823 (type 1; cagA(+)/cagE(+)/VacA(+)), LC20 (type 2; cagA(-), cagE(-), VacA(-)), and cagA, cagE, and vacA isogenic mutants of strain 8823, with some cells receiving subsequent treatment with the Th2 cytokine IL-4, a known Stat6 activator. Immunofluorescence showed a disruption of Stat6-induced nuclear translocation by IL-4 in LC11-infected HEp-2 cells. IL-4-inducible Stat6 DNA binding in HEp-2 and MKN45 cells was abrogated by infection, but MKN45 cell viability was unaffected. A decrease in IL-4-mediated Stat6 tyrosine phosphorylation in nuclear and whole cell lysates was also observed following infection with strains LC11 and LC20, while neither strain altered IL-4 receptor chain alpha or Janus kinase 1 protein expression. Furthermore, parental strain 8823 and its isogenic cagA, cagE, and vacA mutants also suppressed IL-4-induced Stat6 tyrosine phosphorylation to comparable degrees. Thus, H. pylori did not directly activate Stat6, but blocked the IL-4-induced activation of epithelial Stat6. This may represent an evolutionarily conserved strategy to disrupt a Th2 response and evade the host immune system, allowing for successful chronic infection.