Helicobacter pylori CagA-dependent macrophage migration inhibitory factor produced by gastric epithelial cells binds to CD74 and stimulates procarcinogenic events

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that has recently been implicated in carcinogenesis. Helicobacter pylori, which is closely linked to gastric cancer, induces the gastric epithelium to produce proinflammatory cytokines, including MIF. MIF can bind to CD74, which we have previously shown to be highly expressed on the surface of gastric epithelial cells (GEC) during H. pylori infection. In this study, we sought to investigate the role of the H. pylori-induced MIF on epithelial proliferation and procarcinogenic events. Upon establishing a role for the H. pylori CagA virulence factor in MIF production, MIF binding to CD74 on GEC was confirmed. rMIF and H. pylori were shown to increase GEC proliferation, which was decreased when cagA- strains were used and when CD74 was blocked by mAbs. Apoptosis was also decreased by MIF, but increased by cagA- strains that induced much lower amounts of MIF than the wild-type bacteria. Furthermore, MIF binding to CD74 was also shown to decrease p53 phosphorylation and up-regulate Bcl-2 expression. This data describes a novel system in which an H. pylori virulence factor contributes to the production of a host factor that in turn up-regulates procarcinogenic events by the gastric epithelium.     

Differential protein expression profiles of gastric epithelial cells following Helicobacter pylori infection using ProteinChips

Helicobacter pylori infects approximately half of the world's population and the bacterium is associated with gastric cancer and peptic and duodenal ulcers. In this study, Surface Enhanced Laser Desorption /Ionization time-of-flight mass spectrometry (SELDI-TOF-MS) was used to identify the biomarkers from H. pylori infected gastric epithelial cells (GEC) to understand key mechanisms associated with pathogenesis. Using different chip surfaces, differential protein expression profile of GEC was obtained and several upregulated or downregulated biomarkers were detected on GEC, following H. pylori infection. Four different H. pylori infected GECs were compared based on their expression of MHC class II, a receptor reported to trigger apoptosis. One biomarker was identified in H. pylori infected GEC as Annexin A2 (Annexin II) from the flow through of the anion-exchange resin. The increased expression of Annexin II in GEC following H. pylori infection was further confirmed by Western Blot analyses and indicates its involvement in H. pylori pathogenesis.     

Helicobacter pylori-induced IL-8 production by gastric epithelial cells up-regulates CD74 expression

CD74, or the class II MHC-associated invariant chain, is best known for the regulation of Ag presentation. However, recent studies have suggested other important roles for this protein in inflammation and cancer studies. We have shown that CD74 is expressed on the surface of gastric cells, and Helicobacter pylori can use this receptor as a point of attachment to gastric epithelial cells, which lead to IL-8 production. This study investigates the ability of H. pylori to up-regulate one of its receptors in vivo and with a variety of gastric epithelial cell lines during infection with H. pylori. CD74 expression was increased dramatically on gastric biopsies from H. pylori-positive patients and gastric cell lines exposed to the bacteria. Gastric cells exposed to H. pylori-conditioned medium revealed that the host cell response was responsible for the up-regulation of CD74. IL-8 was found to up-regulate CD74 cell surface expression because blocking IL-8Rs or neutralizing IL-8 with Abs counteracted the increased expression of CD74 observed during infection with H. pylori. These studies demonstrate how H. pylori up-regulates one of its own receptors via an autocrine mechanism involving one of the products induced from host cells.     

Helicobacter pylori cag Pathogenicity Island's Role in B7-H1 Induction and Immune Evasion

During Helicobacter pylori (H. pylori) infection CD4⁺ T cells in the gastric lamina propria are hyporesponsive and polarized by Th1/Th17 cell responses controlled by T_reg cells. We have previously shown that H. pylori upregulates B7-H1 expression on GEC, which, in turn, suppress T cell proliferation, effector function, and induce T_reg cells in vitro. In this study, we investigated the underlying mechanisms and the functional relevance of B7-H1 induction by H. pylori infection to chronic infection. Using H. pylori wild type (WT), cag pathogenicity island (cag PAI^-) and cagA ^- isogenic mutant strains we demonstrated that H. pylori requires its type 4 secretion system (T4SS) as well as its effector protein CagA and peptidoglycan (PG) fragments for B7-H1 upregulation on GEC. Our study also showed that H. pylori uses the p38 MAPK pathway to upregulate B7-H1 expression in GEC. In vivo confirmation was obtained when infection of C57BL/6 mice with H. pylori PMSS1 strain, which has a functional T4SS delivery system, but not with H. pylori SS1 strain lacking a functional T4SS, led to a strong upregulation of B7-H1 expression in the gastric mucosa, increased bacterial load, induction of T_reg cells in the stomach, increased IL-10 in the serum. Interestingly, B7-H1^-/- mice showed less T_reg cells and reduced bacterial loads after infection. These studies demonstrate how H. pylori T4SS components activate the p38 MAPK pathway, upregulate B7-H1 expression by GEC, and cause T_reg cell induction; thus, contribute to establishing a persistent infection characteristic of H. pylori.     

Human dendritic cells respond to Helicobacter pylori, promoting NK cell and Th1-effector responses in vitro

Helicobacter pylori infection leads to chronic gastric inflammation. The current study determined the response of human APCs, NK cells, and T cells toward the bacteria in vitro. Human monocyte-derived dendritic cells (DC) were incubated with bacteria for 48 h. Intact H. pylori at a multitude of infection 5 stimulated the expression of MHC class II (4- to 7-fold), CD80, and CD86 B7 molecules (10- to 12-fold) and the CD83 costimulatory molecule (>30-fold) as well as IL-12 secretion (>50-fold) in DCs, and thereby, strongly induced their maturation and activation. CD56(+)/CD4(-) NK cells, as well as CD4(+)/CD45RA(+) naive T cells, were isolated and incubated with DCs pulsed with intact bacteria or different cellular fractions. Coculture of H. pylori-pulsed DCs with NK cells strongly potentiated the secretion of TNF-alpha and IFN-gamma. Coculture of naive T cells with H. pylori-pulsed DCs significantly enhanced TNF-alpha, IFN-gamma, and IL-2 secretion as well as T-bet mRNA levels, while GATA-3 mRNA was lowered. However, the effect appeared attenuated compared with coculture with Escherichia coli. A greater stimulation was seen with naive T cells and DCs pulsed with H. pylori membrane preparations. Intact H. pylori potently induced the maturation and activation of human monocyte-derived DC and thereby promote NK and Th1 effector responses. The strong activation of NK cells may be important for the innate immune response. Th1-polarized T cells were induced especially by incubation with membrane preparations of H. pylori, suggesting that membrane proteins may account for the specific adaptive immune response.     

Suppression of human mononuclear cell response by Helicobacter pylori: Effects on isolated monocytes and lymphocytes

Abstract Helicobacter pylori colonization of the human gastric mucosa causes a long-term, not self-limiting inflammation, suggesting that the microbe has properties to protect itself against the host immune defence system. Recently we were able to demonstrate that H. pylori suppresses the in vitro proliferative response of human peripheral blood mononuclear cells to antigens as well as to mitogens without affecting cell viability. The purpose of this study was to clarify which cell subsets of mononuclear cells are influenced by H. pylori . The use of monocytes which had been pretreated with a soluble cytoplasmic fraction of H. pylori (30 μg ml⁻¹) led to a suppressed proliferation of T cells after PHA-activation. Activation of isolated T cells with PHA and PMA revealed that the proliferative response of lymphocytes could also be inhibited independently of monocytes. The anti-proliferative effect was associated with a reduction of IL-2 receptor (CD25) expression as well as an inhibition of blastogenesis. Furthermore, the spontaneous proliferation of EBV-transformed B cell lines was suppressed in a dose-dependent manner. FACS-analysis of HLA-DR, ICAM-1 and CD14 expression on the surface of monocytes revealed an influence of H. pylori on CD14 expression at a concentration of 30 μg ml⁻¹, while the expression of HLA-DR and ICAM-1 was not affected at this concentration.     

B7-h1 expressed by activated CD8 T cells is essential for their survival

An immunoinhibitory role of B7 homologue 1 (B7-H1) expressed by non-T cells has been established; however, the function of B7-H1 expressed by T cells is not clear. Peak expression of B7-H1 on Ag-primed CD8 T cells was observed during the contraction phase of an immune response. Unexpectedly, B7-H1 blockade at this stage reduced the numbers of effector CD8 T cells, suggesting B7-H1 blocking Ab may disturb an unknown function of B7-H1 expressed by CD8 T cells. To exclusively examine the role of B7-H1 expressed by T cells, we introduced B7-H1 deficiency into TCR transgenic (OT-1) mice. Naive B7-H1-deficient CD8 T cells proliferated normally following Ag stimulation; however, once activated, they underwent more robust contraction in vivo and more apoptosis in vitro. In addition, B7-H1-deficient CD8 T cells were more sensitive to Ca-dependent and Fas ligand-dependent killing by cytotoxic T lymphocytes. Activation-induced Bcl-x(L) expression was lower in activated B7-H1-deficient CD8 T cells, whereas Bcl-2 and Bim expression were comparable to the wild type. Transfer of effector B7-H1-deficient CD8 T cells failed to suppress tumor growth in vivo. Thus, upregulation of B7-H1 on primed T cells helps effector T cells survive the contraction phase and consequently generate optimal protective immunity.     

幽门螺杆菌感染者CD4~+ CD25~+调节性T细胞的检测及其相关性分析

目的检测幽门螺杆菌(Helicobacter pylori,H.pylori)感染阳性的胃部疾病患者外周血中CD4+CD25+调节性T细胞(Treg细胞)的百分含量及转化生长因子-β1(transforming growth factor-β1,TGF-β1)的水平,探讨CD4+CD25+调节性T细胞在H.pylori感染中的免疫调节作用及意义。方法采用流式细胞术检测H.pylori感染的慢性浅表性胃炎、胃癌前病变和胃癌患者外周血中CD4+CD25+调节性T细胞的含量、CD4+CD25+T细胞中表达FOXP3的细胞比例;并采用ELISA方法检测H.pylori感染者血清中TGF-β1的含量,无H.pylori感染的患者作为阴性对照。结果 H.pylori感染的患者外周血中CD4+CD25+调节性T细胞的百分含量及TGF-β1的水平较不伴有H.pylori感染的患者显著升高(P<0.05);H.pylori感染的浅表性胃炎、胃癌前病变及胃癌患者外周血中CD4+CD25+T淋巴细胞的百分含量及CD4+CD25+T细胞中表达FOXP3的细胞比例随病变严重程度的进展逐渐升高,差异有统计学意义(P<0.05);H.pylori感染的患者血清中TGF-β1水平也随病变严重程度的进展逐渐升高,差异有统计学意义(P<0.05)。结论 H.pylori感染可增加CD4+CD25+调节性T细胞的含量和TGF-β1的水平;随着病变严重程度的进展,CD4+CD25+调节性T细胞的含量和TGF-β1的水平逐渐升高,CD4+CD25+调节性T细胞百分含量和TGF-β1水平可作为临床判断病情进展的指标。     

Helicobacter pylori arginase inhibits T cell proliferation and reduces the expression of the TCR zeta-chain (CD3zeta)

Helicobacter pylori infects approximately half the human population. The outcomes of the infection range from gastritis to gastric cancer and appear to be associated with the immunity to H. pylori. Patients developing nonatrophic gastritis present a Th1 response without developing protective immunity, suggesting that this bacterium may have mechanisms to evade the immune response of the host. Several H. pylori proteins can impair macrophage and T cell function in vitro through mechanisms that are poorly understood. We tested the effect of H. pylori extracts and live H. pylori on Jurkat cells and freshly isolated human normal T lymphocytes to identify possible mechanisms by which the bacteria might impair T cell function. Jurkat cells or activated T lymphocytes cultured with an H. pylori sonicate had a reduced proliferation that was not caused by T cell apoptosis or impairment in the early T cell signaling events. Instead, both the H. pylori sonicate and live H. pylori induced a decreased expression of the CD3zeta-chain of the TCR. Coculture of live H. pylori with T cells demonstrated that the wild-type strain, but not the arginase mutant rocF(-), depleted L-arginine and caused a decrease in CD3zeta expression. Furthermore, arginase inhibitors reversed these events. These results suggest that H. pylori arginase is not only important for urea production, but may also impair T cell function during infection.     

Increase of antigen-presenting cells in the gastric mucosa of Helicobacter pylori-infected children

BACKGROUND: Infection with Helicobacter pylori leads to an increase of T cells in the gastric mucosa of children. In contrast to peripheral blood, where monocytes are the most abundant antigen-presenting cells, CD14+ macrophages are very rare in infected gastric mucosa. We postulated that other types of antigen-presenting cells must be present in infected gastric mucosa. MATERIAL AND METHODS: Antral biopsies were obtained from 56 children. The cellular expression of major histocompatibility complex (MHC) class II molecules, CD1a/b, and CD23, which are involved in antigen presentation were analyzed by immunohistochemistry. In addition, T cells (CD4, CD8, CD25, and gamma/delta-TCR), B cells (anti-IgM), macrophages (CD14) and granulocytes (CD15) were quantified. RESULTS: Twenty-eight children were H. pylori-infected. Thirteen children were healthy, 15 had other gastric pathologies. T cells (p<.0001), B cells (p<.0001), CD23+ (p<.0001), and CD1a/b+ (p<.005) cells were significantly increased in the lamina propria of H. pylori-infected children, whereas macrophages were rare without significant differences among the groups. Within the epithelium, CD8+ T lymphocytes predominated clearly over CD4+ cells. H. pylori-negative children had only few MHC class II-positive cells within the gastric epithelium, whereas MHC class II antigens were strongly expressed on epithelial cells (p<.0001) of all H. pylori-infected children. CONCLUSION: Helicobacter pylori infection leads to an enhanced expression of antigen-presenting molecules together with a parallel rise of T cells in the lamina propria. This may represent an effort of the immune system to optimize local immune responses against H. pylori. We speculate that the epithelium participates in the initiation of a local immune response against H. pylori.     

Increased B7-H1 expression on dendritic cells correlates with programmed death 1 expression on T cells in simian immunodeficiency virus-infected macaques and may contribute to T cell dysfunction and disease progression

Suppression of dendritic cell (DC) function in HIV-1 infection is thought to contribute to inhibition of immune responses and disease progression, but the mechanism of this suppression remains undetermined. Using the rhesus macaque model, we show B7-H1 (programmed death [PD]-L1) is expressed on lymphoid and mucosal DCs (both myeloid DCs and plasmacytoid DCs), and its expression significantly increases after SIV infection. Meanwhile, its receptor, PD-1, is upregulated on T cells in both peripheral and mucosal tissues and maintained at high levels on SIV-specific CD8(+) T cell clones in chronic infection. However, both B7-H1 and PD-1 expression in SIV controllers was similar to that of controls. Expression of B7-H1 on both peripheral myeloid DCs and plasmacytoid DCs positively correlated with levels of PD-1 on circulating CD4(+) and CD8(+) T cells, viremia, and declining peripheral CD4(+) T cell levels in SIV-infected macaques. Importantly, blocking DC B7-H1 interaction with PD-1(+) T cells could restore SIV-specific CD4(+) and CD8(+) T cell function as evidenced by increased cytokine secretion and proliferative capacity. Combined, the results indicate that interaction of B7-H1-PD-1 between APCs and T cells correlates with impairment of CD4(+) Th cells and CTL responses in vivo, and all are associated with disease progression in SIV infection. Blockade of this pathway may have therapeutic implications for HIV-infected patients.     

Helicobacter pylori, T cells and cytokines: the "dangerous liaisons"

Helicobacter pylori infection is the major cause of gastroduodenal pathologies, but only a minority of infected patients develop chronic and life threatening diseases, as peptic ulcer, gastric cancer, B-cell lymphoma, or autoimmune gastritis. The type of host immune response against H. pylori is crucial for the outcome of the infection. A predominant H. pylori-specific Th1 response, characterized by high IFN-gamma, TNF-alpha, and IL-12 production associates with peptic ulcer, whereas combined secretion of both Th1 and Th2 cytokines are present in uncomplicated gastritis. Gastric T cells from MALT lymphoma exhibit abnormal help for autologous B-cell proliferation and reduced perforin- and Fas-Fas ligand-mediated killing of B cells. In H. pylori-infected patients with autoimmune gastritis cytolytic T cells infiltrating the gastric mucosa cross-recognize different epitopes of H. pylori proteins and H+K+ ATPase autoantigen. These data suggest that peptic ulcer can be regarded as a Th1-driven immunopathological response to some H. pylori antigens, whereas deregulated and exhaustive H. pylori-induced T cell-dependent B-cell activation can support the onset of low-grade B-cell lymphoma. Alternatively, H. pylori infection may lead in some individuals to gastric autoimmunity via molecular mimicry.     

Helicobacter pylori urease binds to class II MHC on gastric epithelial cells and induces their apoptosis

Infection by Helicobacter pylori leads to injury of the gastric epithelium and a cellular infiltrate that includes CD4+ T cells. H. pylori binds to class II MHC molecules on gastric epithelial cells and induces their apoptosis. Because urease is an abundant protein expressed by H. pylori, we examined whether it had the ability to bind class II MHC and induce apoptosis in class II MHC-bearing cells. Flow cytometry revealed the binding of PE-conjugated urease to class II MHC+ gastric epithelial cell lines. The binding of urease to human gastric epithelial cells was reduced by anti-class II MHC Abs and by staphylococcal enterotoxin B. The binding of urease to class II MHC was confirmed when urease bound to HLA-DR1-transfected COS-1 (1D12) cells but not to untransfected COS-1 cells. Urease also bound to a panel of B cell lines expressing various class II MHC alleles. Recombinant urease induced apoptosis in gastric epithelial cells that express class II MHC molecules, but not in class II MHC- cells. Also, Fab from anti-class II MHC and not from isotype control Abs blocked the induction of apoptosis by urease in a concentration-dependent manner. The adhesin properties of urease might point to a novel and important role of H. pylori urease in the pathogenesis of H. pylori infection.     

Loss of B7-H1 expression by recipient parenchymal cells leads to expansion of infiltrating donor CD8+ T cells and persistence of graft-versus-host disease

Previous experimental studies have shown that acute graft-versus-host disease (GVHD) is associated with two waves of donor CD8(+) T cell expansion. In the current studies, we used in vivo bioluminescent imaging, in vivo BrdU labeling, and three different experimental GVHD systems to show that B7-H1 expression by recipient parenchymal cells controls the second wave of alloreactive donor CD8(+) T cell expansion and the associated second phase of GVHD. Loss of B7-H1 expression by parenchymal cells during the course of GVHD was associated with persistent proliferation of donor CD8(+) T cells in GVHD target tissues and continued tissue injury, whereas persistent expression of B7-H1 expression by parenchymal cells led to reduced proliferation of donor CD8(+) T cells in GVHD target tissues and resolution of GVHD. These studies demonstrate that parenchymal cell expression of B7-H1 is required for tolerizing infiltrating T cells and preventing the persistence of GVHD. Our results suggest that therapies designed to preserve or restore expression of B7-H1 expression by parenchymal tissues in the recipient could prevent or ameliorate GVHD in humans.     

Integrin subunit CD18 Is the T-lymphocyte receptor for the Helicobacter pylori vacuolating cytotoxin

Helicobacter pylori infection is associated with gastritis, ulcerations, and gastric adenocarcinoma. H. pylori secretes the vacuolating cytotoxin (VacA), a major pathogenicity factor. VacA has immunosuppressive effects, inhibiting interleukin-2 (IL-2) secretion by interference with the T cell receptor/IL-2 signaling pathway at the level of calcineurin, the Ca2+-calmodulin-dependent phosphatase. Here, we show that VacA efficiently enters activated, migrating primary human T lymphocytes by binding to the beta2 (CD18) integrin receptor subunit and exploiting the recycling of lymphocyte function-associated antigen (LFA)-1. LFA-1-deficient Jurkat T cells were resistant to vacuolation and IL-2 modulation, and genetic complementation restored sensitivity to VacA. VacA targeted human, but not murine, CD18 for cell entry, consistent with the species-specific adaptation of H. pylori. Furthermore, expression of human integrin receptors (LFA-1 or Mac-1) in murine T cells resulted in VacA-mediated cellular vacuolation. Thus, H. pylori co-opts CD18 as a VacA receptor on human T lymphocytes to subvert the host immune response.     

B7-H1 (CD274) inhibits the development of herpetic stromal keratitis (HSK)

The co-signaling molecule B7-H1 (CD274) functions as both a co-inhibitor through programmed death-1 (PD-1) receptor and a co-stimulator via an as-yet-unidentified receptor on T cells. We investigated the physiological role of endogenous B7-H1 in the pathogenesis of herpetic stromal keratitis (HSK) caused by herpes simplex virus type 1 (HSV-1). Following HSV-1 infection of the cornea of mice, B7-H1 expression was up-regulated in the CD11b+ macrophage population in the draining lymph nodes (dLN) and in the inflamed cornea. In addition, HSV-1 infection significantly increased PD-1 expression on CD4+ T cells in the dLN and inflamed cornea. The administration of antagonistic B7-H1 monoclonal antibody resulted in the proliferation of HSV-specific CD4+ T cells that secreted interferon (INF)-gamma, and inhibited the apoptosis of HSV-specific CD4+ T cells, which exaggerated HSK. These results strongly suggest that the B7-H1 may be involved in suppression of the development of HSK.     

Blockade of B7-H1 (programmed death ligand 1) enhances humoral immunity by positively regulating the generation of T follicular helper cells

T follicular helper (T(FH)) cells are critical initiators in the development of T cell-dependent humoral immunity and the generation of protective immunity. We demonstrate that T(FH) cell accumulation and Ab production are negatively regulated by B7-H1 (programmed death ligand 1) in response to both helminth infection and active immunization. Following immunization of B7-H1(-/-) mice with keyhole limpet hemocyanin or helminth Ags, there is a profound increase in induction of T(FH) cells as a result of increased cell cycling and decreased apoptosis relative to wild-type mice. The increase in T(FH) cells in the absence of B7-H1 was associated with significant elevations in Ag-specific Ig response. Cotransfer experiments in vivo demonstrated that B7-H1 expression on B cells was required for negatively regulating T(FH) cell expansion and production of Ag-specific Ig. Treatment of immunized wild-type mice with anti-B7-H1 or anti-programmed death 1 mAbs, but not anti-B7-DC, led to a significant expansion of the T(FH) cell population and an enhanced Ag-specific Ig response. Our results demonstrate that the coinhibitory B7-H1/programmed death 1 pathway can limit the expansion of T(FH) cells and constrain Ag-specific Ig responses. This finding has direct implications for investigations examining the feasibility of therapeutically manipulating this pathway and reveals new insights into the regulation of the humoral immune response.