吉田 優

The neonatal Fc receptor for IgG (FcRn) plays a major role in regulating host IgG levels and transporting IgG and associated antigens across polarized epithelial barriers. Selective expression of FcRn in the epithelium is shown here to be associated with secretion of IgG into the lumen that allows for defense against an epithelium-associated pathogen (Citrobacter rodentium). This pathway of host resistance to a bacterial pathogen as mediated by FcRn involves retrieval of bacterial antigens from the lumen and initiation of adaptive immune responses in regional lymphoid structures. Epithelial-associated FcRn, through its ability to secrete and absorb IgG, may thus integrate luminal antigen encounters with systemic immune compartments and as such provide essential host defense and immunoregulatory functions at the mucosal surfaces.

Chemical modification of the internal surfaces of cylindrical pores with submicrometer pore diameter in a poly(ethylene terephthalate) (PET) film was examined. The modification involved the alkylation of the carboxylic acid on the surfaces with the alkylation reagent containing a fluorescent probe, and it was monitored by observing the change in fluorescent emission intensity. When the N,N-dimethylformamide solution of 4-(bromomethyl)-6,7-dimethoxycoumarin (BrCU), which bore a coumarin fluorophore, was introduced into the pores, the emission and excitation intensities of the membranes increased proportionally with increases of the pore surface areas. Fluorescent spots about 300 nm in diameter, which were located at the positions of the pores, can be observed in the fluorescence microscope image of the membranes, indicating that highly concentrated fluorescent probes are chemically incorporated on the internal surfaces of the cylindrical pores with 210 nm diameter in the membranes. In the reactions of the PET surfaces with BrCU, the fluorescent intensities increased with increases of the contact angles. This result indicates that the hydrophilicity of the internal pore surfaces can be qualitatively modified by controlling the change in the fluorescent intensities.

Resistance to and control of Cryptosporidium parvum infection in mice in the absence of adaptive immunity appears to be gamma interferon (IFN-gamma) dependent. Using an IFN-gamma-neutralizing antibody in a murine model, we demonstrated increased susceptibility to infection within 24 h. We correlated this early resistance and control with increased mucosal expression of IFN-gamma and demonstrate that CD8+ T-cell receptor alphabeta intestinal intraepithelial lymphocytes express and secrete this cytokine shortly after infection. The rapid kinetics of IFN-gamma expression and secretion by naive CD8+ T cells in response to a protozoan pathogen have not previously been demonstrated.

Resolvin E1 (RvE1; 5S,12R,18R-trihydroxyeicosapentaenoic acid) is an antiinflammatory lipid mediator derived from omega-3 fatty acid eicosapentaenoic acid (EPA). At the local site of inflammation, aspirin treatment enhances EPA conversion to 18R-oxygenated products, including RvE1, which carry potent antiinflammatory signals. Here, we obtained evidence for reduced leukocyte infiltration in a mouse peritonitis model, where the administration of EPA and aspirin initiated the generation of RvE1 in the exudates. Similar results were obtained with the administration of synthetic RvE1, which blocked leukocyte infiltration. RvE1 also protected against the development of 2,4,6-trinitrobenzene sulfonic acid-induced colitis. The beneficial effect was reflected by increased survival rates, sustained body weight, improvement of histologic scores, reduced serum anti-2,4,6-trinitrobenzene sulfonic acid IgG, decreased leukocyte infiltration, and proinflammatory gene expression, including IL-12 p40, TNF-alpha, and inducible nitric oxide synthase. Thus, the endogenous lipid mediator RvE1 counter-regulates leukocyte-mediated tissue injury and proinflammatory gene expression. These findings show an endogenous mechanism that may underlie the beneficial actions of omega-3 EPA and provide targeted approaches for the treatment of intestinal inflammation.

Inflammatory bowel disease is an immune-mediated intestinal inflammatory condition that is associated with an increase in autoantibodies that bind to epithelial cells. However, it is unknown whether the epithelial cell-derived products that are recognized by such autoantibodies are involved in the pathogenic process. Through a combined antigen-screening approach utilizing humoral and cellular immune responses, we identify herein an epithelial lectin, galectin-4, that specifically stimulates IL-6 production by CD4(+) T cells. Interestingly, the reactivity of CD4(+) T cells to galectin-4 is precisely elicited under intestinal inflammatory conditions. The galectin-4-mediated production of IL-6 is MHC class II independent and induced by PKCtheta-associated pathway through the immunological synapse. The galectin-4-mediated stimulation of CD4(+) T cells is shown to exacerbate chronic colitis and delay the recovery from acute intestinal injury. These studies identify the presence of an immunogenic, endogenous lectin in the intestine and dissect the biological role of lectin/CD4(+) T cell interactions under inflammatory conditions.

Mucosal secretions of the human gastrointestinal, respiratory, and genital tracts contain significant quantities of IgG. The mechanism by which IgG reaches luminal secretions and the function of IgG in these locations are unknown. Here, we find that the human neonatal Fc receptor (FcRn) is the vehicle that transports IgG across the intestinal epithelial barrier into the lumen where the IgG can bind cognate antigen. The FcRn can then recycle the IgG/antigen complex back across the intestinal barrier into the lamina propria for processing by dendritic cells and presentation to CD4(+) T cells in regional organized lymphoid structures. These results explain how IgG is secreted onto mucosal surfaces and scavenges luminal antigens for recognition by the immune system.

CD1d is a major histocompatibility complex (MHC) class I-related molecule that functions in glycolipid antigen presentation to distinct subsets of T cells that express natural killer receptors and an invariant T-cell receptor-alpha chain (invariant NKT cells). The acquisition of glycolipid antigens by CD1d occurs, in part, in endosomes through the function of resident lipid transfer proteins, namely saposins. Here we show that microsomal triglyceride transfer protein (MTP), a protein that resides in the endoplasmic reticulum of hepatocytes and intestinal epithelial cells (IECs) and is essential for lipidation of apolipoprotein B, associates with CD1d in hepatocytes. Hepatocytes from animals in which Mttp (the gene encoding MTP) has been conditionally deleted, and IECs in which Mttp gene products have been silenced, are unable to activate invariant NKT cells. Conditional deletion of the Mttp gene in hepatocytes is associated with a redistribution of CD1d expression, and Mttp-deleted mice are resistant to immunopathologies associated with invariant NKT cell-mediated hepatitis and colitis. These studies indicate that the CD1d-regulating function of MTP in the endoplasmic reticulum is complementary to that of the saposins in endosomes in vivo.

While Crohn disease (CD) has been clearly identified as a Th1 inflammation, the immunopathogenesis of its counterpart inflammatory bowel disease, ulcerative colitis (UC), remains enigmatic. Here we show that lamina propria T (LPT) cells from UC patients produce significantly greater amounts of IL-13 (and IL-5) than control cells and little IFN-gamma, whereas comparable cells from CD patients produce large amounts of IFN-gamma and small amounts of IL-13. We then show that stimulation of UC LPT cells bearing an NK marker (CD161) with anti-CD2/anti-CD28 or with B cells expressing transfected CD1d induces substantial IL-13 production. While this provided firm evidence that the IL-13-producing cell is an NK T (NKT) cell, it became clear that this cell does not express invariant NKT cell receptors characteristic of most NKT cells since there was no increase in cells binding alpha-galactosylceramide-loaded tetramers, and alpha-galactosylceramide did not induce IL-13 secretion. Finally, we show that both human NKT cell lines as well as UC CD161(+) LPT cells are cytotoxic for HT-29 epithelial cells and that this cytotoxicity is augmented by IL-13. These studies show that UC is associated with an atypical Th2 response mediated by nonclassical NKT cells producing IL-13 and having cytotoxic potential for epithelial cells.

Colonization of Helicobacter pylori in the stomach leads to chronic gastritis with massive infiltration by Th1 cells. To assess a role played by those T cells in the remodeling of gastric epithelium, we activated gastric T cells utilizing mice with CD4 T cells bearing transgenic TCR with or without deficiency in either IL-4 or IFN-gamma or IL-12. Mice developed gastritis upon injection of an antigen into gastric mucosa. While neutrophil infiltration occurred even with a control antigen, infiltration by transgenic T cells was dependent on the specific antigen. The numbers of epithelial cells undergoing apoptosis and regeneration were increased in the mice with infiltrating T cells producing IFN-gamma and the alignment of those cells in the glands was markedly dysregulated. In contrast, mice deficient in Th1 response showed no increase in cell division and apoptosis of epithelial cells. Thus, Th1 type T cells infiltrating into gastric mucosa play an independent role in controlling turnover of epithelial cells.

BACKGROUND: Helicobacter pylori (H. pylori) infection is associated with chronic infiltration into the stomach by T cells and plasma cells producing IFN-gamma and antibodies of various specificities, respectively. It is unknown whether these lymphocyte-products may play coordinated roles in the gastric pathology of this infection. AIMS: To know how IFN-gamma may relate to anti-H. pylori antibodies in their roles in pathogenesis, we determined the isotype subclass of those antibodies as well as their cross-reactivity and cytotoxicity to gastric epithelium. METHODS AND RESULTS: We infected BALB/c mice with H. pylori (SS1, Sydney Strain 1) and generated monoclonal antibodies, which were comprised of 240 independent clones secreting immunoglobulin and included 80 clones reactive to SS1. Ninety percent of the SS1-reactive clones had IgG2a isotype. Two clones, 2B10 and 1A9, were cross reactive to cell surface antigens in H. pylori and to antigens of 28 KDa and 42 KDa, respectively, which were present on the cell surface of and shared by both mouse and human gastric epithelial cells. The antigens recognized by these monoclonal antibodies localized a distinctive area in the gastric glands. In the presence of complement, 2B10 showed cytotoxicity to gastric epithelial cells. The effect was dose dependant and augmented by IFN-gamma. Finally, administration of 2B10 to mice with SS1 infection aggravated gastritis by increasing cellular infiltration. CONCLUSION: IFN-gamma by gastric T cells may participate in pathogenesis of the H. pylori infected stomach by directing an isotype-switch of anti-H. pylori antibodies to complement-binding subclass and by augmenting cytotoxic activity of a certain autoantibody. This may explain a host-dependent diversity in gastric pathology of the patients with H. pylori infection.

Epstein-Barr virus-induced gene 3 (EBI3) is a widely expressed IL-12p40-related protein that associates as a heterodimer with either IL-12p35 or an IL-12p35 homologue, p28, to create a new cytokine (IL-27). To define the function of EBI3 in vivo, we generated knockout mice in which the ebi3 gene was targeted by homologous recombination. EBI3-/- mice exhibited normal numbers of both naive and mature CD4+ and CD8+ T cells and B cells, but markedly decreased numbers of invariant natural killer T cells (iNKT) as defined by staining with an alpha-galactosylceramide (alphaGalCer)-loaded CD1d-tetramer. iNKT cells from EBI3-/- mice exhibited decreased IL-4 and, to a lesser extent, IFN-gamma production after alphaGalCer stimulation in vitro. A sustained decrease in IL-4 production was also observed in EBI3-/- mice after alphaGalCer stimulation in vivo in contrast to IFN-gamma production, which was only transiently decreased under such stimulation. Notably, EBI3-/- mice were resistant to the induction of immunopathology associated with oxazolone-induced colitis, a colitis model mediated primarily by T helper (Th) 2-type cytokine production by iNKT cells. In contrast, trinitrobenzene sulfonic acid-induced colitis, a predominantly Th1-mediated colitis model, was unaffected. Thus, EBI3 plays a critical regulatory role in the induction of Th2-type immune responses and the development of Th2-mediated tissue inflammation in vivo, which may be mediated through the control of iNKT cell function.

BACKGROUND & AIMS: Clonal expansion of T cells is associated with inflammatory bowel diseases, which indicates antigenic activation of the T cells. We investigated whether the introduction of CD4 T cells specific to a microflora would initiate colitis and assessed the cytokine requirements for colitogenic CD4 T cells. METHODS: Severe combined immunodeficiency disease (SCID) mice were reconstituted with CD4 T cells, which were either deficient in interleukin (IL)-4/interferon (IFN)-gamma production or differentiated in vitro to T-helper (Th) 1/Th 2 and bearing a transgenic T-cell receptor (TCR) specific to ovalbumin (OVA), and then inoculated with an Escherichia coli-producing OVA (ECOVA). Clinical and histologic manifestations of colitis were assessed. RESULTS: Mice with ECOVA colonization and OVA-specific CD4 T cells developed colitis with histologic features of focal infiltration by mononuclear cells, destruction of crypts, and loss of goblet cells. Further, infiltration was initiated in pre-existing lymph follicles. Th1- and IL-4 deficient T cells were diffusely localized in the lamina propria and submucosa, whereas Th2- and IFN-gamma-deficient T cells were localized preferentially in lymph follicles. CONCLUSIONS: A microbe-associated antigen, non-cross-reactive to colonic tissue, can drive antigen-specific CD4 T cells to cause colitis in SCID mice. Although the presence of IFN-gamma and IL-4 in the effector CD4 T cells was not an absolute requirement for the development of colitis, they seemed to regulate it in part by modulating migration of the effector T cells.

The major histocompatibility complex class I-related neonatal Fc receptor, FcRn, assembles as a heterodimer consisting of a heavy chain and beta(2)-microglobulin (beta(2)m), which is essential for FcRn function. We observed that, in Madin-Darby canine kidney (MDCK) cells, the function of human FcRn in mediating the bidirectional transport of IgG was significantly increased upon co-expression of the human isoform of beta(2)m. In MDCK cells, the presence of human beta(2)m endowed upon human FcRn an enhanced ability to exit the endoplasmic reticulum and acquire mature carbohydrate side-chain modifications at steady state, a faster kinetics of maturation, and augmented localization at the cell surface as a mature glycoprotein able to bind IgG. Although human FcRn with immature carbohydrate side-chain modifications was capable of exhibiting pH-dependent binding of IgG, only human FcRn with mature carbohydrate side-chain modifications was detected on the cell surface. These results show that human FcRn travels to the cell surface via the normal secretory pathway and that the appropriate expression and function of human FcRn in MDCK cells depends upon the co-expression of human beta(2)m.

Ags administered orally at a high dose are absorbed in immunogenic forms and perfuse the liver, which raises a question regarding the relevance of hepatic lymphocyte activation to the systemic hyporesponsiveness against the ingested Ag. Oral administration of 100 mg of OVA to the mice led to massive cell death of OVA-specific (KJ1-26+)CD4+ T cells by Fas-Fas ligand (FasL)-mediated apoptosis in the liver, which was associated with the emergence of hepatic KJ1-26+CD4+ T cells expressing FasL. Hepatic CD4+ T cells in OVA-fed mice secreted large amounts of IL-4, IL-10, and TGF-beta(1) upon restimulation in vitro and inhibited T cell proliferation. Adoptive transfer of these hepatic CD4+ T cells to naive mice and subsequent antigenic challenge led to suppression of T cell proliferation as well as IgG Ab responses to OVA; this effect was mostly abrogated by a blocking Ab to FasL. i.p. administration of an Ag at a high dose also generated hepatic CD4+FasL+ T cells with similar cytokine profile as T cells activated by oral administration of Ags at a high dose. Finally, we did not see an increase in FasL+ cells in the hepatic CD4+Vbeta8+ T cell subset of MRL/lpr/lpr mice given staphylococcal enterotoxin B, indicating the requirement for Fas-mediated signals. These hepatic CD4+FasL+ regulatory cells may explain the tolerogenic property of the liver and play roles in systemic hyporesponsiveness induced by an Ag administered at a high dose.