Satoshi Hachimura

Identification of Multiple Isolated Lymphoid Follicles on the Antimesenteric Wall of the Mouse Small Intestine

We have revealed that 100–200 clusters, filled with closely packed lymphocytes, can be found throughout the length of the antimesenteric wall of the mouse small intestine. They are composed of a large B cell area, including a germinal center, and epithelia overlying the clusters contain M cells. A large fraction of B cells displays B220+CD19+CD23+IgMlowIgDhighCD5−Mac-1− phenotype, and the composition of IgA+ B cells is smaller but substantial. To our knowledge, these clusters are the first identification of isolated lymphoid follicles (ILF) in mouse small intestine. ILF can be first detected at 7 (BALB/c mice) and 25 (C57BL/6 mice) days after birth, and lymphoid clusters equivalent in terms of cellular mass to ILF are present in germfree, athymic nude, RAG-2−/−, TCR-β−/−, and Ig μ-chain mutant (μm−/−) mice, although c-kit+ cells outnumber B220+ cells in germfree and athymic nude mice, and most lymphoid residents are c-kit+B220− in RAG-2−/−, TCR-β−/−, and μm−/− mice. ILF develop normally in the progeny of transplacentally manipulated Peyer’s patch (PP)-deficient mice, and decreased numbers of conspicuously atrophied ILF are present in IL-7Rα−/− PPnull mice. Neither ILF nor PP are detectable in lymphotoxin α−/− and aly/aly mice that retain well-developed cryptopatches (CP) and thymus-independent subsets of intraepithelial T cells, whereas ILF, PP, CP, and thymus-independent subsets of intraepithelial T cells disappear from common cytokine receptor γ-chain mutant mice. These findings indicate that ILF, PP, and CP constitute three distinct organized gut-associated lymphoid tissues that reside in the lamina propria of the mouse small intestine.

Lactobacillus casei Inhibits Antigen-Induced IgE Secretion through Regulation of Cytokine Production in Murine Splenocyte Cultures

Background:Lactobacillus casei is a nonpathogenic gram-positive bacterium widely used in dairy products and has been shown to enhance the cellular immunity of the host. Methods: To examine the inhibitory effect of L. casei on IgE production, splenocytes obtained from ovalbumin (OVA)-primed BALB/c mice were restimulated in vitro with the same antigen in the presence of heat-killed L. casei. The effect of this bacterium on T helper (Th) phenotype development was also examined with naive T cells from OVA-specific T cell receptor-transgenic mice. Results:L. casei induced IFN-γ, but inhibited IL-4 and IL-5 secretion, and markedly suppressed total and antigen-specific IgE secretion by OVA-stimulated splenocytes. The inhibitory effect of L. casei on IgE, IL-4, and IL-5 production was partially abrogated by addition of neutralizing antibody to IFN-γ. Augmented IL-12 production was also observed in the cell cultures containing L. casei, and anti-IL-12 monoclonal antibody completely restored the IgE, IL-4, and IL-5 production to the control levels. The IL-12 augmentation by L. casei was macrophage-dependent. The Th cell development assay showed the ability of L. casei to induce Th1 development preferentially. This effect was also completely blocked by anti-IL-12 antibody. Conclusions: This is the first demonstration that a nonpathogenic microorganism, L. casei, can inhibit antigen-induced IgE production through induction of IL-12 secretion by macrophages. The findings suggest a potential use of this organism in preventing IgE-mediated allergy.

CD11b+ Peyer’s Patch Dendritic Cells Secrete IL-6 and Induce IgA Secretion from Naive B Cells

Peyer’s patch (PP) dendritic cells (DCs) have been shown to exhibit a distinct capacity to induce cytokine secretion from CD4+ T cells compared with DCs in other lymphoid organs such as the spleen (SP). In this study, we investigated whether PP DCs are functionally different from DCs in the SP in their ability to induce Ab production from B cells. Compared with SP DCs, freshly isolated PP DCs induced higher levels of IgA secretion from naive B cells in DC-T cell-B cell coculture system in vitro. The IgA production induced by PP DCs was attenuated by neutralization of IL-6. In addition, the induction of IgA secretion by SP DCs, but not PP DCs, was further enhanced by the addition of exogenous IL-6. Finally, we demonstrated that only PP CD11b+ DC subset secreted higher levels of IL-6 compared with other DC subsets in the PP and all SP DC populations, and that PP CD11b+ DC induced naive B cells to produce higher levels of IgA compared with SP CD11b+ DC. These results suggest a unique role of PP CD11b+ DCs in enhancing IgA production from B cells via secretion of IL-6.

CD4+CD25− T Cells That Express Latency-Associated Peptide on the Surface Suppress CD4+CD45RBhigh-Induced Colitis by a TGF-β-Dependent Mechanism

Murine CD4+CD25+ regulatory cells have been reported to express latency-associated peptide (LAP) and TGF-β on the surface after activation, and exert regulatory function by the membrane-bound TGF-β in vitro. We have now found that a small population of CD4+ T cells, both CD25+ and CD25−, can be stained with a goat anti-LAP polyclonal Ab without being stimulated. Virtually all these LAP+ cells are also positive for thrombospondin, which has the ability to convert latent TGF-β to the active form. In the CD4+CD45RBhigh-induced colitis model of SCID mice, regulatory activity was exhibited not only by CD25+LAP+ and CD25+LAP− cells, but also by CD25−LAP+ cells. CD4+CD25−LAP+ T cells were part of the CD45RBlow cell fraction. CD4+CD25−LAP−CD45RBlow cells had minimal, if any, regulatory activity in the colitis model. The regulatory function of CD25−LAP+ cells was abrogated in vivo by anti-TGF-β mAb. These results identify a new TGF-β-dependent regulatory CD4+ T cell phenotype that is CD25− and LAP+.

Lactobacillus casei strain Shirota suppresses serum immunoglobulin E and immunoglobulin G1 responses and systemic anaphylaxis in a food allergy model

Background  Our previous study using allergen‐sensitized murine splenocyte cultures has shown that Lactobacillus casei strain Shirota (LcS), a lactic acid bacterium widely used as a starter for fermented milk products, suppresses IgE production through promoting a dominant Th1‐type response mediated by IL‐12 induction.

Memory B cells in the lung participate in protective humoral immune responses to pulmonary influenza virus reinfection

After pulmonary virus infection, virus-binding B cells ectopically accumulate in the lung. However, their contribution to protective immunity against reinfecting viruses remains unknown. Here, we show the phenotypes and protective functions of virus-binding memory B cells that persist in the lung following pulmonary infection with influenza virus. A fraction of virus-binding B-cell population in the lung expressed surface markers for splenic mature memory B cells (CD73, CD80, and CD273) along with CD69 and CXCR3 that are up-regulated on lung effector/memory T cells. The lung B-cell population with memory phenotype persisted for more than 5 mo after infection, and on reinfection promptly differentiated into plasma cells that produced virus-neutralizing antibodies locally. This production of local IgG and IgA neutralizing antibody was correlated with reduced virus spread in adapted hosts. Our data demonstrates that infected lungs harbor a memory B-cell subset with distinctive phenotype and ability to provide protection against pulmonary virus reinfection.

Monoclonal antibodies as probes for monitoring the denaturation process of bovine β-lactoglobulin

Five monoclonal antibodies (MAbs) of different idiotypes were produced against bovine beta-lactoglobulin (beta-LG). Among them, MAbs 61B4 and 62A6 reacted preferentially to native beta-LG, while MAbs 21B3 and 31A4 reacted more strongly to the reduced carboxymethylated (denatured) beta-LG than to the native material. These two types of MAb were used to analyze the denaturation process of a beta-LG molecule during heating. The binding affinity of MAbs 21B3 and 31A4 with beta-LG was increased by increasing the heating temperature, the transition temperature being 67-68 degrees C, while that of MAbs 61B4 and 62A6 was reduced by increasing the temperature, this transition temperature being about 80 degrees C. Epitopes recognized by MAbs 31A4 and 61B4 were shown to be included in the segments, Lys8-Trp19 (mostly in the random-coil region) and Thr125-Lys135 (helical region), respectively. The heat-induced conformational change of the beta-LG molecule is, therefore, likely to start in random-coil region as Lys8-Trp19, and to be followed by a structural change in a helical region as Thr125-Lys135. This study demonstrates that MAb is a useful probe to monitor local conformational changes of a protein molecule during denaturation.

Growth Factor-induced Phosphorylation of Sterol Regulatory Element-binding Proteins Inhibits Sumoylation, Thereby Stimulating the Expression of Their Target Genes, Low Density Lipoprotein Uptake, and Lipid Synthesis *□

The destiny and activity of sterol regulatory element-binding proteins (SREBPs) in the nucleus are regulated by modification with ubiquitin, small ubiquitin-like modifier (SUMO), or phosphorus. ERK-dependent phosphorylation causes an increase in their transcriptional activity, whereas SUMO modification halts it. We hypothesized a causal linkage between phosphorylation and sumoylation because their sites are very closely located in SREBP-1 and -2 molecules. When Ser455, a phosphorylation site in SREBP-2, was substituted with Ala, this SREBP-2 mutant was more efficiently modified by SUMO-1. On the other hand, substitution of Asp inhibited SUMO conjugation, mimicking phosphoserine. When cells were cultured with insulin-like growth factor-1, sumoylation of SREBP-2 was decreased with an increase in its phosphorylation, but SREBP-2(S455A) was continuously sumoylated. An ERK cascade inhibitor, U0126, inversely augmented SUMO modification of SREBP-2. Insulin-like growth factor-1 treatment stimulated the expression of SREBP target genes such as the low density lipoprotein (LDL) receptor, squalene synthase, and hydroxymethylglutaryl-CoA synthase genes. These results indicate that growth factor-induced phosphorylation of SREBP-2 inhibits sumoylation, thereby facilitating SREBP transcriptional activity. Glutathione S-transferase pulldown assays revealed that wild-type SREBP-2, but not a mutant lacking Lys464, interacts with HDAC3 preferentially among the histone deacetylase family members. HDAC3 small interfering RNA induced gene expression of the LDL receptor and thereby augmented fluorescently labeled LDL uptake in HepG2 cells. In summary, growth factors inhibit sumoylation of SREBPs through their phosphorylation, thus avoiding the recruitment of an HDAC3 corepressor complex and stimulating the lipid uptake and synthesis required for cell growth.

Naive CD4+ T Cells Exhibit Distinct Expression Patterns of Cytokines and Cell Surface Molecules on Their Primary Responses to Varying Doses of Antigen

The amount of an Ag used for stimulation affects the type and magnitude of T cell responses. In this study we have investigated the primary response of naive CD4+ T cells derived from OVA-specific TCR-transgenic mice (OVA23-3) upon stimulation with varying doses of the antigenic peptide, OVA323–339. IL-4 expression was maximal with 50 nM Ag and decreased significantly with increasing doses. In contrast, IFN-γ expression, which was also detected at 50 nM Ag, increased with increasing doses. The expression patterns of mRNA for the Th2-specific transcription factors GATA-3 and c-Maf were parallel to that of IL-4. These expression profiles were not altered by the addition of anti-IL-4 plus anti-IL-12 mAbs, suggesting that cytokine receptor signaling is not essential. Naive CD4+ T cells stimulated with 5 nM Ag elicited IgM secretion from cocultured B cells, whereas those stimulated with 50 nM Ag or more elicited apoptosis of B cells. This may be because at lower doses of Ag (5 nM), naive CD4+ T cells express CD40 ligand and OX40, whereas at higher doses (50 nM), they express Fas ligand. Clearly, the expression of each type of molecule depends on the Ag dose, and different molecules had different expression patterns. Thus, in the primary response, naive CD4+ T cells can exhibit different functions depending on the dose of Ag.

Determinant analysis of IgE and IgG4 antibodies and T cells specific for bovine αs1-casein from the same patients allergic to cow's milk: Existence of αs1-casein–specific B cells and T cells characteristic in cow's-milk allergy

In an effort to clarify the etiology of milk allergy from the standpoint of allergen-specific immune reactions, we investigated the determinants of IgE, IgG4, and T cells specific for bovine alpha(s)1-casein from the same individual patients by using its synthetic peptides and cyanogen bromide-digested fragments. Alpha(s)1-casein is a major allergen in cows milk, and its unique conformation enabled us to investigate the determinants of antibodies without consideration about missing the reactivities because of conformational changes. Nine patients were selected as subjects from among 129 milk-sensitive infants screened by ELISA to assess the anti-alpha(s)1-casein IgE levels in their sera. By using ELISA for epitope mapping, a C-terminal region of alpha(s)1-casein was identified as a common binding site for IgE from all of these patients, whereas those for anti-alpha(s)1-casein IgG4 were located in multiple regions of alpha(s)1-casein. We determined the specificities of seven alpha(s)1-casein-specific T-cell lines established from peripheral blood mononuclear cells of two of the patients. These T cells have been shown to secrete IL-4. All of the T-cell lines had different specificities to alpha(s)1-casein. However, a common amino acid residue use was found among the determinants of various T-cell lines from each patient. The results suggest that patients allergic to cows milk have characteristic B cells recognizing a limited region of alpha(s)1-casein and secreting alpha(s)1-casein-specific IgE. These B cells may interact particularly with T cells recognizing determinants with a common structure.