Expression and significance of peripheral myeloid-derived suppressor cells in chronic hepatitis B patients.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) exert their suppressive effects on multiple immune response and contribute to the development of many diseases. However, limited data is available on the involvement of MDSCs in human chronic HBV infection. OBJECTIVE: To investigate whether the progression of chronic HBV infection was associated with imbalance of MDSCs. METHODS: The percentages of MDSCs, regulatory T (Treg), Th1 and Tc1 cells in the peripheral blood from chronic hepatitis B (CHB) patients and healthy controls (HC) were determined by flow cytometry. Plasma levels of IL-10, TGF-ß and IFN-γ were measured using enzyme-linked immunosorbent assay. The potential association of the frequencies of MDSCs with clinical parameters was assessed. RESULTS: The percentages of MDSCs and Treg cells were significantly higher in CHB patients than those in HC. The percentages of MDSCs were negatively correlated with Th1 cells. Increased plasma IL-10 level and decreased IFN-γ level were found in CHB patients compared with HC. Moreover, the frequencies of MDSCs and plasma IL-10 levels were positively correlated with serum HBV DNA loads, as well as liver function impairment. CONCLUSION: The expanded peripheral MDSCs may contribute to poor viral clearance and disease progression during chronic HBV infection.

Effect of intestinal microbiota on the induction of regulatory CD25+ CD4+ T cells.

When oral tolerance was induced in either specific pathogen-free (SPF) or germ-free (GF) mice, ovalbumin (OVA) feeding before immunization induced oral tolerance successfully in SPF mice. On the other hand, OVA-specific immunoglobulin G1 (IgG1) and IgE titres in OVA-fed GF mice were comparable to those in phosphate-buffered saline-fed GF mice, thus demonstrating that oral tolerance could not be induced in GF mice. The frequencies of CD25(+) CD4(+)/CD4(+) cells in the mesenteric lymph node (MLN) and the absolute number of CD25(+) CD4(+) cells in the Peyer's patches and MLN of naive GF mice were significantly lower than those in naive SPF mice. In an in vitro assay, the CD25(+) CD4(+) cells from the naive SPF mice suppressed more effectively the proliferation of responder cells in a dose-dependent manner than those from the GF mice. In addition, the CD25(+) CD4(+) regulatory T (T(reg)) cells from the naive SPF mice produced higher amounts of interleukin (IL)-10 and transforming growth factor (TGF)-beta than those from the GF mice. When anti-TGF-beta neutralizing antibody, but not anti-IL-10 neutralizing antibody, was added to the in vitro proliferation assay, the suppressive effect of the CD25(+) CD4(+) T(reg) cells from the SPF mice was attenuated to the same level as that of the CD25(+) CD4(+) cells from the GF mice. In conclusion, the TGF-beta-producing CD25(+) CD4(+) T(reg) cells from the MLN of SPF mice played a major role in oral tolerance induction. In addition, as the regulatory function of the CD25(+) CD4(+) cells from the naive GF mice was much lower than that of the CD25(+) CD4(+) T(reg) cells from the SPF mice, indigenous microbiota are thus considered to contribute to the induction and maintenance of CD25(+) CD4(+) T(reg) cells.

Suppression of allergic reaction by lambda-carrageenan: toll-like receptor 4/MyD88-dependent and -independent modulation of immunity.

BACKGROUND: Recognition of foreign substances by innate immunity through pattern recognition receptors (PRRs) regulates acquired immunity such as allergic reaction. Because PRRs recognize heterogeneous ligands, daily food intake can potentially regulate immune allergic reaction. OBJECTIVE: Elucidation of the effect of lambda-carrageenan on allergic reactions was aimed. METHOD: IFN-gamma and IL-4 was measured in in vitro T cell-stimulated culture. Cytokine production from macrophages in response to lambda-carrageenan was measured as indicator for innate immunity activation. Mice were immunized with OVA in alum to induce specific IgE, and then histamine release was induced by systemic injection of OVA. RESULTS: Activation of innate immunity by lambda-carrageenan is dependent on Toll-like receptor-4 (TLR4) and MyD88, in which induction of pro-inflammatory cytokines such as TNF-alpha and IL-6 was largely impaired in macrophages from TLR4- and MyD88-deficient mice. Footpad oedema, a model for in vivo inflammatory reactions, was significantly reduced in these mice. Similar to recent evidence showing a preference for the stimulation of Th1 via TLR/MyD88 signalling, lambda-carrageenan showed enhanced IFN-gamma and decreased IL-4 in stimulated T cell cultures. Interestingly, increased IFN-gamma production was still seen in TLR4- and MyD88-deficient splenocytes. Oral administration of lambda-carrageenan to immunized mice successfully decreased OVA-specific IgE, and lambda-carrageenan was also effective in previously immunized mice. Further, serum histamine release upon systemic challenge of OVA was significantly inhibited. Neither OVA-specific IgG1/IgG2a nor cytokine secretion from in vitro cultures were altered, suggesting the involvement of multiple PRRs as demonstrated by TLR4/MyD88-independent IFN-gamma up-regulation. The simultaneous feeding of OVA with lipopolysaccharide abrogated oral tolerance, but lambda-carrageenan was not only devoid of such an effect but was also found to promote oral tolerance in the absence of TLR4. CONCLUSION: lambda-Carrageenan was suggested to be a useful dietary supplement to ameliorate allergic reactions while maintaining oral tolerance-dependent intestinal homeostasis.

Overexpression of the Wiskott-Aldrich syndrome protein N-terminal domain in transgenic mice inhibits T cell proliferative responses via TCR signaling without affecting cytoskeletal rearrangements.

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia with small platelets, severe eczema, and recurrent infections due to defects in the immune system. The disease arises from mutations in the gene encoding the WAS protein (WASP), which plays a role as an adaptor molecule in signal transduction accompanied by cytoskeletal rearrangement in T cells. To investigate the functional domain of WASP, we developed transgenic mice overexpressing the WASP N-terminal region (exon 1-5) including the Ena/VASP homology 1 (pleckstrin homology/WASP homology 1) domain, in which the majority of mutations in WAS patients have been observed. WASP transgenic mice develop and grow normally under the specific pathogen-free environment, and showed normal lymphocyte development. However, proliferative responses and cytokine production induced by TCR stimulation were strongly inhibited in transgenic mice, whereas Ag receptor capping and actin polymerization were normal. These findings suggest that overexpressed Ena/VASP homology 1 (pleckstrin homology/WASP homology 1) domain of WASP inhibits the signaling from TCR without coupling of cytoskeletal rearrangement. WASP transgenic mice shown here could be valuable tools for further understanding the WASP-mediated processes.

Interleukin-10-secreting Peyer's patch cells are responsible for active suppression in low-dose oral tolerance.

We demonstrate the induction of antigen-specific interleukin-10 (IL-10)-secreting cells in murine Peyer's patches (PPs) after low-dose beta-lactoglobulin (BLG) feeding. In addition, we show that PP cells can inhibit the T-cell proliferative response in vitro as well as T-cell-mediated inflammation in vivo. The active suppression mediated by these regulatory cells was seen only within a narrow range of antigen dosage (feeding), with the most prominent effect at 5 x 1 mg BLG. On either side of this range, T-helper 1-like cytokine responses were observed when PP cells were stimulated with antigen in vitro. This result correlated with reduced production of regulatory cytokines as well as reduced activity of bystander suppression. We found that changes in IL-10 production correlated inversely with changes in interferon-gamma production. Inhibitory effects mediated by CD4(+) PP cells were partially neutralized by antibodies to IL-10 and transforming growth factor-beta. Interestingly, the generation of such regulatory cells after low-dose BLG feeding exhibited organ dependence. Among spleen, lymph node and PP cells derived from orally tolerized mice, PP cells were the most effective in promoting bystander suppression in the presence of BLG, indicating the significance of PPs as an inductive site for antigen-specific regulatory cells upon induction of low-dose oral tolerance. Moreover, PP cells from mice fed 5 x 1 mg BLG were shown to suppress a BLG-specific delayed-type hypersensitivity response induced in footpads, suggesting that IL-10-secreting PP cells regulate systemic inflammation.

Lactococci as probiotic strains: adhesion to human enterocyte-like Caco-2 cells and tolerance to low pH and bile.

There have been few studies on the probiotic activity of Lactococcus strains although they are commonly used as starter bacteria in manufacturing many kinds of fermented dairy products. Nine strains of the genus Lactococcus were examined for their probiotic properties, such as adherence to human enterocyte-like Caco-2 cells and tolerance to acid and bile. Six strains were adhesive and the highest adhesion was observed with Lactcoccus lactis ssp. lactis NIAI527. This strain adhered to the microvilli of cells as observed by scanning electron microscopy and also tolerated low pH and bile. These properties should make strain 527 a potential new probiotic strain.

Anti-DNA IgA autoantibodies are spontaneously generated in mouse Peyer's patches.

IgA antibodies in the mucosal immune system are produced specifically to environmental antigens such as virus and bacteria, and possibly to some food components, which will provide a potential luminal antigen, DNA. To study the immune response to DNA in the gut, we established B-cell hybridomas producing IgA monoclonal antibodies (mAb) from Peyer's patches (PP) of non-immunized, non-autoimmune, specific pathogen-free BALB/c mice, and examined their specificity by enzyme-linked immunosorbent assay (ELISA). Three mAb out of 18 bound strongly to self, bacterial and synthetic DNA, with Kd of about 10-7 m. One of the three mAb also reacted with the histone component and another reacted with some mouse food component. The VH genes of these three mAb have not previously been reported to have anti-DNA specificity, and carry putative somatically mutated sites favouring DNA binding in CDR. The features resemble those of anti-DNA antibodies found in human and murine models of systemic lupus erythmatosus (SLE), and are indicative of an antigen-driven selection process. Our findings suggest that even in normal healthy animals, anti-DNA antibodies of IgA isotype can be produced in certain peripheral environments such as in PP by spontaneous antigenic stimulation.

MHC class II/T-cell receptor interactions potentiate secretion of IgG but not IgM in response to T-dependent antigens.

We have examined whether the interaction of peptide-loaded MHC molecules on the surface of B-cells with antigen-specific T-cell receptors (TCRs) enhances Ig secretion in the presence of other antigen-independent interactions in vitro. B-cells specific for region 25-40 of beta-lactoglobulin (beta-LG) were stimulated in a T-cell dependent manner using plasma membranes (PM) derived from two different T-helper (Th) clones, culture supernatants of activated Th2 cells and beta-LG as a specific antigen. PM were obtained from either the beta-LG-specific T-cell clone H1.1 which can mediate specific TCR/MHC class II interactions as well as antigen-independent ones or from the D10 clone which bears a TCR of an irrelevant specificity and thus, can only mediate antigen-independent interactions. IgG, but not IgM, secretion was specifically enhanced by H1.1 PM, but not D10 PM in the presence of beta-LG. Furthermore, a blockade of TCR/MHC class II interactions using either anti-T-cell receptor, beta or anti-CD4 monoclonal antibodies inhibited this enhanced IgG secretion in response to beta-LG. The results show that while antigen-independent interactions between T- and B-cells can enhance secretion of IgM antibodies, specific interactions between TCRs and peptide:MHC complexes stimulate B-cells to enhance secretion of IgG but not IgM antibodies. This mechanism may contribute to antibody secretion only from B-cells activated through cognate interaction in vivo.

Localization of T-cell determinants on bovine beta-lactoglobulin.

T-cell determinants of bovine beta-lactoglobulin (beta-LG) in BALB/c(H-2d), C57BL/6(H-2b) and C3H/He(H-2k) mice were identified using a set of overlapping synthetic peptides encompassing the entire primary structure of the protein. Lymph node cells from mice immunized with beta-LG were subjected to cell proliferation assay in the presence of these peptides and uptake of 3H-labeled thymidine was measured. Determinant regions were indicated to lie in residues 42-56, 62-76 and 139-153 in BALB/c mice, residues 11-26, 72-86, 100-113 and 119-133 in C57BL/6 mice, residues 72-86, 91-104, 129-143 and 139-153 in C3H/He mice. Some of these fragments included the antigenic motifs predicted by hypotheses according to amphipathicity and sequential patterns of peptides. We reported elsewhere that residues 42-56 and 72-86 represent one of the B-cell antigenic determinants in BALB/c and C3H/He, respectively. These peptides serve as good models of colinear T- and B-cell determinants as they contain both of T- and B-cell determinants.

Establishment of CD4+ T cell clones specific to bovine beta-lactoglobulin and analysis of their specificity.

Bovine beta-lactoglobulin (beta-LG) specific T cell clones, H1.1, 5G, 2.11G, and 6.11G were established from BALB/c mice and characterized. Surface phenotypes of clones were Thy1+, CD3+, CD4+, and CD8-. All clones recognize beta-LG in a I-Ad restricted manner. The T cell determinant region for H1.1 and 5G was identified as residues 42-56 of beta-LG by proliferation assay with overlapping synthetic peptides covering the entire sequence of beta-LG. The T cell determinant region for H1.1 was further delimited to residues 43-52 by using analog peptides. Previously we analyzed the T cell determinant regions of beta-LG in BALB/c, C3H/He, and C57BL/6 mouse by polyclonal T cell proliferation assay and reported that residues 42-56 of beta-LG constitute one of the major T cell determinants in BALB/c mice. Specificities of 2.11G and 6.11G were not identified by these synthetic peptides. Although reduction of disulfide bonds and CNBr cleavage of beta-LG did not affect the antigen recognition by 2.11G and 6.11G, tryptic degradation of the protein caused a clearer decrease of response in 6.11G than in 2.11G.

Preferential suppression of delayed-type hypersensitivity by L-156,602, a C5a receptor antagonist.

In the course of our screening for in vivo immunomodulating substances in which sheep red blood cells (SRBC) and heat-killed Brucella abortus cells (thymus dependent and independent antigens, respectively) for antibody production assays, and trinitrobenzene sulfonic acid (TNBS) for delayed-type hypersensitivity (DTH) assay were adopted as antigens, we detected a DTH-specific suppressive activity. The producing organism was isolated from a soil sample collected in Ushiku City, Ibaraki, Japan and identified with Streptomyces sp. A1502 (FERM P-12448). The active component was identified with L-156,602, a C5a receptor antagonist. L-156,602 suppressed both TNBS-induced and TNP-SRBC-induced DTH while it enhanced antibody production against SRBC, Brucella abortus, and TNP-SRBC. L-156,602 significantly suppressed DTH induced by direct injection of type 1 helper T cells and its relevant antigen into hind-footpads, indicating that the efferent phase of DTH was affected by L-156,602. The results demonstrated that L-156,602 preferentially suppressed the DTH response.