IFN-gamma deficiency worsen Pneumocystis pneumonia with Th17 development in nude mice.

Pneumocystis pneumonia (PCP) occurs frequently in patients with immunodeficiency syndromes, especially AIDS. In order to investigate the role of IFN-gamma on PCP, nude mice deficient in IFN-gamma (GKO nude) and their wild-type ones (WT nude) were infected with murine Pneumocystis. Nine weeks later they were sacrificed, and cytokines in BALF and lung histopathology were compared between them. Cyst burden was greater in GKO than in WT nude mice. Histopathology in the lung was severer and granulomatous lesions were observed more frequently in GKO nude mice. Levels of IL-17 were higher in BALF of GKO than in that of WT nude mice. Greater number of CD4(+) T cells from lungs of infected GKO nude mice produced IL-17 than those from WT ones. These results suggest that deficiency in IFN-gamma induces the differentiation of Th17 and that IL-17 is responsible for inflammatory response in PCP.

Distinct response in maintenance of human naive and memory B cells via IL-21 receptor and TCL1/Akt pathways.

The molecular mechanisms involving in B-cell survival/proliferation are poorly understood. Here we investigated the molecules affecting the survival of human naïve and memory B cells. Without stimulation, naïve B cells survived longer than memory B cells. Moreover, the viability of memory B cells decreased more rapidly than that of naïve B cells following with Staphylococcus aureus Cowan strain (SAC), anti-immunoglobulin (Ig), or anti-CD40 stimulation, but displayed the same levels of survival following CpG DNA stimulation. We analyzed the transcriptional differences between B-cell subsets by gene expression profiling, and identified 15 genes significantly correlated to survival/proliferation. Among them, IL-21 receptor (IL-21R) and T-cell leukemia 1 (TCL1) proto-oncogene were highly expressed in naïve B cells. IL-21 induced the proliferation of both naïve and memory B cells. Marked phosphorylation of Akt was found in naïve B cells compared with memory B cells. This study suggests that naive and memory B cells are regulated by several distinct molecules, and the IL-21R and TCL1/Akt pathways might play crucial roles in naïve B cells for their maintenance.

Fc receptor gamma-chain, a constitutive component of the IL-3 receptor, is required for IL-3-induced IL-4 production in basophils.

The Fc receptor common gamma-chain (FcRgamma) is a widely expressed adaptor bearing an immunoreceptor tyrosine-based activation motif (ITAM) that transduces activation signals from various immunoreceptors. We show here that basophils lacking FcRgamma developed normally and proliferated efficiently in response to interleukin 3 (IL-3) but were very impaired in IL-3-induced production of IL-4 and in supporting T helper type 2 differentiation. Through its transmembrane portion, FcRgamma associated constitutively with the common beta-chain of the IL-3 receptor and signaled by recruiting the kinase Syk. Retrovirus-mediated complementation demonstrated the essential function of the ITAM of FcRgamma in IL-3 signal transduction. Our results identify a previously unknown mechanism whereby FcRgamma functions to 'route' selective cytokine-triggered signals into the ITAM-mediated IL-4 production pathway.

STAT6 signalling is important in CD8 T-cell activation and defence against Toxoplasma gondii infection in the brain.

Signal transducer and activator of transcription (STAT) 6 is a molecule involved in interleukin (IL)-4 and -13 signalling. We investigated the role of STAT6 signalling in Toxoplasma gondii-infected mice using STAT6-deficient (STAT6(-/-)) and wild-type (WT) mice. A significantly larger number of cysts were recovered from the brain in STAT6(-/-) than in WT mice on days 28 and 56 post-infection. CD8(+) T cells in cerebrospinal fluid and spleen stimulated with T. gondii antigen produced higher levels of interferon (IFN)-gamma in WT than in STAT6(-/-) mice. CD8(+) T-cell function, estimated by expression of CD25 and cytotoxic activity, was lower in STAT6(-/-) than in WT mice. Transfer of CD8(+) but not CD4(+) T cells, purified from infected WT mice, into STAT6(-/-) mice successfully prevented formation of cysts in the brain. However, transfer of naïve CD8(+) T cells from WT into STAT6(-/-) mice did not show either activation of CD8(+) T cells or a decrease in the number of cysts in the brain. Transfer of splenic adherent cells from WT into STAT6(-/-) mice induced activation of CD8(+) T cells and decreased the number of cysts in the brain. Expression of CD86 on splenic dendritic cells and IL-12 p40 production were weaker in STAT6(-/-) than in WT mice after T. gondii infection. These results indicate that STAT6 signalling is important in CD8(+) T-cell activation, possibly through regulation of antigen-presenting cells, which could suppress T. gondii infection in the brain.

Interleukin-21 stimulates B-cell immunoglobulin E synthesis in human beings concomitantly with activation-induced cytidine deaminase expression and differentiation into plasma cells.

Interleukin (IL)-21 downregulates immunoglobulin E (IgE) production in murine systems by inhibiting germline epsilon transcription in IL-4-stimulated B cells. We here sought to clarify the function of IL-21 in human B-cell IgE synthesis. IL-21 dramatically enhanced IgE production by human mononuclear cells, or purified total, naive, or memory B cells in the presence of IL-4 plus anti-CD40 mAb cross-linked with CD32-transfectants, and the production was strengthened with further addition of IL-10. It was concomitant to the enhancement of activation-induced cytidine deaminase (AID) mRNA expression, but no increase of germline epsilon transcription. We also observed that IL-21 promoted B-cell differentiation into plasma cells with increase of B-lymphocyte-induced maturation protein-1 (Blimp-1), but not X-box binding protein 1 (XBP-1), which was further accentuated by co-stimulation with IL-4 plus CD40 signaling. Thus, IL-21 is a strong inducer of IgE production in human beings concomitantly with AID expression and the differentiation into plasma cells. Our data suggest that IL-21 plays an important role in occurrence and the treatment of allergic disorders.

Exposure to Bisphenol A prenatally or in adulthood promotes T(H)2 cytokine production associated with reduction of CD4CD25 regulatory T cells.

BACKGROUND: Bisphenol A (BPA) is a widespread endocrine-disrupting chemical that can affect humans and animals. OBJECTIVES: We investigated the effects of adult or prenatal exposure to BPA on T-helper (T(H))1/T(H)2 immune responses and the mechanisms underlying these effects. METHODS: To evaluate the effects of exposure to BPA in adulthood, male Leishmania major-susceptible BALB/c and -resistant C57BL/6 mice were subcutaneously injected with 0.625, 1.25, 2.5, and 5 micromol BPA 1 week before being infected with L. major. To evaluate prenatal exposure, female mice were given BPA-containing drinking water at concentrations of 1, 10, and 100 nM for 2 weeks, then mated, and given BPA for another week. Male 10-week-old offspring were infected with L. major. Footpad swelling was assessed as a measure of the course of infection. RESULTS: Mice exposed to BPA prenatally or in adulthood showed a dose-dependent increase in footpad swelling after being infected with L. major. Exposure to BPA in adulthood significantly promoted antigen-stimulated production of interleukin (IL)-4, IL-10, and IL-13 but not interferon-gamma (IFN-gamma). However, mice prenatally exposed to BPA showed increased production of not only IL-4 but also IFN-gamma. The percentages of CD4(+)CD25(+) cells were decreased in mice exposed to BPA either prenatally or in adulthood. Effects of prenatal BPA exposure were far more pronounced than effects of exposure in adulthood. CONCLUSION: BPA promotes the development of T(H)2 cells in adulthood and both T(H)1 and T(H)2 cells in prenatal stages by reducing the number of regulatory T cells.

Role of tonsillar IgD+CD27+ memory B cells in humoral immunity against pneumococcal infection.

IgD+CD27+ memory B cells are a major compartment of circulating memory B cells. However, the characteristics of these cells in the tonsils have been unclear. In this study, IgD+CD27+ memory B cells residing in the tonsillar marginal zone were found to exhibit similar characteristics as IgD+CD27+ memory B cells in the periphery, namely large morphology, expression of surface molecules, and hypermutated Ig variable region genes. Furthermore, these IgD+CD27+ memory B cells predominantly produced IgM, including IgM specific against pneumococcal polysaccharides. Taken together, these results provide convincing evidence that tonsillar IgD+CD27+ memory B cells impart protective humoral immunity against pneumococcal infection by producing high-affinity IgM.

The ICOS-ligand B7-H2, expressed on human type II alveolar epithelial cells, plays a role in the pulmonary host defense system.

The mechanism of immune defense against pathogens in the lung, has so far been poorly understood. Here, we show that human type II alveolar epithelial cells play a key role in defense via interactions between B7 homolog (B7h), also known as ICOS ligand, and its receptor ICOS expressed on activated T cells. The A549 alveolar type II cell line abundantly expresses B7-H2, CD40 and B7-1, but not B7-2 or hGL50. TNF-alpha significantly induced B7-H2 and CD40 expression by A549 cells, but had no effect on B7-1 or B7-2 expression. TNF-alpha-deficient mice exhibited low B7-H2 expression on alveolar epithelial cells in comparison with wild-type mice. Co-culture of TNF-alpha pre-stimulated A549 cells with CD4+ T cells promoted CD154 expression, CD4+ T cell proliferation and cytokine production, especially IFN-gamma. Monocyte-derived TNF-alpha in combination with IFN-gamma and LPS markedly induced B7-H2 expression in A549 cells. This study thus identifies a unique costimulatory pathway via alveolar epithelial type II cells that preferentially affects T helper cell function, implying that alveolar epithelial type II cells play a crucial role in innate immunity in the lung by regulating IFN-gamma-synthesis via B7-H2/ICOS interactions.

Regulation of aged humoral immune defense against pneumococcal bacteria by IgM memory B cell.

Elderly persons have a high incidence of lethal infections by encapsulated bacteria. However, mechanisms involved in their poor defense and maintenance of immunological memory have been poorly understood. The present study characterized the population of B cells known as IgM memory B cell compartment and their response by pneumococcal vaccine in elderly people. CD27+ memory B cells, particularly IgD+IgM+CD27+ IgM memory B cells, had dramatically declined in the aged. Their Ig syntheses by B cells and the differentiation into plasma cells were diminished in vitro compared with those in adults. A rise of anti-pneumococcal IgM in sera of elderly persons was found with lower levels compared with those in adults after pneumococcal vaccination. Although diminished function itself of aged B cells surely exist, decline of the IgM memory B cell pool is expected to result in a poor humoral immunity against pneumococcal infection in elderly people.

Granulocyte-macrophage colony-stimulating factor induces de novo methylation of the p15 CpG island in hematopoietic cells.

The process of p15 CpG island methylation induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) was investigated, using MO7e cells. The cells proliferating in response to GM-CSF+fetal bovine serum (FBS) were almost fully methylated in the p15 CpG island. The withdrawal of both GM-CSF and FBS for 48 h reduced the cell viability, and increased the frequency of alleles with completely or partially demethylated CpG sites by approximately 50%. Viable cells were responsible for this epigenetic change. The add-back of GM-CSF restored the methylation. Seventy-two hours withdrawal of GM-CSF+FBS followed by 24-h exposure to inhibitors for DNA methyltransferase (DNMT) and histone deacetylase (HDAC) caused the demethylation of nearly all CpG sites in the p15 CpG island on every allele sequenced. When GM-CSF was re-added after 96-h treatment, the cells exhibited p15 transcriptional silencing via the methylation. The initial methylation event encompassed the entire CpG island. No new methylated alleles appeared in the coexistence of the DNMT and HDAC inhibitors. Taken together, GM-CSF may be able to induce de novo methylation of the p15 gene, using HDAC(s) as well as DNMT(s).

CD72-mediated suppression of human naive B cell differentiation by down-regulating X-box binding protein 1.

B cells can differentiate into antibody-secreting plasma cells, however the signals that control the entry into this pathway are not clearly understood. We have investigated the role of human CD72 in mature B cell differentiation. Human CD72 is preferentially expressed in naive B cells, but marginal levels of expression can be found in switched memory B cells. CD72 cross-linking promoted an increase in B cell activation and proliferation. Interestingly, expression of CD27, whose signal induces the differentiation of B cells into plasma cells, was down-modulated by CD72 stimulation. This CD72 signaling also induced tyrosine phosphorylation of various proteins such as Blk. Plasma cell differentiation and Ig syntheses were diminished by CD72 ligation in the presence of Staphylococcus aureus Cowan strain (SAC) plus IL-2 but not in the presence of CD40 signaling or CpG oligodeoxynucleotide. Our results show that CD72 signaling reduces the expression of X-box binding protein 1 in B cells stimulated with SAC plus IL-2, but the expression of PRDI-BF1 was unaffected. Taken together, these data demonstrate that CD72 is a key molecule in regulating mature B cell differentiation, particularly in preventing the differentiation of naive B cells into plasma cells, thus blocking the production of low-affinity antibodies.

Impaired recognition by Toll-like receptor 4 is responsible for exacerbated murine Pneumocystis pneumonia.

We investigated the effect of Toll-like receptor 4 (TLR4) on the progression of murine Pneumocystis pneumonia. TLR4-mutant C3H/HeJ and wild-type C3H/HeN mice were infected with Pneumocystis after depletion of CD4 T cells. Mutant mice lost body weight more quickly and showed exacerbated pulmonary injury even though there was no difference in Pneumocystis organism burden in the lung. Mutant mice showed reduced levels of IL-10, IL-12p40 and MIP-2 accompanied by elevated levels of TNF-alpha and IL-6 in the bronchoalveolar lavage fluid compared with those of wild-type mice 8 weeks after the infection. In response to stimulation with Pneumocystis antigen, the production of IL-10, IL-12p40 and MIP-2 by alveolar macrophages was partially impaired in mutant mice, while that in wild-type mice was suppressed by the anti-TLR4/MD-2 mAb, MTS510. Unlike the response to lipopolysaccharide stimulation, TLR4-reconstituted HEK293 cells showed no elevated NF-kappaB activation after stimulation with Pneumocystis antigen. Taken together, these findings suggest that recognition of Pneumocystis by TLR4 helps to regulate the host inflammatory responses through cytokine and chemokine production by alveolar macrophages.

A serine/threonine kinase, Cot/Tpl2, modulates bacterial DNA-induced IL-12 production and Th cell differentiation.

A serine/threonine protein kinase, Cot/Tpl2, is indispensable for extracellular signal-regulated kinase (ERK) activation and production of TNF-alpha and PGE2 in LPS-stimulated macrophages. We show here that Cot/Tpl2 is also activated by other Toll-like receptor (TLR) ligands. Bacterial DNA rich in the dinucleotide CG (CpG-DNA), unlike LPS or synthetic lipopeptide, activated ERK in a Cot/Tpl2-independent manner. Peritoneal macrophages and bone marrow-derived DCs from Cot/Tpl2-/- mice produced significantly more IL-12 in response to CpG-DNA than those from WT mice. Enhanced IL-12 production in Cot/Tpl2-/- macrophages is, at least partly, regulated at the transcriptional level, and the elevated IL-12 mRNA level in Cot/Tpl2-/- macrophages is accompanied by decreased amounts of IL-12 repressors, such as c-musculoaponeurotic fibrosarcoma (c-Maf) and GATA sequence in the IL-12 promoter-binding protein (GA-12-binding protein; GAP-12) in the nucleus. Consistently, Cot/Tpl2-/- mice showed Th1-skewed antigen-specific immune responses upon OVA immunization and Leishmania major infection in vivo. These results indicate that Cot/Tpl2 is an important negative regulator of Th1-type adaptive immunity, that it achieves this regulation by inhibiting IL-12 production from accessory cells, and that it might be a potential target molecule in CpG-DNA-guided vaccination.

Bisphenol A promotes IL-4 production by Th2 cells.

BACKGROUND: It has been proposed that estrogen plays an important role in modulating the Th1/Th2 cytokine balance. From this viewpoint, chemicals with estrogenic responses were expected to possess similar immunoregulatory roles which have not been defined to date. To address this, we studied the effects of one of the estrogenic chemicals, bisphenol A (BPA), on the in vitro production of Th1 and Th2 cytokines. METHODS: Mesenteric lymph node cells from Trichinella spiralis (Ts)-infected mice were incubated with serialfold dilutions of BPA under stimulation with Ts antigen. The Th2 cytokine production in the supernatant was determined by ELISA. The Th2 cytokine production by mesenteric lymph node cells from Ts-infected mice inoculated orally with BPA was compared with that of uninoculated mice infected with Ts. RESULTS: The antigen-stimulated interleukin (IL)-4 production by Th2-dominant mesenteric lymph node cells from Ts-infected mice increased significantly by addition of 3 microM of BPA. The IL-5 production was not affected. The production of IL-4, but not that of IL-5, by splenocytes of Th2-skewed Leishmania major-infected BALB/c mice increased at concentrations of 3 and 10 microM of BPA. However, the interferon gamma production was not affected by BPA in Th1-skewed L. major-infected C57BL/6 mice. The production of IL-4 and IL-10, but not that of IL-13, markedly increased in Ts-infected mice inoculated orally with BPA. CONCLUSIONS: We demonstrated that the IL-4 production was increased both in vitro and in vivo by treatment with BPA. This suggests that BPA might cause allergic diseases by stimulating the IL-4 production by Th2 cells.

Functional analysis of human memory B-cell subpopulations: IgD+CD27+ B cells are crucial in secondary immune response by producing high affinity IgM.

The number of memory B cells in peripheral blood has been assayed in various diseases by using CD27 as a memory B-cell marker. However, the defining differences of characteristic and function between the two memory B-cell subpopulations separated by immunoglobulin (Ig)D expression remain to be clearly elucidated. We analyzed here IgD(+)CD27(+) B cells (circulating B cells 2, cB2) and IgD(-)CD27(+) memory B cells (cB3) in comparison with IgD(+)CD27(-) naive B cells (cB1). cB2 were found to be morphologically similar to cB3 with abundant cytoplasm, whereas cB3 expressed CD80, CD86, and CD95 on their surface more predominantly than cB2. A majority of cB2 expressed both IgD and IgM, and cB3 expressed IgA or IgG. Mature gamma1 and gamma2 transcripts were found in cB3, but at very low levels in cB2, and activation-induced cytidine deaminase (AID) mRNA expression was recognized only in cB3. The frequencies of somatic hypermutation in cB2 and cB3 were comparable levels studied by VH5. cB2 did not shift to cB3 in vitro by the stimuli such as via B-cell receptor or CD40. cB2 produced large amounts of IgM predominantly and promptly, which is in accordance with the known characteristics of memory B cells. Taken together, although cB2 are unclass-switched, cB2 have the functions of memory B cells and are not in the process of transition from naive to switched memory B cells, playing a crucial role in secondary immune response by producing high-affinity IgM in the early phase of infections.

Absence of memory B cells in patients with common variable immunodeficiency.

The molecular basis of common variable immunodeficiency (CVID) is unknown. To assess humoral immunity in CVID, we selected 24 patients with early or late onset of disease. X-linked agammaglobulinemia (XLA), X-linked hyper-IgM syndrome (XHIM), and non-XHIM were excluded based on clinical phenotype, assessment of the immune response, presence of Bruton's tyrosine kinase (Btk) in monocytes or platelets, and normal expression of CD40 ligand by activated T cells. The number of circulating B cells was within the normal range or reduced. IgD(-) CD27(+) memory B cells were markedly reduced or absent in all 24 patients and IgD(+) CD27(+) B cells were diminished in 8 patients. Circulating B cells from all 6 patients examined, including CVID patients with IgD(+) CD27(+) cells, failed to undergo somatic hypermutation in immunoglobulin-variable (V)-region genes, similar to cord blood B cells. B cells from CVID patients produced IgM and IgG, but not IgA upon the engagement of Ig receptor and CD40 in the presence of IL-2 and IL-10. B cells from all but 5 patients secreted IgE when stimulated by CD40 crosslinking in the presence of IL-4. The observation of defective memory B cells with abnormal cell marker expression and function demonstrates that naive CVID B cells including those expressing IgD(+) CD27(+), in analogy to cord blood and hyper-IgM syndrome B cells, may be responsible for their failure to differentiate into plasma cells and to produce high-affinity antibodies of different isotypes.

The different process of class switching and somatic hypermutation; a novel analysis by CD27(-) naive B cells.

The relationship between class switch recombination (CSR) and somatic hypermutation has been unclear. By using human CD27(-) naive B cells, we investigated the somatic hypermutation and producibility of immunoglobulins (Igs) that occur after CSR. Although neither adult CD27(-) nor cord blood B cells, which showed the unmutated Ig V-region genes, produced IgG, IgM, or IgA in response to conventional stimuli, they produced IgG and IgM but not IgA in the presence of Staphylococcus aureus Cowan strain (SAC) + interleukin-2 (IL-2) + IL-10 + anti-CD40 mAb + CD32 transfectants (CD40/CD32T). The naive B cells also produced IgE when combined with IL-4 + CD40/CD32T. In parallel with IgG production, the expression of mature gamma1 and gamma 2 transcripts was induced from naive B cells by the stimuli. The CD27 expression on human naive B cells was induced remarkably by CD40 signaling or B-cell receptor engagement, but somatic hypermutation could not be induced. The proliferation and differentiation into plasma cells were induced from naive B cells, whereas most of the plasma cells displayed very low levels of mutations in Ig V-region genes. CD27(-) naive B cells expressed activation-induced cytidine deaminase messenger RNA by the stimuli later than CD27(+) memory B cells. Our results demonstrate that CSR, but not noticeable somatic hypermutation, can be induced from CD27(-) naive B cells upon B-cell receptor engagement and CD40 signaling in cooperation with cytokines, suggesting that CSR and somatic hypermutation processes can occur independently, and the antibodies produced in this in vitro system are low-affinity antibodies.