IL-6 is a potent cofactor of IL-1 in IgM synthesis and of IL-5 in IgA synthesis

In these studies we determined the capacity of IL-6 to act as a differentiation cofactor for murine Peyer's patch B cells producing different Ig classes and subclasses. In preliminary studies we determined that sufficient endogenous IL-6 was produced in LPS-induced cell systems to obscure responses to exogenous IL-6. We therefore studied IL-6 effects on Peyer's patch B cells (T cell-depleted cell populations) in the absence of LPS, relying on responses of in vivo-activated cells. rIL-1 alpha or purified IL-6 only slightly enhanced synthesis of IgM over minimal baseline levels in Peyer's patch T cell-depleted cell cultures; however, when IL-6 was added to cultures also containing rIL-1, IgM synthesis was very substantially increased. In addition, rIL-5 alone gave rise to a modest increase in IgM synthesis and its effect was not enhanced by either rIL-1 or IL-6. IgG production (mainly IgG3) followed a similar pattern. In contrast, IgA production was only modestly increased above baseline by rIL-1, rIL-5, or IL-6 alone or by rIL-1 and IL-6 in combination, but was greatly increased by rIL-5 and IL-6 in combination. The effect of IL-6 on Ig synthesis in the above studies was not due to an effect on cell proliferation. In summary, these data indicate that B cells differ in respect to the cytokines supporting maximal terminal differentiation and thus the class of Ig produced may depend on the presence of a particular combination of cytokines and lymphokines.     

Activation of macrophages and the intestinal immune system by an orally administered decoction from cultured mycelia of Cordyceps sinensis

The effects of an orally administered hot-water extract (HW) from cultured mycelia of Cordyceps sinensis on the activation of macrophages and the intestinal immune system were studied in mice. The general composition of HW was 83.9% carbohydrate, 11.8% protein, 1.9% lipid and 2.4% ash, and the carbohydrates were mainly composed of glucose, mannose, galactose and arabinose (molar ratio of 1.0:0.8:0.5:0.1). HW stimulated the activation (1.7-fold of the saline control) of macrophages and IL-6 production (1.5-fold) at 2.0 g/kg/day. Analyzing the culture supernatant of Peyer's patch cells from C3H/HeJ mice that had been fed with HW at 1.0 g/kg/day for 7 days indicated that the bone marrow cells had significantly proliferated (1.9-fold). In addition, the amounts of GM-CSF and IL-6 in the culture supernatant of Peyer's patch cells at the same dose were significantly increased (1.8-fold and 2.2-fold, respectively). These results indicate that an oral administration of HW may modulate IL-6 production by the activation of macrophages, and also enhance the secretion of hematopoietic growth factors such as GM-CSF and IL-6 from Peyer's patch cells. Since such cytokines as GM-CSF and IL-6 from Peyer's patch cells act on the systemic immune system, it can be assumed that orally administered HW modulated not only the local but also systemic immune system.     

Altered expression of inflammatory cytokine receptors in response to LPS challenge through interaction between intestinal epithelial cells and lymphocytes of Peyer's patch

The intense innate immunological activities occurring at the enteric mucosal surface involve interactions between intestinal epithelial cells and immune cells. Our previous studies have indicated that Peyer's patch lymphocytes may modulate intestinal epithelial barrier and ion transport function in homeostasis and host defense via cell-cell contact as well as cytokine signaling. The present study was undertaken using the established co-culture system of Caco-2 epithelial cells with lymphocytes of Peyer's patch to investigate the expression of IL-8 and IL-6 cytokines and cytokine receptors in the co-culture system after challenge with Shigella F2a-12 lipopolysaccharide (LPS). The human colonic epithelial cell line Caco-2 was co-cultured with freshly isolated lymphocytes from the murine Peyer's patch either in the mixed or separated (isolated but permeable compartments) co-culture configuration, and was challenged with Shigella F2a-12 LPS for 8 h. The level of mRNA expressions of human interleukin-8 (hIL-8), human interleukin-8 receptor (hIL-8R), mouse interleukin-8 receptor (mIL-8R), mouse interleukin-6 (mIL-6), mouse interleukin-6 receptor (mIL-6R) and human interleukin-6 receptor (hIL-6R) was examined by semi-quantitative PCR. In both co-culture groups, hIL-8 expression of Caco-2 cells was decreased, and hIL-8R expression was increased compared to the Caco-2 alone group. Upon LPS challenge, hIL-8 expression from the Caco-2 cells of both co-culture groups was higher than in the Caco-2 control group. The increased hIL-8 expression of Caco-2 cells in the separated co-culture group is correlated with a decreased hIL-8R expression and an increased mIL-8R expression. In the mixed co-culture group, the increased expression of hIL-8 was associated with the upregulated hIL-8R expression on Caco-2 cells and downregulated mIL-8R on murine Peyer's patch lymphocytes (PPL). mIL-6 expression from mouse PPL was also upregulated by LPS in mixed co-culture. However, upon the treatment with LPS, hIL-6R expression of Caco-2 cells was decreased in the mixed co-culture, but increased in separated co-culture. The data suggest that release of hIL-8 from epithelial cells may act on lymphocytes through a paracrine pathway, but it may also act on the epithelial cells themselves via an autocrine pathway. The data also suggest that the release of mIL-6 from Peyer's patch lymphocytes affects epithelial cells in a paracrine fashion.     

The expression analysis of inflammatory and antimicrobial genes in the goldfish (Carassius auratus L.) infected with Trypanosoma carassii

We report results of a comprehensive analysis of inflammatory gene expression during the course of infection of Trypanosoma carassii in the goldfish. We observed significant increases in mRNA levels of genes encoding pro-inflammatory cytokines IFN-γ, TNFα1 and TNFα2; IL-1β-1 and IL-1β-2; IL-12-p35 and IL-12-p40; CCL1; CXCL8, anti-inflammatory cytokines IL-10 and TGFβ and iNOS A and iNOS B, using quantitative PCR. Expression levels and profiles of these cytokines and iNOS isoforms varied in the different tissues (kidney, spleen, liver) of goldfish during the course of T.?carassii infection. The expression of majority of genes that encode pro- and anti-inflammatory cytokines were up-regulated during the acute phase of infection (days 7-21 post-infection). The mRNA levels of these cytokines returned to normal levels or were down-regulated during the elimination phase of infection (days 28-56), with exception of IL-10 in the spleen and liver of infected fish. A parallel up-regulation of IFN-γ and IL-10 mRNA levels were observed in all tissues of infected fish during the acute phase of the infection. The expression of iNOS genes (iNOS A and B) was significantly delayed (day 14?pi) in the kidney, liver and spleen of infected fish. These results provide insights into the interaction between T.?carassii and goldfish, and suggest that Th1/Th2-like responses may be important for controlling T.?carassii infection in the goldfish.     

Rotavirus 2/6 virus-like particles administered intranasally in mice, with or without the mucosal adjuvants cholera toxin and Escherichia coli heat-labile toxin, induce a Th1/Th2-like immune response

We investigated the rotavirus-specific lymphocyte responses induced by intranasal immunization of adult BALB/c mice with rotavirus 2/6 virus-like particles (2/6-VLPs) of the bovine RF strain, by assessing the profile of cytokines produced after in vitro restimulation and serum and fecal antibody responses. The cytokines produced by splenic cells were first evaluated. Intranasal immunization with 50 microg of 2/6-VLPs induced a high serum antibody response, including immunoglobulin G1 (IgG1) and IgG2a, a weak fecal antibody response, and a mixed Th1/Th2-like profile of cytokines characterized by gamma interferon and interleukin 10 (IL-10) production and very low levels of IL-2, IL-4, and IL-5. Intranasal immunization with 10 microg of 2/6-VLPs coadministered with the mucosal adjuvants cholera toxin and Escherichia coli heat-labile toxin (LT) considerably enhanced the Th1/Th2-like response; notably, significant levels of IL-2, IL-4, and IL-5 were observed. Since rotavirus is an enteric pathogen, we next investigated the production of IL-2 and IL-5, as being representative of Th1 and Th2 responses, by Peyer's patch and mesenteric lymph node cells from mice immunized intranasally with 2/6-VLPs and LT. The results were compared to those obtained from splenic and cervical lymph node cells. We found that both cytokines were produced by cells from each of these lymphoid tissues. These results confirm the Th1/Th2-like response observed at the systemic level and show, on the assumption that T cells are the primary cells producing the cytokines after in vitro restimulation, that rotavirus-specific T lymphocytes are present in the intestine after intranasal immunization with 2/6-VLPs and LT.     

Pretreatment with granulocyte-colony stimulating factor decreases lipopolysaccharide-induced interferon-gamma production in mice in association with the production of interleukin-18

We studied the effects of pretreatment with granulocyte-colony stimulating factor (G-CSF) on the production of pro- and anti-inflammatory cytokines induced by lipopolysaccharide (LPS). Mice received G-CSF or control saline once a day for 7 days or once at 1 h before the injection of LPS. Cytokines were measured by enzyme-linked immunosorbent assay or antibody-based electrochemiluminescence assay and cytokine mRNA was measured by RNAse protection assay. Mice pretreated with G-CSF for 7 days before LPS had lower serum levels of LPS-induced interferon-gamma (IFN-gamma) and higher levels of interleukin (IL)-6 and IL-10 than controls. G-CSF-pretreated mice also had lower mRNA levels of IFN-gamma and higher mRNA levels of IL-6 and IL-10 in the spleen and/or liver than controls. G-CSF-pretreated mice had serum levels of tumor necrosis factor, IL-12 p70 and IL-12 p40 similar to controls. G-CSF-pretreated mice had lower levels of spleen IL-18 than controls-serum IL-18 being undetectable in mice after LPS-and lower levels of IL-18 mRNA in the spleen. Mice pretreated with G-CSF 1 h before LPS had lower levels of serum IFN-gamma and spleen IL-18 than controls. G-CSF pretreatment alters the expression of LPS-induced cytokines with a decrease in pro-inflammatory IFN-gamma and an increase in anti-inflammatory IL-6 and IL-10. G-CSF decrease of IL-18 production may be a major mechanism explaining the effects of G-CSF on the production of IFN-gamma.     

Expression of alpha(4)beta(7) integrin defines a distinct pathway of lymphoid progenitors committed to T cells, fetal intestinal lymphotoxin producer, NK, and dendritic cells

During embryogenesis, the Peyer's patch anlagen are induced by a cell population that produces lymphotoxin (LT) alpha(1)beta(2) following stimulation of IL-7Ralpha. In this study, we show that the LT-producing cell is localized within the IL-7Ralpha(+) and integrin alpha(4)beta(7) (alpha(4)beta(7))(+) population in the embryonic intestine. Lineage commitment to the LT producer phenotype in the fetal liver coincides with expression of alpha(4)beta(7). Before expression of alpha(4)beta(7), the potential of IL-7Ralpha(+) population to generate B cells is lost. However, the progenitors for T cells and LT producer cells reside in the IL-7Ralpha(+)alpha(4)beta(7)(+) cells, but during subsequent differentiation, the potential to give rise to T cells is lost. This IL-7Ralpha(+)alpha(4)beta(7)(+) population migrates to the intestine, where it induces the Peyer's patch anlagen. When stimulated with IL-15 or IL-3 and TNF, the intestinal IL-7Ralpha(+)alpha(4)beta(7)(+) population can differentiate into fully competent NK1.1(+) NK cells or CD11c(+) APCs. Expression of alpha(4)beta(7) is lost during differentiation of both lineages; IL-7Ralpha expression is lost during NK1.1(+) cells differentiation. A newly discovered lineage(-)IL-7Ralpha(+)c-Kit(+)alpha(4)beta(7)(+) population in the fetal liver is committed to T, NK, dendritic, and fetal intestinal LT producer lineage, the latter being an intermediate stage during differentiation of NK and dendritic cells.     

不同麻醉方法对肝癌手术患者炎性细胞因子基因表达的影响

目的:探讨不同麻醉方法对肝癌手术患者外周血炎性细胞因子基因表达的影响。方法:选择肝癌手术患者48例,随机分为单纯全麻和全麻复合硬膜外麻醉两组,在麻醉前和麻醉后4h抽取静脉血,Trizol法抽提RNA,RT-PCR检测IL-1β、IL-6、IL-8的TNF-α的mRNA表达水平。结果:麻醉前两组患者间外周血炎性细胞因子基因表达无差别,麻醉4h后全麻组外周血IL-1β、IL-6、IL-8的mRNA表达水平显著升高(P<0.05),并高于联合组(P<0.05)。结论:不同麻醉方法对细胞因子的分泌会产生不同的影响,单纯全麻将增强肝癌手术病人外周血炎性细胞因子基因的表达。     

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.     

LysGH15 reduces the inflammation caused by lethal methicillin-resistant Staphylococcus aureus infection in mice

The endolysin LysGH15, derived from staphylococcal phage GH15, has a wide lytic spectrum and strong lytic activity against Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), in vitro and in vivo. Here, the ability of lethal MRSA to induce mRNA levels of interleukin-6 (IL-6), interleukin-4 (IL-4), and interferon-γ (IFN-γ) in spleen tissues of mice was studied. A large number of bacteria were detected in spleens. The bacteria caused elevated expression levels of these three cytokines. Administration of LysGH15 significantly reduced the number of bacteria and the levels of IL-6, IL-4, and IFN-γ mRNA in spleen cells compared with those in untreated mice at 24 h (P < 0.05). LysGH15 can eliminate a large number of bacteria and effectively alleviate inflammation induced by infection with lethal MRSA.     

Effect of azacytidine in the release of leukemia inhibitory factor,oncostatin m,interleukin (IL)-6, and IL-11 by mononuclear cells of patients with refractory anemia

5-azacytidine (AZA) yields hematologic improvement in patients with myelodysplastic syndromes (MDS). Ineffective hemopoiesis in MDS produce the paradox of high intramedullary cellularity with peripheral cytopenias. Leukemia inhibitory factor (LIF), oncostatin M (OSM), interleukin (IL)-6, and IL-11 regulate hemopoiesis and LIF, OSM, and IL-6 also inhibit the proliferation of myeloid leukemic cell lines through the signal-transducing subunit gp130. These IL-6-type cytokines were measured by enzyme-linked immunosorbent assay in cell culture supernatants (SN) obtained from peripheral blood mononuclear cells (MNC) and monocyte-depleted MNC of patients with refractory anemia (RA; n=12) and healthy individuals (n=10). AZA down-regulated OSM, IL-6, and IL-11 release by MNC of patients but not by MNC from healthy individuals. Patient's SN had significantly lower concentrations of LIF, OSM, and IL-11 than SN of normal subjects. When monocyte-depleted MNC of patients were stimulated with phytohemagglutinin a significant increment in OSM levels was observed. In contrast, monocyte depletion in healthy subjects did not cause any significant change in OSM values. We conclude that: (a) AZA inhibits the release of OSM, IL-6, and IL-11 exclusively in RA-diseased MNC, (b) Patient's MNC release subnormal amounts of LIF, OSM, and IL-11, and (c) RA-derived monocytes probably down-regulate OSM release by phytohemagglutinin-activated MNC.     

Diminished cytokines gene expression in lymphoid organs of healthy aged rats

Immunuosenescence-related dysregulation of cytokine production is not fully understood and the roles of cytokines in organ aging have been insufficiently studied. This work aimed to compare the expression of interleukin-2 (IL-2), IL-4, IL-6, interferon-gamma (IFNγ), and transforming growth factor-beta (TGFβ) in lymphoid organs of young (3 months; n = 10), adult (1 year; n = 10), and aged (2 years; n = 7) rats under healthy, steady-state conditions. Cytokine mRNA levels were determined with TaqMan real-time PCR. In the spleen, all cytokine expression gradually declined with age (for representative cytokine IL-2 averages dCt in spleens of 3 months, 1 year and 2 years rats were 13,645, 13,19 and 15,470, respectively). In lymph nodes, all cytokines except TGF-β showed markedly upregulated expression in adult compared to young rats, but all were expressed at very low levels in aged rats. In bone marrow, adult animals showed enhanced IL-2, IFNγ, and TGFβ expression, but similar IL-4 and IL-6 expression, compared to young rats. Bone marrow of aged rats showed very low expression of all the measured cytokines. Our results demonstrated that changes occurred unevenly with aging in different immune system compartments.     

Interleukin-13 and IgE production in rat experimental schistosomiasis

We have previously demonstrated in rat experimental schistosomiasis an upregulation of IL-4 expression at the mRNA and protein levels which could explain, at least in part, the increased IgE production observed during infection. Using this model, we have investigated the expression of IL-13 which is also involved in the induction of the IgE response. In the present study, we have shown a significant increase in IL-13 mRNA expression in spleen, liver and lungs following primary and secondary infection. IL-13 protein was detected by intracellular staining in spleen cells from infected rats, and in the supernatants of antigen-stimulated spleen cells. Furthermore, circulating levels of IL-13 were increased in sera from infected rats as compared to those from non-infected control animals. These findings show that, similarly to IL-4, IL-13 is upregulated and secreted during rat schistosomiasis, suggesting an involvement of both cytokines in IgE induction. In the in vivo experiments, only rats cotreated with neutralizing anti-IL-4 and anti-IL-13 antibodies showed significant decrease in the IgE levels. Moreover, administration of IL-13 enhanced total IgE levels. These results demonstrate the implication of IL-4 and IL-13 in vivo in IgE production, and provide a relevant animal model for a better understanding of the role of IL-4 and IL-13 in humans.     

Interleukin 5 and interleukin 4 produced by Peyer's patch T cells selectively enhance immunoglobulin A expression

Considerable evidence suggests that the high frequency of B cells committed to the IgA isotype in Peyer's patches is regulated by T lymphocytes. To understand more accurately the mechanism of this immunoregulation, an autoreactive T cell line from Peyer's patches was generated by culturing L3T4+ Peyer's patches T cells with syngeneic B cell blasts. The resulting T cell line, designated PT-1, and a clone derived from this line, PT-1.14, stimulated immunoglobulin secretion in spleen B cells with a preferential enhancement of IgA and IgG1 isotypes. Supernatant derived from concanavalin A-stimulated PT-1 or PT-1.14 cells could also enhance IgA secretion if spleen B cells were preactivated with lipopolysaccharide. Peyer's patches T cell supernatant did not contain IgA-specific binding factors. PT-1 supernatant scored positive in lymphokine assays for interleukin (IL)-2, IL-4 (B cell stimulatory factor 1), IL-5 (B cell growth factor II), and interferon-gamma, whereas PT-1.14 supernatant was positive for IL-4 and IL-5 and negative for IL-2 and interferon-gamma. Only IL-5 enhanced IgA secretion in lipopolysaccharide-activated B cells and this response was increased two- to three-fold by IL-4. These results suggest that the type 2 T helper subset which produces both IL-5 and IL-4 plays a primary role in regulating IgA expression.     

IFN-gamma production from liver mononuclear cells of mice in burn injury as well as in postburn bacterial infection models and the therapeutic effect of IL-18

Hosts after severe burn injury are known to have a defect in the Th1 immune response and are susceptible to bacterial infections. We herein show that liver NK cells are potent IFN-gamma producers early after burn injury. However, when mice were injected with LPS 24 h after burn injury, IFN-gamma production from liver mononuclear cells (MNC; which we previously showed to be NK cells) was suppressed, and the serum IFN-gamma concentration did not increase, while serum IL-10 conversely increased compared with control mice. Interestingly, a single injection of IL-18 simultaneously with LPS greatly restored the serum IFN-gamma concentration in mice with burn injury and also increased IFN-gamma production from liver MNC. Nevertheless, a single IL-18 injection into mice simultaneously with LPS was no longer effective in the restoration of serum IFN-gamma and IFN-gamma production from the liver MNC at 7 days after burn injury, when mice were considered to be the most immunocompromised. However, IL-18 injections into mice on alternate days beginning 1 day after burn injury strongly up-regulated LPS-induced serum IFN-gamma levels and IFN-gamma production from liver and spleen MNC of mice 7 days after burn injury and down-regulated serum IL-10. Furthermore, similar IL-18 therapy up-regulated serum IFN-gamma levels in mice with experimental bacterial peritonitis 7 days after burn injury and greatly decreased mouse mortality. Thus, IL-18 therapy restores the Th1 response and may decrease the susceptibility to bacterial infection in mice with burn injury.     

Functional characteristics of Peyer's patch lymphoid cells. IV. Effect of antigen feeding on the frequency of antigen-specific B cells.

The frequency of B cells in Peyer's patches from normal BDF(1) and sheep red blood cell (SRBC)-fed BDF(1) mice that could respond to antigenic determinants on SRBC and trinitrophenyl (TNP) was determined using an in vitro system of limiting dilution analysis. In normal mice, one B cell in 1.9 x 10(4) Peyer's patch cells could be induced to an anti-SRBC response and one B cell in 3.6 x 10(4) Peyer's patch cells could be induced to an anti-TNP response. The frequency of B cells capable of responding to SRBC in normal mice was similar in Peyer's patches and spleen. However, after feeding mice SRBC for 3 weeks, there was a 6-fold reduction in the frequency of B cells in Peyer's patches capable of responding to SRBC but no change in the frequency of B cells capable of responding to TNP. The average clone size of Peyer's patch B cells responding to SRBC was similar in normal and SRBC-fed mice. Although antigen-feeding does not stimulate Peyer's patch B cells in situ to humoral antibody synthesis, antigen-feeding can markedly alter the reactivity of the antigen-sensitive cell population in Peyer's patches. We previously demonstrated that T cells in Peyer's patches could be specifically carrier primed for helper function by SRBC feeding. We have now demonstrated that antigen-feeding reduced significantly the frequency of B cells in Peyer's patches capable of responding to the fed antigen. Peyer's patches appear to serve an important function as a sampling site for intestinal antigens.