Antigen-induced Cytokine Research Articles

Down-modulation of antigen-induced activation of murine cultured mast cells sensitized with a highly cytokinergic IgE clone

Accumulating evidence suggests that several IgE clones can activate mast cells during the sensitization phase even in the absence of antigen. They were found to induce pro-inflammatory cytokine release, histamine synthesis, chemotaxis, adhesion, and accelerated maturation of mast cells, although it remains unknown whether antigen-induced responses can be affected by differences of IgE clones. We compared two IgE clones, which were different in the capacity to activate mast cells during sensitization, in terms of potentials to affect antigen-induced degranulation and cytokine releases using IL-3-dependent murine bone marrow-derived cultured mast cells (BMMCs). Antigen-induced degranulation and pro-inflammatory cytokine release were augmented, when BMMCs were sensitized with elevated concentrations of a clone IgE-3, which did not induce phosphorylation of JNK and cytokine release in the absence of antigen, whereas those were significantly rather decreased, when BMMCs were sensitized with elevated concentrations of a clone SPE-7, one of the most potent cytokinergic IgE clones, which intensively induced phosphorylation of JNK. This attenuated response with SPE-7 was accompanied by decreased tyrosine phosphorylation of the cellular proteins including Syk upon antigen stimulation. SP600125, which is known to inhibit JNK, restored the levels of antigen-induced degranulation and phosphorylation of Syk in BMMCs sensitized with higher concentrations of a clone SPE-7 when it was added before sensitization. Treatment with anisomycin, a potent activator of JNK, before IgE sensitization significantly suppressed antigen-induced degranulation. These findings suggest that differences of sensitizing IgE clones can affect antigen-induced responses and activation of JNK during sensitization might suppress antigen-induced activation of mast cells.

Absolute quantification of murine interleukine-4, interleukine-10 and interferon-γ gene transcripts using real time PCR.

The study of cytokines gene expression is quite important in various conditions of health and disease for the evaluation of clinical responses to new vaccination approaches. An absolute quantification is based on a calibration curve and production of standard controls to achieve more reliable results than in relative system. In this study we attempted to construct standard controls to evaluate the murine immune response. The number of 10 Balb/c mice immunized with hydatid cyst fluid subcutaneously with two week intervals to induce transcription of Th1 and Th2 related cytokines. Three Pairs of primers were designed to amplification of interleukine-4, interleukine-10 and interferon-γ by Oligo software. Partial sequences of three cytokine genes were cloned into pTZ57T vector. Three recombinant plasmids were purified and serial dilutions were prepared. Real time qPCR carried out using SYBR-Green I fluorescence dye and standard curves were provided by the 7500 ABI SDS software based on the exact concentration ofdilutions and the amplification plots. Results showed that this method was ableto evaluate the cytokines mRNA levels less than 0.01pg (~150 copy). We concluded that absolute real-time qPCR can be successfully applied to the quantification of antigen-induced cytokines.

HISPOLON DIFFERENTIALLY MODULATED THE PRODUCTION OF ANTIGEN-INDUCED T CELL CYTOKINES VIA THE REGULATION OF CELLULAR GLUTATHIONE

Hispolon is an active ingredient contained in the medicinal mushroom Phellinus linteus (PL) that is used in traditional Chinese medicine for various remedies, including lymphatic diseases. Previous studies reported that hispolon exhibited anti-inflammatory activities and suppressed mitogen-induced proliferation of splenic lymphocytes. It remains unclear if hispolon influences antigen-specific immunity. The present study investigated the effects of hispolon on cytokine production by antigen-activated T cells. Ovalbumin (OVA)-primed splenocytes were exposed to hispolon, followed by restimulation with OVA. Cell viability was determined by the AlamarBlue® assay and T cell cytokine production was measured by enzyme linked immunosorbent assay (ELISA). The splenocyte viability and the production of interleukin (IL)-4 were unaffected, whereas the production of interferon (IFN)-γ was significantly suppressed by treatment with hispolon (1–5 μM) in a concentration-dependent manner. The suppressive effect of hispolon on the production of IFN-γ was attenuated in the presence of thiol antioxidants, including N-acetyl-L-cysteine (NAC) and glutathione, whereas the non-thiol antioxidant pyruvate was ineffective. Taken together, these results demonstrated that hispolon induced a differential effect on antigen-induced cytokine production by T cells, in which the T helper 1 (Th1) cytokine IFN-γ was sensitive, whereas the Th2 cytokine IL-4 was unaffected. In addition, the suppressive effect of hispolon on IFN-γ production was associated with the diminishment of intracellular glutathione.

The negative regulatory role of ORMDL3 in antigen-induced cytokine production, cyclooxygenase-2 expression, and chemotactic response in mast cells (IRM11P.637)

Abstract Single-nucleotide polymorphism studies have linked the chromosome 17q12-q21 region, where human ORMDL3 gene is localized, to the risk of asthma and other inflammatory diseases. Although mast cells are involved in development of these diseases, the contribution of ORMDL3 to mast cell physiology is unknown. In this study we examined the role of ORMDL3 in mouse bone marrow-derived mast cells with reduced or enhanced ORMDL3 expression. Our data show that in antigen-activated mast cells, reduced expression of the ORMDL3 protein had no effect on degranulation, calcium response, and sarco/endoplasmic reticulum Ca2+ ATPase 2b expression, but significantly enhanced phosphorylation of the IκBα and translocation of the NF-κB p65 subunit into the nucleus. These events were followed by an increased expression of pro-inflammatory cytokines (TNF-α, IL-6, and IL-13), chemokines (CCL3 and CCL4), and cyclooxygenase-2, an enzyme involved in the synthesis of prostanoids. Antigen-mediated chemotaxis was also enhanced in ORMDL3-deficient cells, whereas adhesion and spreading on fibronectin were decreased. On the other hand, increased expression of ORMDL3 had no significant effect on mast cell signaling events, except for reduced antigen-mediated chemotaxis. Our data provide the evidence that ORMDL3 in mast cells acts as a non-inducible suppressor of NF-kB-directed signaling pathways and chemotaxis.

Constitutive and Antigen‐Induced Secretion of Cytokines by Peripheral Blood Mononuclear Cells of Tuberculosis Patients

Cytokines play a major role in protection against and pathogenesis of tuberculosis. Generally, T‐helper (Th)1 and pro‐inflammatory cytokines are considered protective, and anti‐inflammatory and Th2 cytokines are responsible for pathogenesis of tuberculosis. In order to study the release of various cytokines in response to a variety of antigenic stimuli, the in vitro secretion of cytokines was assessed using peripheral blood mononuclear cells from tuberculosis patients (diabetic and non‐diabetic) and healthy subjects in response to various mycobacterial antigens, including whole cells, culture filtrate, cell walls and pools of synthetic peptides corresponding to M. tuberculosis‐specific genomic regions, i.e. the regions of differences (RDs). The results showed that the concentrations of antigen‐induced Th2 cytokines were low/undetected and the pro‐inflammatory cytokines were non‐discriminatory. However, with regard to the Th1 and anti‐inflammatory cytokines, the antigens could be divided into two groups; the first group showed Th1‐bias (culture filtrate, RD1, RD5, RD7, RD9, RD10 and RD15), and the second group had anti‐inflammatory‐bias (whole bacilli and cell walls of M. tuberculosis, RD12 and RD13). Interestingly, the RD1 peptide pool induced the strongest Th1‐bias, and the addition of RD12 and RD13 peptides to the cultures suppressed the secretion of RD1‐induced Th1‐cytokines. Furthermore, when data were compared between diabetic and non‐diabetic TB patients, there was a shift towards pathologic (Th2/anti‐inflammatory) cytokines in diabetic TB patients, which may explain, at least in part, a faster deterioration in clinical condition of diabetic TB patients.Supported by Kuwait University Research Sector grants MI01/10 and SRUL02/13.

Regulation of FcϵRI Signaling in Mast Cells by G Protein-coupled Receptor Kinase 2 and Its RH Domain

Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) by GPCRkinases (GRKs) promotes their desensitization and internalization. Here, we sought to determine the role of GRK2 on Fc∈RI signaling and mediator release in mast cells. The strategies utilized included lentiviral shRNA-mediated GRK2 knockdown, GRK2 gene deletion (GRK2(flox/flox)/cre recombinase) and overexpression of GRK2 and its regulator of G protein signaling homology (RH) domain (GRK2-RH). We found that silencing GRK2 expression caused ~50% decrease in antigen-induced Ca(2+) mobilization and degranulation but resulted in ablation of cytokine (IL-6 and IL-13) generation. The effect of GRK2 on cytokine generation does not require its catalytic activity but is mediated via the phosphorylation of p38 and Akt. Overexpression of GRK2 or its RH domain (GRK2-RH) enhanced antigen-induced mast cell degranulation and cytokine generation without affecting the expression levels of any of the Fc∈RI subunits (α, β, and γ). GRK2 or GRK2-RH had no effect on antigen-induced phosphorylation of Fc∈RIγ or Src but enhanced tyrosine phosphorylation of Syk. These data demonstrate that GRK2 modulates Fc∈RI signaling in mast cells via at least two mechanisms.One involves GRK2-RH and modulates tyrosine phosphorylation of Syk, and the other is mediated via the phosphorylation of p38 and Akt.

Interferon alpha inhibits antigen-specific production of proinflammatory cytokines and enhances antigen-specific transforming growth factor beta production in antigen-induced arthritis

IntroductionInterferon alpha (IFN-α) has a complex role in autoimmunity, in that it may both enhance and prevent inflammation. We have previously shown that the presence of IFN-α at sensitization protects against subsequent antigen-triggered arthritis. To understand this tolerogenic mechanism, we performed a descriptive, hypothesis-generating study of cellular and humoral responses associated with IFN-α-mediated protection against arthritis.MethodsArthritis was evaluated at day 28 in mice given a subcutaneous injection of methylated bovine serum albumin (mBSA), together with Freund adjuvant and 0 to 5,000 U IFN-α at days 1 and 7, followed by intraarticular injection of mBSA alone at day 21. The effect of IFN-α on mBSA-specific IgG1, IgG2a, IgG2b, IgA, and IgE was evaluated by enzyme-linked immunosorbent assay (ELISA). Cytokines in circulation and in ex vivo cultures on mBSA restimulation was evaluated with ELISA and Luminex, and the identity of cytokine-producing cells by fluorescence-activated cell sorting (FACS) analysis.ResultsAdministration of IFN-α protected mice from arthritis in a dose-dependent manner but had no effect on antigen-specific antibody levels. However, IFN-α did inhibit the initial increase of IL-6, IL-10, IL-12, and TNF, and the recall response induced by intraarticular mBSA challenge of IL-1β, IL-10, IL-12, TNF, IFN-γ, and IL-17 in serum. IFN-α decreased both macrophage and CD4+ T cell-derived IFN-γ production, whereas IL-17 was decreased only in CD4+ T cells. Ex vivo, in mBSA-restimulated spleen and lymph node cell cultures, the inhibitory effect of in vivo administration of IFN-α on proinflammatory cytokine production was clearly apparent, but had a time limit. An earlier macrophage-derived, and stronger activation of the antiinflammatory cytokine transforming growth factor beta (TGF-β) was observed in IFN-α-treated animals, combined with an increase in CD4+ T cells producing TGF-β when arthritis was triggered by mBSA (day 21). Presence of IFN-α at immunizations also prevented the reduction in TGF-β production, which was induced by the intraarticular mBSA injection triggering arthritis in control animals.ConclusionsAdministration of IFN-α has a profound effect on the cellular response to mBSA plus adjuvant, but does not affect antigen-specific Ig production. By including IFN-α at immunizations, spleen and lymph node cells inhibit their repertoire of antigen-induced proinflammatory cytokines while enhancing antiinflammatory TGF-β production, first in macrophages, and later also in CD4+ T cells. On intraarticular antigen challenge, this antiinflammatory state is reenforced, manifested as inhibition of proinflammatory recall responses and preservation of TGF-β levels. This may explain why IFN-α protects against antigen-induced arthritis.

Pacific oyster-derived polysaccharides enhance antigen-specific T helper (Th)1 immunity in vitro and in vivo

Bacterial and fungal polysaccharides are well known as being immunoactive. However, evidence pertaining to the immunomodulatory activity of polysaccharides derived from animal origins is scarce. This study investigated the effects of an extract of oyster (Crassostrea gigas) polysaccharides (OPS) on antigen-specific T-cell immunity. For in vitro studies, ovalbumin (OVA)-primed splenocytes were exposed to OPS and re-stimulated with OVA in culture to induce antigen-specific responses. For in vivo studies, mice were administered with OPS prior to OVA sensitization, and the functionality of their splenocytes was examined. Direct exposure of OVA-primed splenocytes to OPS (10–500 μg/mL) resulted in a marked enhancement of the cell metabolic activity and proliferation. Exposure to OPS also augmented the expression of the T helper (Th)1 cytokine interferon (IFN)-γ, whereas the Th2 cytokine interleukin-4 was suppressed. The enhancement of antigen-specific IFN-γ expression was further demonstrated in splenocytes of mice administered with OPS (100 and 200 mg/kg). Moreover, OPS enhanced the mRNA expression of T-bet, a transcription factor governing the development of Th1 cells, by splenocytes. Taken together, these results demonstrated that OPS modulated antigen-specific T cell responses, including antigen-induced splenocyte proliferation and T-cell cytokine expression. In addition, OPS polarized the Th1/Th2 immunobalance toward the Th1-dominant direction, which may be attributed to the up-regulation of Th1 cell development.

Immunological status in patients undergoing in vitro fertilisation: responses to hormone treatment and relationship to outcome

We aimed to prospectively investigate the paternal antigen-induced cytokine secretion by peripheral blood mononuclear cells (PBMCs) in response to hormone treatment in women undergoing in vitro fertilisation (IVF) and to examine the predictive value of the cytokine secretion profile in the outcome of IVF treatment, in a pilot study. Twenty-five women were included and IVF treatment was successful for six and unsuccessful for 19 women. Blood samples were collected before IVF treatment, on four occasions during IVF and four weeks after embryo transfer. The numbers of Th1-, Th2- and Th17-associated cytokine-secreting cells and cytokine levels in cell supernatants were analysed by enzyme-linked immunospot-forming (ELISpot), enzyme-linked immune-sorbent (ELISA) or Luminex assay. None of the cytokines (IFN-γ, IL-4, IL-5, IL-10, IL-12, IL-13, IL-17, TNF and GM-CSF) had any predictive value regarding IVF outcome. The majority of the cytokines reached their peak levels at ovum pick-up, suggesting an enhancing influence of the hormonal stimulation. Pregnancy was associated with a high number of IL-4-, IL-5- and IL-13-secreting cells four weeks after ET. In conclusion, the results do not support our hypothesis of a more pronounced peripheral Th1 and Th17 deviation towards paternal antigens in infertile women with an unsuccessful IVF outcome, although this is based on a small number of observations. A larger study is required to confirm this conclusion. Higher numbers of Th2-associated cytokine-secreting cells in pregnant women four weeks after ET do corroborate the hypothesis of a Th2 deviation during pregnancy.

Reduced IFN-γ and IL-10 responses to paternal antigens during and after pregnancy in allergic women

Normal pregnancy and allergy are both characterized by a T helper (Th) 2 deviation. In the current study, we hypothesized that paternal antigen-induced cytokine responses during pregnancy would be deviated toward Th2 and an anti-inflammatory profile, and that the Th2 deviation would be more pronounced in allergic pregnant women. Blood samples were collected longitudinally on three occasions during pregnancy and two occasions post partum (pp). Of the 86 women initially included, 54 women had a normal pregnancy and completed the sampling procedures. Twelve women fulfilled the criteria for allergy (allergic symptoms and circulating immunoglobulin [Ig] E antibodies to inhalant allergens) and 20 were non-allergic (nonsensitized without symptoms). The levels of Th1- and Th2-associated cytokines and chemokines, the Th17 cytokine IL-17 and the anti-inflammatory cytokine IL-10 of the groups were compared. Paternal antigen-induced IL-4 and IL-10 responses increased between the first and the third trimester. Allergy was associated with decreased paternal antigen-induced IFN-γ and CXCL10 secretion in the nonpregnant state (one year pp) and also decreased IFN-γ/IL-4 and IFN-γ/IL-13 ratios during pregnancy. We also observed a decreased paternal antigen-induced IL-10 response in allergic compared with non-allergic women during pregnancy, along with a decreased IL-10/IL-13 ratio. In conclusion, our findings support the hypothesis of lower Th1 responses toward paternal antigens in allergic than in non-allergic women, but also indicate that allergy is associated with a lower capacity to induce anti-inflammatory IL-10 responses after paternal antigen stimulation during pregnancy.

Antigen-induced cytokine and chemokine release test for tuberculosis infection using adsorption of stimulated whole blood on filter paper and multiplex analysis

Background: In vitro stimulation of whole blood or isolated blood cells with specific antigens is used for several purposes. Immediately following incubation with antigens, samples have to be centrifuged to stop the reactions by remaining cells and the supernatant refrigerated or analysed directly to preserve the analytes of interest, which makes samples difficult to prepare outside laboratories. We have tested whether spotting whole blood on filter paper after activation can be used in one of the tests for Mycobacterium tuberculosis infection (MTI), the QuantiFERON®-TB Gold In Tube test (QFT), where the spotting technique can make it suitable for use in locations without facilities like a centrifuge and a refrigerator. Materials and methods: Samples from 22 individuals undergoing screening for MTI and 10 healthy controls were incubated, centrifuged and IFN-γ measured by Enzyme-linked immunosorbent assay (ELISA), as described in the kit insert. In parallel, activated blood was spotted on filter paper (Schleicher & Schuell) and dried. The dried blood spot samples were analysed for 21 inflammatory markers with an in-house assay based on Luminex technology. Results: Our multiplex measurements of inflammatory markers in samples from suspected MTI patients confirmed the IFN-γ findings in the QFT. IL-2, GM-CSF, IL-5, and IL-1β were also found as useful markers for MTI. We were not able to distinguish between active tuberculosis and latent MTI. Conclusion: Applying blood on filter paper after incubation makes in vitro stimulation tests feasible in locations where heat and electricity is unavailable.

T cell responses in rheumatoid arthritis patients treated with TNF antagonists (107.7)

Abstract Rheumatoid Arthritis (RA) is a chronic inflammatory disease characterized by the overproduction of pro-inflammatory cytokines such as TNFα and IL1. An effective RA treatment is to block the action of TNFα with inhibitors. Due to the central role of TNFα in the inflammatory phase of the immune response, we investigated whether diminishing the TNFα level could lead to an impaired T cell response in RA patients. We first measured a panel of 71 proteins in the serum of healthy donors, early/new RA patients without any treatment and RA patients under anti-TNF therapy. We detected higher levels of several chemokines and cytokines in RA patients. Most levels returned to near-normal values after TNF antagonist therapy, but some chemokines showed no alterations in levels either in RA subjects or after therapy. We then evaluate T cell cytokine secretion by 16-color flow cytometry. A previous in vitro study showed that proliferative responses to recall antigens were reduced in RA patients, but these responses were restored after TNFα antagonist treatment. In contrast, ex vivo analysis of antigen-induced cytokine secretion (after 10 hours) in our studies showed that normal, RA and TNF antagonist-treated patients all showed recall responses against influenza antigens.

Cannabidiol attenuates delayed-type hypersensitivity reactions via suppressing T-cell and macrophage reactivity

to investigate the effects cannabidiol (CBD) on delayed-type hypersensitivity (DTH) reactions and antigen-induced T-cell cytokine expression. DTH was induced by subcutaneous ovalbumin (OVA) challenge to the footpads of mice sensitized with OVA. Inflammatory reactions were measured by footpad swelling and histological analysis. Antigen-induced cytokine expression by OVA-primed splenocytes was measured using ELISA and RT-PCR. CBD (1-10 mg/kg) administration, in a dose-dependent fashion, significantly attenuated inflammatory reactions associated with DTH in the footpads of mice sensitized and challenged with OVA. Histological examination revealed that CBD suppressed the infiltration of T cells and macrophages, and the expression of interferon (IFN)-γ and tumor necrosis factor-α, two pro-inflammatory cytokines implicated in DTH in the inflammatory site. In contrast, the expression of interleukin (IL)-10 in the footpads was enhanced by CBD administration. In addition, CBD at concentrations devoid of cytotoxic effects (1-4 micromol/L) attenuated OVA-induced IFN-γ production by OVA-primed splenocytes, whereas IL-4 was unaffected. CBD curbs DTH reactions via suppressing the infiltration and functional activity of T cells and macrophages in the inflammatory site, suggesting a therapeutic potential for CBD for the treatment of type IV hypersensitivity.

Interleukin-17 regulation: an attractive therapeutic approach for asthma

Interleukin (IL)-17 is recognized to play a critical role in numerous immune and inflammatory responses by regulating the expression of various inflammatory mediators, which include cytokines, chemokines, and adhesion molecules. There is growing evidence that IL-17 is involved in the pathogenesis of asthma. IL-17 orchestrates the neutrophilic influx into the airways and also enhances T-helper 2 (Th2) cell-mediated eosinophilic airway inflammation in asthma. Recent studies have demonstrated that not only inhibitor of IL-17 per se but also diverse regulators of IL-17 expression reduce antigen-induced airway inflammation, bronchial hyperresponsiveness, and Th2 cytokine levels in animal models of asthma. This review will summarize the role of IL-17 in the context of allergic airway inflammation and discuss the therapeutic potential of various strategies targeting IL-17 for asthma.

Gender differences in transcriptional regulation of IL‐5 expression by bronchial lymph node cells in a mouse model of asthma

The severity of asthma after puberty is higher in women than in men. Increased numbers of eosinophils in the airways of female mice after antigen challenge was associated with increased levels of T helper (Th)2 cytokines at the site of inflammation, and in human and mouse studies, the profile of cytokines produced by immune cells from women showed greater Th2 predominance. The aim of this study was to investigate gender differences in the development of Th2 immune responses. Male and female C57BL/6 mice were sensitized with ovalbumin. Cells prepared from bronchial lymph nodes were cultured in the absence or presence of ovalbumin. Cytokine concentrations in the culture supernatants were measured, and IL-5 and GATA-binding protein 3 (GATA-3) gene expression were evaluated. T-cell subsets were analysed using specific surface markers. The concentrations of IL-4, IL-5, IL-13 and IL-10, but not interferon-gamma or transforming growth factor-beta(1), were higher in cell supernatants from female mice than in those from male mice. IL-5 and GATA-3 gene expressions were higher in cells from women than in cells from men. The numbers of CD3(+)CD4(+)T1/ST2(+) cells, but not CD3(+)CD4(+) or CD4(+)CD25(+) cells, were significantly higher in cells from women than in cells from men. Greater antigen-induced Th2 cytokine production by bronchial lymph node cells from female mice was associated with enhanced Th2 cell differentiation and increased expression of the Th2-specific transcription factor, GATA-3.

Intricate regulation of IL-17 response by IFN-γ and IL-27 determines the outcome of the pathogenetic events in experimental autoimmune arthritis (143.18)

Abstract Recent advances in defining the new subsets of T cells and the related newer cytokines have stimulated new undertakings in examining the pathogenesis of autoimmunity beyond the traditional Th1-Th2 paradigm. We report here novel findings regarding the roles of IFN-γ, IL-17 and IL-27 in the adjuvant arthritis (AA) model of human rheumatoid arthritis that challenge the traditional viewpoints. We hypothesized that the temporal kinetics and the relative levels of antigen-induced cytokine responses determined both the propagation of AA as well as resistance to the disease. We determined the cytokine responses of arthritic Lewis rats at different phases of the disease and that of the arthritis-resistant, MHC-compatible Wistar Kyoto (WKY) rats given a potentially arthritogenic challenge. In Lewis rats, IL-17 dominated the early (incubation phase) response, whereas IFN-γ and IL-27 appeared late (peak phase). Intriguingly, in WKY rats, all these 3 cytokines appeared concomitantly and early, showing that IFN-γ and IL-27 successfully pre-empted the pathogenicity of IL-17. This cross-regulation, along with the novel finding of upregulation of IL-27 by IFN-γ, was validated through mechanistic experiments, including the AA-protective effect of IFN-γ injected in vivo, and the differential modulation of specific signaling biomolecules and inflammatory mediators in the joints. These results provide a novel IL-17-IL-27-centered perspective on the pathogenesis of autoimmune arthritis.

Inhibition of degranulation and cytokine production in bone marrow-derived mast cells by hydrolyzed rice bran

We investigated the effects of hydrolyzed rice bran (HRB), an arabinoxylan extracted from rice bran, on mast cell degranulation and cytokine production. HRB was obtained by treating rice bran with an extract obtained from shiitake mushrooms. Bone marrow-derived mast cells (BMMCs) were prepared by culturing bone marrow cells from BALB/c mice in the presence of interleukin-3 and stem cell factor for 4 weeks. BMMCs were pretreated with HRB (0-3 mg/ml) for 30 min and were then antigen activated. Pretreatment of BMMCs with HRB significantly inhibited antigen-induced degranulation and cytokine production (tumor necrosis factor-alpha and interleukin-4) in a dose-dependent manner. HRB also diminished membrane fusion between liposomes in which soluble N-ethyl maleimide-sensitive factor attachment protein receptors were reconstituted. Phosphorylation of RelA and mitogen-activated kinases after antigen stimulation was suppressed by pretreatment of BMMCs with HRB. These findings suggest that HRB may have an anti-inflammatory effect by inhibiting mast cell degranulation and cytokine production.

Role of intestinalBifidobacterium pseudolongumin dietary fructo-oligosaccharide inhibition of 2,4-dinitrofluorobenzene-induced contact hypersensitivity in mice

Strategies to manipulate the gut microbiota have been explored for preventing allergy development. We previously showed that dietary supplementation with fructo-oligosaccharide (FOS) reduced 2, 4-dinitrofluorobenzene (DNFB)-induced contact hypersensitivity (CHS) in BALB/c mice. Because the CHS response was negatively correlated with the number of faecal bifidobacteria, particularly Bifidobacterium pseudolongum, the present study aimed to examine whether oral administration of B. pseudolongum affects CHS response. Viable B. pseudolongum was successfully isolated from mouse faeces. Female BALB/c mice were fed a synthetic diet with or without FOS supplementation, and B. pseudolongum (2 x 10(7) cells) was administered daily throughout the experimental period. Two weeks after starting the test diets, mice received DNFB on the ear auricle twice at 7-d intervals. Conventional cultivation and molecular biological analyses based on 16S rRNA gene sequences showed that administration of FOS and B. pseudolongum resulted in higher excretion of viable bifidobacteria, mainly B. pseudolongum. Although dietary FOS reduced the CHS response as demonstrated by ear swelling, B. pseudolongum administration resulted in a reduction in the initial phase only of the CHS response. B. pseudolongum administration increased hapten-specific IgG1, while dietary FOS decreased IgG2a in sera. Administration of FOS and B. pseudolongum decreased interferon-gamma production and increased IL-10 production in cervical lymph node cells restimulated with hapten in vitro. We conclude that B. pseudolongum proliferation in the intestinal tract is partially responsible for the reduction in DNFB-induced CHS response by dietary supplementation with FOS in mice, which may be mediated by the modulation of antigen-induced cytokine production.

Interleukin-25 Expressed by Brain Capillary Endothelial Cells Maintains Blood-Brain Barrier Function in a Protein Kinase Cϵ-dependent Manner

Interleukin (IL)-25, a member of the IL-17 family of cytokines, is expressed in the brains of normal mice. However, the cellular source of IL-25 and its function in the brain remain to be elucidated. Here, we show that IL-25 plays an important role in preventing infiltration of the inflammatory cells into the central nervous system. Brain capillary endothelial cells (BCECs) express IL-25. However, it is down-regulated by inflammatory cytokines, including tumor necrosis factor (TNF)-alpha, IL-17, interferon-gamma, IL-1beta, and IL-6 in vitro, and is also reduced in active multiple sclerosis (MS) lesions and in the inflamed spinal cord of experimental autoimmune encephalomyelitis, an animal model of MS. Furthermore, IL-25 restores the reduced expression of tight junction proteins, occludin, junction adhesion molecule, and claudin-5, induced by TNF-alpha in BCECs and consequently repairs TNF-alpha-induced blood-brain barrier (BBB) permeability. IL-25 induces protein kinase Cepsilon (PKCepsilon) phosphorylation, and up-regulation of claudin-5 is suppressed by PKCepsilon inhibitor peptide in the IL-25-stimulated BCECs. These results suggest that IL-25 is produced by BCECs and protects against inflammatory cytokine-induced excessive BBB collapse through a PKCepsilon-dependent pathway. These novel functions of IL-25 in maintaining BBB integrity may help us understand the pathophysiology of inflammatory brain diseases such as MS.

Differential LiveMycobacterium tuberculosis-,M. bovisBCG-, Recombinant ESAT6-, and Culture Filtrate Protein 10-Induced Immunity in Tuberculosis

The high prevalence of Mycobacterium tuberculosis makes it imperative that immune responses to evaluate could be predictive of infection. We investigated live Mycobacterium- and recombinant antigen-induced cytokine and chemokine responses in patients with active tuberculosis (TB) compared with those of healthy controls from an area where TB is endemic (ECs). M. tuberculosis-, M. bovis BCG-, ESAT6-, and culture filtrate protein 10 (CFP10)-induced responses were determined in peripheral blood mononuclear cells from patients with pulmonary TB (n = 38) and ECs (n = 39). The levels of the cytokines gamma interferon (IFN-gamma) and interleukin-10 (IL-10) and the chemokines CCL2, CCL3, and CXCL9 were measured. The levels of M. tuberculosis- and BCG-induced IFN-gamma secretion were significantly reduced (P = 0.002 and P < 0.01, respectively), while the amount of IL-10 induced by both virulent (P < 0.01) and avirulent (P = 0.002) mycobacteria was increased in patients with TB. The ESAT6-induced IFN-gamma responses were increased in the patients with TB (P = 0.013) compared with those in the EC group. When tuberculin skin test (TST)-negative (TST(-); induration, <10 mm) and TST-positive (TST(+)) donors were studied separately, both TST(-) and TST(+) individuals showed increased IFN-gamma responses to M. tuberculosis compared with the responses of the patients with TB (P = 0.037 and P = 0.006, respectively). However, only TST(+) ECs showed reduced IFN-gamma responses to ESAT6 (P = 0.008) compared with the responses of the patients with TB. The levels of M. tuberculosis-induced CCL2 (P = 0.006) and CXCL9 (P = 0.017) were greater in the patients with TB. The levels of CCL3 secretion in response to Mycobacterium and antigen stimulation were comparable between the two groups. While the levels of ESAT6-induced chemokines did not differ between the patients with TB and the ECs, the levels of CFP10-induced CCL2 (P = 0.01) and CXCL9 (P = 0.001) were increased in the patients. These data indicate differential host IFN-gamma, CXCL9, and CCL2 responses to live mycobacteria and mycobacterial antigens and have implications for the identification of potential biomarkers of infection which could be used for the diagnosis of TB.