Down-regulated IL-10 Production Research Articles

DNAM-1 regulates the proliferation and function of T regulatory type 1 cells via the IL-2/STAT5 pathway

To explore the role of DNAM-1 in the activation, proliferation and function of type Ⅰ regulatory T cells (Tr1 cells). Anti-CD3/CD28 antibodies were used to stimulate mouse T cells derived from the spleen of wild-type (WT) mice, and the expression level of DNAM-1 in resting and activated Tr1 cells was evaluated with flow cytometry. Na?ve CD4+ T cells isolated by magnetic cell sorting from the spleens of WT mice and DNAM-1 knockout (KO) mice were cultured in Tr1 polarizing conditions for 3 days, after which CD25 and CD69 expressions were measured using flow cytometry. The induced Tr1 cells were labelled with CFSE and cultured in the presence of anti-CD/CD28 antibodies for 3 days, and their proliferative activity was analyzed. The expressions of IL-10 and p-STAT5 in DNAM-1-deficient Tr1 cells were detected before and after IL-2 stimulation. The expression level of DNAM-1 was significantly upregulated in CD4+ T cells and Tr1 cells after stimulation with anti-CD3/CD28 antibodies (P < 0.05). DNAM-1 knockout did not cause significant changes in the number or proportion of Tr1 cells, but but significantly increased the expression levels of the activation markers CD69 and CD25 (P < 0.05). Compared with WT Tr1 cells, DNAM-1-deficient Tr1 cells exhibited reduced proliferative activity in vitro (P < 0.05) with downregulated IL-10 production (P < 0.05) and decreased expressions of Il-10 and Gzmb mRNA (P < 0.05). In DNAM-1-deficient Tr1 cells, IL-2 stimulation significantly reduced IL-10 secretion level and the expression of p-STAT5 as compared with WT Tr1 cells. DNAM-1 participate in the activation and proliferation of Tr1 cells and affect the biological functions of Tr1 cells through the IL-2/STAT5 pathway.

RNASE2 Mediates Age-Associated B Cell Expansion Through Monocyte Derived IL-10 in Patients With Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is characterized by the production of pathogenic autoantibodies. Ribonuclease A family member 2 (RNase2) is known to have antiviral activity and immunomodulatory function. Although RNASE2 level has been reported to be elevated in SLE patients based on mRNA microarray detection, its pathologic mechanism remains unclear. Here, we confirmed that RNASE2 was highly expressed in PBMCs from SLE patients and associated with the proportion of CD11c+T-bet+ B cells, a class of autoreactive B cells also known as age-associated B cells (ABCs). We showed that reduction of RNASE2 expression by small interfering RNA led to the decrease of ABCs in vitro, accompanied by total IgG and IL-10 reduction. In addition, we demonstrated that both RNASE2 and IL-10 in peripheral blood of lupus patients were mainly derived from monocytes. RNASE2 silencing in monocytes down-regulated IL-10 production and consequently reduced ABCs numbers in monocyte-B cell co-cultures, which could be restored by the addition of recombinant IL-10. Based on above findings, we concluded that RNASE2 might induce the production of ABCs via IL-10 secreted from monocytes, thus contributing to the pathogenesis of SLE.

Genome-wide siRNA screening in mouse bone marrow-derived macrophages revealed that knockdown of ribosomal proteins suppresses IL-10 and enhances TNF-α production.

Macrophages play a central role in the immune response, and their diverse functions are attributed to the spectrum of their functional states. To elucidate molecules involved in modulating the balance between the anti-inflammatory cytokine IL-10 and the pro-inflammatory cytokine TNF-α, we conducted genome-wide siRNA screening. First, we established an siRNA screening system using mouse bone marrow-derived macrophages, which are a suitable model for studying functional states of macrophages in vitro. In the primary screen and the subsequent reproducibility assay, 112 siRNA pools demonstrated enhancement of IL-10 production and 497 siRNA pools suppressed IL-10 production. After a deconvolution assay for IL-10-up-regulating siRNA pools, 8 genes were identified as IL-10 repressors, including Cnot1 and Rc3h1, components of the CCR4-NOT complex known to degrade cytokine mRNAs. On the other hand, siRNA pools targeting ribosomal proteins were frequently found among those that down-regulated IL-10 production and up-regulated TNF-α production. Four pools were assayed using deconvoluted siRNAs and identified as high-confidence hits. Thus, we found that the genome-wide knockdown of 19 ribosomal proteins resulted in decreased IL-10 and increased TNF-α production.

Antimicrobial and immunomodulatory activity of host defense peptides, clavanins and LL-37, in vitro: An endodontic perspective

Endodontic treatment is mainly based on root canal disinfection and its failure may be motivated by microbial resistance. Endodontic therapy can be benefitted by host defense peptides (HDPs), which are multifunctional molecules that act against persistent infection and inflammation. This study aimed to evaluate the antimicrobial, cytotoxic and immunomodulatory activity of several HDPs, namely clavanin A, clavanin A modified (MO) and LL-37, compared to intracanal medication Ca(OH)2. HDPs and Ca(OH)2 were evaluated by: (1) antimicrobial assays against Candida albicans and Enterococcus faecalis, (2) cytotoxicity assays and (3) cytokine tumor necrosis factor (TNF)-α, monocyte chemoattractant protein (MCP)-1, interleukin (IL)-1α, IL-6, IL-10 and IL-12 and nitric oxide (NO) production by RAW 264.7 cells incubated with or without heat-killed (HK) C. albicans or E. faecalis combined or not with interferon-γ. The minimum inhibitory concentration (MIC) was established only for E. faecalis (LL-37, 57μM). Considering cytotoxicity, clavanin MO was able to reduce cell viability in many groups and demonstrated lowest LC50. The Ca(OH)2 up-regulated the production of MCP-1, TNF-α, IL-12 and IL-6 and down-regulated IL-1α, IL-10 and NO. Clavanins up-regulated the TNF-α and NO and down-regulated IL-10 production. LL-37 demonstrated up-regulation of IL-6 and TNF-α production and down-regulation in IL-10 and NO production. In conclusion, LL-37 demonstrated better antibacterial potential. In addition, Ca(OH)2 demonstrated a proinflammatory response, while the HDPs modulated the inflammatory response from non-interference with the active cytokines in the osteoclastogenesis process, probably promoting the health of periradicular tissues.

Direct effects of interleukin-8 on growth and functional activity of T lymphocytes

CD3+ T-lymphocytes were isolated from the normal donors by positive magnetic separation. Activation of the T cells with particles conjugated with antibodies to CD3, СD28 and СD2 molecules led to a marked increase in T-cell production of interleukine-8 (IL-8). We present evidence that IL-8 receptor α-chain (CXCR1, CD181) is expressed on the cell surface of 13.3% T cells. Activation of T-lymphocytes resulted in significant enhancement of CD181+ cells both in naive CD4+ T cell and terminally differentiated effector CD4+ T cell compartments with concomitant reduction of CD181+ cells in effector memory CD4+ T cell subset. The level of T cell activation was assessed judging from the surface expression of CD25 (IL-2 receptor α-chain). We demonstrate that IL-8 treatment (0.01–10.0ng/ml concentration range) reduced the activation status of both CD4− and CD4+ effector memory T cells, as well as terminally differentiated effector T cells, without significantly affecting the activation of naive T cells or central memory T cells. In addition, IL-8 up-regulated IL-2 and down-regulated IL-10 production by activated T cells, with no effect on interferon-gamma (IFN-γ) and IL-4 production. Data obtained suggests the importance of IL-8 in the direct regulation of adaptive T cell reactivity.

Dysregulation of peritoneal cavity B1a cells and murine primary biliary cholangitis.

Primary biliary cholangitis (PBC) is a chronic autoimmune liver disease with progressive cholestasis and liver fibrosis. Similar to human patients with PBC, p40−/−IL-2Rα−/− mice spontaneously develop severe autoimmune cholangitis. Although there has been considerable work on immune regulation and autoimmunity, there is a relative paucity of work directed at the functional implications of the key peritoneal cavity (PC) B cell subset, coined B1a cells in PBC. We used flow cytometry and high-resolution microarrays to study the qualitative and quantitative characteristics of B cells, particularly B1a cells, in the PC of p40−/−IL-2Rα−/− mice compared to controls. Importantly, B1a cell proliferation was markedly lower as the expression of Ki67 decreased. Meanwhile, the apoptosis level was much higher. These lead to a reduction of B1a cells in the PC of p40−/−IL-2Rα−/− mice compared to controls. In contrast, there was a dramatic increase of CD4+ and CD8+ T cells accompanied by elevated production of IFN-γ. In addition, we found a negative correlation between the frequency of B1a cells and the presence of autoreactive CD8+ T cells in both liver and PC of p40−/−IL-2Rα−/− mice. From a functional perspective, B cells from p40−/−IL-2Rα−/− mice downregulated IL-10 production and CTLA-4 expression, leading to loss of B cell regulatory function. We suggest that the dysfunction of B1a cells in the PC in this murine model of autoimmune cholangitis results in defective regulatory function. This highlights a new potential therapeutic target in PBC.

ID: 100: Regulation and B-cell help mediated by distinct subsets of IL10-producing T-cells

IL10 is a B-cell growth factor produced by CD4 + helper T-cells, but it also mediates suppression of T-cell responses by regulatory T-cells. We found that IL10-producing regulatory T-cells (Tr1) and IL10-dependent B-helper T-cells are distinct subsets in humans and mice. Tr1 cells that suppressed T cell proliferation and experimental colitis co-expressed CCR5 and PD1, but not CCR6 or IL-7R. They down-regulated IL-10 production in response to pro-inflammatory cytokines, and consequently produced only low levels of IL-10 in the intestine of IBD patients. Tr1 cells failed to up-regulate CD40L and did not promote B-cell responses, but conversely suppressed IgG production. However, Tr1 cells from lupus patients were unable to inhibit B cell responses. In marked contrast, IL10-producing IL-7R + CCR6 + helper T-cells expressed CD40L and induced B-cell antibody production via IL10. Notably, these IL10-dependent B-helper T-cells were distinct from conventional TFH and Th17-cells, were auto-reactive and consequently provided spontaneous B cell help in lupus patients. In summary, we defined a strategy to distinguish IL10 producing T-cell subsets with opposing functions in B-cell responses and in autoimmune diseases.

IL-12p40, IL-23 and IL-10 production from monocytes of patients with colorectal cancer: effect of inhibition of JNK signaling pathway

Event Abstract Back to Event IL-12p40, IL-23 and IL-10 production from monocytes of patients with colorectal cancer: effect of inhibition of JNK signaling pathway Noyko S. Stanilov1*, Zlatka G. Dobreva2 and Spaska A. Stanilova2 1 Trakia University, Medical faculty, Department of Neurosurgery, Surgery and Urology, Bulgaria 2 Trakia University, Medical Faculty, Department of Molecular Biology, Immunology and Medical genetics, Bulgaria Cytokine production in patients with colorectal cancer (CRC) has an important impact on CRC development. We investigated changes of IL-12p40, IL-23 and IL-10 production from monocytes of CRC patients depending on cancer stages. Isolated monocytes were stimulated with LPS or C3bgp. We also explored molecular regulatory mechanisms focusing on the JNK activation by using selective inhibitor SP600125. The quantity determination of IL-12p40, IL-23 and IL-10 was performed by ELISA. IL-12p40 production from patients' monocytes was diminished in early and advanced stages of the disease. Similarly, independent of disease stage, patients’ monocytes secreted significantly lower IL-23 quantity compared to monocytes from healthy individuals. We observed significantly decreased IL-10 production from monocytes of CRC patients in early stage, but not in monocytes from patients with advanced CRC. Moreover, patients in early stages showed significant lower IL-10 in comparison with advanced stages of CRC. Inhibition of JNK is clearly upregulating IL-12p40 and IL-23 production from patients’ monocytes, unlike healthy donors where inhibition of this kinase is slightly reduced IL-12p40 and IL-23 production is significantly downregulated. Regarding IL-10, the inhibition of JNK did not result in significant changes of its production, while the inhibition of JNK in monocytes from healthy donors led to downregulated IL-10 production. In conclusion, this study showed altered functional activity of peripheral blood monocytes of CRC patients which are demonstrated through downregulation of IL-12p40 and IL-23, but not of IL-10. This different response of patients’ monocytes is mediated partly by changes in involvement of JNK signaling pathway. Keywords: colorectal cancer, Monocytes, IL-10, IL-12p40, IL-23 Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Immune-mediated disease pathogenesis Citation: Stanilov NS, Dobreva ZG and Stanilova SA (2013). IL-12p40, IL-23 and IL-10 production from monocytes of patients with colorectal cancer: effect of inhibition of JNK signaling pathway. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00298 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 12 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Noyko S Stanilov, Trakia University, Medical faculty, Department of Neurosurgery, Surgery and Urology, Stara Zagora, Bulgaria, noyko.stanilov@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Noyko S Stanilov Zlatka G Dobreva Spaska A Stanilova Google Noyko S Stanilov Zlatka G Dobreva Spaska A Stanilova Google Scholar Noyko S Stanilov Zlatka G Dobreva Spaska A Stanilova PubMed Noyko S Stanilov Zlatka G Dobreva Spaska A Stanilova Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Abstract 4840: Blockade of IL-10 production in effector B cells significantly increases their therapeutic efficacy in cancer adoptive immunotherapy

Abstract We have previously reported the antitumor reactivity of adoptively transferred effector B cells and the mechanisms by which they mediate tumor regression in a spontaneous metastasis model. 4T1 breast cancer cells were inoculated into syngeneic Balb/c mice to prime tumor-draining lymph node (TDLN). TDLNs were subsequently harvested and B cells activated ex vivo. When adoptively transferred into mice inoculated with 4T1 tumor in the mammary fat pad, these activated B cells alone mediated the inhibition of spontaneous metastases to the lung. In this study, we used IL-10−/− Balb/c mice to generate IL-10−/− TDLN, and evaluated the antitumor immunity of IL-10−/− TDLN B cells. Adoptively transferred IL-10−/− TDLN B cells mediated significantly more effective antitumor immunity than equal numbers of WT TDLN B cells (p&amp;lt;0.05). Activated IL-10−/− effector 4T1 TDLN B cells were capable of killing 4T1 tumor cells in a tumor antigen specific manner in in vitro cytotoxicity assays independent of antibody and complement. Adoptive transfer of IL-10−/− TDLN B cells resulted in the induction of host T and B cell antitumor immunity significantly more than adoptive transfer of WT TDLN B cells (p&amp;lt;0.05). This was evident by significantly modulated cytokine production (e.g. up-regulated IFN-gamma and IL-2 production by host T cells, but down-regulated IL-10 production by host B cells). In addition, adoptively transferred IL-10−/− TDLN B cells increased the production of IgG which bound to 4T1 tumor cells. Furthermore, IL-10−/− TDLN B cell infusion significantly increased tumor cell lysis (CTL) activity mediated by host B cells as well as T cells in an immunologically specific fashion. The molecular and cellular pathways involved in the direct killing of 4T1 tumor cells by IL-10−/- 4T1 TDLN effector B cells and by host B cells subjected to IL-10−/- 4T1 TDLN effector B cell adoptive transfer is under active investigation in our lab. While the role played by B cells in the host immune response to cancer is complex and controversial, our results indicate that removal of IL-10-producing B cell subsets may represent an effective strategy to augment the therapeutic efficacy of TDLN B cells used in adoptive immunotherapy. Mechanistically, adoptive transfer of B cells depleted of IL-10-producing subsets destroys tumor cells directly and confers greater host cellular and humoral antitumor immunity by shifting cytokine production to a Type 1 profile and by producing tumor-reactive antibodies respectively. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4840. doi:1538-7445.AM2012-4840

Exogenous HIV-1 Nef Upsets the IFN-γ-Induced Impairment of Human Intestinal Epithelial Integrity

BackgroundThe mucosal tissues play a central role in the transmission of HIV-1 infection as well as in the pathogenesis of AIDS. Despite several clinical studies reported intestinal dysfunction during HIV infection, the mechanisms underlying HIV-induced impairments of mucosal epithelial barrier are still unclear. It has been postulated that HIV-1 alters enterocytic function and HIV-1 proteins have been detected in several cell types of the intestinal mucosa. In the present study, we analyzed the effect of the accessory HIV-1 Nef protein on human epithelial cell line.Methodology/Principal FindingsWe used unstimulated or IFN-γ-stimulated Caco-2 cells, as a model for homeostatic and inflamed gastrointestinal tracts, respectively. We investigated the effect of exogenous recombinant Nef on monolayer integrity analyzing its uptake, transepithelial electrical resistance, permeability to FITC-dextran and the expression of tight junction proteins. Moreover, we measured the induction of proinflammatory mediators. Exogenous Nef was taken up by Caco-2 cells, increased intestinal epithelial permeability and upset the IFN-γ-induced reduction of transepitelial resistance, interfering with tight junction protein expression. Moreover, Nef inhibited IFN-γ-induced apoptosis and up-regulated TNF-α, IL-6 and MIP-3α production by Caco-2 cells while down-regulated IL-10 production. The simultaneous exposure of Caco-2 cells to Nef and IFN-γ did not affect cytokine secretion respect to untreated cells. Finally, we found that Nef counteracted the IFN-γ induced arachidonic acid cascade.Conclusion/SignificanceOur findings suggest that exogenous Nef, perturbing the IFN-γ-induced impairment of intestinal epithelial cells, could prolong cell survival, thus allowing for accumulation of viral particles. Our results may improve the understanding of AIDS pathogenesis, supporting the discovery of new therapeutic interventions.

CD30 Ligand Is a Target for a Novel Biological Therapy against Colitis Associated with Th17 Responses

We have previously found that CD30 ligand (CD30L; CD153)/CD30 signaling executed by the T-T cell interaction plays a critical role in Th17 cell differentiation, at least partly via downregulation of IL-2 production. In this study, we investigated the role of CD30L in the development of colitis experimentally induced by dextran sulfate sodium (DSS), in which IL-17A is involved in the pathogenesis. CD30L(-/-) mice were resistant to both acute colitis induced by administration of 3 to ∼ 5% DSS and to chronic colitis induced by administration of 1.5% DSS on days 0-5, 10-15, and 20-25 as assessed by weight loss, survival rate, and histopathology. The levels of IFN-γ, IL-17A, and IL-10 were significantly lower but the IL-2 level higher in the lamina propria T lymphocytes of CD30L(-/-) mice than those in lamina propria T lymphocytes of wild-type mice after DSS administration. Soluble murine CD30-Ig fusion protein, which was capable of inhibiting Th17 cell differentiation in vitro, ameliorated both types of DSS-induced colitis in wild-type mice. Modulation of CD30L/CD30 signaling by soluble CD30 could be a novel biological therapy for inflammatory diseases associated with Th17 responses.

Central role of extracellular signal-regulated kinase and Toll-like receptor 4 in IL-10 production in regulatory dendritic cells induced by Trypanosoma cruzi

Several Trypanosoma cruzi molecules that stimulate macrophages activity were described as Toll-like receptor 2 (TLR2) ligands. Besides, the models of dendritic cells (DC) are poorly characterised. We have previously demonstrated that live-trypomastigotes (Tp) plus lipopolysaccharide (LPS) induce DC with tolerogenic properties that produce high levels of interleukin (IL)-10 and an impaired capacity to induce lymphoproliferation. Here, we show that the regulatory phenotype was observed with heat-killed trypomastigotes (Tphk) stimulation, ruling out DC infection. T. cruzi induced a particular DC activation state increasing LPS-activation of extracellular regulated kinase (ERK) 1/2 and signal transducer and activator of transcription (STAT) 3. Inhibition of ERK down-regulated IL-10 production and restored DC stimulatory capacity, showing the importance of this pathway in the DC modulation. A recent work shows that signalling via TLR4 and TLR2 induces a synergism in anti-inflammatory cytokine production in murine DC. Upon TLR2 and TLR4 stimulation using Pam 3Cys or LPS and Tphk in DC from TLR2 knock out (KO) or TLR4-mutant mice, we showed that high levels of IL-10 were independent of TLR2 but associated with TLR4 and NF-κB signallization. Although sialic acid has been described as a molecule responsible of DC inhibition, we determine that it is not associated with T. cruzi-IL-10 modulatory response. In conclusion, all these findings demonstrate a key role of ERK and TLR4 in association with NF-κB in IL-10 modulation induced by T. cruzi and suggest that this regulatory effect involves parasite–DC interactions not described yet.

Inhibition of endogenous MHC class II‐restricted antigen presentation by tacrolimus (FK506) via FKBP51

The effect of tacrolimus (FK506) on down-regulation of IL-2 production by T cells is considered to be mainly responsible for its strong suppression of immunological events. In this study, we show that FK506 also has an affect on antigen presentation by antigen-presenting cells in vitro. FK506 was able to inhibit the presentation of endogenous MHC class II-restricted minor histocompatibility antigens in primary dendritic cells (DC) in vitro, but cyclosporine A (CsA) and rapamycin (RAP) were not. RNA interference (RNAi)-mediated reduction of endogenous FK506-binding protein (FKBP)51 expression resulted in a marked decrease in antigen presentation, suggesting that FKBP51 plays a role in endogenous MHC class II-restricted antigen presentation. Since our model used naturally expressed cytosolic antigens in primary DC, these effects might have been due to novel properties of the immunosuppressive drugs and may allow us to elucidate a new paradigm for the immunosuppressive mechanism of FK506.

Effects of combined therapy with thalidomide and glucantime on leishmaniasis induced by Leishmania major in BALB/c mice

For treating Leishmania major infection in BALB/c mice, we used thalidomide in conjunction with glucantime. Groups of mice were challenged with 510(3) metacyclic promastigotes of L. major subcutaneously. A week after the challenge, drug treatment was started and continued for 12 days. Thalidomide was orally administrated 30 mg/kg/day and glucantime was administrated intraperitoneally (200 mg/kg/day). It was shown that the combined therapy is more effective than single therapies with each one of the drugs since the foot pad swelling in the group of mice received thalidomide and glucantime was significantly decreased (0.9 +/- 0.2 mm) compared to mice treated with either glucantime, thalidomide, or carrier alone (1.2 +/- 0.25, 1.4 +/- 0.3, and 1.7 +/- 0.27 mm, respectively). Cytokine study showed that the effect of thalidomide was not dependent on IL-12; however, it up-regulated IFN-gamma and down-regulated IL-10 production. Conclusively, thalidomide seems promising as a conjunctive therapy with antimony in murine model of visceral leishmaniasis.

PK1/EG-VEGF induces monocyte differentiation and activation

Macrophages exist as sentinels in innate immune response and react by expressing proinflammatory cytokines and up-regulating antigen-presenting and costimulatory molecules. We report a novel function for prokineticin-1 (PK1)/endocrine gland-derived vascular endothelial growth factor. Screening of murine tissue sections and cells for specific binding site leads to the identification of macrophages as an in vivo cellular target for PK1. We demonstrate PK1 induces differentiation of murine and human bone marrow cells into the monocyte/macrophage lineage. Human peripheral blood monocytes respond to PK1 by morphological changes and down-regulation of B7-1, CD14, CC chemokine receptor 5, and CXC chemokine receptor 4. Monocytes treated with PK1 have elevated interleukin (IL)-12 and tumor necrosis factor alpha and down-regulated IL-10 production in response to lipopolysaccharide. PK1 induces a distinct monocyte-derived cell population, which is primed for release of proinflammatory cytokines that favor a T helper cell type 1 response.

Down-regulation of IL-2 production in T lymphocytes by phosphorylated protein kinase A-RIIbeta.

The beta isoform of the type II regulatory subunit (RIIbeta) of protein kinase A suppresses CREB transcriptional activity and c-Fos production in T cells following activation via the TCR. Because CREB is an integral nuclear transcription factor for IL-2 production by T cells, we tested the hypothesis that RIIbeta down-regulates IL-2 expression and IL-2 production in T cells. Stable transfection of RIIbeta in Jurkat T cells led to an approximately 90% reduction in IL-2 mRNA and IL-2 protein following T cell activation. The inhibition of IL-2 production was associated with phosphorylation of the RIIbeta subunit at serine 114 (pRIIbeta) and localization of pRIIbeta in intranuclear clusters. A serine 114 phosphorylation-defective mutant, RIIbeta(S114A), did not form these intranuclear clusters as well as wild-type RIIbeta, and did not inhibit IL-2 mRNA and protein synthesis, indicating that serine 114 phosphorylation is required for both nuclear localization and down-regulation of IL-2 production by RIIbeta. In contrast to its effect on IL-2, RIIbeta induced constitutive up-regulation of CD154 mRNA and cell surface expression. Thus, pRIIbeta differentially regulates gene expression following T cell activation. Unexpectedly, we also found that stable overexpression of another protein kinase A regulatory subunit, RIalpha, had the opposite effect on IL-2 expression, causing a 3- to 4-fold increase in IL-2 production following stimulation. In summary, our data demonstrate a novel mechanism by which serine 114 phosphorylation and nuclear localization of RIIbeta controls the regulation of gene expression in T cells.

The cyclic adenosine 5'-monophosphate response element modulator suppresses IL-2 production in stimulated T cells by a chromatin-dependent mechanism.

The production of IL-2 is tightly controlled by several transcription factors that bind to the IL-2 promoter. The cAMP response element modulator (CREM) is known to form complexes with CREB and bind to the -180 site of the IL-2 promoter in anergic and in systemic lupus erythematosus T cells. In this study we show that CREM is transcriptionally induced in T cells following stimulation through CD3 and CD28, binds to the IL-2 promoter in vivo, and suppresses IL-2 production. Transfection of an antisense CREM plasmid into T cells blocked the expression and binding of CREM to the IL-2 promoter and the decrease of IL-2 production, which follows the early increase after T cell stimulation with CD3 and CD28. In addition, as assessed by chromatin immunoprecipitation experiments, antisense CREM prevented the binding of protein 300 and cAMP response element binding protein and promoted the acetylation of histones. Antisense CREM also enhanced the accessibility of the IL-2 promoter to endonucleases and prevented the condensation of chromatin in vivo. Our data suggest that upon T cell activation, CREM gradually replaces phosphorylated CREB at the -180 site of the IL-2 promoter. CREM, in turn, binds protein 300 and cAMP response element binding protein, but CREM is unable to activate its histone acetyltransferase activity, which results in condensation of chromatin and down-regulation of IL-2 production.

Legionella pneumophila replication in macrophages inhibited by selective immunomodulatory effects on cytokine formation by epigallocatechin gallate, a major form of tea catechins.

Epigallocatechin gallate (EGCg) is a major form of tea catechin and has a variety of biological activities, including antitumor as well as antimicrobial activity against some pathogens. Although the biological activities of EGCg have been extensively studied, its immunological effects are not well known. In the present study, the ability of EGCg to modulate macrophage immune functions in an in vitro Legionella pneumophila infection model of macrophages was examined. The study showed that EGCg inhibited the growth of L. pneumophila in macrophages at a concentration as low as 0.5 microg/ml without any direct antibacterial effect on the organisms. The EGCg selectively upregulated the production of interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-alpha) and downregulated IL-10 production of macrophages induced by L. pneumophila infection in a dose-dependent manner, but did not alter IL-6 production even at a high dose. The upregulation of the levels of macrophage gamma interferon (IFN-gamma) mRNA by EGCg was also demonstrated. Treatment of macrophage cultures with anti-TNF-alpha and anti-IFN-gamma monoclonal antibodies markedly abolished the anti-L. pneumophila activity of macrophages induced by the EGCg treatment. These results indicate that EGCg selectively alters the immune responses of macrophages to L. pneumophila and leads to an enhanced anti-L. pneumophila activity of macrophages mediated by enhanced production of both TNF-alpha and IFN-gamma. However, the enhancement of in vitro anti-L. pneumophila activity by EGCg may not be directly mediated by IL-10 and IL-12 production modulation. Thus, the results of this study revealed the immunomodulatory effect of EGCg on macrophages, which have a critical role in infections.

Mice infected with the larvae of Taenia crassiceps exhibit a Th2-like immune response with concomitant anergy and downregulation of Th1-associated phenomena.

Infection of intermediate hosts with eggs of taeniid parasites results in a larval infestation known as cysticercosis. A number of studies have indicated that cysticercosis is associated with immunosuppression, although little is known about the mechanisms involved. In the present study, mice infected with the larvae of Taenia crassiceps were found to exhibit a pronounced energy, which preferentially affected T-cells located anatomically close to the parasite. This anergy was linked to late events in the T cell activation pathway; that is, stimulation through the T cell receptor(TCR)/CD3 complex by Concanavalin-A, or plate-bound monoclonal antibodies (mAb) to TCR alpha beta or CD3 epsilon, or combinations of phorbol ester and ionomycin (all of which can bypass early membrane-related events), failed to fully activate T lymphocytes. The relative proximity of T cells to the parasite was directly related to upregulation of IL-4 and downregulation of IL-2 production. In addition, the profiles of parasite-specific Abs showed an exclusive increase of serum IgG1 during infection. Taken together, the data suggest that infection of mice with larvae of T. crassiceps alters the balance of CD4+ Th cells by upregulating Th2 and downregulating Th1 cells located in close proximity to the parasite.

Down-regulation of IL-2 production by activation of T cells through Ly-6A/E.

Ly-6A/E molecules are expressed on the surface of T cells and have been shown to function in activation by the capacity of anti-Ly-6A/E mAb to induce T cell hybridomas or normal T cells to produce IL-2. Recent evidence suggests that activation through Ly-6A/E may be linked to the TCR signaling pathway. To further investigate the relationship between Ly-6- and TCR-induced T cell activation, we have examined whether an anti-Ly-6A/E mAb (D7) modulates TCR signaling in vitro. We now report that mAb D7 specifically inhibited IL-2 production by T cells also activated through TCR. Such inhibition was noted for normal T cells stimulated by soluble anti-CD3 or alloantigen and for T hybridomas stimulated by soluble anti-CD3. The ability of D7 to inhibit IL-2 production by T hybridomas was dependent on the nature of the TCR activating signal because IL-2 production was not inhibited when T hybridomas were stimulated with Ag or immobilized anti-CD3. Inhibition of IL-2 production by D7 apparently required cross-linking of the mAb because D7 F(ab')2 fragments were not effective for inhibition of IL-2 production. Similar to its ability to enhance anti-Ly-6A/E-induced activation of T and B cells, IFN-gamma enhanced the D7-induced inhibition of IL-2 production by alloantigen-activated normal T cells. These data further support the notion that Ly-6 and TCR signaling pathways are interrelated.