bluuebunny/crossref_metadata_2025 huggingface dataset:
c-Jun N-Terminal Kinase Negatively Regulates Lipopolysaccharide-Induced IL-12 Production in Human Macrophages: Role of Mitogen-Activated Protein Kinase in Glutathione Redox Regulation of IL-12 Production
Authors: Utsugi Mitsuyoshi , Dobashi Kunio , Ishizuka Tamotsu , Endou Katsuaki , Hamuro Junji , Murata Yukie , Nakazawa Tsugio , Mori Masatomo
Published: April 2014

Although c-Jun N-terminal kinase (JNK) plays an important role in cytokine expression, its function in IL-12 production is obscure. The present study uses human macrophages to examine whether the JNK pathway is required for LPS-induced IL-12 production and defines how JNK is involved in the regulation of IL-12 production by glutathione redox, which is the balance between intracellular reduced (GSH) and oxidized glutathione (GSSG). We found that LPS induced IL-12 p40 protein and mRNA in a time- and concentration-dependent manner in PMA-treated THP-1 macrophages, and that LPS activated JNK and p38 mitogen-activated protein (MAP) kinase, but not extracellular signal-regulated kinase, in PMA-treated THP-1 cells. Inhibition of p38 MAP kinase activation using SB203580 dose dependently repressed LPS-induced IL-12 p40 production, as described. Conversely, inhibition of JNK activation using SP600125 dose dependently enhanced both LPS-induced IL-12 p40 production from THP-1 cells and p70 production from human monocytes. Furthermore, JNK antisense oligonucleotides attenuated cellular levels of JNK protein and LPS-induced JNK activation, but augmented IL-12 p40 protein production and mRNA expression. Finally, the increase in the ratio of GSH/GSSG induced by glutathione reduced form ethyl ester (GSH-OEt) dose dependently enhanced LPS-induced IL-12 p40 production in PMA-treated THP-1 cells. GSH-OEt augmented p38 MAP kinase activation, but suppressed the JNK activation induced by LPS. Our findings indicate that JNK negatively affects LPS-induced IL-12 production from human macrophages, and that glutathione redox regulates LPS-induced IL-12 production through the opposite control of JNK and p38 MAP kinase activation.
CD4+ T Cells from (New Zealand Black × New Zealand White)F1 Lupus Mice and Normal Mice Immunized Against Apoptotic Nucleosomes Recognize Similar Th Cell Epitopes in the C Terminus of Histone H3
Authors: Fournel Sylvie , Neichel Sarah , Dali Hayet , Farci Sandrine , Maillère Bernard , Briand Jean-Paul , Muller Sylviane
Published: April 2014

We have previously reported that peptide 88-99 of histone H4 represents a minimal T cell epitope recognized by Th cells from nonautoimmune BALB/c (H-2d/d) mice immunized with nucleosomes. In this study, we tested a panel of overlapping peptides spanning the whole sequences of H4 and H3 for recognition by CD4+ T cells from unprimed (New Zealand Black (NZB) × New Zealand White (NZW))F1 lupus mice (H-2d/z). None of the 11 H4 peptides was recognized by CD4+ T cells from (NZB × NZW)F1 mice. In contrast, these cells proliferated and secreted IL-2, IL-10, and IFN-γ upon ex vivo stimulation with H3 peptides representing sequences 53-70, 64-78, and 68-85. Peptides 56-73 and 61-78 induced the production of IFN-γ and IL-10, respectively, without detectable proliferation, suggesting that they may act as partial agonist of the TCR. Th cells from unprimed BALB/c mice and other lupus-prone mice such as SNF1 (H-2d/q) and MRL/lpr (H-2k/k) mice did not recognize any peptides present within the H3 region 53-85. We further demonstrated that immunization of normal BALB/c mice with syngeneic liver nucleosomes and spleen apoptotic cells, but not with nonapoptotic syngeneic cells, induced Th cell responses against several peptides of the H3 region 53-85. Moreover, we found that this conserved region of H3, which is accessible at the surface of nucleosomes, is targeted by Abs from (NZB × NZW)F1 mice and lupus patients, and contains motifs recognized by several distinct HLA-DR molecules. It might thus be important in the self-tolerance breakdown in lupus.
Effects of IL-7 on Early Human Thymocyte Progenitor Cells In Vitro and in SCID-hu Thy/Liv Mice
Authors: Napolitano Laura A. , Stoddart Cheryl A. , Hanley Mary Beth , Wieder Eric , McCune Joseph M.
Published: April 2014

IL-7 is a critical component of thymopoiesis in animals and has recently been shown to play an important role in T cell homeostasis. Although there is increasing interest in the use of IL-7 for the treatment of lymphopenia caused by the HIV type 1, evidence that IL-7 may accelerate HIV replication has raised concerns regarding its use in this setting. We sought to identify the effects of IL-7 on human thymocyte survival and to determine the impact of IL-7 administration on in vivo HIV infection of the human thymus. Using in vitro analysis, we show that IL-7 provides potent anti-apoptotic and proliferative signals to early thymocyte progenitors. Analysis of CD34+ subpopulations demonstrates that surface IL-7 receptor is expressed on most CD34highCD5+CD1a− thymocytes and that this subpopulation appears to be one of the earliest maturation stages responsive to the effects of IL-7. Thus, IL-7 provides survival signals to human thymocytes before surface expression of CD1a. CD4+CD8+ thymocytes are relatively unresponsive to IL-7, although IL-7 protects these cells from dexamethasone-induced apoptosis. IL-7 has a predominantly proliferative effect on mature CD4+CD3+CD8− and CD8+CD3+CD4− thymocytes. In contrast to the in vitro findings, we observe that in vivo administration of IL-7 to SCID-hu Thy/Liv mice does not appear to enhance thymocyte survival nor does it appear to accelerate HIV infection. Given the growing interest in the use of IL-7 for the treatment of human immunodeficiency, these findings support additional investigation into its in vivo effects on thymopoiesis and HIV infection.
Attrition of Virus-Specific Memory CD8+ T Cells During Reconstitution of Lymphopenic Environments
Authors: Peacock Craig D. , Kim Sung-Kwon , Welsh Raymond M.
Published: April 2014

Viruses can cause a severe lymphopenia early in infection and a subsequent, lasting loss of pre-existing CD8+ memory T cells. We therefore questioned how well virus Ag-specific memory CD8+ T cells could reconstitute mice rendered lymphopenic as a consequence of genetics, irradiation, or viral or poly(I:C)-induced cytokines. In each case, reconstitution of the CD8+ compartment was associated with limited division of virus-specific memory T cells and a reduction in their proportion. This indicates that foreign Ag-experienced CD44highCD8+ memory T cells may respond differently to homeostatic signals than other CD44highCD8+ cells, and that events inducing lymphopenia may lead to a permanent reduction in T cell memory.
Febrile Temperatures Attenuate IL-1β Release by Inhibiting Proteolytic Processing of the Proform and Influence Th1/Th2 Balance by Favoring Th2 Cytokines
Authors: Boneberg Eva-Maria , Hartung Thomas
Published: April 2014

We investigated possible feedback mechanisms of febrile temperatures on LPS- and staphylococcal enterotoxin B (SEB)-induced cytokine release in human whole blood. LPS-induced IL-1β release was inhibited at temperatures >38°C, whereas intracellular proIL-1β formation as well as the release of other cytokines except IL-18 were only attenuated above 42°C, indicating that febrile temperatures impair the proteolytic processing of proIL-1β. This attenuated processing is not due to either heat inactivation of caspase-1 or structural changes in proIL-1β produced at higher temperatures. Instead, we propose that febrile conditions change cytosolic compartmentation or trafficking, so that synthesized proIL-1β cannot encounter caspase-1. Febrile temperatures also influenced Th1/Th2 cytokine balance. We observed a 3-fold increase in the Th2-cytokines IL-5 and IL-13 and a reduction to 15% of the Th1-cytokine IL-2 when SEB-stimulated whole blood was incubated at 40°C compared with 37°C. These results indicate that fever limits the production of the fever-inducing IL-1β and also influences the adaptive immune response, favoring Th2 cytokine production.
P-Selectin Enhances Generation of CD14+CD16+ Dendritic-Like Cells and Inhibits Macrophage Maturation from Human Peripheral Blood Monocytes
Authors: Li Geling , Kim Young-June , Mantel Charlie , Broxmeyer Hal E.
Published: April 2014

Endothelial cells play a critical role in monocyte differentiation. Platelets also affect terminal maturation of monocytes in vitro. P-selectin is an important adhesion molecule expressed on both endothelial cells and activated platelets. We investigated its effects on human peripheral blood monocyte differentiation under the influence of different cytokines. Generation of dendritic-like cells (DLCs) from peripheral blood monocytes was promoted by immobilized P-selectin in the presence of M-CSF and IL-4 as judged by dendritic cell (DC) morphology; increased expression of CD1a, a DC marker; low phagocytic activity; and high alloreactivity to naive T cells. In contrast to typical DCs, DLCs expressed CD14 and FcγRIII (CD16). These features link the possible identity of DLCs to that of an uncommon CD14+CD16+CD64− monocyte subset found to be expanded in a variety of pathological conditions. Functionally, DLCs generated by P-selectin in combination with M-CSF plus IL-4 primed naive allogeneic CD4+ T cells to produce significantly less IFN-γ than cells generated by BSA in the presence of M-CSF and IL-4. P-selectin effects on enhancing CD14+CD16+ DLC generation were completely abrogated by pretreatment of cells with the protein kinase C δ inhibitor rottlerin, but not by classical protein kinase C inhibitor Gö6976. Immobilized P-selectin also inhibited macrophage differentiation in response to M-CSF alone as demonstrated by morphology, phenotype, and phagocytosis analysis. The effects of P-selectin on macrophage differentiation were neutralized by pretreatment of monocytes with Ab against P-selectin glycoprotein ligand 1. These results suggest a novel role for P-selectin in regulating monocyte fate determination.
Late Immature B Cells (IgMhighIgDneg) Undergo a Light Chain Receptor Editing Response to Soluble Self-Antigen
Authors: Tze Lina E. , Hippen Keli L. , Behrens Timothy W.
Published: April 2014

Receptor editing is an important mechanism to modify the Ag specificity of newly developing B cells that are reactive with self-Ags. Previous studies have suggested that late immature B cells, bearing high levels of IgM on their cell surface, are unable to initiate receptor editing and instead are deleted by apoptosis. Using the hen egg lysozyme transgenic system, we show that IgMhigh late-immature B cells are fully capable of receptor editing to soluble self-Ag. This was demonstrated by the induction of new endogenous light chain locus rearrangements and by a single-cell flow cytometric assay using a recombination-activating gene 2-green fluorescence protein reporter transgene. These studies suggest that the developmental window available for immature B cells to edit their Ig receptors, at least in response to certain soluble Ags, extends through the IgMhigh late immature B cell stage.
IL-15-Mediated Induction of LFA-1 Is a Late Step Required for Cytotoxic Differentiation of Human NK Cells from CD34+Lin− Bone Marrow Cells
Authors: Barao Isabel , Hudig Dorothy , Ascensao Joao L.
Published: April 2014

Optimal differentiation of cytotoxic NK cells is important to provide protective innate immunity to patients after bone marrow transplantation. In vitro differentiation of CD56+CD3− NK cells takes weeks and is supported by several cytokines, including IL-2, IL-7, and IL-15, and thus can be useful for immunotherapy. However, IL-2 therapy is problematic in vivo, and NK cells differentiated in vitro with only IL-7 lack cytotoxicity. We assessed whether human NK cells initially differentiated in vitro from CD34+Lin− bone marrow cells with IL-7 could acquire cytotoxicity after exposure to additional cytokines and what changes promoted cytotoxicity. The cells cultured with IL-7 already had granzyme B as well as perforin, as previously reported, the proteins of cytotoxic granules. The cells also lacked LFA-1. After 1 wk of secondary culture with either IL-2 or IL-15, but not with IL-12 or IL-18, the IL-7-cultured cells acquired cytotoxicity. IL-2 or IL-15 also induced LFA-1. Ab to the LFA-1 subunits CD11a and CD18 blocked lysis by the NK cells, indicating that the new LFA-1 correlated with, and was essential for, the cytotoxic function of the in vitro generated cells. The LFA-1 also participated in target cell binding by the in vitro differentiated cells. In this study, we demonstrated a new function for IL-15, the induction of LFA-1 in NK progenitor cells, and that IL-15 does more than merely support NK progenitor cell proliferation. The efficacy after only 1 wk of IL-15 administration is a positive practical feature that may apply to human therapy.
Immune Modulation by Silencing IL-12 Production in Dendritic Cells Using Small Interfering RNA
Authors: Hill Jonathan A. , Ichim Thomas E. , Kusznieruk Kornel P. , Li Mu , Huang Xuyan , Yan Xiaotao , Zhong Robert , Cairns Ewa , Bell David A. , Min Wei-Ping
Published: April 2014

RNA interference is a mechanism of posttranscriptional gene silencing that functions in most eukaryotic cells, including human and mouse. Specific gene silencing is mediated by short strands of duplex RNA of ∼21 nt in length (termed small interfering RNA or siRNA) that target the cognate mRNA sequence for degradation. We demonstrate here that RNAi can be used for immune modulation by targeting dendritic cell (DC) gene expression. Transfection of DC with siRNA specific for the IL-12 p35 gene resulted in potent suppression of gene expression and blockade of bioactive IL-12 p70 production without affecting unrelated genes or cellular viability. Inhibition of IL-12 was associated with increased IL-10 production, which endowed the DC with the ability to stimulate production of Th2 cytokines from allogenic T cells in vitro. Furthermore, siRNA-silenced DC lacking IL-12 production were poor allostimulators in MLR. IL-12-silenced and KLH-pulsed DC polarized the immune response toward a Th2 cytokine profile in an Ag-specific manner. These data are the first to demonstrate that RNA interference is a potent and specific tool for modulating DC-mediated immune responses.
In Vivo Ligation of CD40 Enhances Priming Against the Endogenous Tumor Antigen and Promotes CD8+ T Cell Effector Function in SV40 T Antigen Transgenic Mice
Authors: Staveley-O’Carroll Kevin , Schell Todd D. , Jimenez Marcela , Mylin Lawrence M. , Tevethia M. Judith , Schoenberger Stephen P. , Tevethia Satvir S.
Published: April 2014

The ability to initiate and sustain CD8+ T cell responses to tumors in vivo is hindered by the development of peripheral T cell tolerance against tumor-associated Ags. Approaches that counter the onset of T cell tolerance may preserve a pool of potentially tumor-reactive CD8+ T cells. Administration of agonist Ab to the CD40 molecule, expressed on APCs, can enhance immunization approaches targeting T lymphocytes in an otherwise tolerance-prone environment. In this report, the effects of anti-CD40 administration on priming of naive CD8+ T cells against an endogenous tumor Ag were investigated. Line 501 mice express the SV40 large T Ag oncoprotein as a transgene from the α-amylase promoter, resulting in the development of peripheral CD8+ T cell tolerance to the H-2-Db-restricted immunodominant epitope I of T Ag by 6 mo of age, before the appearance of osteosarcomas. We demonstrate that naive epitope I-specific TCR transgenic (TCR-I) T cells undergo peripheral tolerance following adoptive transfer into 6-mo-old 501 mice. In contrast, administration of agonistic anti-CD40 Ab led to increased expansion of TCR-I T cells in 501 mice, the acquisition of effector function by TCR-I T cells and the establishment of T cell memory. Importantly, this enhanced priming effect of anti-CD40 administration did not require immunization and was effective even if administered after naive TCR-I T cells had encountered the endogenous T Ag. Thus, anti-CD40 administration can block the onset of peripheral tolerance and enhance the recruitment of functionally competent effector T cells toward an endogenous tumor Ag.
Regulation of Murine Inflammatory Bowel Disease by CD25+ and CD25− CD4+ Glucocorticoid-Induced TNF Receptor Family-Related Gene+ Regulatory T Cells
Authors: Uraushihara Koji , Kanai Takanori , Ko Kwibeom , Totsuka Teruji , Makita Shin , Iiyama Ryoichi , Nakamura Tetsuya , Watanabe Mamoru
Published: April 2014

CD4+CD25+ regulatory T cells in normal animals are engaged in the maintenance of immunological self-tolerance and prevention of autoimmune disease. However, accumulating evidence suggests that a fraction of the peripheral CD4+CD25− T cell population also possesses regulatory activity in vivo. Recently, it has been shown glucocorticoid-induced TNFR family-related gene (GITR) is predominantly expressed on CD4+CD25+ regulatory T cells. In this study, we show evidence that CD4+GITR+ T cells, regardless of the CD25 expression, regulate the mucosal immune responses and intestinal inflammation. SCID mice restored with the CD4+GITR− T cell population developed wasting disease and severe chronic colitis. Cotransfer of CD4+GITR+ population prevented the development of CD4+CD45RBhigh T cell-transferred colitis. Administration of anti-GITR mAb-induced chronic colitis in mice restored both CD45RBhigh and CD45RBlow CD4+ T cells. Interestingly, both CD4+CD25+ and CD4+CD25− GITR+ T cells prevented wasting disease and colitis. Furthermore, in vitro studies revealed that CD4+CD25−GITR+ T cells as well as CD4+CD25+GITR+ T cells expressed CTLA-4 intracellularly, showed anergic, suppressed T cell proliferation, and produced IL-10 and TGF-β. These data suggest that GITR can be used as a specific marker for regulatory T cells controlling mucosal inflammation and also as a target for treatment of inflammatory bowel disease.
Role of the Phosphatidylinositol 3 Kinase-Akt Pathway in the Regulation of IL-10 and IL-12 byPorphyromonas gingivalisLipopolysaccharide
Authors: Martin Michael , Schifferle Robert E. , Cuesta Natalia , Vogel Stefanie N. , Katz Jannet , Michalek Suzanne M.
Published: April 2014

Stimulation of the APC by Porphyromonas gingivalis LPS has been shown to result in the production of certain pro- and anti-inflammatory cytokines. However, the signaling pathways that regulate these processes are currently unknown. In the present study, the role of the phosphatidylinositol 3 kinase (PI3K)-Akt pathway in regulating P. gingivalis LPS-induced production of IL-10, IL-12 p40, and IL-12 p70 by human monocytes was investigated. P. gingivalis LPS selectively activates the PI3K-Akt pathway via Toll-like receptor 2, and inhibition of this pathway results in an abrogation of extracellular signal-regulated kinase 1/2 phosphorylation, whereas the activation of p38 and c-Jun N-terminal kinase 1/2 kinases were unaffected. Analysis of cytokine production following stimulation of monocytes with P. gingivalis LPS revealed that inhibition of the PI3K pathway differentially regulated IL-10 and IL-12 synthesis. IL-10 production was suppressed, whereas IL-12 levels were enhanced. Inhibition of P. gingivalis LPS-mediated activation of the PI3K-Akt pathway resulted in a pronounced augmentation of NF-κB p65 that was independent of IκB-α degradation. Furthermore, the ability of the PI3K-Akt pathway to modulate IL-10 and IL-12 production appears to be mediated by the selective suppression of extracellular signal-regulated kinase 1/2 activity, as the MEK1 inhibitor PD98059 closely mimicked the effects of wortmannin and LY294002 to differentially regulate IL-10 and IL-12 production by P. gingivalis LPS-stimulated monocytes. These studies provide new insight into how engagement of the PI3K-Akt pathway by P. gingivalis LPS affects the induction of key immunoregulatory cytokines that control both qualitative and quantitative aspects of innate and adaptive immunity.
Gr-1+ Myeloid Cells Lacking T Cell Protein Tyrosine Phosphatase Inhibit Lymphocyte Proliferation by an IFN-γ- and Nitric Oxide-Dependent Mechanism
Authors: Dupuis Maryse , de Jesús Ibarra-Sánchez María , Tremblay Michel L. , Duplay Pascale
Published: April 2014

The T cell protein tyrosine phosphatase is involved in the immune system regulation, as evidenced by defective function and development of several hemopoietic cell populations in T cell protein tyrosine phosphatase (TC-PTP)-deficient mice. In particular, B and T cell proliferation is greatly inhibited when total splenocytes are stimulated by LPS or anti-CD3 mAb. To define the functional defect of TC-PTP−/− lymphocytes, we isolated T and B cells from the spleen of TC-PTP−/− mice. We show that the proliferative response of lymphocytes was greatly increased when cultured as a purified population, indicating that an inhibitory population is present in TC-PTP−/− spleen. However, TC-PTP−/− lymphocytes have a 2- to 3-fold lower proliferation rate compared with TC-PTP+/+ lymphocytes, suggesting that, as shown previously in embryonic fibroblasts, TC-PTP is involved in the control of cell cycle in lymphocytes. We have characterized phenotypically and functionally the inhibitory population present in the spleen of TC-PTP−/− mice. We show that a Gr-1+-enriched cell population isolated from TC-PTP−/− mice suppresses the CD3-induced proliferation of T cells in coculture in vitro. The specific inhibition of NO synthesis with NG-monomethyl-l-arginine.monoacetate restored splenocyte responses, and there is a strict correlation between NO levels and the degree of suppression. Neutralization of IFN-γ with specific mAb almost completely abolished the inhibitory activity of Gr-1+ cells and concomitantly high levels of NO secretion. Moreover, inhibition of lymphocyte proliferative responses required cell-cell contact to achieve sufficient levels of NO. These findings demonstrate an important function of TC-PTP in the induction of the NO pathway that mediates inhibition of T cell proliferation.
Induction of Autoantigen-Specific Th2 and Tr1 Regulatory T Cells and Modulation of Autoimmune Diabetes
Authors: Chen Cyndi , Lee Wen-Hui , Yun Pen , Snow Peter , Liu Chih-Pin
Published: April 2014

Autoantigen-based immunotherapy can modulate autoimmune diabetes, perhaps due to the activation of Ag-specific regulatory T cells. Studies of these regulatory T cells should help us understand their roles in diabetes and aid in designing a more effective immunotherapy. We have used class II MHC tetramers to isolate Ag-specific T cells from nonobese diabetic (NOD) mice and BALB/c mice treated with glutamic acid decarboxylase 65 peptides (p206 and p221). Based on their cytokine secretion profiles, immunization of NOD mice with the same peptide induced different T cell subsets than in BALB/c mice. Treatment of NOD mice induced not only Th2 cells but also IFN-γ/IL-10-secreting T regulatory type 1 (Tr1) cells. Adoptive transfer experiments showed that isolated tetramer+ T cells specific for p206 or p221 could inhibit diabetes development. These cells were able to suppress the in vitro proliferation of other NOD mouse T cells without cell-cell contact. They performed their regulatory functions probably by secreting cytokines, and Abs against these cytokines could block their suppressive effect. Interestingly, the presence of both anti-IL-10 and anti-IFN-γ could enhance the target cell proliferation, suggesting that Tr1 cells play an important role. Further in vivo experiments showed that the tetramer+ T cells could block diabetogenic T cell migration into lymph nodes. Therefore, treatment of NOD mice with autoantigen could induce Th2 and Tr1 regulatory cells that can suppress the function and/or block the migration of other T cells, including diabetogenic T cells, and inhibit diabetes development.
CD49d Overexpression and T Cell Autoimmunity
Authors: Mo Ru-Ran , Eisenbraun Julie K. , Sonstein Joanne , Craig Ronald A. , Curtis Jeffrey L. , Stoolman Lloyd M. , Chen Jun , Yung Raymond L.
Published: April 2014

D10.G4.1 (D10) cells, a murine conalbumin-reactive Th2 cell line, made to overexpress the β2 integrin LFA-1 by pharmacological manipulation or by transfection become autoreactive and are capable of inducing in vivo autoimmunity. However, whether this is specific to LFA-1 and whether overexpression of other T cell integrin molecules has the same effect are unknown. We examined the functional consequences of T cell CD49d (α4 integrin) overexpression by transfecting murine CD49d cDNA into D10 cells. Similar to the LFA-1-transfected cells, the CD49d-overexpressing T cells are autoreactive and proliferate in response to APCs in an MHC class II-dependent manner in the absence of nominal Ag. Additionally, CD49d overexpression is associated with increased in vitro adhesion to endothelial cells and increased in vivo splenic homing. However, in contrast to LFA-1 overexpression, increased T cell CD49d expression is not associated with autoreactive cytotoxicity or the ability to induce in vivo autoimmunity. In addition to the novel observation that CD49d overexpression is sufficient to induce T cell autoreactivity, our results also support the hypothesis that the ability to induce in vivo autoimmunity is related to T cell cytotoxicity and not to T cell proliferation function in the D10 murine adoptive transfer model of autoimmunity.
The Slc11a1 (Nramp1) Gene Controls Efficacy of Mycobacterial Treatment of Allergic Asthma
Authors: Smit Joost J. , Van Loveren Henk , Hoekstra Maarten O. , Karimi Khalil , Folkerts Gert , Nijkamp Frans P.
Published: April 2014

Genes controlling antibacterial resistance may be important in the hygiene hypothesis, which states that lack of bacterial infections during childhood would favor development of allergic disease. We, therefore, studied whether Nramp1 (Slc11a1) alleles, which determine susceptibility (Nramp1s) or resistance (Nramp1r) to intracellular bacteria, affect the efficacy of heat-killed Mycobacterium vaccae in the treatment of allergic asthma in a mouse model. Treatment of OVA-sensitized Nramp1s mice with M. vaccae suppressed airway hyperresponsiveness, airway eosinophilia, Ag-specific IgE, and IL-4 and IL-5 production after OVA aerosol challenge. In contrast, M. vaccae hardly affected these parameters in Nramp1r mice. In addition, The Nramp1 gene affected both T cell-mediated responses to M. vaccae in vivo and the level of macrophage activation after stimulation with M. vaccae in vitro. In conclusion, the efficacy of M. vaccae in preventing allergic and asthmatic manifestations in a mouse model is strongly affected by Nramp1 alleles. These findings could have important implications for the future use of mycobacteria and their components in the prevention or treatment of allergic asthma. A new link is described between genes, the environment, and the development of allergy, in which the Nramp1 gene fine tunes the hygiene hypothesis.
NF-κB1 p50 Is Required for BLyS Attenuation of Apoptosis but Dispensable for Processing of NF-κB2 p100 to p52 in Quiescent Mature B Cells
Authors: Hatada Eunice N. , Do Richard K. G. , Orlofsky Amos , Liou Hsiou-Chi , Prystowsky Michael , MacLennan Ian C. M. , Caamano Jorge , Chen-Kiang Selina
Published: April 2014

B lymphocyte stimulator (BLyS), a TNF family protein essential for peripheral B cell development, functions primarily through attenuation of B cell apoptosis. In this study, we show that BLyS activates NF-κB through both classical and alternative pathways with distinct kinetics in quiescent mature B cells. It rapidly and transiently enhances the p50/p65 DNA binding activity and induces phosphorylation of IκBα characteristic of the classical NF-κB pathway, albeit maintaining IκBα at a constant level through ongoing protein synthesis and proteasome-mediated destruction. With delayed kinetics, BLyS promotes the processing of p100 to p52 and sustained formation of p52/RelB complexes via the alternative NF-κB pathway. p50 is dispensable for p100 processing. However, it is required to mediate the initial BLyS survival signals and concomitant activation of Bcl-xL in quiescent mature B cells ex vivo. Although also a target of BLyS activation, at least one of the A1 genes, A1-a, is dispensable for the BLyS survival function. These results suggest that BLyS mediates its survival signals in metabolically restricted quiescent B cells, at least in part, through coordinated activation of both NF-κB pathways and selective downstream antiapoptotic genes.
Initiation and Limitation of Ly-49A NK Cell Receptor Acquisition by T Cell Factor-1
Authors: Ioannidis Vassilios , Kunz Béatrice , Tanamachi Dawn M. , Scarpellino Léonardo , Held Werner
Published: April 2014

The establishment of clonally variable expression of MHC class I-specific receptors by NK cells is not well understood. The Ly-49A receptor is used by ≈20% of NK cells, whereby most cells express either the maternal or paternal allele and few express simultaneously both alleles. We have previously shown that NK cells expressing Ly-49A were reduced or almost absent in mice harboring a single or no functional allele of the transcription factor T cell factor-1 (TCF-1), respectively. In this study, we show that enforced expression of TCF-1 in transgenic mice yields an expanded Ly-49A subset. Even though the frequencies of Ly-49A+ NK cells varied as a function of the TCF-1 dosage, the relative abundance of mono- and biallelic Ly-49A cells was maintained. Mono- and biallelic Ly-49A NK cells were also observed in mice expressing exclusively a transgenic TCF-1, i.e., expressing a fixed amount of TCF-1 in all NK cells. These findings suggest that Ly-49A acquisition is a stochastic event due to limiting TCF-1 availability, rather than the consequence of clonally variable expression of the endogenous TCF-1 locus. Efficient Ly-49A acquisition depended on the expression of a TCF-1 isoform, which included a domain known to associate with the TCF-1 coactivator β-catenin. Indeed, the proximal Ly-49A promoter was β-catenin responsive in reporter gene assays. We thus propose that Ly-49A receptor expression is induced from a single allele in occasional NK cells due to a limitation in the amount of a transcription factor complex requiring TCF-1.
Induction of CD4+ T Cell Apoptosis as a Consequence of Impaired Cytoskeletal Rearrangement in UVB-Irradiated Dendritic Cells
Authors: Wachter Tina , Averbeck Marco , Hara Hisamichi , Tesmann Jens P. , Simon Jan C. , Termeer Christian C. , Denfeld Ralf W.
Published: April 2014

Low dose UVB irradiation of dendritic cells (DC) dose-dependently decreases their allostimulatory capacity and inhibits alloreactive T cell proliferation. The reduction of the stimulatory capacity is not associated with a perturbation of CD28 costimulation. To examine the underlying mechanism, cell cycle analysis of T cells from cocultures with UVB-irradiated DC (UVB-DC) was performed, revealing no cell cycle arrest, but an increased number of apoptotic T cells in sub-G0 phase. We confirmed T cells to undergo apoptosis after coincubation with UVB-DC by TUNEL staining and DNA laddering. To analyze whether T cell apoptosis requires the Fas/Fas ligand (FasL) pathway, MLRs were performed with Fas-, FasL-deficient, and wild-type DC and T cells. No differences were found on comparison of wild-type DC with Fas-/FasL-deficient DC or T cells. Likewise, addition of a neutralizing anti-TNF-α mAb to cocultures could not overcome inhibition of T cell proliferation by UVB-DC, excluding involvement of the TNF-α/TNF-αR pathway. FACS analysis of CD69 and CD25 revealed no up-regulation on T cells cocultured with UVB-DC, suggesting a perturbation of early T cell activation. Analysis of UVB-DC by confocal microscopy demonstrated impaired filamentous actin bundling, a process critical for T cell stimulation. To investigate the functional relevance of these observations, time lapse video microscopy was performed. Indeed, calcium signaling in CD4+ T cells was significantly diminished after interaction with UVB-DC. In conclusion, UVBR of DC impairs their cytoskeletal rearrangement and induces apoptosis in CD4+ T cells by disruption of early DC-T cell interaction, resulting in a reduced Ca2+ influx in T cells.
Regulatory Roles of IL-2 and IL-4 in H4/Inducible Costimulator Expression on Activated CD4+ T Cells During Th Cell Development
Authors: Yagi Junji , Arimura Yutaka , Dianzani Umberto , Uede Toshimitsu , Okamoto Toshihiro , Uchiyama Takehiko
Published: April 2014

We found a tight correlation among the levels of H4/inducible costimulator (ICOS) expression, IL-4 production, and GATA-3 induction, using activated CD4+ T cells obtained from six different murine strains. BALB/c-activated CD4+ T cells expressed ∼10-fold more H4/ICOS on their surfaces and produced ∼10-fold more IL-4 upon restimulation than C57BL/6-activated CD4+ T cells. BALB/c naive CD4+ T cells were shown to produce much higher amounts of IL-2 and IL-4 upon primary stimulation than C57BL/6 naive CD4+ T cells. Neutralization of IL-4 with mAbs in culture of BALB/c naive CD4+ T cells strongly down-regulated both H4/ICOS expression on activated CD4+ T cells and IL-4 production upon subsequent restimulation. Conversely, exogenous IL-4 added to the culture of BALB/c or C57BL/6 naive CD4+ T cells up-regulated H4/ICOS expression and IL-4 production upon restimulation. In addition, retroviral expression of GATA-3 during the stimulation of naive CD4+ T cells from C57BL/6 or IL-4−/− mice increased H4/ICOS expression on activated CD4+ T cells. A similar effect of IL-2 in the primary culture of BALB/c naive CD4+ T cells appeared to be mediated by IL-4, the production of which was regulated by IL-2. These data suggest that IL-4 induced by IL-2 is critical to the maintenance of high H4/ICOS expression on BALB/c-activated CD4+ T cells.
Innate Immunity Together with Duration of Antigen Persistence Regulate Effector T Cell Induction
Authors: Storni Tazio , Ruedl Christiane , Renner Wolfgang A. , Bachmann Martin F.
Published: April 2014

Proliferation of T cells is important for the expansion of specific T cell clones during immune responses. In addition, for the establishment of protective immunity against viruses, bacteria, and tumors, the expanded T cells must differentiate into effector T cells. Here we show that effector T cell generation is driven by activation of APCs and duration of antigenic stimulation. Adoptively transferred TCR-transgenic T cells extensively proliferated upon immunization. However, these T cells failed to differentiate into effector cells and died within 1 wk after immunization unless antigenic peptides persisted for >1 day or were presented by activated APCs. The induction of protective immunity in a nontransgenic system was more stringent, since activation of APCs or prolonged Ag persistence alone was not sufficient to drive immunity. In contrast, Ag had to be presented for several days by activated APCs to trigger protective T cell responses. Thus, activation of APCs and duration of Ag presentation together regulate the induction of protective T cell responses.
Characterization of Transcriptional Regulation During Negative Selection In Vivo
Authors: DeRyckere Deborah , Mann Derrick L. , DeGregori James
Published: April 2014

Negative selection is the process whereby immature thymocytes expressing TCRs with high affinity for self-peptide:MHC complexes are induced to undergo apoptosis. The transcriptional events that occur as a result of TCR signaling during negative selection are not well-characterized. Using oligonucleotide arrays, we have identified 33 genes that exhibit changes in RNA levels in CD4+CD8+ thymocytes during negative selection in vivo. Of 18 genes that have been further characterized, 13 are regulated in response to stimulation with Ag or anti-CD3 and anti-CD28 Abs ex vivo, indicating that these genes are regulated independently of activation of the peripheral immune system. These data also support the idea that anti-CD3/CD28-mediated thymocyte apoptosis is a valid model for negative selection in vivo. A detailed examination of the regulation of many of the identified genes in response to treatment with dexamethasone or gamma-radiation or in response to anti-CD3/anti-CD28 stimulation in the presence of pharmacological inhibitors of mitogen-activated protein kinase kinase kinase 1, p38 mitogen-activated protein kinase, phosphatidylinositol 3-kinase, calcineurin, and cyclin-dependent kinase 2 has facilitated the elucidation of a map of the transcriptional events that occur downstream of the TCR. These studies support a model whereby similar signal transduction pathways are activated by stimuli that induce positive and negative selection and are consistent with the idea that the balance between opposing proapoptotic and antiapoptotic pathways determines cell fate. The data presented in this study also suggest that calcineurin functions to amplify TCR signals by promoting sustained increases in the levels of specific transcripts.
Modular Organization of the Carboxyl-Terminal, Globular Head Region of Human C1q A, B, and C Chains
Authors: Kishore Uday , Gupta Sanjeev K. , Perdikoulis Michael V. , Kojouharova Mihaela S. , Urban Britta C. , Reid Kenneth B. M.
Published: April 2014

The first step in the activation of the classical complement pathway, by immune complexes, involves the binding of the globular heads of C1q to the Fc regions of aggregated IgG or IgM. Located C-terminal to the collagen region, each globular head is composed of the C-terminal halves of one A (ghA), one B (ghB), and one C chain (ghC). To dissect their structural and functional autonomy, we have expressed ghA, ghB, and ghC in Escherichia coli as soluble proteins linked to maltose-binding protein (MBP). The affinity-purified fusion proteins (MBP-ghA, -ghB, and -ghC) bound differentially to heat-aggregated IgG and IgM, and also to three known C1q-binding peptides, derived from HIV-1, HTLV-I, and β-amyloid. In the ELISAs, the MBP-ghA bound to heat-aggregated IgG and IgM as well as to the HIV-1 gp41 peptide; the MBP-ghB bound preferentially to IgG rather than IgM, in addition to binding β-amyloid peptide, whereas the MBP-ghC showed a preference for IgM and the HTLV-I gp21 peptide. Both MBP-ghA and MBP-ghB also inhibited C1q-dependent hemolysis of IgG- and IgM-sensitized sheep erythrocytes. However, for IgM-coated erythrocytes, MBP-ghC was a better inhibitor of C1q than MBP-ghB. The recombinant forms of ghA, ghB, and ghC also bound specifically to apoptotic PBMCs. We conclude that the C1q globular head region is likely to have a modular organization, being composed of three structurally and functionally independent modules, which retains multivalency in the form of a heterotrimer. The heterotrimeric organization thus offers functional flexibility and versatility to the whole C1q molecule.
Functional Cooperation of Simian Virus 40 Promoter Factor 1 and CCAAT/Enhancer-Binding Protein β and δ in Lipopolysaccharide-Induced Gene Activation of IL-10 in Mouse Macrophages
Authors: Liu Yi-Wen , Tseng Hui-Ping , Chen Lei-Chin , Chen Ben-Kuen , Chang Wen-Chang
Published: April 2014

Previous studies have revealed that LPS can activate transcription of the IL-10 gene promoter through an SV40 promoter factor 1 (Sp1) binding site in mouse macrophage RAW264.7. In this study, we determined that, in addition to Sp1, C/EBPβ and δ were also involved in LPS-induced gene expression of IL-10. By transient transfection with 5′-deletion mutants of the IL-10 promoter, we found that there were two LPS-responsive elements in the promoter of the mouse IL-10 gene. Analysis of these two regions by gel shift assay suggested that Sp1 and C/EBPβ and δ were bound to these two regions, respectively. By site-directed mutagenesis, we found that disruption at both the Sp1 and C/EBP binding sites almost completely blocked the LPS response. By gel shift assay and Western blotting, we found that the DNA binding complex and protein expression of C/EBPβ and δ were increased by LPS treatment, but these results were not found for Sp1. Overexpression of C/EBPβ or C/EBPδ, respectively, activated the promoter of the IL-10 gene, and they were enhanced by LPS. Coimmunoprecipitation experiments in intact cells indicated that LPS stimulated interaction between Sp1 and C/EBPβ and δ. These results suggested that the interaction between Sp1 and C/EBPβ and δ induced by LPS cooperatively activated expression of the IL-10 gene. The increase of C/EBPβ and δ proteins and the enhancement of transactivation activity of C/EBPβ and δ by LPS treatment, at least in part, explain the activation of IL-10 gene expression.
Increase of TCR Vβ Accessibility within Eβ Regulatory Region Influences its Recombination Frequency But Not Allelic Exclusion
Authors: Senoo Makoto , Wang Lili , Suzuki Daisuke , Takeda Naoki , Shinkai Yoichi , Habu Sonoko
Published: April 2014

Seventy percent of the murine TCRβ locus (475 kb) was deleted to generate a large deleted TCRβ (βLD) allele to investigate a possible linkage between germline transcription, recombination frequency, and allelic exclusion of the TCR Vβ genes. In these βLD/LD mice, the TCRβ gene locus contained only four Vβ genes at the 5′ side of the locus, and consequently, the Vβ10 gene was located in the original Dβ1-Jβ1cluster within the Eβ regulatory region. We showed that the frequency of recombination and expression of the Vβ genes are strongly biased to Vβ10 in these mutant mice even though the proximity of the other three 5′Vβ genes was also greatly shortened toward the Dβ-Jβ cluster and the Eβ enhancer. Accordingly, the germline transcription of the Vβ10 gene in βLD/LD mice was exceptionally enhanced in immature double negative thymocytes compared with that in wild-type mice. During double negative-to-double positive transition of thymocytes, the level of Vβ10 germline transcription was prominently increased in βLD/LD recombination activating gene 2-deficient mice receiving anti-CD3ε Ab in vivo. Interestingly, however, despite the increased accessibility of the Vβ10 gene in terms of transcription, allelic exclusion of this Vβ gene was strictly maintained in βLD/LD mice. These results provide strong evidence that increase of Vβ accessibility influences frequency but not allelic exclusion of the TCR Vβ rearrangement if the Vβ gene is located in the Eβ regulatory region.
Hyperconservation of theN-Formyl Peptide Binding Site ofM3: Evidence thatM3Is an Old Eutherian Molecule with Conserved Recognition of a Pathogen-Associated Molecular Pattern
Authors: Doyle C. Kuyler , Davis Beckley K. , Cook Richard G. , Rich Robert R. , Rodgers John R.
Published: April 2014

The mouse MHC class I-b molecule H2-M3 has unique specificity for N-formyl peptides, derived from bacteria (and mitochondria), and is thus a pathogen-associated molecular pattern recognition receptor (PRR). To test whether M3 was selected for this PRR function, we studied M3 sequences from diverse murid species of murine genera Mus, Rattus, Apodemus, Diplothrix, Hybomys, Mastomys, and Tokudaia and of sigmodontine genera Sigmodon and Peromyscus. We found that M3 is highly conserved, and the 10 residues coordinating the N-formyl group are almost invariant. The ratio of nonsynonymous and synonymous substitution rates suggests the Ag recognition site of M3, unlike the Ag recognition site of class I-a molecules, is under strong negative (purifying) selection and has been for at least 50–65 million years. Consistent with this, M3 α1α2 domains from Rattus norvegicus and Sigmodon hispidus and from the “null” allele H2-M3b specifically bound N-formyl peptides. The pattern of nucleotide substitution in M3 suggests M3 arose rapidly from murid I-a precursors by an evolutionary leap (“saltation”), perhaps involving intense selective pressure from bacterial pathogens. Alternatively, M3 arose more slowly but prior to the radiation of eutherian (placental) mammals. Older dates for the emergence of M3, and the accepted antiquity of CD1, suggest that primordial class I MHC molecules could have evolved originally as monomorphic PRR, presenting pathogen-associated molecular patterns. Such MHC PRR molecules could have been preadaptations for the evolution of acquired immunity during the early vertebrate radiation.
T Cell Antagonism is Functionally Uncoupled from the 21- and 23-kDa Tyrosine-Phosphorylated TCR ζ Subunits
Authors: Pitcher Lisa A. , Ohashi Pamela S. , van Oers Nicolai S. C.
Published: April 2014

The functional effects of altered peptide ligands on T cells is proposed to involve differential intracellular signaling mediated by the 21- and 23-kDa tyrosine-phosphorylated derivatives of the TCR ζ subunit (p21 and p23). To understand the functional contribution of p21 and p23 to T cell development and T cell antagonism, we generated selected TCR ζ transgenic mice maintained on the P14 αβ TCR transgenic line such that p23 or both p21 and p23 were selectively eliminated. Importantly, one line (YF1,2) retains the constitutively tyrosine-phosphorylated p21 in the complete absence of inducible p23. We determined that T cell development was uncoupled from p21 and/or p23. Using a series of agonist, weak agonist, and antagonist peptides, we analyzed the role of each of the phosphorylated forms of TCR ζ on T cell activation and antagonism. In this study, we report that the proliferative responses of αβ P14 T cells to agonist peptides and the inhibition of proliferation resulting from antagonist peptide treatments was functionally uncoupled from p21 and/or p23. These results suggest that the mechanism of T cell antagonism is independent of the two phosphorylated TCR ζ derivatives.
Editing of an Immunodominant Epitope of Glutamate Decarboxylase by HLA-DM
Authors: Lich John D. , Jayne Jennifer A. , Zhou Delu , Elliott John F. , Blum Janice S.
Published: April 2014

HLA-DM stabilizes peptide-receptive class II αβ dimers and facilitates the capture of high affinity peptides, thus influencing the peptide repertoire presented by class II molecules. Variations in DM levels may therefore have a profound effect on the antigenic focus of T cell-mediated immune responses. Specifically, DM expression may influence susceptibility and resistance to autoimmune diseases. In this study the role of DM in HLA-DR4-restricted presentation of an insulin-dependent diabetes mellitus autoantigen, glutamate decarboxylase (GAD), was tested. Presentation of immunodominant GAD epitope 273–285 was regulated by endogenous DM levels in human B lymphoblasts. T cell responses to exogenous GAD as well as an endogenous cytoplasmic form of this Ag were significantly diminished with increasing cellular expression of DM. Epitope editing by DM was observed only using Ag and not small synthetic peptides, suggesting that this process occurred within endosomes. Results with cytoplasmic GAD also indicated that peptides from this compartment intersect class II proteins in endocytic vesicles where DM editing was facilitated. Changes in DM levels within APC may therefore influence the presentation of autoantigens and the development of autoimmune disorders such as type I diabetes.