bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential Gene Expression Modulated by the Cytoplasmic Domain of FcγRIa (CD64) α-Chain
Authors: Qin Hongwei , Edberg Jeffrey C. , Gibson Andrew W. , Page Grier P. , Teng Lihong , Kimberly Robert P.
Published: April 2014

The cytoplasmic domain (CY) of the ligand-binding α-chain of the γ-chain-associated FcRs can modulate receptor function such as phagocytosis, endocytosis, and intracellular trafficking of receptor-Ag complexes. To assess the potential role of the CY domain of human FcγRIa (CD64) α-chain in the transcriptional regulation of receptor-induced gene expression, we developed stably transfected murine macrophage cell lines expressing a full-length or a CY deletion mutant (tail-less) of human FcγRIa to analyze gene expression in response to receptor-specific cross-linking. Using the Affymetrix murine genome U74Av2 GeneChip array, we observed >100 candidate genes having ≥2-fold difference expression at 1.5 and 3 h after stimulation. Focusing on several immunologically related genes, we confirmed differential expression of M-CSF, macrophage inhibitory cytokine-1, leukocyte-specific protein 1, MIP-2, and IL-1R antagonist by RT-PCR and RNase protection assays. Analysis of mRNA stability indicated that the differential regulation of gene expression by the CY of the CD64 α-chain is at the level of gene transcription. Our results indicate that the CY of the CD64 α-chain modulates transcriptional activity induced by receptor-specific engagement in macrophages and provides a framework for understanding distinct expression profiles elicited by different Fc γ-chain-associated receptors.
The 3′ Untranslated Region of the Membrane-Bound IL-1R Accessory Protein mRNA Confers Tissue-Specific Destabilization
Authors: Jensen Liselotte E. , Whitehead Alexander S.
Published: April 2014

IL-1α and IL-1β are proinflammatory cytokines that promote activation of intracellular signaling cascades, leading to stabilization of certain mRNAs and activation of transcription factors. IL-1R type I (IL-1RI) binds IL-1α and IL-1β, and subsequent recruitment of the membrane-bound IL-1R accessory protein (mIL-1RAcP) facilitates signal transduction. Two alternatively spliced isoforms, soluble IL-1RAcP (sIL-1RAcP) and sIL-1RAcP-β, which lack transmembrane and intracellular domains, have been described. The sIL-1RAcP and possibly sIL-1RAcP-β can inhibit IL-1 signaling. Proportional expression of the different IL-1RAcP splice variants may be an important determinant of responsiveness to IL-1. We show that although both mIL-1RAcP and sIL-1RAcP mRNAs are widely expressed in human tissue, their relative proportions differ significantly in a tissue-specific manner. Turnover studies revealed that the sIL-1RAcP mRNA has a half-life of ∼48 h in both the kidney cell line 293 and the hepatoma cell line HepG2. The mIL-1RAcP mRNA has a similar half-life in 293 cells, but a considerably shorter half-life of ∼5 h in HepG2 cells. Using luciferase reporter constructs, we demonstrated that this specific destabilization of the mIL-1RAcP mRNA in the latter cell type is mediated by its 2.8-kb 3′-untranslated region. Deletion analysis further established that the cell line-specific instability does not involve AU-rich elements, but is mediated by several novel elements that appear to act independently; such elements may be recognized by proteins expressed specifically in some, but not all, tissues. These data demonstrate that the cellular capacity to respond to IL-1 is tightly regulated in a tissue-specific manner.
Genetic Manipulation of Telomerase in HIV-Specific CD8+ T Cells: Enhanced Antiviral Functions Accompany the Increased Proliferative Potential and Telomere Length Stabilization
Authors: Dagarag Mirabelle , Evazyan Tandik , Rao Nagesh , Effros Rita B.
Published: April 2014

A large proportion of the CD8+ T cell pool in persons chronically infected with HIV consists of cells that show features of replicative senescence, an end stage characterized by irreversible cell cycle arrest, multiple genetic and functional changes, and shortened telomeres. The objective of our research was to determine whether constitutive expression of the gene for the human telomerase (hTERT) can prevent senescence-induced impairments in human virus-specific CD8+ T cells, particularly in the context of HIV-1 disease. Our results indicate that hTERT-expressing HIV-specific CD8+ lymphocytes show both an enhanced and sustained capacity to inhibit HIV-1 replication in in vitro coculture experiments, as well as prolonged ability to produce IFN-γ and TNF-α in response to stimulation with HIV-1-derived peptides, as compared with vector-transduced controls. Loss of CD28 expression, the signature change of replicative senescence in cell culture, was retarded in those CD8+ T cell cultures that had high levels of CD28 at the time of hTERT transduction. These findings suggest that telomere shortening may be the primary driving force behind several aspects of CD8+ T cell dysfunction associated with replicative senescence. We also demonstrate reduced accumulation of the p16INK4a and p21WAF1 cell cycle inhibitors in hTERT-transduced lymphocytes, providing a possible mechanism by which stable hTERT expression is able to circumvent the senescence barrier in CD8+ T cells. Given the key role of CD8+ T cell function in controlling a variety of acute and latent viral infections, approaches to retard the functional decrements associated with replicative senescence may lead to novel types of immunotherapy.
Helminth Infection Protects Mice from Anaphylaxis via IL-10-Producing B Cells
Authors: Mangan Niamh E. , Fallon Rosemary E. , Smith Philip , van Rooijen Nico , McKenzie Andrew N. , Fallon Padraic G.
Published: April 2014

Modulation of the immune system by infection with helminth parasites, including schistosomes, is proposed to reduce the levels of allergic responses in infected individuals. In this study we investigated whether experimental infection with Schistosoma mansoni could alter the susceptibility of mice to an extreme allergic response, anaphylaxis. We formally demonstrate that S. mansoni infection protects mice from an experimental model of systemic fatal anaphylaxis. The worm stage of infection is shown to mediate this protective effect. In vivo depletion studies demonstrated an imperative role for B cells and IL-10 in worm-mediated protection. Furthermore, worm infection of mice increases the frequency of IL-10-producing B cells compared with that in uninfected mice. However, transfer of B cells from worm-infected mice or in vitro worm-modulated B cells to sensitized recipients exacerbated anaphylaxis, which was attributed to the presence of elevated levels of IL-4-producing B cells. Worm-modulated, IL-10-producing B cells from IL-4-deficient, but not IL-5-, IL-9- or IL-13-deficient, mice conferred complete resistance to anaphylaxis when transferred to naive mice. Therefore, we have dissected a novel immunomodulatory mechanism induced by S. mansoni worms that is dependent on an IL-10-producing B cell population that can protect against allergic hypersensitivity. These data support a role for helminth immune modulation in the hygiene hypothesis and further illustrate the delicate balance between parasite induction of protective regulatory (IL-10) responses and detrimental (IL-4) allergic responses.
Apolipoprotein E Modulates Clearance of Apoptotic Bodies In Vitro and In Vivo, Resulting in a Systemic Proinflammatory State in Apolipoprotein E-Deficient Mice
Authors: Grainger David J. , Reckless Jill , McKilligin Elaine
Published: April 2014

Apolipoprotein E (apoE) is a 34-kDa glycoprotein involved in lipoprotein transport through interaction with the low-density lipoprotein receptor and related receptors. Recently, it has become clear that apoE binding to its receptors plays a role both in development and in control of the immune system. In this study, we show that apoE modulates the rate of uptake of apoptotic cells by macrophages. In vitro, apoE-deficient macrophages ingest less apoptotic thymocytes (but not latex beads) than wild-type macrophages, and this defect can be corrected by addition of exogenous apoE protein. In vivo, the number of dying macrophages is increased in a range of tissues, including lung and brain. Possibly in response to the larger numbers of persistent apoptotic bodies, the number of live macrophages in these tissues are also increased compared with those of wild-type control mice. In addition to the significant changes in macrophage population dynamics we observed, levels of the proinflammatory cytokine TNF-α and the positive acute phase reactant fibrinogen are also elevated in the livers from apoE-deficient mice. In contrast, neither deletion of the gene encoding the LDL receptor nor cholesterol feeding of wild-type mice affected either the number of apoptotic bodies or the number of live macrophages. We conclude that apoE deficiency results in impaired clearance of apoptotic cell remnants and a functionally relevant systemic proinflammatory condition in mice, independent of its role in lipoprotein metabolism. Any similar reduction of apoE activity in humans may contribute to the pathogenesis of a wide range of chronic diseases including atherosclerosis, dementia, and osteoporosis.
Polymorphisms in the FcεRIβ Promoter Region Affecting Transcription Activity: A Possible Promoter-Dependent Mechanism for Association between FcεRIβ and Atopy
Authors: Nishiyama Chiharu , Akizawa Yushiro , Nishiyama Makoto , Tokura Tomoko , Kawada Hiroshi , Mitsuishi Kouichi , Hasegawa Masanari , Ito Tomonobu , Nakano Nobuhiro , Okamoto Atsushi , Takagi Atsushi , Yagita Hideo , Okumura Ko , Ogawa Hideoki
Published: April 2014

The β subunit of the high-affinity IgE receptor (FcεRI) plays an important role in IgE-mediated allergic reactions as an amplifier for cell surface expression and signal transduction of FcεRI. FcεRIβ is presumed to be one of the genes linked with atopic diseases. However, the validity of the associations previously found between single nucleotide polymorphisms (SNPs) in FcεRIβ and atopic diseases is questionable. In the present study, we found correlation between the SNP of FcεRIβ at +6960A/G, resulting in a Glu237Gly amino acid substitution, and the cell surface expression level of FcεRI on blood basophils, although it has been shown that the Glu237Gly mutation itself does not affect the surface expression or function of FcεRI. We additionally found four SNPs in the promoter region of FcεRIβ, among which −426T/C and −654C/T were tightly linked with +6960A/G. Reporter plasmids carrying the −426C and −654T promoter displayed higher transcriptional activity than those carrying the −426T and −654C promoter. We found that transcription factor YY1 preferentially bound and transactivated the −654T promoter. Furthermore, expression of FcεRI β-chain mRNA in basophils from individuals who have the minor heterozygous genotype was significantly higher than that of the major homozygous genotype. These results suggest that the SNPs in the FcεRIβ promoter are causally linked with atopy via regulation of FcεRI expression.
Immune Responses of Breast Cancer Patients to Mutated Epidermal Growth Factor Receptor (EGF-RvIII, ΔEGF-R, and de2–7 EGF-R)
Authors: Purev Enkhtsetseg , Cai Dewei , Miller Eric , Swoboda Rolf , Mayer Ted , Klein-Szanto Andres , Marincola Francesco M. , Mick Rosemarie , Otvos Laszlo , Wunner William , Birebent Brigitte , Somasundaram Rajasekharan , Wikstrand Carol J. , Bigner Darell , DeMichele Angela , Acs Geza , Berlin Jesse A. , Herlyn Dorothee
Published: April 2014

Mutated epidermal growth factor receptor (EGF-RvIII, ΔEGF-R, and de2–7 EGF-R) is the result of an 801-bp deletion within the extracellular domain of wild-type EGF-R and is expressed by breast carcinomas, but not by normal breast tissues. EGF-RvIII is expressed both on the surface and in the cytoplasm of tumor cells. Thus, EGF-RvIII is a potential tumor-specific target for both Abs and T cells. However, it is not known whether breast cancer patients can raise immune responses to EGF-RvIII expressed by their tumors. The demonstration of EGF-RvIII-specific immune responses in patients would suggest that immunization of patients with EGF-RvIII vaccines is feasible, because these vaccines may boost a pre-existing immune response. We have evaluated humoral and cellular immune responses to EGF-RvIII in 16 breast cancer patients and three healthy donors. Seven of 16 patients developed EGF-RvIII-specific Abs that bound to isolated EGF-RvIII protein or the protein expressed by EGF-RvIII-transfected mouse fibroblasts. The Abs that bound to EGF-RvIII did not bind to wild-type EGF-R, and anti-EGF-RvIII Abs were not found in the sera of healthy donors. Three patients had EGF-RvIII peptide-specific lymphoproliferative responses, and two of these patients also had humoral immune responses. Humoral and cellular immune responses correlated with EGF-RvIII expression by patients’ tumors in most cases. These studies demonstrate that breast cancer patients specifically recognize EGF-RvIII with an overall immune response rate of 50%, suggesting that patients may benefit from vaccination against EGF-RvIII, boosting pre-existing immune responses.
Constitutive Expression of CCR7 Directs Effector CD8 T Cells into the Splenic White Pulp and Impairs Functional Activity
Authors: Unsoeld Heike , Voehringer David , Krautwald Stefan , Pircher Hanspeter
Published: April 2014

Antigenic stimulation down-regulates CCR7 on effector T cells. To analyze the importance of CCR7 down-regulation, transgenic (tg) mice constitutively expressing CCR7 were generated. CD8 T cells with defined Ag specificity were obtained by breeding CCR7-tg mice with P14 TCR-tg mice specific for lymphocytic choriomeningitis virus. Transgenic CCR7 expression did not impair proliferation of P14.CCR7 T cells induced by lymphocytic choriomeningitis virus infection, but prevented CCR7 down-regulation. Compared with wild-type P14 effector cells, P14.CCR7 effector cells, expressing the CCR7 transgene, were increased in the spleen, but decreased in blood and peripheral tissues. Moreover, P14.CCR7 effector cells localized almost exclusively in the splenic white pulp, whereas P14 effector cells were excluded from splenic white pulp cords and were found preferentially in the red pulp. Functional experiments further revealed that P14.CCR7 effector cells were impaired in rapid viral clearance and in inducing Ag-specific delayed-type hypersensitivity reactions. Thus, the present study demonstrates that down-regulation of CCR7 during CD8 T cell activation is important to release effector cells from the white pulp of the spleen, and highlights the importance of effector cell localization in providing rapid immunity.
p21Cip1 and p27Kip1 Act in Synergy to Alter the Sensitivity of Naive T Cells to TGF-β-Mediated G1 Arrest through Modulation of IL-2 Responsiveness
Authors: Wolfraim Lawrence A. , Walz Thomas M. , James Zakiya , Fernandez Tania , Letterio John J.
Published: April 2014

Induction of G1 arrest by TGF-β correlates with the regulation of p21Cip1 and p27Kip1, members of the Cip/Kip family of cyclin-dependent kinase inhibitors (cki). However, no definitive evidence exists that these proteins play a causal role in TGF-β1-induced growth arrest in lymphocytes. In this report we show the suppression of cell cycle progression by TGF-β is diminished in T cells from mice deficient for both p21Cip1 and p27Kip1 (double-knockout (DKO)) only when activated under conditions of optimal costimulation. Although there is an IL-2-dependent enhanced proliferation of CD8+ T cells from DKO mice, TGF-β is able to maximally suppress the proliferation of DKO T cells when activated under conditions of low costimulatory strength. We also show that the induction of p15Ink4b in T cells stimulated in the presence of TGF-β is not essential, as TGF-β also efficiently suppressed proliferation of T cells from p15Ink4b−/− mice. Finally, although these cki are dispensable for the suppression of T cell proliferation by TGF-β, we now describe a Smad3-dependent down-regulation of cdk4, suggesting a potential mechanism underlying to resistance of Smad3−/− T cells to the induction of growth arrest by TGF-β. In summary, the growth suppressive effects of TGF-β in naive T cells are a function of the strength of costimulation, and alterations in the expression of cki modify the sensitivity to TGF-β by lowering thresholds for a maximal mitogenic response.
Environmental and Endogenous Peroxisome Proliferator-Activated Receptor γ Agonists Induce Bone Marrow B Cell Growth Arrest and Apoptosis: Interactions between Mono(2-ethylhexyl)phthalate, 9-cis-Retinoic Acid, and 15-Deoxy-Δ12,14-prostaglandin J2
Authors: Schlezinger Jennifer J. , Howard Gregory J. , Hurst Christopher H. , Emberley Jessica K. , Waxman David J. , Webster Thomas , Sherr David H.
Published: April 2014

The common commercial use of phthalate esters has resulted in significant human exposure to these bioactive compounds. The facts that phthalate ester metabolites, like endogenous PGs, are peroxisome proliferator-activated receptor (PPAR) agonists, and that PPARγ agonists induce lymphocyte apoptosis suggest that phthalate esters are immunosuppressants that could act together with PGs to modulate early B cell development. In this study we examined the effects of a metabolite of one environmental phthalate, mono(2-ethylhexyl)phthalate (MEHP), and 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2), on developing B cells. MEHP inhibited [3H]thymidine incorporation by primary murine bone marrow B cells and a nontransformed murine pro/pre-B cell line (BU-11). Cotreatment with a retinoid X receptor α ligand, 9-cis-retinoic acid, decreased [3H]thymidine incorporation synergistically, thereby implicating activation of a PPARγ-retinoid X receptor α complex. These results were similar to those obtained with the natural PPARγ ligand 15d-PGJ2. At moderate MEHP concentrations (25 or 100 μM for primary pro-B cells and a pro/pre-B cell line, respectively), inhibition of [3H]thymidine incorporation resulted primarily from apoptosis induction, whereas at lower concentrations, the inhibition probably reflected growth arrest without apoptosis. Cotreatment of bone marrow B cells with 15d-PGJ2 and MEHP significantly enhanced the inhibition of [3H]thymidine incorporation seen with MEHP alone, potentially mimicking exposure in the bone marrow microenvironment where PG concentrations are high. Finally, MEHP- and 15d-PGJ2-induced death does not result from a decrease in NF-κB activation. These data demonstrate that environmental phthalates can cooperate with an endogenous ligand, 15d-PGJ2, to inhibit proliferation of and induce apoptosis in developing bone marrow B cells, potentially via PPARγ activation.
Folate Deficiency Inhibits the Proliferation of Primary Human CD8+ T Lymphocytes In Vitro
Authors: Courtemanche Chantal , Elson-Schwab Ilan , Mashiyama Susan T. , Kerry Nicole , Ames Bruce N.
Published: April 2014

Folate is required for one-carbon transfer reactions and the formation of purines and pyrimidines for DNA and RNA synthesis. Deficiency of folate can lead to many clinical abnormalities, including macrocytic anemia, cardiovascular diseases, birth defects, and carcinogenesis. The nucleotide imbalance due to folate deficiency causes cell cycle arrest in the S phase and uracil misincorporation into DNA, which may result in DNA double-strand breaks during repair. The role of folate in the immune system has not been fully characterized. We cultured PHA-activated human T lymphocytes in varying concentrations of folate, and measured proliferation, cell cycle, apoptosis, uracil misincorporation, and proportions of Th cells (CD4+) and cytotoxic T (CD8+) cells. Folate deficiency reduced proliferation of T lymphocytes, induced cell cycle arrest in the S phase, induced apoptosis, and increased the level of uracil in DNA. Folate deficiency also increased the CD4+ to CD8+ ratio due to a marked reduction of CD8+ cell proliferation. Folate or nucleoside repletion of folate-deficient cells rapidly restored T lymphocyte proliferation and normal cell cycle, reduced the DNA uracil content, and lowered the CD4+ to CD8+ ratio. These data suggest that folate status may affect the immune system by reducing the capacity of CD8+ cells to proliferate in response to activation.
Single-Stranded DNA Breaks Adjacent to Cytosines Occur during Ig Gene Class Switch Recombination
Authors: Arudchandran Arulvathani , Bernstein Ralph M. , Max Edward E.
Published: April 2014

Class switch recombination (CSR) at the DNA level underlies ability of B lymphocytes to switch from expressing IgM to expressing IgG, IgA, or IgE. The mechanism of CSR is largely unknown, but it is clear that CSR is stimulated by T cell signals and is mediated in part by activation-induced deaminase (AID), an enzyme that is also required for somatic hypermutation of Ig genes. In one current model, AID is proposed to initiate CSR by deaminating cytosines in the unpaired nontemplate strand of DNA displaced from its complementary strand by the “sterile” RNA transcript across the switch region. We have used LM-PCR to analyze single-strand breaks in CH12F3-2, a murine cell line that switches in vitro to IgA expression. In contrast to the above model, we have detected CSR-associated ssDNA breaks in the template strand of the H chain α switch region, the strand thought to be complexed with RNA. Most breaks are adjacent to cytosines, consistent with mediation by AID, and occur within the novel consensus sequence C*AG, which occurs much more frequently on the template strand than on the putatively displaced nontemplate strand. These results suggest that AID may target the DNA strand bound to RNA, perhaps resembling APOBEC-3G, a cytosine deaminase related to AID that inhibits HIV replication by mutating viral DNA. Furthermore, the absence of detectable breaks in the nontemplate strand within the DNA segment under study suggests that the two DNA strands are handled differently in the generation or processing of strand breaks.
Arthropod-Derived Histamine-Binding Protein Prevents Murine Allergic Asthma
Authors: Couillin Isabelle , Maillet Isabelle , Vargaftig B. Boris , Jacobs Muazzam , Paesen Guido C. , Nuttall Patricia A. , Lefort Jean , Moser René , Weston-Davies Wynne , Ryffel Bernhard
Published: April 2014

Because histamine receptor type I blockade attenuates allergic asthma, we asked whether complete neutralization of histamine by an arthropod-derived, high affinity histamine-binding protein (EV131) would prevent allergic asthma. Intranasal administration of EV131 given before Ag challenge in immunized mice prevented airway hyperreactivity by 70%, and abrogated peribronchial inflammation, pulmonary eosinophilia, mucus hypersecretion, and IL-4 and IL-5 secretion. Saturation with histamine abrogated the inhibitory effect of EV131 on bronchial hyperreactivity. The inhibitory effect of EV131 on bronchial hyperreactivity was comparable to that of glucocorticosteroids. These results demonstrate that histamine is a critical mediator of allergic asthma. Therefore, complete neutralization of histamine, rather than specific histamine receptor blockade, may have a profound effect on allergic asthma.
Vaccine-Induced Immunity againstHelicobacter pyloriInfection Is Impaired in IL-18-Deficient Mice
Authors: Akhiani Ali A. , Schön Karin , Lycke Nils
Published: April 2014

Protective immunity against Helicobacter pylori infection in mice has been associated with a strong Th1 response, involving IL-12 as well as IFN-γ, but recent studies have also demonstrated prominent eosinophilic infiltration, possibly linked to local Th2 activity in the gastric mucosa. In this study we investigated the role of IL-18, because this cytokine has been found to be a coregulator of Th1 development as well as involved in Th2-type responses with local eotaxin production that could influence gastric eosinophilia and resistance to infection. We found that IL-18−/− mice failed to develop protection after oral immunization with H. pylori lysate and cholera toxin adjuvant, indicating an important role of IL-18 in protection. Well-protected C57BL/6 wild-type (WT) mice demonstrated substantial influx of CD4+ T cells and eosinophilic cells in the gastric mucosa, whereas IL-18−/− mice had less gastritis, few CD4+ T cells, and significantly reduced numbers of eosinophilic cells. T cells in well-protected WT mice produced increased levels of IFN-γ and IL-18 to recall Ag. By contrast, unprotected IL-18−/− mice exhibited significantly reduced gastric IFN-γ and specific IgG2a Ab levels. Despite differences in gastric eosinophilic cell infiltration, protected WT and unprotected IL-18−/− mice had comparable levels of local eotaxin, suggesting that IL-18 influences protection via Th1 development and IFN-γ production rather than through promoting local production of eotaxin and eosinophilic cell infiltration.
Shaping Phenotype, Function, and Survival of Dendritic Cells by Cytomegalovirus-Encoded IL-10
Authors: Raftery Martin J. , Wieland Dörte , Gronewald Stefanie , Kraus Annette A. , Giese Thomas , Schönrich Günther
Published: April 2014

Human dendritic cells (DCs) are essential for the antiviral immune response and represent a strategically important target for immune evasion of viruses, including human CMV (HCMV). Recently, HCMV has been discovered to encode a unique IL-10 homologue (cmvIL-10). In this study we investigated the capacity of cmvIL-10 to shape phenotype, function, and survival of DCs. For comparison we included human IL-10 and another IL-10 homologue encoded by EBV, which does not directly target DCs. Interestingly, cmvIL-10 strongly activated STAT3 in immature DCs despite its low sequence identity with human IL-10. For most molecules cmvIL-10 blocked LPS-induced surface up-regulation, confirming its role as an inhibitor of maturation. However, a small number of molecules on LPS-treated DCs including IDO, a proposed tolerogenic molecule, showed a different behavior and were up-regulated in response to cmvIL-10. Intriguingly, the expression of C-type lectin DC-specific ICAM-grabbing nonintegrin, a receptor for HCMV infection found exclusively on DCs, was also enhanced by cmvIL-10. This phenotypic change was mirrored by the efficiency of HCMV infection. Moreover, DCs stimulated with LPS and simultaneously treated with cmvIL-10 retained the function of immature DCs. Finally, cmvIL-10 increased apoptosis associated with DC maturation by blocking up-regulation of the antiapoptotic long form cellular FLIP. Taken together, these findings show potential mechanisms by which cmvIL-10 could assist HCMV to infect DCs and to impair DC function and survival.
Regulation of Found in Inflammatory Zone 1 Expression in Bleomycin-Induced Lung Fibrosis: Role of IL-4/IL-13 and Mediation via STAT-6
Authors: Liu Tianju , Jin Hong , Ullenbruch Matthew , Hu Biao , Hashimoto Naozumi , Moore Bethany , McKenzie Andrew , Lukacs Nicholas W. , Phan Sem H.
Published: April 2014

Found in inflammatory zone (FIZZ)1, also known as resistin-like molecule α, belongs to a novel class of cysteine-rich secreted protein family, named FIZZ/resistin-like molecule, with unique tissue expression patterns. FIZZ1 is induced in alveolar type II epithelial cells (AECs) in bleomycin (BLM)-induced lung fibrosis, and found to induce myofibroblast differentiation in vitro. The objective of this study was to elucidate the regulation of AEC FIZZ1 expression in pulmonary fibrosis. AECs were isolated from rat lungs and the effects of a number of cytokines on FIZZ1 expression were evaluated by RT-PCR. Of all cytokines examined, only IL-4 and IL-13 were effective in stimulating FIZZ1 expression in AECs. Stimulation by IL-4/IL-13 was accompanied by increases in phosphorylated STAT6 and JAK1. FIZZ1 expression was also stimulated by transfection with a STAT6 expression plasmid, but was inhibited by antisense oligonucleotides directed against STAT6. In vivo studies showed that compared with wild-type controls, both IL-4- and IL-13-deficient mice showed reduced BLM-induced lung FIZZ1 expression and fibrosis, which were essentially abolished in IL-4 and IL-13 doubly deficient mice. Furthermore, STAT6-deficient mice showed marked reduction in BLM-induced lung FIZZ1 expression. Thus, IL-4 and IL-13 are potent inducers of AEC FIZZ1 expression via STAT6 and play key roles in BLM-induced lung FIZZ1 expression and fibrosis. This represents a potential mechanism by which IL-4/IL-13 could play a role in the pathogenesis of lung fibrosis.
A Critical Regulatory Role of Leucin Zipper Transcription Factor c-Maf in Th1-Mediated Experimental Colitis
Authors: Weigmann Benno , Nemetz Andrea , Becker Christoph , Schmidt Jan , Strand Dennis , Lehr Hans A. , Galle Peter R. , Ho I.-Cheng , Neurath Markus F.
Published: April 2014

In this study, we investigated the role of c-Maf, a transcription factor known to induce IL-4 production, in inflammatory bowel diseases and experimental colitis. Although Crohn′s disease (CD) is associated with low IL-4 production by T-bet-expressing Th1 cells in the lamina propria, surprisingly a higher expression of c-Maf in these cells was found as compared with control patients. The relevance of this finding was further evaluated in an animal model of CD induced by adoptive transfer of CD4+CD62L+ T cells in RAG-deficient mice. In this Th1-mediated model, an increase of c-Maf-expressing T lymphocytes in the lamina propria over time was observed. Interestingly, adoptive transfer of c-Maf transgenic CD4+CD62L+ T cells in RAG-1-deficient mice resulted in an IL-4-dependent inability to induce colitis and suppressed colitis activity induced by wild-type CD4+CD62L+ T cells. In contrast, transfer of CD4+CD62L− T cells from c-Maf transgenic, but not wild-type mice induced colitis and augmented colitis induced by CD4+CD62L+ T cells from wild-type mice in an IL-4-independent pathway, as determined by macroscopic, histologic, and endoscopic criteria. This was associated with an accumulation of CD4+ T-bet+ CD25+ effector Th1 cells in the lamina propria of colitic mice. Our results reveal a novel regulatory role of c-Maf in colitis. Although overexpression of c-Maf in naive T cells prevents Th1-mediated colitis, overexpression of c-Maf in memory T-bet+ Th1 cells regulates CD25 expression and augments such colitis. Targeting of c-Maf in memory T cells in CD appears to be an attractive target for therapeutic interventions.
Cyclic Adenosine 5′-Monophosphate Response Element Modulator Is Responsible for the Decreased Expression of c-fos and Activator Protein-1 Binding in T Cells from Patients with Systemic Lupus Erythematosus
Authors: Kyttaris Vasileios C. , Juang Yuang-Taung , Tenbrock Klaus , Weinstein Arthur , Tsokos George C.
Published: April 2014

T cells from patients with systemic lupus erythematosus express increased levels of the cAMP response element modulator (CREM) that has been shown to bind to the IL-2 promoter and suppress its activity. In this study, we demonstrate that CREM binds to the proximal promoter of the c-fos proto-oncogene in live systemic lupus erythematosus T cells and represses its expression following stimulation in vitro. Decreased levels of c-fos protein result in decreased AP-1 activity, as determined in shift assays. Blockade of the translation of CREM mRNA with an antisense CREM vector increases the expression of c-fos and the AP-1 activity. The levels of c-fos mRNA vary with disease activity. We conclude that CREM represses the expression of c-fos and limits the activity of the enhancer AP-1. Thus, CREM is involved indirectly in the modulation of transcriptional regulation of multiple genes including IL-2.
Cutting Edge: Direct Interaction of TLR4 with NAD(P)H Oxidase 4 Isozyme Is Essential for Lipopolysaccharide-Induced Production of Reactive Oxygen Species and Activation of NF-κB
Authors: Park Hye Sun , Jung Hye Young , Park Eun Young , Kim Jaesang , Lee Won Jae , Bae Yun Soo
Published: April 2014

LPS, the primary constituent of the outer membrane of Gram-negative bacteria, is recognized by TLR4. Binding of TLR4 to LPS triggers various cell signaling pathways including NF-κB activation and reactive oxygen species (ROS) production. In this study, we present the data that LPS-induced ROS generation and NF-κB activation are mediated by a direct interaction of TLR4 with (NAD(P)H oxidase 4 (Nox) 4), a protein related to gp91phox (Nox2) of phagocytic cells, in HEK293T cells. Yeast two hybrid and GST pull-down assays indicated that the COOH-terminal region of Nox4 interacted with the cytoplasmic tail of TLR4. Knockdown of Nox4 by transfection of small interference RNA specific to the Nox4 isozyme in HEK293T cells expressing TLR4 along with MD2 and CD14 resulted in inhibition of LPS-induced ROS generation and NF-κB activation. Taken together, these results indicate that direct interaction of TLR4 with Nox4 is involved in LPS-mediated ROS generation and NF-κB activation.
Adoptive Transfer of Paternal Antigen-Hyporesponsive T Cells Induces Maternal Tolerance to the Allogeneic Fetus in Abortion-Prone Matings
Authors: Jin Li-Ping , Li Da-Jin , Zhang Jin-Ping , Wang Ming-Yan , Zhu Xiao-Yong , Zhu Ying , Meng Yi , Yuan Min-Min
Published: April 2014

The embryo expresses paternal Ags foreign to the mother and therefore has been viewed as an allograft. It has been shown that anergic T cells generated by blocking of the CD28/B7 costimulatory pathway with anti B7-1 and anti B7-2 mAbs can be transferred as suppresser cells to prevent allograft rejection. Little is known, however, about the in vivo function of anti-B7-treated T cells after their transfer into abortion-prone mice in the maintenance of materno-fetal tolerance. In the present study, abortion-prone CBA/J females mated with DBA/2 males were administered anti-B7-1 and anti-B7-2 mAbs on day 4 of gestation (murine implantation window). The anti-B7-treated T cells subsequently were adoptively transferred into abortion-prone CBA/J mice. We demonstrated that costimulation blockade with anti-B7 mAbs at the time of implantation resulted in altered allogeneic T cell response and overcame increased maternal rejection to the fetus in the CBA/J×DBA/2 system. The transferred anti-B7-treated T cells appeared to be regulatory, decreasing responsiveness and generating clonal deviation in maternal recipient T cells. The transferred CFSE-labeled T cells were found to reside in the spleen and uterine draining lymph nodes, and a few were localized to the materno-fetal interface of the maternal recipient. Our findings suggest that the anti-B7-treated T cells not only function as potent suppresser cells, but also exert an immunoregulatory effect on the maternal recipient T cells, which cosuppresses maternal rejection to the fetus. This procedure might be considered potentially useful for fetal survival when used as an immunotherapy for human recurrent spontaneous abortion.
Fas-Fas Ligand Interactions Are Essential for the Binding to and Killing of Activated Macrophages by γδ T Cells
Authors: Dalton Jane E. , Howell Gareth , Pearson Jayne , Scott Phillip , Carding Simon R.
Published: April 2014

γδ T cells have a direct role in resolving the host immune response to infection by eliminating populations of activated macrophages. Macrophage reactivity resides within the Vγ1/Vδ6.3 subset of γδ T cells, which have the ability to kill activated macrophages following infection with Listeria monocytogenes (Lm). However, it is not known how γδ T cell macrophage cytocidal activity is regulated, or what effector mechanisms γδ T cells use to kill activated macrophages. Using a macrophage-T cell coculture system in which peritoneal macrophages from naive or Lm-infected TCRδ−/− mice were incubated with splenocytes from wild-type and Fas ligand (FasL)-deficient mice (gld), the ability of Vγ1 T cells to bind macrophages was shown to be dependent upon Fas-FasL interactions. Combinations of anti-TCR and FasL Abs completely abolished binding to and killing of activated macrophages by Vγ1 T cells. In addition, confocal microscopy showed that Fas and the TCR colocalized on Vγ1 T cells at points of contact with macrophages. Collectively, these studies identify an accessory or coreceptor-like function for Fas-FasL that is essential for the interaction of Vγ1 T cells with activated macrophages and their elimination during the resolution stage of pathogen-induced immune responses.
CD59a Is the Primary Regulator of Membrane Attack Complex Assembly in the Mouse
Authors: Baalasubramanian Sivasankar , Harris Claire L. , Donev Rossen M. , Mizuno Masashi , Omidvar Nader , Song Wen-Chao , Morgan B. Paul
Published: April 2014

Gene-deleted mice have provided a potent tool in efforts to understand the roles of complement and complement-regulating proteins in vivo. In particular, mice deficient in the membrane regulators complement receptor 1-related gene/protein y, decay-accelerating factor, or CD59 have demonstrated homeostatic relevance and backcrossing between the strains has revealed cooperativity in regulation. In mouse, genes encoding decay-accelerating factor and CD59 have been duplicated and show differential expression in tissues, complicating interpretation and extrapolation of findings to man. The first described form of CD59, CD59a, is broadly distributed and deletion of the cd59a gene causes a mild hemolytic phenotype with increased susceptibility in complement-mediated disease models. The distribution of the second form, CD59b, was originally described as testis specific, but later by some as widespread. Deletion of the cd59b gene caused a severe hemolytic and thrombotic phenotype. To apply data from these mouse models to man it is essential to know the relative distribution and functional roles of these two forms of CD59. We have generated new specific reagents and used them in sensitive quantitative analyses to comprehensively characterize expression of mRNA and protein and functional roles of CD59a and CD59b in wild-type (wt) and CD59a-negative mice. cd59b mRNA was detected only in testis and, at very low levels, in bone marrow. CD59b protein was present on mature spermatozoa and precursors and, in trace amounts, erythrocytes. Erythrocyte CD59b did not inhibit complement lysis except when CD59a was absent or blocked. These data confirm that CD59a is the primary regulator of complement membrane attack in mouse.
Differential Requirement for Tapasin in the Presentation of Leader- and Insulin-Derived Peptide Antigens to Qa-1b-Restricted CTLs
Authors: Li LiQi , Sullivan Barbara A. , Aldrich Carla J. , Soloski Mark J. , Forman James , Grandea Andres G. , Jensen Peter E. , Van Kaer Luc
Published: April 2014

The loading of MHC class I molecules with peptides involves a variety of accessory proteins, including TAP-associated glycoprotein (tapasin), which tethers empty MHC class I molecules to the TAP peptide transporter. We have evaluated the role of tapasin for the assembly of peptides with the class Ib molecule Qa-1b. In normal cells, Qa-1b is predominantly bound by a peptide, the Qa-1 determinant modifier (Qdm), derived from the signal sequence of class Ia molecules. Our results show that tapasin links Qa-1b to the TAP peptide transporter, and that tapasin facilitates the delivery of Qa-1b molecules to the cell surface. Tapasin was also required for the presentation of endogenous Qdm peptides to Qdm-specific, Qa-1b-restricted CTLs. In sharp contrast, tapasin expression was dispensable for the presentation of an insulin peptide to insulin-specific, Qa-1b-restricted CTL isolated from TCR transgenic mice. However, tapasin deficiency significantly impaired the positive selection of these insulin-specific, Qa-1b-restricted transgenic CD8+ T cells. These findings reveal that tapasin plays a differential role in the loading of Qdm and insulin peptides onto Qa-1b molecules, and that tapasin is dispensable for retention of empty Qa-1b molecules in the endoplasmic reticulum, and are consistent with the proposed peptide-editing function of tapasin.
Expansion and Function of CD8+ T Cells Expressing Ly49 Inhibitory Receptors Specific for MHC Class I Molecules
Authors: Anfossi Nicolas , Robbins Scott H. , Ugolini Sophie , Georgel Philippe , Hoebe Kasper , Bouneaud Cécile , Ronet Catherine , Kaser Arthur , DiCioccio Catherine B. , Tomasello Elena , Blumberg Richard S. , Beutler Bruce , Reiner Steven L. , Alexopoulou Lena , Lantz Olivier , Raulet David H. , Brossay Laurent , Vivier Eric
Published: April 2014

MHC class I-specific Ly49 inhibitory receptors regulate NK cell activation, thereby preventing autologous damage to normal cells. Ly49 receptors are also expressed on a subset of CD8+ T cells whose origin and function remain unknown. We report here that, despite their phenotypic and cytolytic similarities, Ly49+CD8+ T cells and conventional Ly49−CD44high memory-phenotype CD8+ T cells present strikingly distinct features. First, under steady state conditions Ly49+CD8+ T cells are poor cytokine producers (TNF-α and IFN-γ) upon TCR triggering. Second, Ly49+CD8+ T cells are not induced upon various settings of Ag immunization or microbial challenge. However, Ly49 can be induced on a fraction of self-specific CD8+ T cells if CD4+ T cells are present. Finally, the size of the Ly49+CD8+ T cell subset is selectively reduced in the absence of STAT1. These results indicate that Ly49 expression is associated with a differentiation program of cytolytic CD8+ T cells triggered upon chronic antigenic exposure. They further suggest that the size of the Ly49+CD8+ T cell subset marks a history of CD8+ T cell activation that might preferentially result from endogenous inducers of inflammation rather than from microbial infections.
Enhanced Responses of Glycosylphosphatidylinositol Anchor-Deficient T Lymphocytes
Authors: Hazenbos Wouter L. W. , Murakami Yoshiko , Nishimura Jun-ichi , Takeda Junji , Kinoshita Taroh
Published: April 2014

The functions of GPI-anchored proteins in T lymphocyte activation have been controversial. This issue was addressed by studying the responses of T lymphocytes from T lymphocyte-specific GPI anchor-deficient mice to different stimuli that normally allow coligation of TCR and GPI-anchored proteins. Stimulation of GPI anchor-deficient T lymphocytes with ConA induced 2-fold higher proliferative responses than did normal cells. In response to allogeneic stimulation, proliferation of GPI anchor-deficient T lymphocytes was enhanced 2- to 3-fold. The response to ConA of a GPI anchor-deficient anti-OVA T lymphocyte clone generated from these mice was ∼3-fold higher than that of cells from the same clone in which GPI anchor expression was restored by retroviral transduction. The response of the GPI anchor-deficient cloned anti-OVA T lymphocytes to antigenic stimulation was similar to that of the retrovirally restored cells. These results indicate that coligation with GPI-anchored proteins counteracts the response to TCR stimulation by ConA or alloantigen but not protein Ag.
HIV-1 Tat Protein Modulates the Generation of Cytotoxic T Cell Epitopes by Modifying Proteasome Composition and Enzymatic Activity
Authors: Gavioli Riccardo , Gallerani Eleonora , Fortini Cinzia , Fabris Marina , Bottoni Arianna , Canella Alessandro , Bonaccorsi Angela , Marastoni Mauro , Micheletti Fabiola , Cafaro Aurelio , Rimessi Paola , Caputo Antonella , Ensoli Barbara
Published: April 2014

Tat, the trans activation protein of HIV, is produced early upon infection to promote and expand HIV replication and transmission. However, Tat appears to also have effects on target cells, which may affect Ag recognition both during infection and after vaccination. In particular, Tat targets dendritic cells and induces their maturation and Ag-presenting functions, increasing Th1 T cell responses. We show in this work that Tat modifies the catalytic subunit composition of immunoproteasomes in B and T cells either expressing Tat or treated with exogenous biological active Tat protein. In particular, Tat up-regulates latent membrane protein 7 and multicatalytic endopeptidase complex like-1 subunits and down-modulates the latent membrane protein 2 subunit. These changes correlate with the increase of all three major proteolytic activities of the proteasome and result in a more efficient generation and presentation of subdominant MHC-I-binding CTL epitopes of heterologous Ags. Thus, Tat modifies the Ag processing and modulates the generation of CTL epitopes. This may have an impact on both the control of virally infected cells during HIV-1 infection and the use of Tat for vaccination strategies.
An Indispensable Role for STAT1 in IL-27-Induced T-bet Expression but Not Proliferation of Naive CD4+ T Cells
Authors: Kamiya Sadahiro , Owaki Toshiyuki , Morishima Noriko , Fukai Fumio , Mizuguchi Junichiro , Yoshimoto Takayuki
Published: April 2014

IL-27 is a novel IL-12 family member that plays a role in the early regulation of Th1 initiation, induces proliferation of naive CD4+ T cells, and synergizes with IL-12 in IFN-γ production. It has been recently reported that IL-27 induces T-bet and IL-12Rβ2 expression through JAK1/STAT1 activation. In the present study, we further investigated the JAK/STAT signaling molecules activated by IL-27 and also the role of STAT1 in IL-27-mediated responses using STAT1-deficient mice. In addition to JAK1 and STAT1, IL-27-activated JAK2, tyrosine kinase-2, and STAT2, -3, and -5 in naive CD4+ T cells. The activation of STAT2 and STAT5, but not of STAT3, was greatly diminished in STAT1-deficient naive CD4+ T cells. Comparable proliferative response to IL-27 was observed between STAT1-deficient and wild-type naive CD4+ T cells. In contrast, IL-27 hardly induced T-bet and subsequent IL-12Rβ2 expression, and synergistic IFN-γ production by IL-27 and IL-12 was impaired in STAT1-deficient naive CD4+ T cells. Moreover, IL-27 augmented the expression of MHC class I on naive CD4+ T cells in a STAT1-dependent manner. These results suggest that IL-27 activates JAK1 and -2, tyrosine kinase-2, STAT1, -2, -3, and -5 in naive CD4+ T cells and that STAT1 plays an indispensable role in IL-27-induced T-bet and subsequent IL-12Rβ2 expression and MHC class I expression as well but not proliferation, while STAT3 presumably plays an important role in IL-27-induced proliferation.
Unraveling the Consecutive Recombination Events in the HumanIGKLocus
Authors: Langerak Anton W. , Nadel Bertrand , de Torbal Anneke , Wolvers-Tettero Ingrid L. M. , van Gastel-Mol Ellen J. , Verhaaf Brenda , Jäger Ulrich , van Dongen Jacques J. M.
Published: April 2014

In addition to the classical Vκ-Jκ, Vκ-κ deleting element (Kde), and intron-Kde gene rearrangements, atypical recombinations involving Jκ recombination signal sequence (RSS) or intronRSS elements can occur in the Igκ (IGK) locus, as observed in human B cell malignancies. In-depth analysis revealed that atypical JκRSS-intronRSS, Vκ-intronRSS, and JκRSS-Kde recombinations not only occur in B cell malignancies, but rather reflect physiological gene rearrangements present in normal human B cells as well. Excision circle analysis and recombination substrate assays can discriminate between single-step vs multistep rearrangements. Using this combined approach, we unraveled that the atypical Vκ-intronRSS and JκRSS-Kde pseudohybrid joints most probably result from ongoing recombination following an initial aberrant JκRSS-intronRSS signal joint formation. Based on our observations in normal and malignant human B cells, a model is presented to describe the sequential (classical and atypical) recombination events in the human IGK locus and their estimated relative frequencies (0.2–1.0 vs <0.03). The initial JκRSS-intronRSS signal joint formation (except for Jκ1RSS-intronRSS) might be a side event of an active V(D)J recombination mechanism, but the subsequent formation of Vκ-intronRSS and JκRSS-Kde pseudohybrid joints can represent an alternative pathway for IGK allele inactivation and allelic exclusion, in addition to classical Cκ deletions. Although usage of this alternative pathway is limited, it seems essential for inactivation of those IGK alleles that have undergone initial aberrant recombinations, which might otherwise hamper selection of functional Ig L chain proteins.