bluuebunny/crossref_metadata_2025 huggingface dataset:
Human Vγ2Vδ2 T Cells Produce IFN-γ and TNF-α with an On/Off/On Cycling Pattern in Response to Live Bacterial Products
Authors: Wang Lisheng , Das Hiranmoy , Kamath Arati , Bukowski Jack F.
Published: April 2014

Whereas cytokine production in αβ T cells is rapidly regulated by exposure to peptide Ag, the mechanisms regulating cytokine production by γδ T cells are unknown. In this study, we demonstrate that human Vγ2Vδ2 T cells produce IFN-γ and TNF-α as early as 2 h after Ag exposure, and that they produce these cytokines in a dose- and time- dependent manner in response to stimulation with a live bacterial product, iso-butylamine (IBA), but not to dead bacteria or LPS. γδ T cells began, ceased, and then resumed IFN-γ and TNF-α generation in an on/off/on cycling pattern, both in vitro and in vivo, depending on the presence or absence of IBA. IFN-γ and TNF-α, whose optimum production was dependent on IBA-stimulated γδ T cells, were critical for monocyte-mediated killing of Escherichia coli. By limiting cytokine production to periods of direct contact with live bacteria, γδ T cells focus their resources at the site of infection, while limiting systemic immunopathology. Thus, human γδ T cells may mediate innate resistance to extracellular bacteria via tightly regulated cytokine production without necessarily expanding in number.
TCRβ Chain Influences But Does Not Solely Control Autoreactivity of Vα14J281T Cells
Authors: Gui Ming , Li Jin , Wen Li-Jun , Hardy Richard R. , Hayakawa Kyoko
Published: April 2014

CD1d-dependent accumulation of αβ T cells bearing a canonical Vα14Jα281 α-chain (Vα14+ T cells) is thought to model positive selection of lipid-specific T cells, based on their ability to recognize CD1d-presented self glycolipid(s). However, it has been difficult to demonstrate self ligand specificity in this system, as most Vα14+ T cells do not exhibit significant autoreactivity despite high reactivity to α-galactosylceramide presented by CD1d (α-GalCer/CD1d). To assess the role of TCRβ chain in determining the α-GalCer/CD1d vs autoreactive specificity of Vα14+ T cells, we conducted TCRα or TCRβ chain transduction experiments. In this study we demonstrate, by combining different TCRβ chains with the Vα14 α-chain in retrovirally transduced T cell lines, that the Vα14 α-chain plays a primary role, necessary but not sufficient for imparting α-GalCer/CD1d recognition. β-Chain usage alone is not the sole factor that controls the extent of autoreactivity in Vα14+ T cells, since transduction of TCRαβ chains from a high CD1d autoreactive Vα14+ T cell line conferred the α-GalCer/CD1d specificity without induction of autoreactivity. Thus, heterogeneity of Vα14+ T cell reactivity is due to both β-chain diversity and control mechanism(s) beyond primary TCR structure.
Evidence for a Compartmentalized B Cell Response as Characterized by IgG Epitope Specificity in Human Ocular Toxoplasmosis
Authors: Klaren Vincent N. A. , Peek Ron
Published: April 2014

Infectious agents in the eye induce both a local and a systemic humoral immune response. Previously, differences in Ag recognition were observed between systemic and ocular derived IgG of patients with ocular toxoplasmosis. This finding implied a nonrandom distribution of IgG-producing B cells in the inflamed eye. In the present study, we compared the intraocular and systemic B cell responses of patients with ocular toxoplasmosis to a single Toxoplasma gondii Ag. Two series of C-terminally deleted recombinant T. gondii GRA-2 proteins were constructed to delineate IgG B cell epitopes of paired ocular and serum samples. Differences in epitope region recognition between the ocular and systemic compartment were detected in 9 of 13 patients. The difference in distribution of GRA-2 epitopes between paired samples is indicative of a local GRA-2 specific B cell population functionally different from the systemic GRA-2-specific B cell population. Our results suggest a selective activation of a subset of B cells locally in nonlymphoid tissue.
Receptor Modulation by FcγRI-Specific Fusion Proteins Is Dependent on Receptor Number and Modified by IgG
Authors: Guyre Cheryl A. , Keler Tibor , Swink Sharon L. , Vitale Laura A. , Graziano Robert F. , Fanger Michael W.
Published: April 2014

The high-affinity IgG receptor, FcγRI (CD64), is constitutively expressed exclusively on professional APCs. Human FcγRI binds monomeric IgG with high affinity and is, therefore, saturated in vivo. The binding of IgG to FcγRI causes receptor recycling, while Abs that cross-link FcγRI cause rapid down-modulation of surface FcγRI. Because studies performed in the absence of ligand may not be representative of FcγRI modulation in vivo, we investigated the ability of FcγRI-cross-linking Abs and non-cross-linking derivatives to modulate FcγRI in the presence and absence of ligand. In the absence of ligand mAb H22 and wH22xeGFP, an enhanced green fluorescent protein (eGFP)-labeled fusion protein of H22, cross-linked and rapidly down-modulated surface FcγRI on the human myeloid cell line, U937, and its high FcγRI-expressing subclone, 10.6. This effect was dependent on the concentration of fusion protein and the level of FcγRI expression and correlated with internalization of both wH22xeGFP and FcγRI, itself, as assessed by confocal microscopy. A single-chain Fv version, sFv22xeGFP, which does not cross-link FcγRI, was unable to modulate FcγRI in the absence of IgG. However, if ligand was present, treatment with either monovalent or cross-linking fusion protein led to intracellular receptor accumulation. These findings suggest at least two alternate mechanisms of internalization that are influenced by ligand and demonstrate the physiologic potential of FcγRI to transport a large antigenic load into APCs for processing. These studies may lead to the development of better FcγRI-targeted vaccines, as well as therapies to down-modulate FcR involved in autoimmune diseases.
The Amount of Scurfin Protein Determines Peripheral T Cell Number and Responsiveness
Authors: Khattri Roli , Kasprowicz Deborah , Cox Tom , Mortrud Marty , Appleby Mark W. , Brunkow Mary E. , Ziegler Steven F. , Ramsdell Fred
Published: April 2014

In the absence of the recently identified putative transcription factor scurfin, mice develop a lymphoproliferative disorder resulting in death by 3 wk of age from a pathology that resembles TGF-β or CTLA-4 knockout mice. In this report, we characterize mice that overexpress the scurfin protein and demonstrate that these animals have a dramatically depressed immune system. Mice transgenic for the Foxp3 gene (which encodes the scurfin protein) have fewer T cells than their littermate controls, and those T cells that remain have poor proliferative and cytolytic responses and make little IL-2 after stimulation through the TCR. Although thymic development appears normal in these mice, peripheral lymphoid organs, particularly lymph nodes, are relatively acellular. In a separate transgenic line, forced expression of the gene specifically in the thymus can alter thymic development; however, this does not appear to affect peripheral T cells and is unable to prevent disease in mice lacking a functional Foxp3 gene, indicating that the scurfin protein acts on peripheral T cells. The data indicate a critical role for the Foxp3 gene product in the function of the immune system, with both the number and functionality of peripheral T cells under the aegis of the scurfin protein.
SF20/IL-25, a Novel Bone Marrow Stroma-Derived Growth Factor That Binds to Mouse Thymic Shared Antigen-1 and Supports Lymphoid Cell Proliferation
Authors: Tulin Edgardo E. , Onoda Nobuhisa , Nakata Yasuhiko , Maeda Masatsugu , Hasegawa Masakazu , Nomura Hitoshi , Kitamura Toshio
Published: April 2014

Using a forward genetic approach and phenotype-based complementation screening to search for factors that stimulate cell proliferation, we have isolated a novel secreted bone marrow stroma-derived growth factor, which we termed SF20/IL-25. This protein signals cells to proliferate via its receptor, which we have identified as mouse thymic shared Ag-1 (TSA-1). Enforced expression of TSA-1 in IL-3-dependent Ba/F3 cells that do not express endogenous TSA-1 rendered cells to proliferate in a dose-dependent manner when stimulated with SF20/IL-25. FDCP2, a factor-dependent hemopoietic cell line that expresses endogenous TSA-1, could also be stimulated to proliferate with SF20/IL-25. Binding of SF20 to TSA-1 was blocked by anti-TSA-1 Ab and SF20-induced proliferation of TSA-1-expressing cells was inhibited by anti-TSA-1. In vitro assay revealed that SF20/IL-25 has no detectable myelopoietic activity but supports proliferation of cells in the lymphoid lineage.
The Association of Aiolos Transcription Factor and Bcl-xL Is Involved in the Control of Apoptosis
Authors: Rebollo Angelita , Ayllón Verónica , Fleischer Aarne , Martínez-A. Carlos , Zaballos Angel
Published: April 2014

We have analyzed the mechanism implicated in the control of the anti-apoptotic role of Bcl-xL. We show that IL-4 deprivation induces apoptosis, but does not modulate Bcl-xL expression. Because Bcl-xL does not promote cell survival in the absence of IL-4, we investigate the mechanism by which Bcl-xL was unable to inhibit apoptosis. Using yeast two-hybrid system, coimmunoprecipitation, and indirect immunofluorescence techniques, we found that Bcl-xL interacts with the transcription factor Aiolos in IL-4-stimulated cells, increasing upon IL-4 deprivation. IL-4 does not promote translocation of Aiolos or Bcl-xL, but induces tyrosine phosphorylation of Aiolos, which is required for dissociation from Bcl-xL. Transfection experiments confirm that cells overexpressing Bcl-xL are able to prevent apoptosis in the absence of IL-4. On the contrary, cells that overexpress Bcl-xL and Aiolos are unable to block apoptosis in the absence of IL-4. We propose a model for the regulation of the Bcl-xL anti-apoptotic role via Aiolos.
Characterization of a CD40-Dominant Inhibitory Receptor Mutant
Authors: Mehl Anja M. , Jones Matthew , Rowe Martin , Brennan Paul
Published: April 2014

CD40 is an important mediator of immune and inflammatory responses. It is a costimulatory molecule for B cell proliferation and survival. Blockade of CD40 has been shown to induce tolerance and its role in other pathogenic conditions has led to the proposal that CD40 inhibition could be valuable therapeutically. As a first step to this end, we have characterized a CD40-dominant negative receptor. This inhibitory mutant lacks the identified CD40 signaling domains. It inhibits both cotransfected and endogenous CD40 activation of NF-κB. This mutant is specific, as it does not affect TNF or latent membrane protein 1 signaling. Its potential usefulness is illustrated by its ability to inhibit the CD40 ligand-stimulated increases of HLA and CD54 expression, molecules involved in Ag recognition and lymphocyte recruitment leading to organ rejection. The inhibitory mutant has no TNFR-associated factor 2-binding capabilities and inhibits the recruitment of TNFR-associated factor 2 to the CD40 signaling complex after stimulation. These studies show that the CD40 inhibitory receptor molecule is effective, specific, and useful both for research and potentially as a clinical tool. And furthermore, it is likely that similar dominant inhibitory receptors can be generated for all of the members of the TNFR superfamily.
A Novel NF-κB/Rel Site in Intron 1 Cooperates with Proximal Promoter Elements to Mediate TNF-α-Induced Transcription of the Human Polymeric Ig Receptor
Authors: Schjerven Hilde , Brandtzaeg Per , Johansen Finn-Eirik
Published: April 2014

Secretory Abs constitute the first line of specific immune defense at mucosal surfaces. Such Abs are generated by the active transport of polymeric Ig (pIg) across secretory epithelia mediated by the pIgR, also known as transmembrane secretory component (SC). The proinflammatory cytokine TNF-α is a key mediator of host responses to infections, and it can stimulate protein synthesis-dependent transcriptional up-regulation of pIgR/SC in the HT-29 intestinal adenocarcinoma cell line. By reporter gene assay we identified a novel TNF-α-responsive region located within a 748-bp fragment in intron 1 of the human pIgR/SC gene which depended on an NF-κB/Rel site for full responsiveness. EMSAs demonstrated preferential binding of the NF-κB/Rel family member p65 (RelA) to this DNA element after TNF-α stimulation, with weaker and more delayed binding of p50. Furthermore, the TNF-α-responsive region in intron 1 required cooperation with DNA elements located in the proximal promoter region of the gene. Mutational analysis demonstrated that an IFN-stimulated response element near the transcriptional start site in exon 1 was involved in the TNF-α responsiveness. Thus, DNA elements located >4 kb apart were found to cooperate in TNF-α-induced pIgR/SC up-regulation. The intronic TNF-α-responsive enhancer overlapped with a recently identified IL-4-responsive enhancer. Several intronic DNA elements found to be functionally important in the human gene are highly conserved between the human and mouse pIgR/SC genes, suggesting the presence of a conserved cytokine-responsive enhancer region.
The Carboxyl Terminus of the Granulocyte Colony- Stimulating Factor Receptor, Truncated in Patients with Severe Congenital Neutropenia/Acute Myeloid Leukemia, Is Required for SH2-Containing Phosphatase-1 Suppression of Stat Activation
Authors: Dong Fan , Qiu Yaling , Yi Taolin , Touw Ivo P. , Larner Andrew C.
Published: April 2014

The G-CSF receptor transduces signals that regulate the proliferation, differentiation, and survival of myeloid cells. A subgroup of patients with severe congenital neutropenia (SCN) has been shown to harbor mutations in the G-CSF receptor gene that resulted in the truncation of the receptor’s carboxyl-terminal region. SCN patients with mutations in the G-CSF receptor gene are predisposed to acute myeloid leukemia. The truncated receptors from SCN/acute myeloid leukemia patients mediate augmented and sustained activation of Stat transcription factors and are accordingly hyperactive in inducing cell proliferation and survival but are defective in inducing differentiation. Little is known about the molecular mechanisms underlying the negative role of the receptor’s carboxyl terminus in the regulation of Stat activation and cell proliferation/survival. In this study, we provide evidence that SH2-containing phosphatase-1 (SHP-1) plays a negative regulatory role in G-CSF-induced Stat activation. We also demonstrate that the carboxyl terminus of the G-CSF receptor is required for SHP-1 down-regulation of Stat activation induced by G-CSF. Our results indicate further that this regulation is highly specific because SHP-1 has no effect on the activation of Akt and extracellular signal-related kinase1/2 by G-CSF. The data together strongly suggest that SHP-1 may represent an important mechanism by which the carboxyl terminus of the G-CSF receptor down-regulates G-CSF-induced Stat activation and thereby inhibits cell proliferation and survival in response to G-CSF.
Efficient Delivery of Antennapedia Homeodomain Fused to CTL Epitope with Liposomes into Dendritic Cells Results in the Activation of CD8+ T Cells
Authors: Chikh Ghania G. , Kong Spencer , Bally Marcel B. , Meunier Jean-Claude , Schutze-Redelmeier Marie-Paule M.
Published: April 2014

The in vivo induction of a CTL response using Antennapedia homeodomain (AntpHD) fused to a poorly immunogenic CTL epitope requires that the Ag is given in presence of SDS, an unacceptable adjuvant for human use. In the present report, we developed a hybrid CTL epitope delivery system consisting of AntpHD peptide vector formulated in liposomes as an alternative approach to bypass the need for SDS. It is proposed that liposomes will prevent degradation of the Ag in vivo and will deliver AntpHD recombinant peptide to the cytosol of APCs. We show in this work that dendritic cells incubated with AntpHD-fused peptide in liposomes can present MHC class I-restricted peptide and induce CTL response with a minimal amount of Ag. Intracellular processing studies have shown that encapsulated AntpHD recombinant peptide is endocytized before entering the cytosol, where it is processed by the proteasome complex. The processed liposomal peptides are then transported to the endoplasmic reticulum. The increase of the CTL response induced by AntpHD-fused peptide in liposomes correlates with this active transport to the class I-processing pathway. In vivo studies demonstrated that positively charged liposomes increase the immunogenicity of AntpHD-Cw3 when injected s.c. in mice in comparison to SDS. Moreover, addition of CpG oligodeoxynucleotide immunostimulatory sequences further increase the CD8+ T cell response. This strategy combining lipid-based carriers with AntpHD peptide to target poorly immunogenic Ags into the MHC class I processing pathway represents a novel approach for CTL vaccines that may have important applications for development of cancer vaccines.
Two Listeria monocytogenes Vaccine Vectors That Express Different Molecular Forms of Human Papilloma Virus-16 (HPV-16) E7 Induce Qualitatively Different T Cell Immunity That Correlates with Their Ability to Induce Regression of Established Tumors Immortalized by HPV-16
Authors: Gunn George R. , Zubair Abba , Peters Christian , Pan Zhen-Kun , Wu Tzyy-Choou , Paterson Yvonne
Published: April 2014

Two recombinant Listeria monocytogenes (rLm) strains were produced that secrete the human papilloma virus-16 (HPV-16) E7 protein expressed in HPV-16-associated cervical cancer cells. One, Lm-E7, expresses and secretes E7 protein, whereas a second, Lm-LLO-E7, secretes E7 as a fusion protein joined to a nonhemolytic listeriolysin O (LLO). Lm-LLO-E7, but not Lm-E7, induces the regression of the E7-expressing tumor, TC-1, established in syngeneic C57BL/6 mice. Both recombinant E7-expressing rLm vaccines induce measurable anti-E7 CTL responses that stain positively for H-2Db E7 tetramers. Depletion of the CD8+ T cell subset before treatment abrogates the ability of Lm-LLO-E7 to impact on tumor growth. In addition, the rLm strains induce markedly different CD4+ T cell subsets. Depletion of the CD4+ T cell subset considerably reduces the ability of Lm-LLO-E7 to eliminate established TC-1 tumors. Surprisingly, the reverse is the case for Lm-E7, which becomes an effective anti-tumor immunotherapeutic in mice lacking this T cell subset. Ab-mediated depletion of TGF-β and CD25+ cells improves the effectiveness of Lm-E7 treatment, suggesting that TGF-β and CD25+ cells are in part responsible for this suppressive response. CD4+ T cells from mice immunized with Lm-E7 are capable of suppressing the ability of Lm-LLO-E7 to induce the regression of TC-1 when transferred to tumor-bearing mice. These studies demonstrate the complexity of L. monocytogenes-mediated tumor immunotherapy targeting the human tumor Ag, HPV-16 E7.
Involvement of Nicotinic Acetylcholine Receptors in Suppression of Antimicrobial Activity and Cytokine Responses of Alveolar Macrophages to Legionella pneumophila Infection by Nicotine
Authors: Matsunaga Kazuto , Klein Thomas W. , Friedman Herman , Yamamoto Yoshimasa
Published: April 2014

Although nicotine is thought to be one of the major immunomodulatory components of cigarette smoking, how nicotine alters the host defense of the lung and, in particular, immune responses of alveolar macrophages, which are critical effector cells in the lung defense to infection, is poorly understood. Nicotinic acetylcholine receptors (nAChRs) are the receptor for nicotine and may be involved in the modulation of macrophage function by nicotine. In this study, therefore, nicotine-induced suppression of antimicrobial activity and cytokine responses of alveolar macrophages mediated by nAChRs to Legionella pneumophila, a causative agent for pneumonia, were examined. The murine MH-S alveolar macrophage cell line cells expressed the messages for α4 and β2 subunits of nAChRs, but not α7 subunits, determined by RT-PCR. The nicotine treatment of MH-S alveolar macrophages after infection with L. pneumophila significantly enhanced the replication of bacteria in the macrophages and selectively down-regulated the production of IL-6, IL-12, and TNF-α, but not IL-10, induced by infection. These effects were completely blocked by a nonselective antagonist, d-tubocurarine, for nAChRs, but not by a selective antagonist, α-bungarotoxin, for α7-nAChRs. Furthermore, the stimulation of nAChRs with another agonist, 1,1-dimethyl-4-phenylpiperazinium iodide, showed the same effects, which were blocked by the antagonist d-tubocurarine, on the bacterial replication and cytokine regulation with that of nicotine. Thus, the results revealed that nAChRs, the major exogenous ligands of which are nicotine, are involved in the regulation of macrophage immune function by nicotine and may contribute to the cigarette-induced risk factors for respiratory infections in smokers.
Monocyte Chemoattractant Protein-1-Dependent Increase of Vα14 NKT Cells in Lungs and Their Roles in Th1 Response and Host Defense in Cryptococcal Infection
Authors: Kawakami Kazuyoshi , Kinjo Yuki , Uezu Kaori , Yara Satomi , Miyagi Kazuya , Koguchi Yoshinobu , Nakayama Toshinori , Taniguchi Masaru , Saito Atsushi
Published: April 2014

To elucidate the role of NKT cells in the host defense to cryptococcal infection, we examined the proportion of these cells, identified by the expression of CD3 and NK1.1, in lungs after intratracheal infection with Cryptococcus neoformans. This population increased on day 3 after infection, reached a peak level on days 6–7, and decreased thereafter. In Vα14 NKT cell-deficient mice, such increase was significantly attenuated. The proportion of Vα14 NKT cells, detected by binding to α-galactosylceramide-loaded CD1d tetramer, and the expression of Vα14 mRNA increased after infection with a similar kinetics. The delayed-type hypersensitivity response and differentiation of the fungus-specific Th1 cells was reduced in Vα14 NKT cell-deficient mice, compared with control mice. Additionally, elimination of this fungal pathogen from lungs was significantly delayed in Vα14 NKT cell-deficient mice. Production of monocyte chemoattractant protein (MCP)-1 in lungs, detected at both mRNA and protein levels, increased on day 1, reached a peak level on day 3, and decreased thereafter, which preceded the increase in NKT cells. Finally, the increase of total and Vα14+ subset of NKT cells after infection was significantly reduced in MCP-1-deficient mice. Our results demonstrated that NKT cells, especially Vα14+ subset, accumulated in a MCP-1-dependent manner in the lungs after infection with C. neoformans and played an important role in the development of Th1 response and host resistance to this fungal pathogen.
Differential Regulation of Nitric Oxide Synthase-2 and Arginase-1 by Type 1/Type 2 Cytokines In Vivo: Granulomatous Pathology Is Shaped by the Pattern ofl-Arginine Metabolism
Authors: Hesse Matthias , Modolell Manuel , La Flamme Anne C. , Schito Marco , Fuentes José Manuel , Cheever Allen W. , Pearce Edward J. , Wynn Thomas A.
Published: April 2014

Type 2 cytokines regulate fibrotic liver pathology in mice infected with Schistosoma mansoni. Switching the immune response to a type 1-dominant reaction has proven highly effective at reducing the pathologic response. Activation of NOS-2 is critical, because type 1-deviated/NO synthase 2 (NOS-2)-deficient mice completely fail to control their response. Here, we demonstrate the differential regulation of NOS-2 and arginase type 1 (Arg-1) by type 1/type 2 cytokines in vivo and for the first time show a critical role for arginase in the pathogenesis of schistosomiasis. Using cytokine-deficient mice and two granuloma models, we show that induction of Arg-1 is type 2 cytokine dependent. Schistosome eggs induce Arg-1, while Mycobacterium avium-infected mice develop a dominant NOS-2 response. IFN-γ suppresses Arg-1 activity, because type 1 polarized IL-4/IL-10-deficient, IL-4/IL-13-deficient, and egg/IL-12-sensitized animals fail to up-regulate Arg-1 following egg exposure. Notably, granuloma size decreases in these type-1-deviated/Arg-1-unresponsive mice, suggesting an important regulatory role for Arg-1 in schistosome egg-induced pathology. To test this hypothesis, we administered difluoromethylornithine to block ornithine-aminodecarboxylase, which uses the product of arginine metabolism, l-ornithine, to generate polyamines. Strikingly, granuloma size and hepatic fibrosis increased in the ornithine-aminodecarboxylase-inhibited mice. Furthermore, we show that type 2 cytokine-stimulated macrophages produce proline under strict arginase control. Together, these data reveal an important regulatory role for the arginase biosynthetic pathway in the regulation of inflammation and demonstrate that differential activation of Arg-1/NOS-2 is a critical determinant in the pathogenesis of granuloma formation.
Forced Expression of Murine IL-17E Induces Growth Retardation, Jaundice, a Th2-Biased Response, and Multiorgan Inflammation in Mice
Authors: Pan Guohua , French Dorothy , Mao Weiguang , Maruoka Miko , Risser Philip , Lee James , Foster Jessica , Aggarwal Sudeepta , Nicholes Katrina , Guillet Susan , Schow Peter , Gurney Austin L.
Published: April 2014

IL-17 is a proinflammatory cytokine, and its in vivo expression induces neutrophilia in mice. IL-17E is a recently described member of an emerging family of IL-17-related cytokines. IL-17E has been shown to bind IL-17Rh1, a protein distantly related to the IL-17R, suggesting that IL-17E probably possesses unique biological functions. In this study, we have identified the murine ortholog of IL-17E and developed transgenic mice to characterize its actions in vivo. Biological consequences of overexpression of murine (m)IL-17E, both unique to IL-17E and similar to IL-17, were revealed. Exposure to mIL-17E resulted in a Th2-biased response, characterized by eosinophilia, increased serum IgE and IgG1, and a Th2 cytokine profile including elevated serum levels of IL-13 and IL-5 and elevated gene expression of IL-4, IL-5, IL-10, and IL-13 was observed in many tissues. Increased gene expression of IFN-γ in several tissues and elevated serum TNF-α were also noted. In addition, IL-17E induces G-CSF production in vitro and mIL-17E-transgenic mice had increased serum G-CSF and exhibit neutrophilia, a property shared by IL-17. Moreover, exposure to mIL-17E elicited pathological changes in multiple tissues, particularly liver, heart, and lungs, characterized by mixed inflammatory cell infiltration, epithelial hyperplasia, and hypertrophy. Taken together, these findings suggest that IL-17E is a unique pleiotropic cytokine and may be an important mediator of inflammatory and immune responses.
Surfactant Protein A Regulates Complement Activation
Authors: Watford Wendy T. , Wright Jo Rae , Hester C. Garren , Jiang Haixiang , Frank Michael M.
Published: April 2014

Complement proteins aid in the recognition and clearance of pathogens from the body. C1, the first protein of the classical pathway of complement activation, is a calcium-dependent complex of one molecule of C1q and two molecules each of C1r and C1s, the serine proteases that cleave complement proteins. Upon binding of C1q to Ag-bound IgG or IgM, C1r and C1s are sequentially activated and initiate the classical pathway of complement. Because of structural and functional similarities between C1q and members of the collectin family of proteins, including pulmonary surfactant protein A (SP-A), we hypothesized that SP-A may interact with and regulate proteins of the complement system. Previously, SP-A was shown to bind to C1q, but the functional significance of this interaction has not been investigated. Binding studies confirmed that SP-A binds directly to C1q, but only weakly to intact C1. Further investigation revealed that the binding of SP-A to C1q prevents the association of C1q with C1r and C1s, and therefore the formation of the active C1 complex required for classical pathway activation. This finding suggests that SP-A may share a common binding site for C1r and C1s or Clq. SP-A also prevented C1q and C1 from binding to immune complexes. Furthermore, SP-A blocked the ability of C1q to restore classical pathway activity to C1q-depleted serum. SP-A may down-regulate complement activity through its association with C1q. We hypothesize that SP-A may serve a protective role in the lung by preventing C1q-mediated complement activation and inflammation along the delicate alveolar epithelium.
IFN-γ Affects Homing of Diabetogenic T Cells
Authors: Savinov Alexei Y. , Wong F. Susan , Chervonsky Alexander V.
Published: April 2014

IFN-γ is a cytokine with pleiotropic functions that participates in immune and autoimmune responses. The lack of IFN-γ is known to delay the development of autoimmune diabetes in nonobese diabetic (NOD) mice. Splenocytes from diabetic NOD and IFN-γ knockout (KO) NOD mice transfer diabetes into NOD recipients equally well. However, adoptive transfer of diabetogenic T cells from NOD mice into NOD.IFN-γ-KO or NOD mice lacking β-chain of IFN-γ receptor (NOD.IFN-γRβ-KO) appeared to be much less efficient. We found that IFN-γ influences the ability of diabetogenic cells to penetrate pancreatic islets. Tracing in vivo of insulin-specific CD8+ T cells has shown that homing of these cells to the islets of Langerhans was affected by the lack of IFN-γ. While adhesion of insulin-specific CD8+ cells to microvasculature was normal, the diapedesis was significantly impaired. This effect was reversible by treatment of the animals with rIFN-γ. Thus, IFN-γ may, among other effects, influence immune and autoimmune responses by supporting the homing of activated T cells.
Expression and Regulation of Fas and Fas Ligand on Thyrocytes and Infiltrating Cells During Induction and Resolution of Granulomatous Experimental Autoimmune Thyroiditis
Authors: Wei Yongzhong , Chen Kemin , Sharp Gordon C. , Yagita Hideo , Braley-Mullen Helen
Published: April 2014

Granulomatous experimental autoimmune thyroiditis (G-EAT) is induced by mouse thyroglobulin-sensitized spleen cells activated in vitro with mouse thyroglobulin, anti-IL-2R, and IL-12. G-EAT lesions reach maximal severity 19–21 days after cell transfer, and lesions almost completely resolve by day 35. Depletion of CD8+ cells delays resolution and reduces Fas ligand (FasL) mRNA expression in thyroids. This study was undertaken to analyze Fas and FasL protein expression in the thyroid during induction and resolution of G-EAT and to determine whether CD8+ cells might regulate Fas or FasL expression in the thyroid. Fas and FasL expression was analyzed by immunohistochemical staining or in situ hybridization in thyroids of mice with or without depletion of CD8+ cells. Fas and FasL proteins were not detectable in normal thyroids, but expression of both proteins increased during development of G-EAT. Fas was expressed primarily by inflammatory cells; some enlarged thyrocytes were also Fas+. Thyrocytes had intense FasL immunoreactvity, and many CD8+ cells were also FasL positive. Depletion of CD8+ cells resulted in decreased FasL expression by thyrocytes and inflammatory cells, but had no effect on Fas expression. TUNEL assay detected many apoptotic inflammatory cells in proximity to thyrocytes. CD8-depleted thyroids had ongoing inflammation with fewer apoptotic infiltrating cells at day 35. Administration of a neutralizing anti-FasL mAb had no apparent effects on development of G-EAT, but anti-FasL was as effective as anti-CD8 in preventing G-EAT resolution. These results suggested that CD8+ T cells and thyrocytes may kill inflammatory cells through the Fas pathway, contributing to G-EAT resolution.
Differential Secretion of Fas Ligand- or APO2 Ligand/TNF-Related Apoptosis-Inducing Ligand-Carrying Microvesicles During Activation-Induced Death of Human T Cells
Authors: Monleón Inmaculada , Martínez-Lorenzo María José , Monteagudo Luis , Lasierra Pilar , Taulés Marta , Iturralde María , Piñeiro Andrés , Larrad Luis , Alava María Angeles , Naval Javier , Anel Alberto
Published: April 2014

Preformed Fas ligand (FasL) and APO2 ligand (APO2L)/TNF-related apoptosis-inducing ligand (TRAIL) are stored in the cytoplasm of the human Jurkat T cell line and of normal human T cell blasts. The rapid release of these molecules in their bioactive form is involved in activation-induced cell death. In this study, we show by confocal microscopy that FasL and APO2L/TRAIL are mainly localized in lysosomal-like compartments in these cells. We show also by immunoelectron microscopy that FasL and APO2L/TRAIL are stored inside cytoplasmic compartments ∼500 nm in diameter, with characteristics of multivesicular bodies. Most of these compartments share FasL and APO2L/TRAIL, although exclusive APO2L/TRAIL labeling can be also observed in separate compartments. Upon PHA activation, the mobilization of these compartments toward the plasma membrane is evident, resulting in the secretion of the internal microvesicles loaded with FasL and APO2L/TRAIL. In the case of activation with anti-CD59 mAb, the secretion of microvesicles labeled preferentially with APO2L/TRAIL predominates. These data provide the basis of a new and efficient mechanism for the rapid induction of autocrine or paracrine cell death during immune regulation and could modify the interpretation of the role of FasL and APO2L/TRAIL as effector mechanisms in physiological and pathological situations.
Regulation and Possible Function of β-Catenin in Human Monocytes
Authors: Thiele Andrea , Wasner Mark , Müller Claudia , Engeland Kurt , Hauschildt Sunna
Published: April 2014

In this study, we demonstrate that adherence factors, serum constituents, LPS, and zymosan are capable of inducing a cellular accumulation of β-catenin in human monocytes. Whereas adherence-dependent accumulation of β-catenin can be blocked by wortmannin, an inhibitor of phosphatidylinositol 3-kinase, accumulation induced by the remaining stimuli cannot be prevented by inhibition of phosphatidylinositol 3-kinase, implying the involvement of β-catenin in other not yet described signal transduction pathways. A role of β-catenin in adherence-dependent processes by interacting with classical cadherins can be excluded as we could not detect cadherins in monocytes. To test whether it is possible that β-catenin interacts with LEF/TCF (lymphoid enhancer factor/T cell factor) transcription factors, we studied the expression of this protein family. TCF-4 was identified as the LEF/TCF transcription factor present in human monocytes. However, neither cellular induction of β-catenin nor cotransfection experiments with β-catenin conducted in the monocytic cell line THP-1 resulted in the activation of a LEF/TCF-dependent promoter, suggesting the requirement of additional signals. Concurrent with this suggestion, we found that LPS and zymosan, two physiological inducers of β-catenin, caused an increase in the expression of genes that are positively regulated by β-catenin.
TNF Type 2 Receptor (p75) Lowers the Threshold of T Cell Activation
Authors: Kim Edward Y. , Teh Hung-Sia
Published: April 2014

T cell activation requires a threshold amount of TCR-mediated signals, an amount that is reduced by signals mediated through costimulatory molecules expressed on the T cell surface. Here the role of TNFR2 (p75) as a putative costimulatory receptor for T cell activation was examined. It was found that p75 deficiency in CD8+ T cells increased the requirements for TCR agonist approximately 5-fold. Furthermore, p75−/− T cells display a marked reduction in the proliferative response to TCR agonist. This hypoproliferative response was associated with delayed kinetics of induction of the acute activation markers CD25 and CD69 as well as a marked decrease in the production of IL-2 and IFN-γ. The net result is that very few cells are recruited into the dividing population. Interestingly, CD28 costimulation was only partially effective in rescuing the proliferative defect of p75−/−CD8+ T cells. Thus, p75 provides an important costimulatory signal in addition to that provided by CD28 toward optimal T cell proliferation.
Immunoproteasomes Largely Replace Constitutive Proteasomes During an Antiviral and Antibacterial Immune Response in the Liver
Authors: Khan Selina , van den Broek Maries , Schwarz Katrin , de Giuli Rita , Diener Pierre-André , Groettrup Marcus
Published: April 2014

The proteasome is critically involved in the production of MHC class I-restricted T cell epitopes. Proteasome activity and epitope production are altered by IFN-γ treatment, which leads to a gradual replacement of constitutive proteasomes by immunoproteasomes in vitro. However, a quantitative analysis of changes in the steady state subunit composition of proteasomes during an immune response against viruses or bacteria in vivo has not been reported. Here we show that the infection of mice with lymphocytic choriomeningitis virus or Listeria monocytogenes leads to an almost complete replacement of constitutive proteasomes by immunoproteasomes in the liver within 7 days. Proteasome replacements were markedly reduced in IFN-γ−/− mice, but were only slightly affected in IFN-αR−/− and perforin−/− mice. The proteasome regulator PA28α/β was up-regulated, whereas PA28γ was reduced in the liver of lymphocytic choriomeningitis virus-infected mice. Proteasome replacements in the liver strongly altered proteasome activity and were unexpected to this extent, since an in vivo half-life of 12 days had been previously assigned to constitutive proteasomes in the liver. Our results suggest that during the peak phase of viral and bacterial elimination the antiviral cytotoxic T lymphocyte response is directed mainly to immunoproteasome-dependent T cell epitopes, which would be a novel parameter for the design of vaccines.
Differential IL-10R1 Expression Plays a Critical Role in IL-10-Mediated Immune Regulation
Authors: Ding Yaozhong , Qin Lihui , Zamarin Dmitriy , Kotenko Sergei V. , Pestka Sidney , Moore Kevin W. , Bromberg Jonathan S.
Published: April 2014

In this study, we characterized the differential receptor-binding specificity, affinity, and Janus kinase-STAT activation of cellular IL-10 (cIL-10) compared with viral IL-10 (vIL-10). Only cells expressing IL-10R1 bind human IL-10 or vIL-10. IL-10R2 does not bind to cIL-10 or vIL-10 alone and its presence does not enhance the receptor-binding affinity of cIL-10 or vIL-10, but it is essential for both cIL-10- and vIL-10-mediated signal transduction and immune regulation. Responses initiated by cIL-10 and vIL-10 were compared in B cell and mast cell lines, and demonstrated that the inability of vIL-10 to stimulate immune responses, as compared with human IL-10, is due to failure to initiate signaling. Absent signal transduction is due to low level expression of cell surface IL-10R1, since overexpressing IL-10R1 allows vIL-10 to initiate cIL-10-like signals and subsequent biological responses. These results are similar in primary cells, since splenocytes respond to both cIL-10 and vIL-10, while thymocytes respond only to cIL-10 and have very low mouse IL-10R1 but not mouse IL-10R2 expression. These data demonstrate that IL-10R1 expression plays a critical role in determining whether cells respond to IL-10. Modulation of cell surface IL-10R1 density might be an important mechanism for determining whether IL-10 leads to immunostimulation or immunosuppression in vivo.
Enteric Bacteria Counteract Lipopolysaccharide Induction of Antimicrobial Peptide Genes
Authors: Lindmark Hans , Johansson Karin C. , Stöven Svenja , Hultmark Dan , Engström Ylva , Söderhäll Kenneth
Published: April 2014

The humoral immunity of Drosophila involves the production of antimicrobial peptides, which are induced by evolutionary conserved microbial molecules, like LPS. By using Drosophila mbn-2 cells, we found that live bacteria, including E. coli, Salmonella typhimurium, Erwinia carotovora, and Pseudomonas aeruginosa, prevented LPS from inducing antimicrobial peptide genes, while Micrococcus luteus and Streptococcus equi did not. The inhibitory effect was seen at bacterial levels from 20 per mbn-2 cell, while antimicrobial peptides were induced at lower bacterial concentrations (≤2 bacteria per cell) also in the absence of added LPS. Gel shift experiment suggests that the inhibitory effect is upstream or at the level of the activation of the transcription factor Relish, a member of the NF-κB/Rel family. The bacteria have to be in physical contact with the cells, but not phagocytosed, to prevent LPS induction. Interestingly, the inhibiting mechanism is, at least for E. coli, independent of the type III secretion system, indicating that the inhibitory mechanism is unrelated to the one earlier described for YopJ from Yersinia.
Hydrogen Bond Integrity Between MHC Class II Molecules and Bound Peptide Determines the Intracellular Fate of MHC Class II Molecules
Authors: Arneson Lynne S. , Katz John F. , Liu Michael , Sant Andrea J.
Published: April 2014

MHC class II molecules associate with peptides through pocket interactions and the formation of hydrogen bonds. The current paradigm suggests that the interaction of side chains of the peptide with pockets in the class II molecule is responsible for the formation of stable class II-peptide complexes. However, recent evidence has shown that the formation of hydrogen bonds between genetically conserved residues of the class II molecule and the main chain of the peptide contributes profoundly to peptide stability. In this study, we have used I-Ak, a class II molecule known to form strong pocket interactions with bound peptides, to probe the general importance of hydrogen bond integrity in peptide acquisition. Our studies have revealed that abolishing hydrogen bonds contributed by positions 81 or 82 in the β-chain of I-Ak results in class II molecules that are internally degraded when trafficked through proteolytic endosomal compartments. The presence of high-affinity peptides derived from either endogenous or exogenous sources protects the hydrogen bond-deficient variant from intracellular degradation. Together, these data indicate that disruption of the potential to form a complete hydrogen bond network between MHC class II molecules and bound peptides greatly diminishes the ability of class II molecules to bind peptides. The subsequent failure to stably acquire peptides leads to protease sensitivity of empty class II molecules, and thus to proteolytic degradation before export to the surface of APCs.
Control of Advanced Choroid Plexus Tumors in SV40 T Antigen Transgenic Mice Following Priming of Donor CD8+ T Lymphocytes by the Endogenous Tumor Antigen
Authors: Schell Todd D. , Tevethia Satvir S.
Published: April 2014

Mouse models in which tumors arise spontaneously due to the transgenic expression of an oncoprotein provide an opportunity to test approaches that target the immune-mediated control of tumor progression. In this report we investigated the role of SV40 Tag-specific CD8+ T cells in the control of advanced choroid plexus tumor progression using large tumor Ag (Tag) transgenic mice. Since mice of the SV11 line are tolerant to the immunodominant SV40 Tag-derived CTL epitopes, mice with advanced stage tumors were reconstituted with naive C57BL/6 spleen cells following a low dose of γ-irradiation. This led to the priming of CTLs specific for the H2-Kb-restricted epitope IV by the endogenous Tag and a significant increase in the life span of Tag transgenic mice. Epitope IV-specific CD8+ T cells accumulated and persisted in the brains and tumors of SV11 mice, as determined by analysis with epitope-specific MHC class I tetramers. Brain-infiltrating epitope IV-specific T cells were capable of producing IFN-γ as well as lysing syngeneic Tag-transformed cells in vitro. In addition, the adoptive transfer of spleen cells from Tag-immune C57BL/6 mice resulted in a dramatic increase in the control of tumor progression in SV11 mice and was associated with the accumulation of CD8+ T cells specific for multiple Tag epitopes in the brain. These results indicate that the control of advanced stage spontaneous choroid plexus tumors is associated with the induction of a strong and persistent CD8+ T cell response to Tag.
Bacterial Peptidoglycan-Inducedtnf-αTranscription Is Mediated Through the Transcription Factors Egr-1, Elk-1, and NF-κB
Authors: Xu Zhaojun , Dziarski Roman , Wang Qiuling , Swartz Kevin , Sakamoto Kathleen M. , Gupta Dipika
Published: April 2014

Bacteria and their ubiquitous cell wall component peptidoglycan (PGN) activate the innate immune system of the host and induce the release of inflammatory molecules. TNF-α is one of the highest induced cytokines in macrophages stimulated with PGN; however, the regulation of tnf-α expression in PGN-activated cells is poorly understood. This study was done to identify some of the transcription factors that regulate the expression of the tnf-α gene in macrophages stimulated with PGN. Our results demonstrated that PGN-induced expression of human tnf-α gene is regulated by sequences proximal to −182 bp of the promoter. Mutations within the binding sites for cAMP response element, early growth response (Egr)-1, and κB3 significantly reduced this induction. The transcription factor c-Jun bound the cAMP response element site, Egr-1 bound the Egr-1 motif, and NF-κB p50 and p65 bound to the κB3 site on the tnf-α promoter. PGN rapidly induced transcription of egr-1 gene and this induction was significantly reduced by specific mutations within the serum response element-1 domain of the egr-1 promoter. PGN also induced phosphorylation and activation of Elk-1, a member of the Ets family of transcription factors. Elk-1 and serum response factor proteins bound the serum response element-1 domain on the egr-1 promoter, and PGN-induced expression of the egr-1 was inhibited by dominant-negative Elk-1. These results indicate that PGN induces activation of the transcription factors Egr-1 and Elk-1, and that PGN-induced expression of tnf-α is directly mediated through the transcription factors c-Jun, Egr-1, and NF-κB, and indirectly through the transcription factor Elk-1.
Salmonella Flagellin-Dependent Proinflammatory Responses Are Localized to the Conserved Amino and Carboxyl Regions of the Protein
Authors: Eaves-Pyles Tonyia D. , Wong Hector R. , Odoms Kelli , Pyles Richard B.
Published: April 2014

Flagellin, the monomeric subunit of flagella, is an inducer of proinflammatory mediators. Bacterial flagellin genes have conserved domains (D1 and D2) at the N terminus and C terminus and a middle hypervariable domain (D3). To identify which domains induced proinflammatory activity, r6-histidine (6HIS)-tagged fusion constructs were generated from the Salmonella dublin (SD) fliC flagellin gene. A full-length r6HIS SD flagellin (6HIS flag) induced IκBα loss poststimulation and NF-κB activation in Caco-2BBe cells and was as potent as native-purified SD flagellin. IFN-γ-primed DLD-1 cells stimulated with 1 μg/ml of 6HIS flag induced high levels of NO (60 ± 0.95 μM) comparable to the combination of IL-1β and IFN-γ (77 ± 1.2) or purified native SD flag (66.3 ± 0.98). Selected rSD flagellin proteins representing the D1, D2, or D3 domains alone or in combination were tested for proinflammatory properties. Fusion proteins representing the D3, amino, or carboxyl regions alone did not induce proinflammatory mediators. The results with a recombinant protein containing the amino D1 and D2 and carboxyl D1 and D2 separated by an Escherichia coli hinge (ND1-2/ECH/CD2) indicated that D1 and D2 were bioactive when coupled to an ECH element to allow protein folding. This chimera, but not the hinge alone, induced IκBα degradation, NF-κB activation, and NO and IL-8 production in two intestinal epithelial cell lines. ND1–2/ECH/CD2–1 also induced high levels of TNF-α (900 pg/ml) in human monocytes comparable to native SD flagellin (991.5 pg/ml) and 6HIS flag (987 pg/ml). The potent proinflammatory activity of flagellin, therefore, resides in the highly conserved N and C D1 and D2 regions.