bluuebunny/crossref_metadata_2025 huggingface dataset:
Inducible Nitric Oxide Synthase Inhibitors Suppress Airway Inflammation in Mice Through Down-Regulation of Chemokine Expression
Authors: Trifilieff Alexandre , Fujitani Yasushi , Mentz Franck , Dugas Bernard , Fuentes Maria , Bertrand Claude
Published: April 2014

Growing evidence demonstrates that inducible NO synthase (iNOS) is induced in the airways of asthmatic patients. However, the precise role of NO in the lung inflammation is unknown. This study investigated the effect of both selective and nonselective iNOS inhibitors in an allergen-driven murine lung inflammation model. OVA challenge resulted in an accumulation of eosinophils and neutrophils in the airways. Expression of iNOS immunostaining in lung sections together with an increase in calcium-independent NOS activity in lung homogenates was also observed after OVA challenge. Treatment with iNOS inhibitors from the day of challenge to the day of sacrifice resulted in an inhibition of the inflammatory cell influx together with a down-regulation of macrophage inflammatory protein-2 and monocyte chemoattractant protein-1 production. In contrast, eosinophilic and neutrophilic inhibition was not observed with treatment during the sensitization. Both treatments induced an increased production of Th2-type cytokines (IL-4 and IL-5) with a concomitant decrease in production of Th1-type cytokine (IFN-γ). In vitro exposure of primary cultures of murine lung fibroblasts to a NO donor, hydroxylamine, induced a dose-dependent release of macrophage inflammatory protein-2 and monocyte chemoattractant protein-1. Our results suggest that lung inflammation after allergen challenge in mice is partially dependent on NO produced mainly by iNOS. NO appears to increase lung chemokine expression and, thereby, to facilitate influx of inflammatory cells into the airways.
Quantification of Self-Recognition in Multiple Sclerosis by Single-Cell Analysis of Cytokine Production
Authors: Pelfrey Clara M. , Rudick Richard A. , Cotleur Anne C. , Lee Jar-Chi , Tary-Lehmann Magdalena , Lehmann Paul V.
Published: April 2014

Identifying and quantifying autoaggressive responses in multiple sclerosis (MS) has been difficult in the past due to the low frequency of autoantigen-specific T cells, the high number of putative determinants on the autoantigens, and the different cytokine signatures of the autoreactive T cells. We used single-cell resolution enzyme-linked immunospot (ELISPOT) assays to study, directly ex vivo, proteolipid protein (PLP)-specific memory cell reactivity from MS patients and controls. Overlapping 9-aa-long peptides, spanning the entire PLP molecule in single amino acid steps, were used to determine the frequency and fine specificity of PLP-specific lymphocytes as measured by their IFN-γ and IL-5 production. MS patients (n = 22) responded to 4 times as many PLP peptides as did healthy controls (n = 22). The epitopes recognized in individual patients, up to 22 peptides, were scattered throughout the PLP molecule, showing considerable heterogeneity among MS patients. Frequency measurements showed that the number of PLP peptide-specific IFN-γ-producing cells averaged 11 times higher in MS patients than in controls. PLP peptide-induced IL-5-producing T cells occurred in very low frequencies in both MS patients and controls. This first comprehensive assessment of the anti-PLP-Th1/Th2 response in MS shows a greatly increased Th1 effector cell mass in MS patients. Moreover, the highly IFN-γ-polarized, IL-5-negative cytokine profile of the PLP-reactive T cells suggests that these cells are committed Th1 cells. The essential absence of uncommitted Th0 cells producing both cytokines may explain why therapeutic strategies that aim at the induction of immune deviation show little efficacy in the established disease.
Linkage Analysis of Systemic Lupus Erythematosus Induced in Diabetes-Prone Nonobese Diabetic Mice byMycobacterium bovis
Authors: Jordan Margaret A. , Silveira Pablo A. , Shepherd Darren P. , Chu Clara , Kinder Simon J. , Chen Jianhe , Palmisano Linda J. , Poulton Lynn D. , Baxter Alan G.
Published: April 2014

Systemic lupus erythematosus induced by Mycobacterium bovis in diabetes-prone nonobese diabetic mice was mapped in a backcross to the BALB/c strain. The subphenotypes—hemolytic anemia, antinuclear autoantibodies, and glomerular immune complex deposition—did not cosegregate, and linkage analysis for each trait was performed independently. Hemolytic anemia mapped to two loci: Bah1 at the MHC on chromosome 17 and Bah2 on distal chromosome 16. Antinuclear autoantibodies mapped to three loci: Bana1 at the MHC on chromosome 17, Bana2 on chromosome 10, and Bana3 on distal chromosome 1. Glomerular immune complex deposition did not show significant linkage to any genomic region. Mapping of autoantibodies (Coombs’ or antinuclear autoantibodies) identified two loci: Babs1 at the MHC and Babs2 on distal chromosome 1. It has previously been reported that genes conferring susceptibility to different autoimmune diseases map nonrandomly to defined regions of the genome. One possible explanation for this clustering is that some alleles at loci within these regions confer susceptibility to multiple autoimmune diseases—the “common gene” hypothesis. With the exception of the H2, this study failed to provide direct support for the common gene hypothesis, because the loci identified as conferring susceptibility to systemic lupus erythematosus did not colocalize with those previously implicated in diabetes. However, three of the four regions identified had been previously implicated in other autoimmune diseases.
Early Growth Response (Egr)-1 Gene Induction in the Thymus in Response to TCR Ligation During Early Steps in Positive Selection Is Not Required for CD8 Lineage Commitment
Authors: Basson M. Albert , Wilson Timothy J. , Legname Giuseppe A. , Sarner Nitza , Tomlinson Peter D. , Tybulewicz Victor L. J. , Zamoyska Rose
Published: April 2014

The early growth response gene 1 (Egr-1) is induced during positive selection in the thymus and has been implicated in the differentiation of CD4+ thymocytes. Here, we show that signals that specifically direct CD8 lineage commitment also induce Egr-1 DNA-binding activity in the nucleus. However, we find that pharmacological inhibition of mitogen-activated protein kinase/extracellular signal-related kinase kinase activity potently inhibits Egr-1 DNA-binding function at concentrations that promote differentiation of CD8+ thymocytes, suggesting Egr-1 activity is not essential for CD8 commitment. To further determine the role of Egr-1 in thymocyte development, we compare steady-state Egr-1 DNA-binding activity in thymocytes from mice with defined defects in positive selection. The data indicate that the appearance of functional Egr-1 is downstream of signals induced by TCR/MHC engagement, whereas it is less sensitive to alterations in Lck-mediated signals, and does not correlate directly with proficient positive selection. Egr-1 is one of the earliest transcription factors induced upon TCR ligation on immature thymocytes, and plays a potential role in the transcription of genes involved in thymocyte selection.
B Lymphocyte-Derived IL-16 Attracts Dendritic Cells and Th Cells
Authors: Kaser Arthur , Dunzendorfer Stefan , Offner Felix A. , Ludwiczek Othmar , Enrich Barbara , Koch Robert O. , Cruikshank William W. , Wiedermann Christian J. , Tilg Herbert
Published: April 2014

Interaction of B lymphocytes with Th cells is a fundamental step in the establishment of humoral immunity, and recent evidence suggests that direct interaction between B lymphocytes and dendritic cells (DCs) is also an important prerequisite. Factors involved in the selective recruitment of Th cells and DCs by B lymphocytes are insufficiently defined. We set out to delineate the role of IL-16, the soluble ligand of CD4, which is expressed on Th cells and DCs. B lymphocytes express IL-16 mRNA and synthesize bioactive IL-16 protein, and IL-16 is expressed in lymph node follicles in situ. B lymphocyte supernatant efficiently induces migration of CD4+ Th cells, monocyte-derived DCs, and circulating blood DCs in nitrocellulose filter-based assays. Neutralization of IL-16 bioactivity strongly inhibits this migratory response, suggesting that IL-16 might be a major chemotactic factor derived from B cells. The present data further support the idea that IL-16 might have a role in the initiation of cellular as well as humoral immunity by mediating the cellular cross-talk among T lymphocytes, B cells, and DCs, leading to recruitment of these cell types at common anatomical sites.
Evidence for Two Subgroups of CD4−CD8− NKT Cells with Distinct TCRαβ Repertoires and Differential Distribution in Lymphoid Tissues
Authors: Apostolou Irina , Cumano Ana , Gachelin Gabriel , Kourilsky Philippe
Published: April 2014

NKT cells are a subset of T lymphocytes that is mainly restricted by the nonclassical MHC class I molecule, CD1d, and that includes several subpopulations, in particular CD4+ and CD4−CD8− (DN) cells. In the mouse, differential distribution of these subpopulations as well as heterogeneity in the expression of various markers as a function of tissue localization have been reported. We have thus undertaken a detailed study of the DN NKT cell subpopulation. With a highly sensitive semiquantitative RT-PCR technique, its TCR repertoire was characterized in various tissues. We found that mouse DN NKT cells are a variable mixture of two subgroups, one bearing the invariant Vα14 chain paired to rearranged Vβ2, Vβ7, Vβ8.1, Vβ8.2, or Vβ8.3 β-chains and the other exhibiting unskewed α- and β-chains. The proportion of these subgroups varies from about 100:0 in thymus, 80:20 in liver, and 50:50 in spleen to 20:80% in bone marrow, respectively. Finally, further heterogeneity in the tissue-derived DN NKT cells was discovered by sequencing extensively Vβ8.2-Jβ2.5 rearrangements in individual mice. Despite a few recurrences in TCR sequences, we found that each population exhibits its own and broad TCRβ diversity.
Signal Transduction of IL-6, Leukemia-Inhibitory Factor, and Oncostatin M: Structural Receptor Requirements for Signal Attenuation
Authors: Anhuf Dirk , Weissenbach Manuela , Schmitz Jochen , Sobota Radoslaw , Hermanns Heike M. , Radtke Simone , Linnemann Sonja , Behrmann Iris , Heinrich Peter C. , Schaper Fred
Published: April 2014

Stimulation of the IL-6R complex leads to Src homology domain containing tyrosine phosphatase 2 (SHP2) recruitment to the receptor subunit gp130 and its subsequent tyrosine phosphorylation. SHP2 is a two-SH2 domain-containing protein tyrosine phosphatase that is activated by many cytokines and growth factors. SHP2 counteracts the activation of transcription factors of the STAT family and the induction of IL-6-responsive genes. Tyrosine 759 of gp130, the signal transducing subunit of the IL-6R complex, is essential for the phosphorylation of SHP2. Mutation of tyrosine 759 to phenylalanine leads to an enhanced inducibility of IL-6-dependent genes. Here we demonstrate that no further tyrosines in the cytoplasmic part of gp130 are required for the phosphorylation of SHP2. We also tested whether the tyrosine 759 motifs in both subunits of the gp130 dimer are required for SHP2 association and tyrosine phosphorylation. Interestingly, one SHP2-recruiting phosphotyrosine motif in a single chain of the gp130 dimer is sufficient to mediate SHP2 association to the gp130 receptor subunit and its tyrosine phosphorylation as well as to attenuate IL-6-dependent gene induction. Furthermore, we show that repression of gene induction via Y759 does not require the presence of the SHP2 and STAT recruitment sites within the same receptor subunit, but within the same receptor complex. The Y759 motif in gp130 also attenuates gene induction mediated by the oncostatin M and leukemia inhibitory factor receptor complexes, which both contain gp130 as the shared subunit.
Saturation, Competition, and Specificity in Interaction of Heat Shock Proteins (hsp) gp96, hsp90, and hsp70 with CD11b+ Cells
Authors: Binder Robert J. , Harris Michelle L. , Ménoret Antoine , Srivastava Pramod K.
Published: April 2014

Heat shock proteins (hsp(s)) have been postulated to interact with APCs through specific receptors, although the receptors are yet to be identified. Specificity, saturation, and competition are the three defining attributes of a receptor-ligand interaction. We demonstrate here that the interaction of the heat shock proteins gp96 and hsp90 with CD11b+ cells is specific and saturable and that gp96 can compete with itself in gp96-macrophage interaction. Interestingly, the phylogenetically related hsp90 also competes quite effectively with gp96 for binding to macrophages, whereas the unrelated hsp70 does so relatively poorly, although it binds CD11b+ cells just as effectively. These data provide evidence that the heat shock proteins interact with APCs with specificity and for the existence of at least two distinct receptors, one for gp96 and hsp90 and the other for hsp70.
Relevance of the Tumor Antigen in the Validation of Three Vaccination Strategies for Melanoma
Authors: Bellone Matteo , Cantarella Daniela , Castiglioni Paola , Crosti Maria Cristina , Ronchetti Anna , Moro Monica , Garancini Maria Paola , Casorati Giulia , Dellabona Paolo
Published: April 2014

Many preclinical studies of cancer immunotherapy are based on the testing of a single vaccination strategy in several tumor models. Moreover, most of those studies used xenogeneic Ags, which, owing to their high immunogenicity, may not represent realistic models for the validation of cancer immunotherapies. To address these issues, we compared the vaccination efficacy of three well established strategies (i.e., naked DNA; peptide-pulsed dendritic cells (DC), or a mixture of peptide and the Escherichia coli toxin LTR72) using the xenogeneic OVA or the naturally expressed tyrosinase-related protein 2 (TRP-2) tumor Ag in the B16 melanoma model. C57BL/6 mice received one to three s.c. injections of peptide-pulsed DC or DNA, or one to four mucosal administrations of peptide-toxin mixture. One to 2 wk later, the animals were challenged s.c. with B16 or B16 cells expressing OVA (B16-OVA). Vaccination of mice with OVA induced in all cases melanoma-specific CTL and protection against B16-OVA. When TRP-2 was used, all three vaccines elicited B16-specific CTL, but only DC pulsed with the immunodominant T cell epitope TRP-2181–188 allowed protection against B16. Even more importantly, a vaccination regimen with TRP-2-pulsed DC, started 24 h after the injection of a lethal number of B16 cells, caused a therapeutic effect in 60% of the challenged animals. Our results strongly emphasize the relevance of the tumor Ag in the definition of immunotherapeutic strategies for cancer, and support the use of peptide-pulsed DC as cancer vaccine in humans.
Regulation of Infection withHistoplasma capsulatumby TNFR1 and -2
Authors: Allendoerfer Ruth , Deepe George S.
Published: April 2014

The concerted action of several cytokines is necessary for resolution of both primary and secondary infection with Histoplasma capsulatum. Among the soluble factors that contribute to tissue sterilization, TNF-α stands as a central mediator of protective immunity to this fungus. In this study, we explored the regulation of protective immunity by TNFR1 and -2. In primary pulmonary infection, both TNFR1−/− and -2−/− mice manifested a high mortality after infection with H. capsulatum, although TNFR1−/− mice were more susceptible than TNFR2 −/− mice. Overwhelming infection in the former was associated with a pronounced decrement in the number of inflammatory cells in the lungs and elevated IFN-γ and TNF-α levels in the lungs. In contrast, IFN-γ levels were markedly decreased in TNFR2−/− mice, and treatment with this cytokine restored protective immunity. Lung macrophages from both groups of knockout mice released substantial amounts of NO. Upon secondary infection, TNFR2−/− mice survived rechallenge and cleared infection as efficiently as C57BL/6 animals. In contrast, mice given mAb to TNFR1 succumbed to reexposure, and the high mortality was accompanied by a significant increase in fungal burden in the lungs. Both IL-4 and IL-10 were elevated in the lungs of these mice. The results demonstrate the pivotal influence of TNFR1 and -2 in controlling primary infection and highlight the differences between these receptors for regulation reexposure histoplasmosis.
Involvement of Lipopolysaccharide Binding Protein, CD14, and Toll-Like Receptors in the Initiation of Innate Immune Responses byTreponemaGlycolipids
Authors: Schröder Nicolas W. J. , Opitz Bastian , Lamping Norbert , Michelsen Kathrin S. , Zähringer Ulrich , Göbel Ulf B. , Schumann Ralf R.
Published: April 2014

Culture supernatants from Treponema maltophilum associated with periodontitis in humans and Treponema brennaborense found in a bovine cattle disease accompanied with cachexia caused a dose-dependent TNF-α synthesis in human monocytes increasing with culture time. This activity could be reduced significantly by blocking the CD14-part of the LPS receptor using the My 4 mAb and by polymyxin B. In the murine macrophage cell line RAW 264.7, Treponema culture supernatants induced TNF-α secretion in a LPS binding protein (LBP)-dependent fashion. To enrich for active compounds, supernatants were extracted with butanol, while whole cells were extracted using a phenol/water method resulting in recovery of material exhibiting a similar activity profile. An LPS-LBP binding competition assay revealed an interaction of the treponeme phenol/water extracts with LBP, while precipitation studies implied an affinity to polymyxin B and endotoxin neutralizing protein. Macrophages obtained from C3H/HeJ mice carrying a Toll-like receptor (TLR)-4 mutation were stimulated with treponeme extracts for NO release to assess the role of TLRs in cell activation. Furthermore, NF-κB translocation in TLR-2-negative Chinese hamster ovary (CHO) cells was studied. We found that phenol/water-extracts of the two strains use TLRs differently with T. brennaborense-stimulating cells in a TLR-4-dependent fashion, while T. maltophilum-mediated activation apparently involved TLR-2. These results indicate the presence of a novel class of glycolipids in Treponema initiating inflammatory responses involving LBP, CD14, and TLRs.
Association of Deficient Type II Protein Kinase A Activity with Aberrant Nuclear Translocation of the RIIβ Subunit in Systemic Lupus Erythematosus T Lymphocytes
Authors: Mishra Nilamadhab , Khan Islam U. , Tsokos George C. , Kammer Gary M.
Published: April 2014

Systemic lupus erythematosus (SLE) is an autoimmune disorder of indeterminate etiology characterized by abnormal T cell signal transduction and altered T cell effector functions. We have previously observed a profound deficiency of total protein kinase A (PKA) phosphotransferase activity in SLE T cells. Here we examined whether reduced total PKA activity in SLE T cells is in part the result of deficient type II PKA (PKA-II) isozyme activity. The mean PKA-II activity in SLE T cells was 61% of normal control T cells. The prevalence of deficient PKA-II activity in 35 SLE subjects was 37%. Deficient isozyme activity was persistent over time and was unrelated to SLE disease activity. Reduced PKA-II activity was associated with spontaneous dissociation of the cytosolic RIIβ2C2 holoenzyme and translocation of the regulatory (RIIβ) subunit from the cytosol to the nucleus. Confocal immunofluorescence microscopy revealed that the RIIβ subunit was present in ∼60% of SLE T cell nuclei compared with only 2–3% of normal and disease controls. Quantification of nuclear RIIβ subunit protein content by immunoprecipitation and immunoblotting demonstrated a 54% increase over normal T cell nuclei. Moreover, the RIIβ subunit was retained in SLE T cell nuclei, failed to relocate to the cytosol, and was associated with a persistent deficiency of PKA-II activity. In conclusion, we describe a novel mechanism of deficient PKA-II isozyme activity due to aberrant nuclear translocation of the RIIβ subunit and its retention in the nucleus in SLE T cells. Deficient PKA-II activity may contribute to impaired signaling in SLE T cells.
Adenoviral-Mediated Transfer of the TNF-Related Apoptosis-Inducing Ligand/Apo-2 Ligand Gene Induces Tumor Cell Apoptosis
Authors: Griffith Thomas S. , Anderson Richard D. , Davidson Beverly L. , Williams Richard D. , Ratliff Timothy L.
Published: April 2014

TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily of cytokines that induces apoptosis in a variety of cancer cells. The results presented in this study demonstrate that introduction of the human TRAIL gene into TRAIL-sensitive tumor cells using an adenoviral vector leads to the rapid production and expression of TRAIL protein, and subsequent death of the tumor cells. Tumor cell death was mediated by an apoptotic mechanism, as evidenced by the activation of caspase-8, cleavage of poly(ADP-ribose) polymerase, binding of annexin V, and inhibition by caspase inhibitor zVAD-fmk. These results define a novel method of using TRAIL as an antitumor therapeutic, and suggest the potential use for an adenovirus-encoding TRAIL as a method of gene therapy for numerous cancer types in vivo.
Cutting Edge: Lipid Raft Integrity Affects the Efficiency of MHC Class I Tetramer Binding and Cell Surface TCR Arrangement on CD8+ T Cells
Authors: Drake Donald R. , Braciale Thomas J.
Published: April 2014

Physically distinct cholesterol/sphingolipid-rich plasma membrane microdomains, so-called lipid rafts, have been recognized to play an important regulatory role in various cellular processes, from membrane trafficking to signal transduction, in a number of cell types. We report here that the ability of TCR on activated, functional CD8+ T lymphocytes to efficiently bind MHC class I tetramer complexes is dependent on the integrity of lipid rafts on the T lymphocyte membrane. We further provide evidence that TCR interact (associate) with lipid raft elements on the T cell surface before receptor engagement and that the topological arrangement of TCR on the cell surface is likewise influenced by lipid raft integrity.
Cutting Edge: IL-18-Transgenic Mice: In Vivo Evidence of a Broad Role for IL-18 in Modulating Immune Function
Authors: Hoshino Tomoaki , Kawase Yusuke , Okamoto Masaki , Yokota Koichi , Yoshino Kohichiro , Yamamura Ken-ichi , Miyazaki Jun-ichi , Young Howard A. , Oizumi Kotaro
Published: April 2014

IL-18 has been shown to be a strong cofactor for Th1 T cell development. However, we previously demonstrated that when IL-18 was combined with IL-2, there was a synergistic induction of a Th2 cytokine, IL-13, in both T and NK cells. More recently, we and other groups have reported that IL-18 can potentially induce IgE, IgG1, and Th2 cytokine production in murine experimental models. Here, we report on the generation of IL-18-transgenic (Tg) mice in which mature mouse IL-18 cDNA was expressed. CD8+CD44high T cells and macrophages were increased, but B cells were decreased in these mice while serum IgE, IgG1, IL-4, and IFN-γ levels were significantly increased. Splenic T cells in IL-18 Tg mice produced higher levels of IFN-γ, IL-4, IL-5, and IL-13 than control wild-type mice. Thus, aberrant expression of IL-18 in vivo results in the increased production of both Th1 and Th2 cytokines.
Neutrophil FcγRI as Target for Immunotherapy of Invasive Candidiasis
Authors: van Spriel Annemiek B. , van den Herik-Oudijk Ingrid E. , van de Winkel Jan G. J.
Published: April 2014

Invasive candidiasis represents a life-threatening disease for immunocompromised patients. This study focused on new immunotherapeutic approaches for systemic Candida albicans infections in a human FcγRI-transgenic mouse model. FcγRI (CD64) is a potent immunoactivating receptor on phagocytic and dendritic cells. In vivo targeting of C. albicans toward neutrophil-FcγRI by bispecific Abs and G-CSF effectively protected FcγRI-transgenic mice from lethal candidiasis. Nontransgenic mice were not protected, and treatment with bispecific Ab or G-CSF alone did not reduce mortality. Furthermore, infected FcγRI-transgenic mice developed high titers of anti-C. albicans IgG, and survival was extended on secondary infection without further treatment. These findings document the capacity of FcγRI to initiate potent anti-C. albicans immunity and support the development of FcγRI-directed immunotherapy of invasive fungal disease.
A Dual Role for TNF-α in Type 1 Diabetes: Islet-Specific Expression Abrogates the Ongoing Autoimmune Process When Induced Late but Not Early During Pathogenesis
Authors: Christen Urs , Wolfe Tom , Möhrle Ursula , Hughes Anna C. , Rodrigo Evelyn , Green E. Allison , Flavell Richard A. , von Herrath Matthias G.
Published: April 2014

We report here that islet-specific expression of TNF-α can play a dual role in autoimmune diabetes, depending on its precise timing in relation to the ongoing autoimmune process. In a transgenic model (rat insulin promoter-lymphocytic choriomeningitis virus) of virally induced diabetes, TNF-α enhanced disease incidence when induced through an islet-specific tetracycline-dependent promoter system early during pathogenesis. Blockade of TNF-α during this phase prevented diabetes completely, suggesting its pathogenetic importance early in disease development. In contrast, TNF-α expression abrogated the autoimmune process when induced late, which was associated with a reduction of autoreactive CD8 lymphocytes in islets and their lytic activities. Thus, the fine-tuned kinetics of an autoreactive process undergo distinct stages that respond in a differential way to the presence of TNF-α. This observation has importance for understanding the complex role of inflammatory cytokines in autoimmunity.
Infection of Human Macrophages and Dendritic Cells withMycobacterium tuberculosisInduces a Differential Cytokine Gene Expression That Modulates T Cell Response
Authors: Giacomini Elena , Iona Elisabetta , Ferroni Lucietta , Miettinen Minja , Fattorini Lanfranco , Orefici Graziella , Julkunen Ilkka , Coccia Eliana M.
Published: April 2014

Macrophages and dendritic cells (DC) play an essential role in the initiation and maintenance of immune response to pathogens. To analyze early interactions between Mycobacterium tuberculosis (Mtb) and immune cells, human peripheral blood monocyte-derived macrophages (MDM) and monocyte-derived dendritic cells (MDDC) were infected with Mtb. Both cells were found to internalize the mycobacteria, resulting in the activation of MDM and maturation of MDDC as reflected by enhanced expression of several surface Ags. After Mtb infection, the proinflammatory cytokines TNF-α, IL-1, and IL-6 were secreted mainly by MDM. As regards the production of IFN-γ-inducing cytokines, IL-12 and IFN-α, was seen almost exclusively from infected MDDC, while IL-18 was secreted preferentially by macrophages. Moreover, Mtb-infected MDM also produce the immunosuppressive cytokine IL-10. Because IL-10 is a potent inhibitor of IL-12 synthesis from activated human mononuclear cells, we assessed the inhibitory potential of this cytokine using soluble IL-10R. Neutralization of IL-10 restored IL-12 secretion from Mtb-infected MDM. In line with these findings, supernatants from Mtb-infected MDDC induced IFN-γ production by T cells and enhanced IL-18R expression, whereas supernatants from MDM failed to do that. Neutralization of IFN-α, IL-12, and IL-18 activity in Mtb-infected MDDC supernatants by specific Abs suggested that IL-12 and, to a lesser extent, IFN-α and IL-18 play a significant role in enhancing IFN-γ synthesis by T cells. During Mtb infection, macrophages and DC may have different roles: macrophages secrete proinflammatory cytokines and induce granulomatous inflammatory response, whereas DC are primarily involved in inducing antimycobacterial T cell immune response.
Liver-Derived DEC205+B220+CD19− Dendritic Cells Regulate T Cell Responses
Authors: Lu Lina , Bonham C. Andrew , Liang Xiaoyan , Chen Zongyou , Li Wei , Wang Liangfu , Watkins Simon C. , Nalesnik Michael A. , Schlissel Mark S. , Demestris Anthony J. , Fung John J. , Qian Shiguang
Published: April 2014

Leukocytes resident in the liver may play a role in immune responses. We describe a cell population propagated from mouse liver nonparenchymal cells in IL-3 and anti-CD40 mAb that exhibits a distinct surface immunophenotype and function in directing differentiation of naive allogeneic T cells. After culture, such cells are DEC-205brightB220+CD11c−CD19−, and negative for T (CD3, CD4, CD8α), NK (NK 1.1) cell markers, and myeloid Ags (CD11b, CD13, CD14). These liver-derived DEC205+B220+ CD19− cells have a morphology and migratory capacity similar to dendritic cells. Interestingly, they possess Ig gene rearrangements, but lack Ig molecule expression on the cell surface. They induce low thymidine uptake of allogeneic T cells in MLR due to extensive apoptosis of activated T cells. T cell proliferation is restored by addition of the common caspase inhibitor peptide, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk). T cells stimulated by liver-derived DEC205+B220+D19− cells release both IL-10 and IFN-γ, small amounts of TGF-β, and no IL-2 or IL-4, a cytokine profile resembling T regulatory type 1 cells. Expression of IL-10 and IFN-γ, but not bioactive IL-12 in liver DEC205+B220+CD19− cells was demonstrated by RNase protection assay. In vivo administration of liver DEC205+B220+CD19− cells significantly prolonged the survival of vascularized cardiac allografts in an alloantigen-specific manner.
Aspirin Inhibits In Vitro Maturation and In Vivo Immunostimulatory Function of Murine Myeloid Dendritic Cells
Authors: Hackstein Holger , Morelli Adrian E. , Larregina Adriana T. , Ganster Raymond W. , Papworth Glenn D. , Logar Alison J. , Watkins Simon C. , Falo Louis D. , Thomson Angus W.
Published: April 2014

Aspirin is the most commonly used analgesic and antiinflammatory agent. In this study, at physiological concentrations, it profoundly inhibited CD40, CD80, CD86, and MHC class II expression on murine, GM-CSF + IL-4 stimulated, bone marrow-derived myeloid dendritic cells (DC). CD11c and MHC class I expression were unaffected. The inhibitory action was dose dependent and was evident at concentrations higher than those necessary to inhibit PG synthesis. Experiments with indomethacin revealed that the effects of aspirin on DC maturation were cyclooxygenase independent. Nuclear extracts of purified, aspirin-treated DC revealed a decreased NF-κB DNA-binding activity, whereas Ab supershift analysis indicated that aspirin targeted primarily NF-κB p50. Unexpectedly, aspirin promoted the generation of CD11c+ DC, due to apparent suppression of granulocyte development. The morphological and ultrastructural appearance of aspirin-treated cells was consistent with immaturity. Aspirin-treated DC were highly efficient at Ag capture, via both mannose receptor-mediated endocytosis and macropinocytosis. By contrast, they were poor stimulators of naive allogeneic T cell proliferation and induced lower levels of IL-2 in responding T cells. They also exhibited impaired IL-12 expression and did not produce IL-10 after LPS stimulation. Assessment of the in vivo function of aspirin-treated DC, pulsed with the hapten trinitrobenzenesulfonic acid, revealed an inability to induce normal cell-mediated contact hypersensitivity, despite the ability of the cells to migrate to T cell areas of draining lymphoid tissue. These data provide new insight into the immunopharmacology of aspirin and suggest a novel approach to the manipulation of DC for therapeutic application.
TAP-Independent Presentation of CTL Epitopes by Trojan Antigens
Authors: Lu Jun , Wettstein Peter J. , Higashimoto Yuichiro , Appella Ettore , Celis Esteban
Published: April 2014

The majority of CTL epitopes are derived from intracellular proteins that are degraded in the cytoplasm by proteasomes into peptides that are transported into the endoplasmic reticulum by the TAP complex. These peptides can be further processed into the optimal size (8–10 residues) for binding with nascent MHC class I molecules, generating complexes that are exported to the cell surface. Proteins or peptides containing CTL epitopes can be introduced into the cytoplasm of APCs by linking them to membrane-translocating Trojan carriers allowing their incorporation into the MHC class I Ag-processing pathway. The present findings suggest that these “Trojan” Ags can be transported into the endoplasmic reticulum in a TAP-independent way where they are processed and trimmed into CTL epitopes. Furthermore, processing of Trojan Ags can also occur in the trans-Golgi compartment, with the participation of the endopeptidase furin and possibly with the additional participation of a carboxypeptidase. We believe that these findings will be of value for the design of CTL-inducing vaccines for the treatment or prevention of infectious and malignant diseases.
IFN-γ Alters the Pathology of Graft Rejection: Protection from Early Necrosis
Authors: Halloran Philip F. , Miller Leslie W. , Urmson Joan , Ramassar Vido , Zhu Lin-Fu , Kneteman Norman M. , Solez Kim , Afrouzian Marjan
Published: April 2014

We studied the effect of host IFN-γ on the pathology of acute rejection of vascularized mouse heart and kidney allografts. Organs from CBA donors (H-2k) were transplanted into BALB/c (H-2d) hosts with wild-type (WT) or disrupted (GKO, BALB/c mice with disrupted IFN-γ genes) IFN-γ genes. In WT hosts, rejecting hearts and kidneys showed mononuclear cell infiltration, intense induction of donor MHC products, but little parenchymal necrosis at day 7. Rejecting allografts in GKO recipients showed infiltrate but little or no induction of donor MHC and developed extensive necrosis despite patent large vessels. The necrosis was immunologically mediated, since it developed during rejection, was absent in isografts, and was prevented by immunosuppressing the recipient with cyclosporine or mycophenolate mofetil. Rejecting kidneys in GKO hosts showed increased mRNA for heme oxygenase 1, and decreased mRNA for NO synthase 2 and monokine inducible by IFN-γ (MIG). The mRNA levels for CTL genes (perforin, granzyme B, and Fas ligand) were similar in rejecting kidneys in WT and GKO hosts, and the host Ab responses were similar. The administration of recombinant IFN-γ to GKO hosts reduced but did not fully prevent the effects of IFN-γ deficiency: MHC was induced, but the prevention of necrosis and induction of MIG were incomplete compared with WT hosts. Thus, IFN-γ has unique effects in vascularized allografts, including induction of MHC and MIG, and protection against parenchymal necrosis, probably at the level of the microcirculation. This is probably a local action of IFN-γ produced in large quantities in the allograft.
Suppressors of Cytokine Signaling (SOCS)-1 and SOCS-3 Are Induced by CpG-DNA and Modulate Cytokine Responses in APCs
Authors: Dalpke Alexander H. , Opper Sandra , Zimmermann Stefan , Heeg Klaus
Published: April 2014

During infection, the functional status of the innate immune system is tightly regulated. Although signals resulting in activation have been well characterized, counterregulative mechanisms are poorly understood. Suppressor of cytokine signaling (SOCS) proteins have been characterized as cytokine-inducible negative regulators of Janus kinase/STAT signaling in cells of hemopoietic origin. To analyze whether SOCS proteins could also be induced by pathogen-derived stimuli, we investigated the induction of SOCS-1 and SOCS-3 after triggering of macrophage cell lines, bone marrow-derived dendritic cells, and peritoneal macrophages with CpG-DNA. In this study, we show that CpG-DNA, but not GpC-DNA, induces expression of mRNA for SOCS-1 and SOCS-3 in vitro and in vivo. SOCS mRNA expression could be blocked by chloroquine and was independent of protein synthesis. Inhibitors of the mitogen-activated protein kinase pathway triggered by CpG-DNA were able to impede induction of SOCS mRNA. CpG-DNA triggered synthesis of SOCS proteins that could be detected by Western blotting. SOCS proteins were functional because they inhibited IFN-γ as well as IL-6- and GM-CSF-induced phosphorylation of STAT proteins. Furthermore, IFN-γ-induced up-regulation of MHC class II molecules was also prevented. The same effects could be achieved by overexpression of SOCS-1. Hence, the results indicate a substantial cross-talk between signal pathways within cells. They provide evidence for regulative mechanisms of Janus kinase/STAT signaling after triggering Toll-like receptor signal pathways.
Cloning and Characterization of IL-22 Binding Protein, a Natural Antagonist of IL-10-Related T Cell-Derived Inducible Factor/IL-22
Authors: Dumoutier Laure , Lejeune Diane , Colau Didier , Renauld Jean-Christophe
Published: April 2014

The class II cytokine receptor family includes the receptors for IFN-αβ, IFN-γ, IL-10, and IL-10-related T cell-derived inducible factor/IL-22. By screening genomic DNA databases, we identified a gene encoding a protein of 231 aa, showing 33 and 34% amino acid identity with the extracellular domains of the IL-22 receptor and of the IL-20R/cytokine receptor family 2-8, respectively, but lacking the transmembrane and cytoplasmic domains. A lower but significant sequence identity was found with other members of this family such as the IL-10R (29%), cytokine receptor family 2-4/IL-10Rβ (30%), tissue factor (26%), and the four IFN receptor chains (23–25%). This gene is located on chromosome 6q24, at 35 kb from the IFNGR1 gene, and is expressed in various tissues with maximal expression in breast, lungs, and colon. The recombinant protein was found to bind IL-10-related T cell-derived inducible factor/IL-22, and to inhibit the activity of this cytokine on hepatocytes and intestinal epithelial cells. We propose to name this natural cytokine antagonist IL-22BP for IL-22 binding protein.
Identification, Cloning, and Characterization of a Novel Soluble Receptor That Binds IL-22 and Neutralizes Its Activity
Authors: Kotenko Sergei V. , Izotova Lara S. , Mirochnitchenko Olga V. , Esterova Elena , Dickensheets Harold , Donnelly Raymond P. , Pestka Sidney
Published: April 2014

With the use of a partial sequence of the human genome, we identified a gene encoding a novel soluble receptor belonging to the class II cytokine receptor family. This gene is positioned on chromosome 6 in the vicinity of the IFNGR1 gene in a head-to-tail orientation. The gene consists of six exons and encodes a 231-aa protein with a 21-aa leader sequence. The secreted mature protein demonstrates 34% amino acid identity to the extracellular domain of the IL-22R1 chain. Cross-linking experiments demonstrate that the protein binds IL-22 and prevents binding of IL-22 to the functional cell surface IL-22R complex, which consists of two subunits, the IL-22R1 and the IL-10R2c chains. Moreover, this soluble receptor, designated IL-22-binding protein (BP), is capable of neutralizing IL-22 activity. In the presence of the IL-22BP, IL-22 is unable to induce Stat activation in IL-22-responsive human lung carcinoma A549 cells. IL-22BP also blocked induction of the suppressors of cytokine signaling-3 (SOCS-3) gene expression by IL-22 in HepG2 cells. To further evaluate IL-22BP action, we used hamster cells expressing a modified IL-22R complex consisting of the intact IL-10R2c and the chimeric IL-22R1/γR1 receptor in which the IL-22R1 intracellular domain was replaced with the IFN-γR1 intracellular domain. In these cells, IL-22 activates biological activities specific for IFN-γ, such as up-regulation of MHC class I Ag expression. The addition of IL-22BP neutralizes the ability of IL-22 to induce Stat activation and MHC class I Ag expression in these cells. Thus, the soluble receptor designated IL-22BP inhibits IL-22 activity by binding IL-22 and blocking its interaction with the cell surface IL-22R complex.
Differential Regulation of Chemokine Gene Expression by 15-Deoxy-Δ12,1412,14 Prostaglandin J2
Authors: Zhang Xia , Wang Ji Ming , Gong Wang Hua , Mukaida Naofumi , Young Howard A.
Published: April 2014

Ligands for peroxisome proliferator-activated receptor γ (PPARγ), such as 15-deoxy-Δ12,14PGJ2 (15d-PGJ2) have been proposed as a new class of antiinflammatory compounds with possible clinical applications. As there is some controversy over the inhibitory effects of 15d-PGJ2 on chemokine gene expression, we investigated whether 15d-PGJ2 itself affected chemokine gene expression in human monocytes/macrophages and two monocytic cell lines. Here we demonstrate that the 15d-PGJ2 can induce IL-8 gene expression. In contrast, monocyte chemoattractant protein-1 gene expression was suppressed by 15d-PGJ2, while the expression of RANTES was unaltered. Furthermore, concomitant treatment of monocytes/macrophages with 15d-PGJ2 (2.5 × 10−6 M) potentiated LPS-induced gene expression of IL-8 mRNA, but suppressed PMA-induction of IL-8 mRNA. In addition, treatment of U937 and THP-1 cells with 15d-PGJ2 also resulted in induction of IL-8 gene expression. Further studies demonstrated that 15d-PGJ2 regulated IL-8 gene expression via a ligand-specific and PPARγ-dependent pathway. Our observations revealed a previous unappreciated function and mechanism of 15d-PGJ2-mediated regulation of cytokine gene expression in monocytes/macrophages.
Opposite Regulation of Tissue Factor Expression by Calcineurin in Monocytes and Endothelial Cells
Authors: Hölschermann Hans , Rascher Christine , Oelschläger Christian , Stapfer Gerald , Langenstein Andreas , Staubitz Anne , Maus Ulrich , Tillmanns Harald , Bang Holger , Haberbosch Werner
Published: April 2014

Tissue factor (TF), the primary initiator of blood coagulation with structural homology to the cytokine receptor family, has been implicated in various vascular processes including metastasis, angiogenesis, and atherosclerosis. Within the vasculature, monocytes and endothelial cells (EC) can be activated to synthesize TF depending on the induction of NF-κB. Despite the undisputed value of cyclosporin A (CsA) as an immunosuppressant, problems have emerged due to induction of vascular changes by a poorly understood mechanism. We demonstrate that CsA has opposite effects on TF gene expression, inhibiting NF-κB-mediated TF gene transcription in monocytes but enhancing it in EC. To test whether CsA binding proteins (cyclophilins) can mediate these CsA effects we used a nonimmunosuppressant analog of CsA that binds to cyclophilins but does not inhibit the Ca2+/calmodulin-dependent phosphatase calcineurin (Cn). This drug lacked regulatory function for NF-κB and TF expression suggesting that Cn is responsible for the inverse gene regulation. The key function of Cn was supported by experiments demonstrating that other phosphatase inhibitors also either positively or negatively regulated NF-κB in monocytes and EC. Calcineurin was demonstrated to regulate NF-κB activation at the level of IκBα degradation, because agonist-induced phosphorylation and subsequent degradation of IκBα is prevented by Cn inhibitors in monocytes but enhanced in EC. These data identify Cn as an opposite regulator in generating transcriptionally active NF-κB, and they confirm the presumption that the ability of Cn to participate in NF-κB transactivation is not T cell specific.
Augmentation of RANTES-Induced Extracellular Signal-Regulated Kinase Mediated Signaling and T Cell Adhesion by Elastase-Treated Fibronectin
Authors: Brill Alexander , Hershkoviz Rami , Vaday Gayle G. , Chowers Yehuda , Lider Ofer
Published: April 2014

T cells migrating across extracellular matrix (ECM) barriers toward their target, the inflammatory site, should respond to chemoattractant cytokines and to the degradation of ECM by specific enzymes. In this study, we examined the effects of RANTES and ECM proteins treated with human leukocyte elastase on T cell activation and adhesion to the ECM. We found that human peripheral blood T cells briefly suspended with RANTES (0.1–100 ng/ml) had increased phosphorylation of their intracellular extracellular signal-regulated kinase (ERK), a mitogen-activated protein kinase involved in the activation of several intracellular downstream effector molecules implicated in cell adhesion and migration. Consequently, a small portion (12–20%) of the responding cells adhered to fibronectin (FN). However, when the T cells were exposed to RANTES in the presence of native immobilized FN, laminin, or collagen type I, ERK phosphorylation was partially inhibited, suggesting that this form of the ECM proteins can down-regulate RANTES-induced intracellular signaling. In contrast, when the T cells were exposed to RANTES in the presence of elastase-treated immobilized FN, but not to elastase-treated laminin, ERK phosphorylation was markedly increased. Furthermore, a large percentage (30%) of RANTES-activated T cells adhered to the enzymatically treated FN in a β1 integrin-dependent fashion. Thus, while migrating along chemotactic gradients within the ECM, T cells can adapt their adhesive performance according to the level of cleavage induced by enzymes to the matrix.
An Essential Role of the NF-κB/Toll-Like Receptor Pathway in Induction of Inflammatory and Tissue-Repair Gene Expression by Necrotic Cells
Authors: Li Ming , Carpio David F. , Zheng Ye , Bruzzo Peter , Singh Veena , Ouaaz Fateh , Medzhitov Ruslan M. , Beg Amer A.
Published: April 2014

Tissue damage induced by infection or injury can result in necrosis, a mode of cell death characterized by induction of an inflammatory response. In contrast, cells dying by apoptosis do not induce inflammation. However, the reasons for underlying differences between these two modes of cell death in inducing inflammation are not known. Here we show that necrotic cells, but not apoptotic cells, activate NF-κB and induce expression of genes involved in inflammatory and tissue-repair responses, including neutrophil-specific chemokine genes KC and macrophage-inflammatory protein-2, in viable fibroblasts and macrophages. Intriguingly, NF-κB activation by necrotic cells was dependent on Toll-like receptor 2, a signaling pathway that induces inflammation in response to microbial agents. These results have identified a novel mechanism by which cell necrosis, but not apoptosis, can induce expression of genes involved in inflammation and tissue-repair responses. Furthermore, these results also demonstrate that the NF-κB/Toll-like receptor 2 pathway can be activated both by exogenous microbial agents and endogenous inflammatory stimuli.
Mammalian and Viral IL-10 Enhance C-C Chemokine Receptor 5 but Down-Regulate C-C Chemokine Receptor 7 Expression by Myeloid Dendritic Cells: Impact on Chemotactic Responses and In Vivo Homing Ability
Authors: Takayama Takuya , Morelli Adrian E. , Onai Nobuyuki , Hirao Motohiro , Matsushima Kouji , Tahara Hideaki , Thomson Angus W.
Published: April 2014

The immunosuppressive and anti-inflammatory cytokine IL-10 inhibits the phenotypic and functional maturation of dendritic cells (DC) and has been reported to confer tolerogenic properties on these important professional APC. Here, we exposed murine bone marrow-derived myeloid DC to either mouse (m) or viral (v) IL-10 early during their in vitro generation in response to GM-CSF and IL-4. Both mIL-10 and vIL-10 down-regulated the expression of CCR7 mRNA determined by RT-PCR, while mIL-10 up-regulated the expression of CCR5 transcripts. These changes in CCR7 and CCR5 expression were associated with inhibition and augmentation, respectively, of DC chemotaxis toward their respective agonists, macrophage inflammatory proteins 3β and 1α, while in vivo homing of DC from peripheral s.c. sites to secondary lymphoid tissue of syngeneic or allogeneic recipients was significantly impaired. Anti-mIL-10R mAb reversed the effects of mIL-10 on CCR expression and restored DC homing ability. Retroviral transduction of mIL-10- and vIL-10-treated DC to overexpress transgenic CCR7 partially restored the cells’ lymphoid tissue homing ability in allogeneic recipients. However, CCR7 gene transfer did not reinstate the capacity of IL-10-treated DC to prime host naive T cells for ex vivo proliferative responses or Th1 cytokine (IFN-γ) production in response to rechallenge with (donor) alloantigen. These findings suggest that in addition to their capacity to subvert DC maturation/function and confer tolerogenic potential on these cells, mIL-10 and vIL-10 regulate DC migratory responses via modulation of CCR expression.