bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential Regulation of Killer Cell Lectin-Like Receptor G1 Expression on T Cells
Authors: Robbins Scott H. , Terrizzi Stephanie C. , Sydora Beate C. , Mikayama Toshifumi , Brossay Laurent
Published: April 2014

The killer cell lectin-like receptor G1 (KLRG1) is the mouse homologue of the rat mast cell function-associated Ag and contains a tyrosine-based inhibitory motif in its cytoplasmic domain. It has been demonstrated that KLRG1 is induced on activated NK cells and that KLRG1 can inhibit NK cell effector functions. In this study, we show that in naive C57BL/6 mice KLRG1 is expressed on a subset of CD44highCD62Llow T cells. KLRG1 expression can be detected on a small number of Vα14i NK T cells but not on CD8αα+ intraepithelial T cells that are either TCRγδ+ or TCRαβ+. We also show that KLRG1 expression is dramatically induced on ∼50% of the CD8+ T cells during both a viral and a parasitic infection. Interestingly, during Toxoplasma gondii infection, KLRG1 is up-regulated on CD4+ T cells. Although KLRG1 expression can be induced on both NK cells and T cells, the molecular mechanism leading to the induction of KLRG1 differs in these two subsets of cells. Indeed, the up-regulation of KLRG1 on NK cells can be driven in vivo by cytokines, whereas KLRG1 cannot be induced on CD8+ T cells by cytokines. In addition, although induction of KLRG1 on T cells appears to require TCR engagement in vivo, TCR engagement is not sufficient for KLRG1 induction in vitro. Taken together, these data suggest that the expression and induction of KLRG1 on T cells are tightly regulated. This could have important biological consequences on T cell activation and homeostasis.
Transfection of Dendritic Cells with RNA Induces CD4- and CD8-Mediated T Cell Immunity Against Breast Carcinomas and Reveals the Immunodominance of Presented T Cell Epitopes
Authors: Müller Martin R. , Grünebach Frank , Nencioni Alessio , Brossart Peter
Published: April 2014

Transfection of dendritic cells (DC) with tumor-derived RNA has recently been shown to elicit tumor-specific CTL capable of recognizing and lysing a variety of tumor cells. In our study we analyzed the induction of HLA class I- and II-restricted T cell responses against MCF-7 breast cancer cells. Using this approach we were able to elicit CD4- and CD8-mediated antitumor responses. The CTL specifically lysed MCF-7 cells and DC electroporated with MCF-7 RNA, but spared control cell lines. The specificity of the cytotoxic activity was confirmed in cold target inhibition assays and using mAbs blocking HLA class I molecules. Interestingly, these polyclonal cytotoxic T cells recognized selectively two epitopes derived from the MUC1 and Her-2/neu tumor Ags. The induced Th cells were found to be entirely HLA class II restricted and showed a significant cross-reactivity to a renal cell carcinoma cell line, similar to the results obtained with cytotoxic T cells.
Potentiation of a Tumor Cell Susceptibility to Autologous CTL Killing by Restoration of Wild-Type p53 Function
Authors: Thiery Jérôme , Dorothée Guillaume , Haddada Hedi , Echchakir Hamid , Richon Catherine , Stancou Rodica , Vergnon Isabelle , Benard Jean , Mami-Chouaib Fathia , Chouaib Salem
Published: April 2014

Inactivation of p53 has been implicated in many types of tumors particularly in non-small cell lung carcinoma, one of the most common cancers in which p53 mutation has been frequently identified. The aim of this study was to investigate the influence of p53 status on the regulation of tumor susceptibility to specific CTL-mediated cell death. For this purpose, we used a cytotoxic T lymphocyte clone, Heu127, able to lyse the human autologous lung carcinoma cell line, IGR-Heu, in a HLA-A2-restricted manner. Direct genomic DNA sequencing revealed that IGR-Heu expresses a mutated p53 at codon 132 of the exon 5 which results in the loss of p53 capacity to induce the expression of the p53-regulated gene product p21waf/CIP1. Initial experiments demonstrated that IGR-Heu was resistant to Fas, TNF, and TRAIL apoptotic pathways. This correlated with the lack of p55 TNFRI, Fas, DR4, and DR5 expression. The effect of wild-type (wt) p53 restoration on the sensitization of IGR-Heu to autologous CTL clone lysis was investigated following infection of the tumor cell line with a recombinant adenovirus encoding the wt p53 (Adwtp53). We demonstrate that the restoration of wt p53 expression and function resulted in a significant potentiation of target cell susceptibility to CTL-mediated lysis. The wt p53-induced optimization of tumor cell killing by specific CTL involves at least in part Fas-mediated pathway via induction of CD95 expression by tumor cells but does not appear to interfere with granzyme B cytotoxic pathway.
Differentiation of Monocytic Cell Clones into CD8α+ Dendritic Cells (DC) Suggests that Monocytes Can Be Direct Precursors for Both CD8α+ and CD8α− DC in the Mouse
Authors: Gao Jian-Xin , Liu Xingluo , Wen Jing , Zhang Huiming , Durbin Joan , Liu Yang , Zheng Pan
Published: April 2014

Dendritic cells (DC) are the professional APCs that initiate T cell immune responses. DC can develop from both myeloid and lymphoid progenitors. In the mouse, the CD8α+ DC had been designated as “lymphoid” DC, and CD8α− DC as “myeloid” DC until recently when it was demonstrated that common myeloid progenitors can also give rise to CD8α+ DC in bone marrow chimera mice. However, it is still not clear which committed myeloid lineages differentiate into CD8α+ DC. Because monocytes can differentiate into DC in vivo, the simplest hypothesis is that the CD8α+ DC can be derived from the monocyte/macrophage. In this study we show that cell clones, isolated from CD8α+ DC lymphoma but with a monocytic phenotype (CD11clow/−D11bhighCD8α−I-Alow), can redifferentiate into CD8α+ DC either when stimulated by LPS and CD40L or when they migrate into the lymphoid organs. Maturation of DC in vivo correlated with strong priming of allogeneic T cells. Moreover, the monocytes from cultured splenocytes or peritoneal exudates macrophages of wild-type mice are also capable of differentiating into CD11c+CD8α+ DC after their migration into the draining lymph nodes. Our results suggest that monocytes can be direct precursors for CD11c+CD8α+ DC in vivo. In addition, the monocyte clones described in this study may be valuable for studying the differentiation and function of CD8α+ DC that mediate cross-presentation of Ag to CD8 T cells specific for cell-associate Ags.
Mechanisms of Endotoxin Tolerance in Human Intestinal Microvascular Endothelial Cells
Authors: Ogawa Hitoshi , Rafiee Parvaneh , Heidemann Jan , Fisher Pamela J. , Johnson Nathan A. , Otterson Mary F. , Kalyanaraman Balaraman , Pritchard Kirkwood A. , Binion David G.
Published: April 2014

Lipopolysaccharide (endotoxin) tolerance is well described in monocytes and macrophages, but is less well characterized in endothelial cells. Because intestinal microvascular endothelial cells exhibit a strong immune response to LPS challenge and play a critical regulatory role in gut inflammation, we sought to characterize the activation response of these cells to repeated LPS exposure. Primary cultures of human intestinal microvascular endothelial cells (HIMEC) were stimulated with LPS over 6–60 h and activation was assessed using U937 leukocyte adhesion, expression of E-selectin, ICAM-1, VCAM-1, IL-6, IL-8, manganese superoxide dismutase, HLA-DR, and CD86. Effect of repeat LPS stimulation on HIMEC NF-κB and mitogen-activated protein kinase (MAPK) activation, generation of superoxide anion, and Toll-like receptor 4 expression was characterized. LPS pretreatment of HIMEC for 24–48 h significantly decreased leukocyte adhesion after subsequent LPS stimulation. LPS pretreatment inhibited expression of E-selectin, VCAM-1, IL-6, and CD86, while ICAM-1, IL-8, and HLA-DR were not altered. Manganese superoxide dismutase expression increased with repeated LPS stimulation, with a reduction in intracellular superoxide. NF-κB activation was transiently inhibited by LPS pretreatment for 6 h, but not at later time points. In contrast, p44/42 MAPK, p38 MAPK, and c-Jun N-terminal kinase activation demonstrated inhibition by LPS pretreatment 24 or 48 h prior. Toll-like receptor 4 expression on HIMEC was not altered by LPS. HIMEC exhibit endotoxin tolerance after repeat LPS exposure in vitro, characterized by diminished activation and intracellular superoxide anion concentration, and reduced leukocyte adhesion. HIMEC possess specific mechanisms of immunoregulatory hyporesponsiveness to repeated LPS exposure.
A Model for Antigen-Specific T-Cell Anergy: Displacement of CD4-p56lckSignalosome from the Lipid Rafts by a Soluble, Dimeric Peptide-MHC Class II Chimera
Authors: Thomas Sunil , Kumar Rajeev , Preda-Pais Anca , Casares Sofia , Brumeanu Teodor-D.
Published: April 2014

Soluble, dimeric peptide-MHC chimeras were shown to induce Ag-specific T cell anergy in vitro and in vivo. In this study, we describe a mechanism by which a soluble, dimeric peptide MHC class II chimera (DEF) induces Ag-specific T cell anergy. The anergic cells showed a displacement of the CD4-p56lck signaling module from the GM1-rich plasma membrane microdomains (lipid rafts), and subsequently an increase in p59fyn kinase activity, a dominant expression of p21 inhibitory TCR ζ-chain, and a poor phosphorylation and recruitment of ζ-associated protein of 70 kDa kinase to the TCR’s immunoreceptor tyrosine-based activation motifs. The Th1 and Th2 transcription was suppressed and the cells were arrested in the Th0 stage of differentiation. Recovery from DEF anergy occurred late and spontaneously at the expense of low thresholds for activation-induced cell death. In contrast to DEF, a combination of TCR and CD4 mAbs did not induce such alterations or anergy, indicating that the ligand-mediated topology of TCR and CD4 coengagement can differentially affect the T cell function. Our results argue for a model of anergy in which the defective partitioning of signaling molecules in lipid rafts is an early, negative signaling event in T cells. Physiological ligands like DEF chimeras may provide new tools for silencing the autoimmune processes, and may also help in deciphering new mechanisms of negative regulation in T cells.
Acquisition of Viral Receptor by NK Cells Through Immunological Synapse
Authors: Tabiasco Julie , Vercellone Alain , Meggetto Fabienne , Hudrisier Denis , Brousset Pierre , Fournié Jean-Jacques
Published: April 2014

Occasional EBV infection of human NK cells may lead to malignant diseases such as naso-pharyngeal NK lymphoma although NK cells do not express CD21, the primary receptor for EBV. Here we show that during early EBV infection in patients, NK cells attacked EBV-infected autologous B cells. In vitro, NK cells activated by conjugation to CD21+ B-EBV cell targets transiently acquired a weak CD21+ phenotype by synaptic transfer of few receptor molecules onto their own membrane. In the presence of viral particles, these ectopic receptors allowed EBV binding to the novel NK cell host. Hence, trans-synaptic acquisition of viral receptor from target cells might constitute an unsuspected mode of infection for otherwise unreachable lymphoid hosts.
Ephrin-A1 Induces c-Cbl Phosphorylation and EphA Receptor Down-Regulation in T Cells
Authors: Sharfe Nigel , Freywald Andrew , Toro Ana , Roifman Chaim M.
Published: April 2014

Eph receptor tyrosine kinases are expressed by T lineage cells, and stimulation with their ligands, the ephrins, has recently been shown to modulate T cell behavior. We show that ephrin-A1 stimulation of Jurkat T cells induces tyrosine phosphorylation of EphA3 receptors and cytoplasmic proteins, including the c-cbl proto-oncogene. Cbl phosphorylation was also observed in peripheral blood T cells. In contrast, stimulation of Jurkat cells with the EphB receptor ligand ephrin-B1 does not cause Cbl phosphorylation. EphA activation also induced Cbl association with Crk-L and Crk-II adapters, but not the related Grb2 protein. Induction of Cbl phosphorylation upon EphA activation appeared to be dependent upon Src family kinase activity, as Cbl phosphorylation was selectively abrogated by the Src family inhibitor 4-amino-5(4-chlorophenyl-7-(tert-butyl)pyrazolo[3,4-d]pyrimidine, while EphA phosphorylation was unimpaired. Ephrin-A1 stimulation of Jurkat cells was also found to cause down-regulation of endogenous EphA3 receptors from the cell surface and their degradation. In accordance with the role of Cbl as a negative regulator of receptor tyrosine kinases, overexpression of wild-type Cbl, but not its 70-Z mutant, was found to down-regulate EphA receptor expression. Receptor down-regulation could also be inhibited by blockage of Src family kinase activity. Our findings show that EphA receptors can actively signal in T cells, and that Cbl performs multiple roles in this signaling pathway, functioning to transduce signals from the receptors as well as regulating activated EphA receptor expression.
Hepatocyte NF-1 and STAT6 Cooperate with Additional DNA-Binding Factors to Activate Transcription of the Human Polymeric Ig Receptor Gene in Response to IL-4
Authors: Schjerven Hilde , Brandtzaeg Per , Johansen Finn-Eirik
Published: April 2014

Secretory IgA and IgM, which protect the mucosal surfaces, are generated by selective transport of locally produced polymeric (p)Igs through the epithelial barrier by the pIgR. The expression of this receptor, and hence the generation of secretory Igs, is modulated by numerous extracellular factors. We have previously identified a STAT6 site in intron 1 of the human pIgR gene that is required for the slow and de novo protein synthesis-dependent IL-4-mediated transcriptional activation of the gene. In this study, we show that this intronic IL-4-responsive enhancer is confined to a 250-bp region that is highly conserved in the murine pIgR gene. The enhancer was dependent on the cooperation between the STAT6 site and at least four additional DNA elements. EMSA experiments demonstrated binding by hepatocyte NF-1 to one of these DNA elements. Extensive overlap in the tissue distribution of hepatocyte NF-1 and pIgR suggests that this transcription factor contributes to tissue-specific pIgR expression. Changing the helical phase between the STAT6 site and downstream DNA elements greatly reduced the strength of the IL-4 response, suggesting that the precise organization of this enhancer is important for its proper function. Thus, several transcription factors cooperate in this enhanceosome to mediate IL-4 responsiveness in HT-29 epithelial cells.
Characterization and Analysis of the Proximal Janus Kinase 3 Promoter
Authors: Aringer Martin , Hofmann Sigrun R. , Frucht David M. , Chen Min , Centola Michael , Morinobu Akio , Visconti Roberta , Kastner Daniel L. , Smolen Josef S. , O’Shea John J.
Published: April 2014

Janus kinase 3 (Jak3) is a nonreceptor tyrosine kinase essential for signaling via cytokine receptors that comprise the common γ-chain (γc), i.e., the receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Jak3 is preferentially expressed in hemopoietic cells and is up-regulated upon cell differentiation and activation. Despite the importance of Jak3 in lymphoid development and immune function, the mechanisms that govern its expression have not been defined. To gain insight into this issue, we set out to characterize the Jak3 promoter. The 5′-untranslated region of the Jak3 gene is interrupted by a 3515-bp intron. Upstream of this intron and the transcription initiation site, we identified an ∼1-kb segment that exhibited lymphoid-specific promoter activity and was responsive to TCR signals. Truncation of this fragment revealed that core promoter activity resided in a 267-bp fragment that contains putative Sp-1, AP-1, Ets, Stat, and other binding sites. Mutation of the AP-1 sites significantly diminished, whereas mutation of the Ets sites abolished, the inducibility of the promoter construct. Chromatin immunoprecipitation assays showed that histone acetylation correlates with mRNA expression and that Ets-1/2 binds this region. Thus, transcription factors that bind these sites, especially Ets family members, are likely to be important regulators of Jak3 expression.
Spontaneous Clustering and Tyrosine Phosphorylation of NK Cell Inhibitory Receptor Induced by Ligand Binding
Authors: Faure Mathias , Barber Domingo F. , Takahashi Stephenie M. , Jin Tian , Long Eric O.
Published: April 2014

Inhibition of NK cell cytotoxicity by killer cell Ig-like receptors (KIR) depends on phosphorylation of cytoplasmic tyrosines in KIR, which recruit tyrosine phosphatase Src homology protein tyrosine phosphatase 1. It is not clear how KIR, whose function lies downstream of a tyrosine kinase, succeeds in blocking proximal NK cell activation signals upon binding HLA class I on target cells. Here we show that mixing NK cells with insect cells expressing HLA-C was sufficient to induce clustering of KIR, and phosphorylation of KIR and SHP-1. Transient phosphorylation of KIR was detected in the presence of pervanadate, an inhibitor of protein tyrosine phosphatases, at suboptimal concentration. Phosphorylation of KIR was specifically induced by ligand binding because it was detected only when HLA-C was loaded with a peptide that permits KIR binding. KIR phosphorylation was not dependent on ICAM-1-mediated adhesion and was not blocked by inhibition of actin polymerization, but required Zn2+. Fluorescence resonance energy transfer between HLA-C molecules revealed close molecular interactions induced by KIR binding. These results demonstrate tight clustering of KIR and rapid KIR phosphorylation induced simply by binding to HLA-C. The unique property of KIR to become phosphorylated in the absence of adhesion and of actin cytoskeleton rearrangement explains how KIR can efficiently block early activation signals during NK-target cell contacts.
Salmonella typhimurium Strains Carrying Independent Mutations Display Similar Virulence Phenotypes Yet Are Controlled by Distinct Host Defense Mechanisms
Authors: Raupach Bärbel , Kurth Nicole , Pfeffer Klaus , Kaufmann Stefan H. E.
Published: April 2014

The outcome of Salmonella infection in the mammalian host favors whoever succeeds best in disturbing the equilibrium between coordinate expression of bacterial (virulence) genes and host defense mechanisms. Intracellular persistence in host cells is critical for pathogenesis and disease, because Salmonella typhimurium strains defective in this property are avirulent. We examined whether similar host defense mechanisms are required for growth control of two S. typhimurium mutant strains. Salmonella pathogenicity island 2 (SPI2) and virulence plasmid-cured Salmonella mutants display similar virulence phenotypes in immunocompetent mice, yet their gene loci participate in independent virulence strategies. We determined the role of TNF-α and IFN-γ as well as different T cell populations in infection with these Salmonella strains. After systemic infection, IFN-γ was essential for growth restriction of plasmid-cured S. typhimurium, while SPI2 mutant infections were controlled in the absence of IFN-γ. TNFRp55-deficiency restored systemic virulence to both Salmonella mutants. After oral inoculation, control of plasmid-cured bacteria substantially relied on both IFN-γ and TNF-α signaling while control of SPI2 mutants did not. However, for both mutants, ultimate clearance of bacteria from infected mice depended on αβ T cells.
Overexpression of Toll-Like Receptor 4 Amplifies the Host Response to Lipopolysaccharide and Provides a Survival Advantage in Transgenic Mice
Authors: Bihl Franck , Salez Laurent , Beaubier Magali , Torres David , Larivière Line , Laroche Line , Benedetto Alexandre , Martel Dominic , Lapointe Jean-Martin , Ryffel Bernhard , Malo Danielle
Published: April 2014

Toll-like receptors are transmembrane proteins that are involved in the innate immune recognition of microbial constituents. Among them, Toll-like receptor 4 (Tlr4) is a crucial signal transducer for LPS, the major component of Gram-negative bacteria outer cell membrane. The contribution of Tlr4 to the host response to LPS and to infection with virulent Salmonella typhimurium was studied in four transgenic (Tg) strains including three overexpressing Tlr4. There was a good correlation between the level of Tlr4 mRNA expression and the sensitivity to LPS both in vitro and in vivo: Tg mice possessing the highest number of Tlr4 copies respond the most to LPS. Overexpression of Tlr4 by itself appears to have a survival advantage in Tg mice early during infection: animals possessing more than two copies of the gene survived longer and in a greater percentage to Salmonella infection. The beneficial effect of Tlr4 overexpression is greatly enhanced when the mice present a wild-type allele at natural resistance-associated macrophage protein 1, another critical innate immune gene involved in resistance to infection with Salmonella. Tlr4 and natural resistance-associated macrophage protein 1 exhibit functional epistatic interaction to improve the capacity of the host to control bacterial replication. However, this early improvement in disease resistance is not conducted later during infection, because mice overexpressing Tlr4 developed an excessive inflammatory response detrimental to the host.
The Autoreactivity of Anti-Phosphorylcholine Antibodies for Atherosclerosis-Associated Neo-Antigens and Apoptotic Cells
Authors: Shaw Peter X. , Goodyear Carl S. , Chang Mi-Kyung , Witztum Joseph L. , Silverman Gregg J.
Published: April 2014

Abs specific for phosphorylcholine (PC) are known to contribute to the immune defense against a variety of microbial infections. To assess for other types of binding interactions, we performed surveys of anti-PC Abs of diverse biologic origins and structural diversity and demonstrated a common autoreactivity for oxidatively modified low density lipoprotein and other oxidation-specific structures containing PC-Ags. We also found that cells undergoing apoptosis sequentially express a range of oxidation-specific neo-self PC determinants. Whereas natural Abs to PC recognized cells at early stages of apoptosis, by contrast, an IgG anti-PC Ab, representative of a T cell-dependent response, recognized PC determinants primarily associated with late stages of apoptosis. Cumulatively, these results demonstrate a fundamental paradigm in which Abs from both the innate and the T cell-dependent tiers of the B cell compartment recognize a minimal molecular motif arrayed both on microbes and as neo-self Ags linked to atherosclerosis and autoimmune disease.
Both Lymphotoxin-α and TNF Are Crucial for Control ofToxoplasma gondiiin the Central Nervous System
Authors: Schlüter Dirk , Kwok Lai-Yu , Lütjen Sonja , Soltek Sabine , Hoffmann Sigrid , Körner Heinrich , Deckert Martina
Published: April 2014

Immunity to Toxoplasma gondii critically depends on TNFR type I-mediated immune reactions, but the precise role of the individual ligands of TNFR1, TNF and lymphotoxin-α (LTα), is still unknown. Upon oral infection with T. gondii, TNF−/−, LTα−/−, and TNF/LTα−/− mice failed to control intracerebral T. gondii and succumbed to an acute necrotizing Toxoplasma encephalitis, whereas wild-type (WT) mice survived. Intracerebral inducible NO synthase expression and–early after infection–splenic NO levels were reduced. Additionally, peritoneal macrophages produced reduced levels of NO upon infection with T. gondii and had significantly reduced toxoplasmastatic activity in TNF−/−, LTα−/−, and TNF/LTα−/− mice as compared with WT animals. Frequencies of parasite-specific IFN-γ-producing T cells, intracerebral and splenic IFN-γ production, and T. gondii-specific IgM and IgG titers in LTα−/− and TNF/LTα−/− mice were reduced only early after infection. In contrast, intracerebral IL-10 and IL-12p40 mRNA expression and splenic IL-2, IL-4, and IL-12 production were identical in all genotypes. In addition, TNF−/−, LTα−/−, and TNF/LTα−/−, but not WT, mice succumbed to infection with the highly attenuated ts-4 strain of T. gondii or to a subsequent challenge infection with virulent RH toxoplasms, although they had identical frequencies of IFN-γ-producing T cells as compared with WT mice. Generation and infection of bone marrow reconstitution chimeras demonstrated an exclusive role of hematogeneously produced TNF and LTα for survival of toxoplasmosis. These findings demonstrate the crucial role of both LTα and TNF for control of intracerebral toxoplasms.
Variations in Eosinophil Chemokine Responses: An Investigation of CCR1 and CCR3 Function, Expression in Atopy, and Identification of a Functional CCR1 Promoter
Authors: Phillips Rhian M. , Stubbs Victoria E. L. , Henson Mandy R. , Williams Timothy J. , Pease James E. , Sabroe Ian
Published: April 2014

We previously showed in a small group of donors that eosinophils from a subgroup of individuals responded equipotently to CC chemokine ligand (CCL)11/eotaxin and CCL3/macrophage-inflammatory protein-1α in assays of eosinophil shape change (CCL3/macrophage-inflammatory protein-1α-highly responsive (MHR) donors). In this study, we investigated the functional role of CCL3 in eosinophil responses in 73 donors. MHR donors, identified by their eosinophil shape change responses, represented ∼19% of the donor pool. Eosinophils from these donors showed increased eosinophil CCR1 expression and also underwent CCL3-mediated chemotaxis and up-regulation of CD11b. All MHR donors gave a history of atopy-associated diseases. In a further study, we prospectively recruited 110 subjects, subdivided into nonatopics or atopics, and investigated expression of CCR1 and CCR3 on eosinophils, basophils, monocytes, and neutrophils. Eosinophil CCR1 expression was non-normally distributed in atopics, although higher CCR1 expression levels were not predictive of a diagnosis of atopy or atopic disease. We identified the CCR1 promoter and investigated its function. We found a minimal promoter within 177 bp of the transcription start site, and an upstream enhancer region that facilitated expression in leukocyte cell lines. Collectively, these data demonstrate that MHR individuals form an important subgroup that, when associated with a diagnosis of allergic disease, may require tailored therapy to modulate eosinophil recruitment. Identification of a functional CCR1 promoter will facilitate the study of possible genetic determinants underlying this potentially important clinical phenotype.
Regulation of Chemokine mRNA Stability by Lipopolysaccharide and IL-10
Authors: Biswas Roopa , Datta Shyamasree , Gupta Jaydip Das , Novotny Michael , Tebo Julie , Hamilton Thomas A.
Published: April 2014

IL-10 has been reported to inhibit the expression of LPS-induced proinflammatory cytokines and chemokines by altering the rate of specific mRNA decay although the molecular target(s) for its action remain unknown. In the present study, using primary peritoneal exudate macrophages and a cell culture model in which a tetracycline-responsive promoter controls transcription of CXC ligand 1 (KC) mRNA, we demonstrate that LPS promotes a time-dependent increase in KC mRNA stability. Although IL-10 had no direct effect on mRNA decay, this treatment antagonized the stabilizing action of LPS. The mechanisms involved were further explored using a cell-free mRNA degradation system. A 5′-capped, polyadenylated in vitro transcript derived from the 3′-untranslated region of KC mRNA exhibited time-dependent decay in the presence of protein extracts prepared from untreated RAW264.7 macrophages. Extracts prepared from LPS-treated RAW264.7 cells had reduced decay activity and this change was antagonized if the cells were costimulated with IL-10. A substrate in which the AU-rich element motifs were mutated exhibited minimal decay that did not vary using extracts prepared from cells treated with LPS or LPS and IL-10. A nonadenylated RNA substrate was also degraded and that activity was diminished by LPS. In concert, these findings demonstrate that KC mRNA stability is regulated by LPS-induced alterations in activities that govern both deadenylation and degradation of the mRNA body. The effects of IL-10 on KC mRNA stability reflect antagonism of the response to LPS.
Induction of Macrophage Nitric Oxide Production by Gram-Negative Flagellin Involves Signaling Via Heteromeric Toll-Like Receptor 5/Toll-Like Receptor 4 Complexes
Authors: Mizel Steven B. , Honko Anna N. , Moors Marlena A. , Smith Pameeka S. , West A. Phillip
Published: April 2014

The induction of cytokine synthesis by flagellin is mediated by a Toll-like receptor 5 (TLR5) signaling pathway. Although flagellin activation of the IL-1R-associated kinase and induction of TNF-α synthesis are dependent on TLR5 and not TLR4, we have found that flagellin stimulates NO in macrophages via a pathway that requires TLR5 and TLR4. Flagellin induced NO synthesis in HeNC2 cells, a murine macrophage cell line that expresses wild-type TLR4, but not in TLR4-mutant or -deficient GG2EE and 10ScNCr/23 cells. Flagellin stimulated an increase in inducible NO synthase (iNOS) mRNA and activation of the iNOS promoter. TLR5 forms heteromeric complexes with TLR4 as well as homomeric complexes. IFN-γ permitted GG2EE and 10ScNCr/23 cells to produce NO in response to flagellin. Flagellin stimulated IFN-β synthesis and Stat1 activation. The effect of flagellin on iNOS gene expression was inhibited by a Stat1 mutant protein. Taken together, these results support the conclusions that flagellin induces distinct patterns of inflammatory mediators depending on the nature of the TLR5 signaling complex and that the induction of NO by flagellin involves signaling via TLR5/TLR4 complexes.
Activation of NF-κB in Virus-Infected Macrophages Is Dependent on Mitochondrial Oxidative Stress and Intracellular Calcium: Downstream Involvement of the Kinases TGF-β-Activated Kinase 1, Mitogen-Activated Kinase/Extracellular Signal-Regulated Kinase Kinase 1, and IκB Kinase
Authors: Mogensen Trine H. , Melchjorsen Jesper , Höllsberg Per , Paludan Søren R.
Published: April 2014

Efficient clearance of virus infections depends on the nature of the host response raised by the infected organism. A proinflammatory cell-mediated immune response is important for elimination of many viruses, including herpesviruses. Macrophages are intimately involved in generation of a proinflammatory response, the initiation of which involves activation of the transcription factor NF-κB. However, the mechanisms of HSV-induced NF-κB activation are poorly understood. In this study we demonstrate that activation of NF-κB by HSV in macrophages is dependent on a functional viral genome and proceeds through a mechanism involving the cellular IκB kinase, as well as the upstream kinases TGF-β-activated kinase 1, mitogen-activated kinase/extracellular signal-regulated kinase kinase 1, and possibly NF-κB-inducing kinase. Furthermore, we show that HSV triggers NF-κB activation by a signaling pathway involving oxidative stress in mitochondria and intracellular calcium, because specific inhibition of mitochondria-derived reactive oxygen intermediates, as well as mitochondrial calcium channels, prevented NF-κB activation. Together, these results point to mitochondria as cellular checkpoints able to initiate NF-κB activation after virus infection and also show that the cellular NF-κB-regulating kinases IκB kinase, TGF-β-activated kinase 1, mitogen-activated kinase/extracellular signal-regulated kinase kinase 1, and possibly NF-κB-inducing kinase, are essential components in the HSV-induced signaling pathway.
Corneal IL-8 Expression Following Adenovirus Infection Is Mediated by c-Src Activation in Human Corneal Fibroblasts
Authors: Natarajan Kanchana , Rajala Maitreyi S. , Chodosh James
Published: April 2014

Emerging evidence indicates that intracellular signaling cascades mediate entry of pathogenic adenoviruses into target host cells as well as some of the undesirable inflammatory responses to adenoviral gene vectors. We found that Ad19 infection of cultured human corneal fibroblasts induced IL-8 gene transcription independently of IL-1β, TNF-α, and viral gene expression, suggesting that intracellular signaling events might mediate early inflammatory events in adenovirus keratitis. Heat but not UV light inactivation of the virus abrogated the effect of infection on IL-8 mRNA and protein levels, consistent with a viral binding-mediated mechanism. The tyrosine kinase inhibitor herbimycin blocked Ad19-induced IL-8 expression. Western blot analysis revealed tyrosine phosphorylation of the functionally related kinases c-Src and extracellular signal-regulated kinase (ERK) 1/2 in corneal fibroblasts within 15 min after infection. Respective inhibitors of these kinases, PP2 and PD98059, also blocked Ad19-induced IL-8 mRNA and protein expression. Application of inhibitors to Src and ERK kinase assays suggested an upstream relationship of c-Src to ERK. Finally, DNA microarray studies performed 1 h after Ad19 or mock infection of corneal fibroblasts in the presence or absence of the Src-specific inhibitor PP2 confirmed a relationship between c-Src and IL-8 expression in Ad19-infected corneal cells. c-Src may act as a global regulator of early proinflammatory host responses to Ad19 infection of the human cornea.
Differential Regulation of Lipopolysaccharide-Induced Monocyte Matrix Metalloproteinase (MMP)-1 and MMP-9 by p38 and Extracellular Signal-Regulated Kinase 1/2 Mitogen-Activated Protein Kinases
Authors: Lai Wan-Ching , Zhou Min , Shankavaram Uma , Peng Gang , Wahl Larry M.
Published: April 2014

Signal transduction events in monocyte matrix metalloproteinase (MMP) production have been shown to include a PGE2-cAMP-dependent step. To determine earlier pathway components, we examined the role of mitogen-activated protein kinases (MAPKs) in the regulation of monocyte MMP-1 and MMP-9, two major MMPs induced by LPS. Stimulation with LPS resulted in the activation of the extracellular signal-regulated kinase 1 and 2 (ERK1/2) and mitogen-activated kinase p38. The p38-specific inhibitor SB203580 suppressed p38 activity and MMP-1 mRNA and protein, but increased ERK activity and MMP-9 mRNA and protein. In contrast, the MAPK kinase 1/2-specific inhibitor PD98059 inhibited MMP-1 and MMP-9. However, both MAPK inhibitors decreased the production of cyclooxygenase-2 and PGE2, but only the inhibition of MMP-1 by SB203580 was reversed by PGE2 or dibutyryl cAMP. Examination of the effect of these MAPK inhibitors on the promoters of MMP-1 and MMP-9 revealed that PD98059 inhibited the binding of transcription factors to all of the MMP promoter-specific complementary oligonucleotides tested. However, SB203580 only inhibited the binding of MMP-1-specific CREB and SP 1 oligonucleotides, which was reversed by PGE2. Additionally, SB203580 enhanced transcription factor binding to the oligonucleotides complementary to a NF-κB site in the promoter of MMP-9. Thus, LPS induction of MMP-1 production by monocytes is regulated by both ERK1/2 and p38, whereas MMP-9 stimulation occurred mainly through the ERK1/2 pathway. Moreover, p38 regulates MMP-1 mainly through a PGE2-dependent pathway, whereas ERK1/2-mediated MMP-1 and MMP-9 production involves the activation of additional MMP promoter sites through a PGE2-independent mechanism.
A Prominent Role for Airway Epithelial NF-κB Activation in Lipopolysaccharide-Induced Airway Inflammation
Authors: Poynter Matthew E. , Irvin Charles G. , Janssen-Heininger Yvonne M. W.
Published: April 2014

To reveal the causal role of airway epithelial NF-κB activation in evoking airway inflammation, a transgenic mouse was created expressing a mutant version of the inhibitory protein I-κBα. This I-κBα superrepressor (I-κBαSR) acts to repress NF-κB activation exclusively in airway epithelial cells, under the transcriptional control of the rat CC10 promoter (CC10-I-κBαSR). Compared with transgene-negative littermates, intranasal instillation of LPS did not induce nuclear translocation of NF-κB in airway epithelium of CC10-I-κBαSR transgenic mice. Consequently, the influx of neutrophils into the airways and secretion of the NF-κB-regulated neutrophilic chemokine, macrophage-inflammatory protein-2, and the inflammatory cytokine, TNF-α, were markedly reduced in CC10-I-κBαSR mice relative to the transgene-negative mice exposed to LPS. Despite an inability to activate NF-κB in airway epithelium, resident alveolar macrophages from transgene-positive mice were capable of activating NF-κB in a manner indistinguishable from transgene-negative mice. These findings demonstrate that airway epithelial cells play a prominent role in orchestrating the airway inflammatory response to LPS and suggest that NF-κB signaling in these cells is important for modulating innate immune responses to microbial products.
Differential Activation of NF-κB, AP-1, and C/EBP in Endotoxin-Tolerant Rats: Mechanisms for In Vivo Regulation of Glomerular RANTES/CCL5 Expression
Authors: Pocock Johanna , Gómez-Guerrero Carmen , Harendza Sigrid , Ayoub Murwan , Hernández-Vargas Purificación , Zahner Gunther , Stahl Rolf A. K. , Thaiss Friedrich
Published: April 2014

Chemokines play a pivotal role in the regulation of inflammatory cell infiltration in glomerular immune injury. To characterize mechanisms relevant for the regulation of chemokine expression in vivo, the LPS-mediated model of renal inflammation in rats was used in which we have previously demonstrated that the chemokine RANTES/CCL5 is expressed and secreted in glomeruli. Glomerular RANTES/CCL5 expression in this model correlated with an increased glomerular binding activity of the transcription factors AP-1, C/EBP, and NF-κB. To gain further insight into the functional roles of these transcription factors in the regulation of glomerular RANTES/CCL5 expression, we cloned the rat RANTES/CCL5 promoter and established the model of in vivo LPS tolerance. In tolerant rats, LPS-induced glomerular RANTES/CCL5 expression and activation of the transcription factors AP-1 and C/EBP were significantly reduced using both consensus and rat RANTES/CCL5-specific oligonucleotides. Reduced glomerular NF-κB binding activity after LPS injection could be demonstrated in tolerant rats only when using rat RANTES/CCL5-specific oligonucleotides. Reduced binding activity to this RANTES/CCL5-specific NF-κB binding site in the context of broad NF-κB activation might be due to changes in transcription factor interactions or chromatin remodeling processes.
Fas Is Detectable on β Cells in Accelerated, But Not Spontaneous, Diabetes in Nonobese Diabetic Mice
Authors: Darwiche Rima , Chong Mark M. W. , Santamaria Pere , Thomas Helen E. , Kay Thomas W. H.
Published: April 2014

Fas (CD95) is a potential mechanism of pancreatic β cell death in type 1 diabetes. β cells do not constitutively express Fas but it is induced by cytokines. The hypothesis of this study is that Fas expression should be measurable on β cells for them to be killed by this mechanism. We have previously reported that up to 5% of β cells isolated from nonobese diabetic (NOD) mice are positive for Fas expression by flow cytometry using autofluorescence to identify β cells. We have now found that these are not β cells but contaminating dendritic cells, macrophages, and B lymphocytes. In contrast β cells isolated from NODscid mice that are recipients of T lymphocytes from diabetic NOD mice express Fas 18–25 days after adoptive transfer but before development of diabetes. Fas expression on β cells was also observed in BDC2.5, 8.3, and 4.1 TCR-transgenic models of diabetes in which diabetes occurs more rapidly than in unmodified NOD mice. In conclusion, Fas is observed on β cells in models of diabetes in which rapid β cell destruction occurs. Its expression is likely to reflect differences in the intraislet cytokine environment compared with the spontaneous model and may indicate a role for this pathway in β cell destruction in rapidly progressive models.
Chemokines Determine Local Lymphoneogenesis and a Reduction of Circulating CXCR4+ T and CCR7 B and T Lymphocytes in Thyroid Autoimmune Diseases
Authors: Armengol Maria-Pilar , Cardoso-Schmidt Cristina B. , Fernández Marco , Ferrer Xavier , Pujol-Borrell Ricardo , Juan Manel
Published: April 2014

Chemokines and their corresponding receptors are crucial for the recruitment of lymphocytes into the lymphoid organs and for its organization acting in a multistep process. Tissues affected by autoimmune disease often contain ectopic lymphoid follicles which, in the case of autoimmune thyroid disorders, are highly active and specific for thyroid Ags although its pathogenic role remains unclear. To understand the genesis of these lymphoid follicles, the expression of relevant cytokines and chemokines was assessed by real time PCR, immunohistochemistry and by in vitro assays in autoimmune and nonautoimmune thyroid glands. Lymphotoxin α, lymphotoxin β, C-C chemokine ligand (CCL) 21, CXC chemokine ligand (CXCL) 12, CXCL13, and CCL22 were increased in thyroids from autoimmune patients, whereas CXCL12, CXCL13, and CCL22 levels were significantly higher in autoimmune glands with ectopic secondary lymphoid follicles than in those without follicles. Interestingly, thyroid epithelium produced CXCL12 in response to proinflammatory cytokines providing a possible clue for the understanding of how tissue stress may lead to ectopic follicle formation. The finding of a correlation between chemokines and thyroid autoantibodies further suggests that intrathyroidal germinal centers play a significant role in the autoimmune response. Unexpectedly, the percentage of circulating CXCR4+ T cells and CCR7+ B and T cells (but not of CXCR5) was significantly reduced in PBMCs of patients with autoimmune thyroid disease when they were compared with their intrathyroidal lymphocytes. This systemic effect of active intrathyroidal lymphoid tissue emerges as a possible new marker of thyroid autoimmune disease activity.
Irradiation of Tumor Cells Up-Regulates Fas and Enhances CTL Lytic Activity and CTL Adoptive Immunotherapy
Authors: Chakraborty Mala , Abrams Scott I. , Camphausen Kevin , Liu Kebin , Scott Tamalee , Coleman C. Norman , Hodge James W.
Published: April 2014

CD8+ CTL play important roles against malignancy in both active and passive immunotherapy. Nonetheless, the success of antitumor CTL responses may be improved by additional therapeutic modalities. Radiotherapy, which has a long-standing use in treating neoplastic disease, has been found to induce unique biologic alterations in cancer cells affecting Fas gene expression, which, consequently, may influence the overall lytic efficiency of CTL. Here, in a mouse adenocarcinoma cell model, we examined whether exposure of these tumor cells to sublethal doses of irradiation 1) enhances Fas expression, leading to more efficient CTL killing via Fas-dependent mechanisms in vitro; and 2) improves antitumor activity in vivo by adoptive transfer of these Ag-specific CTL. Treatment of carcinoembryonic Ag-expressing MC38 adenocarcinoma cells with irradiation (20 Gy) in vitro enhanced Fas expression at molecular, phenotypic, and functional levels. Furthermore, irradiation sensitized these targets to Ag-specific CTL killing via the Fas/Fas ligand pathway. We examined the effect of localized irradiation of s.c. growing tumors on the efficiency of CTL adoptive immunotherapy. Irradiation caused up-regulation of Fas by these tumor cells in situ, based on immunohistochemistry. Moreover, localized irradiation of the tumor significantly potentiated tumor rejection by these carcinoembryonic Ag-specific CTL. Overall, these results showed for the first time that 1) regulation of the Fas pathway in tumor cells by irradiation plays an important role in their sensitization to Ag-specific CTL; and 2) a combination regimen of tumor-targeted irradiation and CTL promotes more effective antitumor responses in vivo, which may have implications for the combination of immunotherapy and radiation therapy.
Identification of a Mutated Receptor-Like Protein Tyrosine Phosphatase κ as a Novel, Class II HLA-Restricted Melanoma Antigen
Authors: Novellino Luisa , Renkvist Nicolina , Rini Francesca , Mazzocchi Arabella , Rivoltini Licia , Greco Angela , Deho Paola , Squarcina Paola , Robbins Paul F. , Parmiani Giorgio , Castelli Chiara
Published: April 2014

Recent studies increasingly point to a pivotal role of CD4+ T cells in human anti-tumor immune response. Here we show that lymphocytes purified from a tumor-infiltrated lymph node of a melanoma patient that had remained disease free for 10 years after surgical resection of a lymph node metastasis comprised oligoclonal class II HLA-restricted CD4+ T cells recognizing the autologous tumor cells in vitro. In fact, the CD4+ T cell clones isolated from these lymphocytes displayed a tumor-specific, cytotoxic activity in addition to a Th1-like cytokine profile. By a genetic approach, a peptide derived from a mutated receptor-like protein tyrosine phosphatase κ was identified as a novel HLA-DR10-restricted epitope for all the melanoma-specific CD4+ T cell clones. The immunogenic peptide was shown to contain the mutated residue that was crucial for T cell recognition and activation. Moreover, a systemic immunity against the mutated peptide was detectable in the patient’s peripheral blood T lymphocytes obtained during the disease-free period of follow-up. These findings further support the relevance of CD4+ T cells directed against mutated epitopes in tumor immunity and provide the rationale for a possible usage of mutated, tumor-specific Ags for immunotherapy of human cancer.
IL-10 Modulates Host Responses and Lung Damage Induced byPneumocystis cariniiInfection
Authors: Qureshi Mahboob H. , Harmsen Allen G. , Garvy Beth A.
Published: April 2014

Host responses to Pneumocystis carinii infection mediate impairment of pulmonary function and contribute to the pathogenesis of pneumonia. IL-10 is known to inhibit inflammation and reduce the severity of pathology caused by a number of infectious organisms. In the present studies, IL-10-deficient (IL-10 knockout (KO)) mice were infected with P. carinii to determine whether the severity of pathogenesis and the efficiency of clearance of the organisms could be altered in the absence of IL-10. The clearance kinetics of P. carinii from IL-10 KO mice was significantly enhanced compared with that of wild-type (WT) mice. This corresponded to a more intense CD4+ and CD8+ T cell response as well as an earlier neutrophil response in the lungs of IL-10 KO mice. Furthermore, IL-12, IL-18, and IFN-γ were found in the bronchoalveolar lavage fluids at earlier time points in IL-10 KO mice suggesting that alveolar macrophages were activated earlier than in WT mice. However, when CD4+ cells were depleted from P. carinii-infected IL-10 KO mice, the ability to enhance clearance was lost. Furthermore, CD4-depleted IL-10 KO mice had significantly more lung injury than CD4-depleted WT mice even though the intensity of the inflammatory responses was similar. This was characterized by increased vascular leakage, decreased oxygenation, and decreased arterial pH. These data indicate that IL-10 down-regulates the immune response to P. carinii in WT mice; however, in the absence of CD4+ T cells, IL-10 plays a critical role in controlling lung damage independent of modulating the inflammatory response.