bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of the Mitogen-Activated Protein Kinase Signaling Pathway Is Instrumental in Determining the Ability ofMycobacterium aviumto Grow in Murine Macrophages
Authors: Tse Hubert M. , Josephy Steven I. , Chan Edward D. , Fouts Darren , Cooper Andrea M.
Published: April 2014

Of the two common morphotypes of Mycobacterium avium, designated smooth transparent (SmT) or smooth opaque (SmO), the SmO morphotype is avirulent, whereas the SmT morphotype is virulent. The role of the host macrophage in determining these different virulence phenotypes was analyzed using an in vitro model of macrophage infection. Initial studies confirmed previous reports of the increased ability of the SmT bacteria to grow in macrophages; this increased virulence correlated with reduced induction of inflammatory cytokines. Examination of the response of the mitogen-activated protein kinase (MAPK) pathway following infection with either morphotype revealed that all three members of the MAPK pathway were activated. Pharmacologic inhibition of either the extracellular signal-regulated kinase (ERK) or p38MAPK pathways resulted in distinct consequences for the growth of the two morphotypes. In particular, inhibition of the p38MAPK resulted in attenuated growth of the SmT morphotype, which correlated with reduced PGE2 production. Inhibition of cyclooxygenase 2 by indomethacin also inhibited growth of SmT, substantiating the role for PGE2 in promoting the growth of SmT. In contrast, SmO induction of the ERK pathway was increased compared with the SmT morphotype, and inhibition of ERK resulted in decreased TNF-α synthesis and enhanced SmO growth. Pharmacologic inhibitors of the MAPK pathway were present for only the first 4 h of infection and yet had consequences for bacterial growth at 7 days. Therefore, the data suggest that induction of the MAPK pathway during uptake of bacteria is instrumental in determining the eventual fate of the bacteria.
Progression of Armed CTL from Draining Lymph Node to Spleen Shortly After Localized Infection with Herpes Simplex Virus 1
Authors: Coles Richard M. , Mueller Scott N. , Heath William R. , Carbone Francis R. , Brooks Andrew G.
Published: April 2014

We have examined the generation of CTL immunity immediately after localized footpad infection with herpes simplex virus 1 (HSV-1) using three coordinated in vivo T cell tracking methodologies. Tetrameric MHC class I containing the immunodominant peptide from HSV-1 glycoprotein B (gB) showed that after infection the proportion of Ag-specific T cells peaked at day 5 within draining popliteal lymph nodes and 2 days later in the spleen. Preferential expression of the activation marker CD25 by tetramer-positive cells in draining popliteal nodes but not spleen suggested that gB-specific T cells were initially activated within the lymph node. In vivo cytotoxicity assays showed that Ag-specific effector cells were present within the draining lymph nodes as early as day 2 after infection, with a further 2-day lag before detection in the spleen. Consistent with the very early arming of effector CTL in the draining lymph node, adoptive transfer of CFSE-labeled gB-specific transgenic T cells showed that they had undergone one to four rounds of cell division by day 2 after infection. In contrast, proliferating T cells were first detected in appreciable numbers in the spleen on day 4, at which time they had undergone extensive cell division. These data demonstrate that HSV-1-specific T cells are rapidly activated and armed within draining lymph nodes shortly after localized HSV-1 infection. This is followed by their dissemination to other compartments such as the spleen, where they further proliferate in an Ag-independent fashion.
IL-4 Is a Potent Modulator of Ion Transport in the Human Bronchial Epithelium In Vitro
Authors: Galietta Luis J. V. , Pagesy Patrick , Folli Chiara , Caci Emanuela , Romio Leila , Costes Bruno , Nicolis Elena , Cabrini Giulio , Goossens Michel , Ravazzolo Roberto , Zegarra-Moran Olga
Published: April 2014

Recent data show that proinflammatory stimuli may modify significantly ion transport in the airway epithelium and therefore the properties of the airway surface fluid. We have studied the effect of IL-4, a cytokine involved in the pathogenesis of asthma, on transepithelial ion transport in the human bronchial epithelium in vitro. Incubation of polarized bronchial epithelial cells with IL-4 for 6–48 h causes a marked inhibition of the amiloride-sensitive Na+ channel as measured in short circuit current experiments. On the other hand, IL-4 evokes a 2-fold increase in the current activated by a cAMP analog, which reflects the activity of the cystic fibrosis transmembrane conductance regulator (CFTR). Similarly, IL-4 enhances the response to apical UTP, an agonist that activates Ca2+-dependent Cl− channels. These effects are mimicked by IL-13 and blocked by an antagonist of IL-4Rα. RT-PCR experiments show that IL-4 elicits a 7-fold decrease in the level of the γ amiloride-sensitive Na+ channel mRNA, one of the subunits of the amiloride-sensitive Na+ channel, and an increase in CFTR mRNA. Our data suggest that IL-4 may favor the hydration of the airway surface by decreasing Na+ absorption and increasing Cl− secretion. This could be required to fluidify the mucus, which is hypersecreted during inflammatory conditions. On the other hand, the modifications of ion transport could also affect the ion composition of airway surface fluid.
CCL7 and CXCL10 Orchestrate Oxidative Stress-Induced Neutrophilic Lung Inflammation
Authors: Michalec Lidia , Choudhury Barun K. , Postlethwait Edward , Wild James S. , Alam Rafeul , Lett-Brown Michael , Sur Sanjiv
Published: April 2014

Oxidative stress from ozone (O3) exposure augments airway neutrophil recruitment and chemokine production. We and others have shown that severe and sudden asthma is associated with airway neutrophilia, and that O3 oxidative stress is likely to augment neutrophilic airway inflammation in severe asthma. However, very little is known about chemokines that orchestrate oxidative stress-induced neutrophilic airway inflammation in vivo. To identify these chemokines, three groups of BALB/c mice were exposed to sham air, 0.2 ppm O3, or 0.8 ppm O3 for 6 h. Compared with sham air, 0.8 ppm O3, but not 0.2 ppm O3, induced pronounced neutrophilic airway inflammation that peaked at 18 h postexposure. The 0.8 ppm O3 up-regulated lung mRNA of CXCL1,2,3 (mouse growth-related oncogene-α and macrophage-inflammatory protein-2), CXCL10 (IFN-γ-inducible protein-10), CCL3 (macrophage-inflammatory protein-1α), CCL7 (monocyte chemoattractant protein-3), and CCL11 (eotaxin) at 0 h postexposure, and expression of CXCL10, CCL3, and CCL7 mRNA was sustained 18 h postexposure. O3 increased lung protein levels of CXCL10, CCL7, and CCR3 (CCL7R). The airway epithelium was identified as a source of CCL7. The role of up-regulated chemokines was determined by administering control IgG or IgG Abs against six murine chemokines before O3 exposure. As expected, anti-mouse growth-related oncogene-α inhibited neutrophil recruitment. Surprisingly, Abs to CCL7 and CXCL10 also decreased neutrophil recruitment by 63 and 72%, respectively. These findings indicate that CCL7 and CXCL10, two chemokines not previously reported to orchestrate neutrophilic inflammation, play a critical role in mediating oxidative stress-induced neutrophilic airway inflammation. These observations may have relevance in induction of neutrophilia in severe asthma.
Activation of the Stem Cell-Derived Tyrosine Kinase/RON Receptor Tyrosine Kinase by Macrophage-Stimulating Protein Results in the Induction of Arginase Activity in Murine Peritoneal Macrophages
Authors: Morrison Amy C. , Correll Pamela H.
Published: April 2014

Regulation of macrophage activities in response to inflammatory stimuli must be finely tuned to promote an effective immune response while, at the same time, preventing damage to the host. Our lab and others have previously shown that macrophage-stimulating protein (MSP), through activation of its receptor RON, negatively regulates NO production in response to IFN-γ and LPS by inhibiting the expression of inducible NO synthase (iNOS). Furthermore, activated macrophages from mice harboring targeted mutations in RON produce increased levels of NO both in vitro and in vivo, rendering them more susceptible to LPS-induced endotoxic shock. In this study, we demonstrate that stimulation of murine peritoneal macrophages with MSP results in the RON-dependent up-regulation of arginase, an enzyme associated with alternative activation that competes with iNOS for the substrate l-arginine, the products of which are involved in cell proliferation and matrix synthesis. Expression of other genes associated with alternative activation, including scavenger receptor A and IL-1R antagonist, is also up-regulated in MSP-stimulated murine macrophages. Stimulation of cells with IFN-γ and LPS blocks the ability of MSP to induce arginase activity. However, pretreatment of cells with MSP results in the up-regulation of arginase and inhibits their ability to produce NO in response to IFN-γ and LPS, even in the presence of excess substrate, suggesting that the inhibition of NO by MSP occurs primarily through its ability to regulate iNOS expression.
IL-6 Secretion by Human Pancreatic Periacinar Myofibroblasts in Response to Inflammatory Mediators
Authors: Shimada Mitsue , Andoh Akira , Hata Kazunori , Tasaki Kazuhito , Araki Yoshio , Fujiyama Yoshihide , Bamba Tadao
Published: April 2014

There is increasing evidence that IL-6 plays an important role in the pathophysiology of acute pancreatitis via its broad proinflammatory actions. To identify the local biosynthetic site for IL-6 in human pancreas, we investigated IL-6 secretion in human pancreatic periacinar myofibroblasts. IL-6 secretion was determined by ELISA and Northern blotting. The activation of NF-κB was assessed by EMSA. The activation of mitogen-activated protein kinase (MAPK) was assessed by immunoblotting. IL-6 secretion was rapidly induced by IL-17, IL-1β, and TNF-α. EMSAs demonstrated that IL-17, IL-1β, and TNF-α induced NF-κB activation within 1.5 h after stimulation, and a blockade of NF-κB activation by the pyrrolidine derivative of dithiocarbamate and tosyl-phe-chloromethylketone markedly reduced the IL-17-, IL-1β-, or TNF-α-induced IL-6 gene expression. Furthermore, IL-17, IL-1β, and TNF-α induced a rapid activation of extracellular signal-related kinase p42/44 and p38 MAPKs, and specific MAPK inhibitors (SB203580, PD98059, and U0216) significantly reduced IL-17-, IL-1β-, or TNF-α-induced IL-6 secretion, indicating the role of MAPKs in the induction of IL-6. The combination of either IL-17 plus IL-1β or IL-17 plus TNF-α enhanced IL-6 secretion and IL-6 mRNA expression; in particular, the effects of IL-17 plus TNF-α were much stronger than those induced by IL-17 plus IL-1β. TNF-α-induced IL-6 mRNA degraded rapidly at any concentrations, and the combination of IL-17 and TNF-α markedly enhanced IL-6 mRNA stability. This indicates that the effects of IL-17 plus TNF-α were regulated at the post-transcriptional level. In conclusion, pancreatic periacinar myofibroblasts secreted a large amount of IL-6 in response to proinflammatory cytokines. These cells might play an important role in the pathogenesis of acute pancreatitis via IL-6 secretion.
129X1/SvJ Mouse Strain Has a Novel Defect in Inflammatory Cell Recruitment
Authors: White Peter , Liebhaber Stephen A. , Cooke Nancy E.
Published: April 2014

Vitamin D-binding protein (DBP) has been reported to contribute to innate immunity. To verify prior in vitro and cell-based observations supporting this role, we assessed the ability of a recently developed DBP-null mouse line to recruit neutrophils and macrophages to a site of chemical inflammation. The interrupted DBP allele had been generated by homologous recombination in 129X1/SvJ embryonic stem cells and these cells were subsequently used to generate a line of DBP−/− (null) mice. Initial studies revealed a marked defect in the ability of these DBP−/− mice to recruit cells to the peritoneum after localized thioglycolate injection. However, progressive outcrossing of the DBP−/− mice to the C57BL/6J strain, conducted to provide a uniform genetic background for comparison of DBP-null and control mice, resulted in a progressive increase in cell recruitment by the DBP−/− mice and a loss in their apparent recruitment defect when compared with the DPB wild-type controls. These data suggested that the observed recruitment phenotype initially attributed to the absence of DBP was not linked to the DBP locus, but instead reflected the underlying genetic composition of the 129X1/SvJ ES cells used for the initial DBP gene disruption. A profound cell recruitment defect was confirmed in the 129X1/SvJ mice by direct analysis. Each of three commonly used inbred lines was discovered to have a distinct level of cell recruitment to a uniform stimulus (C57BL/6J > BALB/c > CD1 > 129X1/SvJ). Thus, this study failed to support a unique role for DBP in cellular recruitment during a model inflammatory response. Instead, the data revealed a novel and profound defect of cell recruitment in 129X1/SvJ mice, the strain most commonly used for gene deletion studies.
Leptin Signaling Deficiency Impairs Humoral and Cellular Immune Responses and Attenuates Experimental Arthritis
Authors: Busso Nathalie , So Alexander , Chobaz-Péclat Véronique , Morard Carole , Martinez-Soria Eduardo , Talabot-Ayer Dominique , Gabay Cem
Published: April 2014

Leptin is produced almost exclusively by adipocytes and regulates body weight at the hypothalamic level. In addition, recent studies showed that leptin plays an important role in T lymphocyte responses. To examine the role of leptin in Ag-induced arthritis, the development of joint inflammation was assessed in immunized leptin-deficient mice (ob/ob), +/?, and wild-type mice (+/+) following the administration of methylated BSA into the knees. The results showed that ob/ob mice developed less severe arthritis compared with control mice. The levels of IL-1β and TNF-α mRNA in the synovium of arthritic knees were lower in ob/ob than in +/? mice. In vitro Ag-specific T cell proliferative responses were significantly decreased in ob/ob mice with lower IFN-γ and higher IL-10 production, suggesting a shift toward a Th2-type response in ob/ob mice. The serum levels of anti-methylated BSA Abs of any isotype were significantly decreased in arthritic ob/ob mice compared with controls. Essentially identical results were obtained in db/db mice, which lack the expression of the long isoform of leptin receptor. By RT-PCR, we observed that B lymphocytes express leptin receptor mRNA, indicating that in addition to its effect on the cellular response, leptin may exert a direct effect on B cell function. In conclusion, leptin contributes to the mechanisms of joint inflammation in Ag-induced arthritis by regulating both humoral and cell-mediated immune responses.
Selective Intracellular Delivery of Dexamethasone into Activated Endothelial Cells Using an E-Selectin-Directed Immunoconjugate
Authors: Everts Maaike , Kok Robbert J. , Ásgeirsdóttir Sigridur A. , Melgert Barbro N. , Moolenaar Tom J. M. , Koning Gerben A. , van Luyn Marja J. A. , Meijer Dirk K. F. , Molema Grietje
Published: April 2014

In chronic inflammatory diseases, the endothelium is an attractive target for pharmacological intervention because it plays an important role in leukocyte recruitment. Hence, inhibition of endothelial cell activation and consequent leukocyte infiltration may improve therapeutic outcome in these diseases. We report on a drug targeting strategy for the selective delivery of the anti-inflammatory drug dexamethasone to activated endothelial cells, using an E-selectin-directed drug-Ab conjugate. Dexamethasone was covalently attached to an anti-E-selectin Ab, resulting in the so-called dexamethasone-anti-E-selectin conjugate. Binding of the conjugate to E-selectin was studied using surface plasmon resonance and immunohistochemistry. Furthermore, internalization of the conjugate was studied using confocal laser scanning microscopy and immuno-transmission electron microscopy. It was demonstrated that the dexamethasone-anti-E-selectin conjugate, like the unmodified anti-E-selectin Ab, selectively bound to TNF-α-stimulated endothelial cells and not to resting endothelial cells. After binding, the conjugate was internalized and routed to multivesicular bodies, which is a lysosome-related cellular compartment. After intracellular degradation, pharmacologically active dexamethasone was released, as shown in endothelial cells that were transfected with a glucocorticoid-responsive reporter gene. Furthermore, intracellularly delivered dexamethasone was able to down-regulate the proinflammatory gene IL-8. In conclusion, this study demonstrates the possibility to selectively deliver the anti-inflammatory drug dexamethasone into activated endothelial cells, using an anti-E-selectin Ab as a carrier molecule.
Allergic Inflammatory Response to Short Ragweed Allergenic Extract in HLA-DQ Transgenic Mice LackingCD4Gene
Authors: Chapoval Svetlana P. , Iijima Koji , Marietta Eric V. , Smart Michele K. , Chapoval Andrei I. , Andrews Amy G. , David Chella S.
Published: April 2014

To investigate the role of HLA-DQ molecules and/or CD4+ T cells in the pathogenesis of allergic asthma, we generated HLA-DQ6 and HLA-DQ8 transgenic mice lacking endogenous class II (Aβnull) and CD4 genes and challenged them intranasally with short ragweed allergenic extract (SRW). We found that DQ6/CD4null mice developed a strong eosinophilic infiltration into the bronchoalveolar lavage and lung tissue, while DQ8/CD4null mice were normal. However, neither cytokines nor eosinophil peroxidase in the bronchoalveolar lavage of DQ6/CD4null mice was found. In addition, the airway reactivity to methacholine was elevated moderately in DQ6/CD4null mice compared with the high response in DQ/CD4+ counterparts and was only partially augmented by CD4+ T cell transfer. The DQ6/CD4null mice showed Th1/Th2-type cytokines and SRW-specific Abs in the immune sera in contrast to a direct Th2 response observed in DQ6/CD4+ mice. The proliferative response of spleen mononuclear cells and peribronchial lymph node cells demonstrated that the response to SRW in DQ6/CD4null mice was mediated by HLA-DQ-restricted CD4−CD8−NK1.1− T cells. FACS analysis of PBMC and spleen mononuclear cells demonstrated an expansion of double-negative (DN) CD4−CD8−TCRαβ+ T cells in SRW-treated DQ6/CD4null mice. These cells produced IL-4, IL-5, IL-13, and IFN-γ when stimulated with immobilized anti-CD3. IL-5 ELISPOT assay revealed that DN T cells were the cellular origin of IL-5 in allergen-challenged DQ6/CD4null mice. Our study shows a role for HLA-DQ-restricted CD4+ and DN T cells in the allergic response.
The Interrelated Roles of TGF-β and IL-10 in the Regulation of Experimental Colitis
Authors: Fuss Ivan J. , Boirivant Monica , Lacy Brian , Strober Warren
Published: April 2014

In the present study, we define the relation between TGF-β and IL-10 in the regulation of the Th1-mediated inflammation occurring in trinitrobenzene sulfonic acid (TNBS)-colitis. In initial studies, we showed that the feeding of trinitrophenol-haptenated colonic protein to SJL/J mice induces CD4+ regulatory T cells that transfer protection from induction of TNBS-colitis, and that such protection correlates with cells producing TGF-β, not IL-10. Further studies in which SJL/J mice were fed haptenated colonic protein, and then administered either anti-TGF-β or anti-IL-10 at the time of subsequent TNBS administration per rectum, showed that while both Abs abolished protection, anti-TGF-β administration prevented TGF-β secretion, but left IL-10 secretion intact; whereas anti-IL-10 administration prevented both TGF-β secretion and IL-10 secretion. Thus, it appeared that the protective effect of IL-10 was an indirect consequence of its effect on TGF-β secretion. To establish this point further, we conducted adoptive transfer studies and showed that anti-IL-10 administration had no effect on induction of TGF-β producing T cells in donor mice. However, it did inhibit their subsequent expansion in recipient mice, probably by regulating the magnitude of the Th1 T cell response which would otherwise inhibit the TGF-β response. Therefore, these studies suggest that TGF-β production is a primary mechanism of counter-regulation of Th1 T cell-mediated mucosal inflammation, and that IL-10 is necessary as a secondary factor that facilitates TGF-β production.
Generation of EBV-Specific CD4+ Cytotoxic T Cells from Virus Naive Individuals
Authors: Savoldo Barbara , Cubbage Michael L. , Durett April G. , Goss John , Huls M. Helen , Liu Zhensheng , Teresita Lopez , Gee Adrian P. , Ling Paul D. , Brenner Malcolm K. , Heslop Helen E. , Rooney Cliona M.
Published: April 2014

Adoptive immunotherapy with EBV-specific CTL (EBV-CTL) effectively prevents and treats EBV-driven lymphoproliferation in immunocompromised hosts. EBV-seronegative solid organ transplant recipients are at high risk of EBV-driven lymphoproliferation because they lack EBV-specific memory T cells. For the same reason, standard techniques for generating EBV-CTL in vitro from EBV-naive individuals are unsuccessful. To overcome this problem, we compared several methods of expanding EBV-CTL from seronegative adults and children. First, the standard protocol, using EBV-transformed lymphoblastoid B cell lines (LCL) as the source of APC, was compared with protocols using EBV-Ag-loaded dendritic cells as APC. Surprisingly, the standard protocol effectively generated CTL from all seronegative adults. The additional finding of EBV-DNA in the peripheral blood of three of these four adults suggested that some individuals may develop cellular, but not humoral, immune responses to EBV. By contrast, LCL failed to reactivate EBV-CTL from any of the six EBV-seronegative children. EBV-Ag-loaded dendritic cells could expand EBV-CTL, but only in a minority of children. However, the selective expansion of CD25-expressing T cells, 9–11 days after activation with LCL alone, proved to be a simple and reliable method for generating EBV-CTL from all seronegative children. The majority of these CTL were CD4+ (71 ± 26%) and demonstrated HLA class II-restricted, EBV-specific killing. Our results suggest that a negative EBV serology does not accurately identify EBV-negative individuals. In addition, our method for selecting EBV-specific CTL from naive individuals by precursor cell enrichment may be applicable to the immunotherapy of cancer patients with a low frequency of tumor- or virus-specific CTL.
Influence ofMycobacterium bovisBacillus Calmette-Guérin on Antibody and Cytokine Responses to Human Neonatal Vaccination
Authors: Ota Martin O. C. , Vekemans Johan , Schlegel-Haueter Susanna E. , Fielding Katherine , Sanneh Mariama , Kidd Michael , Newport Melanie J. , Aaby Peter , Whittle Hilton , Lambert Paul-Henri , McAdam Keith P. W. J. , Siegrist Claire-Anne , Marchant Arnaud
Published: April 2014

The immaturity of the immune system increases the susceptibility of young infants to infectious diseases and prevents the induction of protective immune responses by vaccines. We previously reported that Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccination induces a potent Th1 response to mycobacterial Ags in newborns. In this study, we evaluated the influence of BCG on the response to unrelated vaccines given in early life. Newborns were randomly allocated to one of three study groups receiving BCG at birth, when infants received their first dose of hepatitis B and oral polio vaccines; at 2 mo of age, when infants received their first dose of diphtheria and tetanus vaccines; or at 4.5 mo of age, when immune responses to vaccines were measured. Administration of BCG at the time of priming markedly increased the cellular and Ab responses to the hepatitis B vaccine, but had only a limited influence on the cytokine response to tetanus toxoid and no effect on the Ab responses to tetanus and diphtheria toxoids. Although BCG induced a potent Th1-type response to mycobacterial Ags, it promoted the production of both Th1- and Th2-type cytokines in response to unrelated vaccines. The effect of BCG was apparent at the systemic level, as it increased the Ab response to oral polio vaccine. These results demonstrate that BCG influences the immune response to unrelated Ags in early life, likely through its influence on the maturation of dendritic cells.
Monophosphoryl Lipid A Activates Both Human Dendritic Cells and T Cells
Authors: Ismaili Jamila , Rennesson Joëlle , Aksoy Ezra , Vekemans Johan , Vincart Benoit , Amraoui Zoulikha , Van Laethem Francois , Goldman Michel , Dubois Patrice M.
Published: April 2014

The induction of dendritic cell (DC) maturation is critical for the induction of Ag-specific T lymphocyte responses and may be essential for the development of human vaccines relying on T cell immunity. In this study, we have investigated the effects of monophosphoryl lipid A (MPL) on human monocyte-derived DC as well as peripheral blood T cells. Calcium mobilization, mitogen-activated protein kinase activation, and the NF-κB transcription factor were induced after MPL stimulation of DC and required high doses of MPL (100 μg/ml). Maturation parameters such as production of IL-12 and increases in cell surface expression of HLA-DR, CD80, CD86, CD40, and CD83 were observed following DC treatment with MPL. However, lower levels of IL-12 were induced by MPL when compared with lipopolysaccharide. This is likely to be related to differences in the kinetics of extracellular signal-related kinase 1/2 and p-38 phosphorylation induced by both molecules. Although maturation induced by MPL was weaker when compared with lipopolysaccharide, it appeared to be sufficient to support optimal activation of allogeneic naive CD45RA+ T cell and anti-tetanus toxoid CD4 T cells. MPL at low doses (5 μg/ml) had no impact on DC maturation, while its addition to DC-T cell cocultures induced full T cell activation. The observed effect was related to the fact that MPL also acts directly on T cells, likely through their Toll-like receptors, by increasing their intracellular calcium and up-regulating their CD40 ligand expression. Together, these data support a model where MPL enhances T cell responses by having an impact on DC and T cells.
Nonmitogenic CD3 Antibody Reverses Virally Induced (Rat Insulin Promoter-Lymphocytic Choriomeningitis Virus) Autoimmune Diabetes Without Impeding Viral Clearance
Authors: von Herrath Matthias G. , Coon Bryan , Wolfe Tom , Chatenoud Lucienne
Published: April 2014

Treatment with nonmitogenic CD3 Ab reverses established autoimmune diabetes in nonobese diabetic mice by restoring self-tolerance, and is currently under clinical evaluation in patients presenting recent onset type I diabetes. Due to the immunosuppressive potential of this strategy, it was relevant to explore how this treatment would influence the outcome of concomitant viral infections. In this study, we used a transgenic model of virally induced autoimmune diabetes (rat insulin promoter-lymphocytic choriomeningitis virus) that allows for more precise tracking of the autoaggressive response and choice of the time point for initiation of autoimmunity. CD3 was most effective during a clearly defined prediabetic phase and prevented up to 100% of diabetes by drastically lowering activation of autoaggressive CD8 lymphocytes and their production of inflammatory cytokines. Interestingly, reversion of established disease could be achieved as well, when nonmitogenic CD3 was administered late during pathogenesis to overtly diabetic recipients. Most importantly, competence to clear viral infections was maintained. Thus, administration of nonmitogenic CD3 prevents diabetes by sufficient systemic reduction of (auto)aggressive lymphocytes, but without compromising antiviral immune competence.
Igs from Patients with Graves’ Disease Induce the Expression of T Cell Chemoattractants in Their Fibroblasts
Authors: Pritchard Jane , Horst Noah , Cruikshank William , Smith Terry J.
Published: April 2014

Thyroid-associated ophthalmopathy and dermopathy are connective tissue manifestations of Graves’ disease (GD). Tissue remodeling is a prominent feature of both and is apparently driven by recruited T cells. In this study, we report that IgG isolated from patients with GD (GD-IgG) up-regulates T lymphocyte chemoattractant activity in GD-derived fibroblasts from orbit, thyroid, and several regions of skin. This chemoattractant activity, absent in fibroblasts from donors without known thyroid disease, is partially susceptible to neutralization by anti-IL-16 and anti-RANTES Abs. IL-16 is a CD4+-specific chemoattractant and RANTES is a C-C-type chemokine. IL-16 and RANTES protein levels, as determined by specific ELISAs, are substantially increased by GD-IgG in GD fibroblasts. Addition of the macrolide, rapamycin, to fibroblast culture medium blocked the up-regulation by GD-IgG of IL-16, implicating the FRAP/mTOR/p70s6k pathway in the induction of IL-16 expression. These findings suggest a specific mechanism for activation of fibroblasts in GD resulting in the recruitment of T cells. They may provide insight into a missing link between the glandular and extrathyroidal manifestations of GD.
Pre-Existing Immunity to Tyrosinase-Related Protein (TRP)-2, a New TRP-2 Isoform, and the NY-ESO-1 Melanoma Antigen in a Patient with a Dramatic Response to Immunotherapy
Authors: Khong Hung T. , Rosenberg Steven A.
Published: April 2014

We have performed a detailed analysis of the recognition of melanoma Ags by the tumor-infiltrating lymphocytes (TIL) 1790, isolated from a patient who experienced a dramatic tumor regression following immunization with peptides from the gp100, MART-1, and tyrosinase Ags. This TIL was found to recognize HLA-A2-restricted CTL epitopes in tyrosinase-related protein (TRP)-2 (clone MR7) and NY-ESO-1 (clone M8). These epitopes were the same as the previously identified nonapeptide TRP-2: 180–188, and the overlapping NY-ESO-1 peptides, obtained by using lymphocytes from in vitro stimulation. We also cloned a previously unknown TRP-2 mRNA isoform (TRP-2-6b) that contained two novel exons alternatively spliced from the sixth intron between exons 6 and 7 of TRP-2 mRNA. The isoform encoded an HLA-A2-restricted antigenic epitope recognized by TIL clone MB4. An immunologic analysis of the patient’s PBMC obtained before treatment showed the presence of high reactivity against NY-ESO-1 and both TRP-2 Ags, but not the Ags used for immunization. Because immune response against these Ags was less pronounced, it is possible that NY-ESO-1, TRP-2, and TRP-2-6b may be of importance in the generation of CTL-mediated tumor destruction and may have played a role in the dramatic tumor regression seen in this patient.
Efficient In Vivo Priming of Specific Cytotoxic T Cell Responses by Neonatal Dendritic Cells
Authors: Dadaglio Gilles , Sun Cheng-Ming , Lo-Man Richard , Siegrist Claire Anne , Leclerc Claude
Published: April 2014

In early life, a high susceptibility to infectious diseases as well as a poor capacity to respond to vaccines are generally observed as compared with observations in adults. The mechanisms underlying immune immaturity have not been fully elucidated and could be due to the immaturity of the T/B cell responses and/or to a defect in the nature and quality of Ag presentation by the APC. This prompted us to phenotypically and functionally characterize early life murine dendritic cells (DC) purified from spleens of 7-day-old mice. We showed that neonatal CD11c+ DC express levels of costimulatory molecules and MHC molecules similar to those of adult DC and are able to fully maturate after LPS activation. Furthermore, we demonstrated that neonatal DC can efficiently take up, process, and present Ag to T cells in vitro and induce specific CTL responses in vivo. Although a reduced number of these cells was observed in the spleen of neonatal mice as compared with adults, this study clearly shows that neonatal DC have full functional capacity and may well prime Ag-specific naive T cells in vivo.
HIV Antigens Can Induce TGF-β1-Producing Immunoregulatory CD8+ T Cells
Authors: Garba Mohammed L. , Pilcher Christopher D. , Bingham Andrea L. , Eron Joseph , Frelinger Jeffrey A.
Published: April 2014

HIV-infected individuals may progressively lose both HIV-specific and unrelated CTL responses despite the high number of circulating CD8+ T cells. In this study, we report that ∼25% of HIV+ donors produced TGF-β1 in response to stimulation with HIV proteins or peptides. The production of TGF-β1 was sufficient to significantly reduce the IFN-γ response of CD8+ cells to both HIV and vaccinia virus proteins. Ab to TGF-β reversed the suppression. We found the source of the TGF-β1 to be predominantly CD8+ cells. Different peptide pools stimulated TGF-β1 and IFN-γ in the same individual. The TGF-β1 secreting cells have distinct peptide specificity from the IFN-γ producing cells. This represents an important mechanism by which an HIV-specific response can nonspecifically suppress both HIV-specific and unrelated immune responses.
Cyclooxygenase-2-Issued Prostaglandin E2 Enhances the Production of Endogenous IL-10, Which Down-Regulates Dendritic Cell Functions
Authors: Harizi Hedi , Juzan Monique , Pitard Vincent , Moreau Jean-François , Gualde Norbert
Published: April 2014

PGE2 is a well-known immunomodulator produced in the immune response by APCs, such as dendritic cells (DCs), the most potent APC of the immune system. We investigated the PGE2 biosynthetic capacity of bone marrow-derived DC (BM-DC) and the effects of PG on the APC. We observed that BM-DC produce PGE2 and other proinflammatory mediators, such as leukotriene B4 and NO, after LPS exposure. Constitutively present in BM-DC, cyclooxygenase (COX)-1 did not contribute significantly to the total pool of PGE2 compared with the LPS-induced COX-2-produced PGE2. Treatment of BM-DC with exogenous PGE2 induced the production of large amounts of IL-10 and less IL-12p70. In addition, selective inhibition of COX-2, but not COX-1, was followed by significant decrements in PGE2 and IL-10, a concomitant restoration of IL-12 production, and an enhancement of DC stimulatory potential. In contrast, we found no demonstrable role for leukotriene B4 or NO. In view of the potential of PGE2 to stimulate IL-10, we examined the possibility that the suppressive effect of PGE2 is mediated via IL-10. We found that exogenous IL-10 inhibits IL-12p70 production in the presence of NS-398, a COX-2 selective inhibitor, while the inhibitory effects of PGE2 were totally reversed by anti-IL-10. We conclude that COX-2-mediated PGE2 up-regulates IL-10, which down-regulates IL-12 production and the APC function of BM-DC.
Expression of Thrombospondin in TGFβ-Treated APCs and Its Relevance to Their Immune Deviation-Promoting Properties
Authors: Masli Sharmila , Turpie Bruce , Hecker Karl H. , Streilein J. Wayne
Published: April 2014

APCs deployed within iris/ciliary body are responsible for promoting anterior chamber-associated immune deviation following injection of Ag into the eye. TGFβ-2, a constituent of the ocular microenvironment, converts conventional APCs that are pulsed with Ag into cells that induce immune deviation when injected into naive mice. TGFβ-2-treated APCs under-express IL-12 and CD40, and over-express active TGFβ. We have examined transcriptional changes within macrophage hybridoma no. 59, which promotes Th1 cell differentiation, and TGFβ-2-treated no. 59 as well as macrophage hybridoma no. 63, both of which induce immune deviation similar to anterior chamber-associated immune deviation. Immune deviation-inducing hybridomas up-regulated expression of thrombospondin, TGFβ, IFN-α and β, murine macrophage elastase, and macrophage-inflammatory protein-2 genes, while down-regulating expression of the genes for NF-κB and CD40. Based on the known properties of these gene products, a model is proposed in which these gene products, alone and through interacting signaling pathways, confer upon conventional APCs the capacity to create and surround themselves with an immunomodulatory microenvironment. The model proposes that the pleiotropic effects of thrombospondin are primarily responsible for creating this microenvironment that is stabile, rich in active TGFβ and IFN-α and β, deficient in IL-12, and chemoattractant via macrophage-inflammatory protein-2 for NK T cells. It is further proposed that presentation of Ag to T cells in this microenvironment leads to their differentiation into regulatory cells that suppress Th1 cell-dependent immunogenic inflammation.
The Apoptotic Protease-Activating Factor 1-Mediated Pathway of Apoptosis Is Dispensable for Negative Selection of Thymocytes
Authors: Hara Hiromitsu , Takeda Atsunobu , Takeuchi Michiyo , Wakeham Andrew C. , Itié Annick , Sasaki Masafumi , Mak Tak W. , Yoshimura Akihiko , Nomoto Kikuo , Yoshida Hiroki
Published: April 2014

Negative selection is a process to delete potentially autoreactive clones in developing thymocytes. Programmed cell death or apoptosis is thought to play an important role in this selection process. In this study, we investigated the role of apoptotic protease-activating factor 1 (Apaf1), a mammalian homologue of CED-4, in programmed cell death during the negative selection in thymus. There was no developmental abnormality in thymocytes from newborn Apaf1−/− mice in terms of CD4 and CD8 expression pattern and thymocyte number. Clonal deletion by endogenous male H-Y Ag of Apaf1-deficient thymocytes with transgenic expression of H-Y Ag-specific TCRs (H-Y Tg/Apaf1−/− thymocytes) was normally observed in lethally irradiated wild-type mice reconstituted with fetal liver-derived hemopoietic stem cells. Clonal deletion induced in vitro by a bacterial superantigen was also normal in fetal thymic organ culture. Thus, Apaf1-mediated pathway of apoptosis is dispensable for the negative selection of thymocytes. However, H-Y Tg/Apaf1−/− thymocytes showed partial resistance to H-Y peptide-induced deletion in vitro as compared with H-Y Tg/Apaf1+/− thymocytes, implicating the Apaf1-mediated apoptotic pathway in the negative selection in a certain situation. In addition, the peptide-induced deletion was still observed in H-Y Tg/Apaf1−/− thymocytes in the presence of a broad spectrum caspase inhibitor, z-VAD-fmk, suggesting the presence of caspase-independent cell death pathway playing roles during the negative selection. We assume that mechanisms for the negative selection are composed of several cell death pathways to avoid failure of elimination of autoreactive clones.
Transgenic Expression of the p16INK4a Cyclin-Dependent Kinase Inhibitor Leads to Enhanced Apoptosis and Differentiation Arrest of CD4−CD8− Immature Thymocytes
Authors: Lagresle Chantal , Gardie Betty , Eyquem Stéphanie , Fasseu Magali , Vieville Jean-Claude , Pla Marika , Sigaux François , Bories Jean-Christophe
Published: April 2014

In the thymus, T cell development proceeds by successive steps of differentiation, expansion, and selection. Control of thymocyte proliferation is critical to insure the full function of the immune system and to prevent T cells from transformation. Deletion of the cell cycle inhibitor p16INK4a is frequently observed in human T cell neoplasias and, in mice, gene targeted inactivation of the Ink4a locus enhances thymocyte expansion and predisposes mutant animal to tumorigenesis. Here, we investigate the mechanism by which p16Ink4a controls thymocyte development by analyzing transgenic mice expressing the human p16INK4a into the T cell lineage. We show that forced expression of p16INK4a in thymocytes blocked T cell differentiation at the early CD4−CD8−CD3−CD25+ stage without significantly affecting the development of γδ T cells. Pre-TCR function was mimicked by the induction of CD3 signaling in thymocytes of recombinase activating gene (RAG)-2-deficient mice (RAG-2−/−). Upon anti-CD3ε treatment in vivo, p16INK4a-expressing RAG-2−/− thymocytes were not rescued from apoptosis, nor could they differentiate. Our data demonstrate that expression of p16INK4a prevents the pre-TCR-mediated expansion and/or survival of differentiating thymocytes.
Dendritic Cells Transduced with Protein Antigens Induce Cytotoxic Lymphocytes and Elicit Antitumor Immunity
Authors: Shibagaki Naotaka , Udey Mark C.
Published: April 2014

Dendritic cell (DC)-based vaccines are being developed for treatment of patients with cancer, in part because DC are potent inducers of CD8+ CTL. DC MHC class I:antigenic peptide complexes that are required for CTL elicitation are most often generated by incubating DC with peptides or by transfecting (or transducing) DC with cDNAs (or viral vectors) that encode protein Ags. The former approach is feasible when MHC class I Ags and relevant peptides are known. The latter approach may be hampered by inefficient DC transfection (transduction) and/or difficulties associated with preparation and use of viral vectors. Herein we demonstrate that a bacterial recombinant model tumor-associated Ag (OVA) that contains the HIV TAT protein transduction domain (PTD) was readily engineered and purified, efficiently transduced murine lymphocytes and DC, and was processed by proteasomes for MHC class I-restricted presentation to CTL. In addition, PTD-containing rOVA was processed and presented by DC to CD4 T cells as efficiently as native OVA or rOVA lacking the PTD. PTD-OVA-transduced DC induced CTL in vivo in a Th cell-independent fashion and vaccinated against OVA-expressing tumors. In contrast, rOVA lacking the PTD did not enter the DC MHC class I presentation pathway and DC treated with this protein did not prime OVA-specific CTL in vivo. Treatment of mice harboring clinically apparent OVA-expressing tumors with PTD-OVA-transduced DC resulted in tumor regression in some animals. This straightforward vaccination strategy may translate into DC-based treatments for patients with cancer and other serious diseases.
Response toNeisseria gonorrhoeaeby Cervicovaginal Epithelial Cells Occurs in the Absence of Toll-Like Receptor 4-Mediated Signaling
Authors: Fichorova Raina N. , Cronin Amanda O. , Lien Egil , Anderson Deborah J. , Ingalls Robin R.
Published: April 2014

Toll-like receptors (TLRs) have recently been identified as fundamental components of the innate immune response to bacterial pathogens. We investigated the role of TLR signaling in immune defense of the mucosal epithelial cells of the lower female genital tract. This site provides first line defense against microbial pathogens while remaining tolerant to a complex biosystem of resident microbiota. Epithelial cells derived from normal human vagina, ectocervix, and endocervix expressed mRNA for TLR1, -2, -3, -5, and -6. However, they failed to express TLR4 as well as MD2, two essential components of the receptor complex for LPS in phagocytes and endothelial cells. Consistent with this, endocervical epithelial cells were unresponsive to protein-free preparations of lipooligosaccharide from Neisseria gonorrhoeae and LPS from Escherichia coli. However, they were capable of responding to whole Gram-negative bacteria and bacterial lysates, as demonstrated by NF-κB activation and proinflammatory cytokine production. The presence of soluble CD14, a high-affinity receptor for LPS and other bacterial ligands, enhanced the sensitivity of genital tract epithelial cells to both low and high concentrations of bacteria, suggesting that soluble CD14 can act as a coreceptor for non-TLR4 ligands. These data demonstrate that the response to N. gonorrhoeae and other Gram-negative bacteria at the mucosal surface of the female genital tract occurs in the absence of endotoxin recognition and TLR4-mediated signaling.
Cooperation of C1q Receptors and Integrins in C1q-Mediated Endothelial Cell Adhesion and Spreading
Authors: Feng Xiaodong , Tonnesen Marcia G. , Peerschke Ellinor I. B. , Ghebrehiwet Berhane
Published: April 2014

The interaction of C1q with endothelial cells elicits a multiplicity of biologic responses. Although these responses are presumed to be mediated by the interaction of C1q with endothelial cell surface proteins, the identity of the participants is not known. In this study we examined the roles of two C1q binding proteins, cC1q-R/calreticulin and gC1q-R/p33, in C1q-mediated adhesion and spreading of human dermal microvascular endothelial cells (HDMVEC). When HDMVEC were cultured in microtiter plate wells coated with concentrations of C1q ranging from 0 to 50 μg/ml, a specific and dose-dependent adhesion and spreading was observed. The extent of adhesion and spreading was similar to the adhesion seen on collagen-coated wells. Spreading (68 ± 12%) and to a moderate extent adhesion (47 ± 9%) were inhibited by anti-gC1q-R mAb 60.11. Similar effects were noted with polyclonal anti-cC1q-R but not with control nonimmune IgG. The two Abs had a slight additive effect (75 ± 13% inhibition) when mixed together in the proportion of 100 μg/ml anti-gC1q-R and 30 μg/ml anti-cC1q-R. More importantly, a 100% inhibition of spreading, but not adhesion, to C1q-coated wells was observed when HDMVEC were cultured in the presence of 30 μM of the peptide GRRGDSP but not GRRGESP. Furthermore, while anti-β1 integrin Ab blocked both adhesion and spreading, anti-α5 integrin blocked only spreading and not adhesion. Ag capture ELISA of endothelial cell membrane proteins using polyclonal anti-gC1q-R showed the presence of not only β1 and α5 integrins but also CD44. Taken together these results suggest that endothelial cell adhesion and spreading require the cooperation of both C1qRs and β1 integrins and possibly other membrane-spanning molecules.
IL-4 Pretreatment Selectively Enhances Cytokine and Chemokine Production in Lipopolysaccharide-Stimulated Mouse Peritoneal Macrophages
Authors: Major Jennifer , Fletcher Julia E. , Hamilton Thomas A.
Published: April 2014

Although well recognized for its anti-inflammatory effect on gene expression in stimulated monocytes and macrophages, IL-4 is a pleiotropic cytokine that has also been shown to enhance TNF-α and IL-12 production in response to stimulation with LPS. In the present study we expand these prior studies in three areas. First, the potentiating effect of IL-4 pretreatment is both stimulus and gene selective. Pretreatment of mouse macrophages with IL-4 for a minimum of 6 h produces a 2- to 4-fold enhancement of LPS-induced expression of several cytokines and chemokines, including TNF-α, IL-1α, macrophage-inflammatory protein-2, and KC, but inhibits the production of IL-12p40. In addition, the production of TNF-α by macrophages stimulated with IFN-γ and IL-2 is inhibited by IL-4 pretreatment, while responses to both LPS and dsRNA are enhanced. Second, the ability of IL-4 to potentiate LPS-stimulated cytokine production appears to require new IL-4-stimulated gene expression, because it is time dependent, requires the activation of STAT6, and is blocked by the reversible protein synthesis inhibitor cycloheximide during the IL-4 pretreatment period. Finally, IL-4-mediated potentiation of TNF-α production involves specific enhancement of mRNA translation. Although TNF-α protein is increased in IL-4-pretreated cells, the level of mRNA remains unchanged. Furthermore, LPS-stimulated TNF-α mRNA is selectively enriched in actively translating large polyribosomes in IL-4-pretreated cells compared with cells stimulated with LPS alone.