bluuebunny/crossref_metadata_2025 huggingface dataset:
Secretion of Oncostatin M by Infiltrating Neutrophils: Regulation of IL-6 and Chemokine Expression in Human Mesothelial Cells
Authors: Hurst Suzanne M. , McLoughlin Rachel M. , Monslow James , Owens Sara , Morgan Llinos , Fuller Gerald M. , Topley Nicholas , Jones Simon A.
Published: April 2014

Recently, we identified that regulation of leukocyte recruitment by IL-6 requires shedding of the IL-6R from infiltrating neutrophils. In this study, experiments have examined whether other IL-6-related cytokines possess similar properties. Levels of oncostatin M (OSM) and leukemia inhibitory factor were analyzed in patients with overt bacterial peritonitis during the first 5 days of infection. Although no change in leukemia inhibitory factor was observed throughout the duration of infection, OSM was significantly elevated on day 1 and rapidly returned to baseline by days 2–3. The source of OSM was identified as the infiltrating neutrophils, and OSM levels correlated both with leukocyte numbers and i.p. soluble IL-6R (sIL-6R) levels. FACS analysis revealed that OSM receptor β expression was restricted to human peritoneal mesothelial cells. Stimulation of human peritoneal mesothelial cells with OSM induced phosphorylation of gp130 and OSM receptor β, which was accompanied by activation of STAT3 and secretion of CC chemokine ligand 2/monocyte chemoattractant protein-1 and IL-6. Although OSM itself did not modulate CXC chemokine ligand 8/IL-8 release, it effectively suppressed IL-1β-mediated expression of this neutrophil-activating CXC chemokine. Moreover, OSM synergistically blocked IL-1β-induced CXC chemokine ligand 8 secretion in combination with the IL-6/sIL-6R complex. Thus suggesting that OSM and sIL-6R release from infiltrating neutrophils may contribute to the temporal switch between neutrophil influx and mononuclear cell recruitment seen during acute inflammation.
Adenosine Up-Regulates Cyclooxygenase-2 in Human Granulocytes: Impact on the Balance of Eicosanoid Generation
Authors: Pouliot Marc , Fiset Marie-Élaine , Massé Mireille , Naccache Paul H. , Borgeat Pierre
Published: April 2014

Polymorphonuclear neutrophils (granulocytes; PMNs) are often the first blood cells to migrate toward inflammatory lesions to perform host defense functions. PMNs respond to specific stimuli by releasing several factors and generate lipid mediators of inflammation from the 5-lipoxygenase and the inducible cyclooxygenase (COX)-2 pathways. In view of adenosine’s anti-inflammatory properties and suppressive impact on the 5-lipoxygenase pathway, we addressed in this study the impact of this autacoid on the COX-2 pathway. We observed that adenosine up-regulates the expression of the COX-2 enzyme and mRNA. Production of PGE2 in response to exogenous arachidonic acid was also increased by adenosine and correlated with COX-2 protein levels. The potentiating effect of adenosine on COX-2 could be mimicked by pharmacological increases of intracellular cAMP levels, involving the latter as a putative second messenger for the up-regulation of COX-2 by adenosine. Specific COX-2 inhibitors were used to confirm the predominant role of the COX-2 isoform in the formation of prostanoids by stimulated PMNs. Withdrawal of extracellular adenosine strikingly emphasized the inhibitory potential of PGE2 on leukotriene B4 formation and involved the EP2 receptor subtype in this process. Thus, adenosine may promote a self-limiting regulatory process through the increase of PGE2 generation, which may result in the inhibition of PMN functions. This study identifies a new aspect of the anti-inflammatory properties of adenosine in leukocytes, introducing the concept that this autacoid may exert its immunomodulatory activities in part by modifying the balance of lipid mediators generated by PMNs.
NF-κB and TNF-α: A Positive Autocrine Loop in Human Lung Mast Cells?
Authors: Coward William R. , Okayama Yoshimichi , Sagara Hironori , Wilson Susan J. , Holgate Stephen T. , Church Martin K.
Published: April 2014

The generation of cytokines, particularly TNF-α, by mast cells is crucial for the initiation of the allergic response. A key transcription factor involved in the synthesis of TNF-α is NF-κB. Using a mAb specific for the activated form of NF-κB, immunocytochemistry, confocal microscopy, and gel shift assays have been used in conjunction to localize this transcription factor to human lung mast cells and to study its activation. Activation of mast cells with stem cell factor (10 ng/ml) and anti-IgE (1 μg/ml) induced maximal activation of NF-κB at 4 and 2 h, respectively. In contrast, with TNF-α (5 ng/ml) maximal activation occurred within 15 min. Parallel falls in IκB were demonstrated. Confocal microscopy demonstrated the localization of the activated form of NF-κB to the nuclei of activated mast cells. NF-κB activation was verified using a gel shift assay. A supershift assay showed mast cell NF-κB to be composed primarily of p50 with smaller amounts of p65. No interaction with Abs for Rel-A, c-Rel, Rel-B, and p52 was seen. Immunocytochemistry and ELISAs showed TNF-α to be stored within mast cells and released into the extracellular environment following activation. The possible participation of TNF-α generated by mast cells in NF-κB activation by anti-IgE was investigated using a blocking Ab for TNF-α. The blocking Ab reduced NF-κB activation by anti-IgE by >50%, suggesting that the release of preformed mast cell-associated TNF-α acts as a positive autocrine feedback signal to augment NF-κB activation and production of further cytokine, including GM-CSF and IL-8.
Down-Regulation of Intestinal Lymphocyte Activation and Th1 Cytokine Production by Antibiotic Therapy in a Murine Model of Crohn’s Disease
Authors: Bamias Giorgos , Marini Marco , Moskaluk Christopher A. , Odashima Masaru , Ross William G. , Rivera-Nieves Jesús , Cominelli Fabio
Published: April 2014

Resident intestinal bacteria likely play an important role in the pathogenesis of Crohn’s disease through their interaction with the gut immune system. SAMP1/YitFc mice spontaneously develop chronic, discontinuous, transmural ileitis with many features similar to Crohn’s disease. The aim of this study was to determine the effects and elucidate the mechanisms of action of antibiotic treatment in the SAMP1/YitFc mouse model of ileitis. Mice were treated orally with ciprofloxacin and metronidazole before the development of ileitis (prevention protocol) or after ileitis was fully established (treatment protocol). Terminal ilea were harvested for histological scoring, and lamina propria and mesenteric lymph node cells were isolated for analysis of activation markers and cytokine production. Antibiotic therapy significantly decreased the severity of ileitis both in the prevention (40% reduction, p < 0.05) and the treatment (25% reduction, p < 0.01) protocols, compared with untreated, control mice. These effects were associated with a decreased percentage of CD4+/CD45RBhigh lymphocytes in mesenteric lymph nodes of antibiotic-treated mice, as well as decreased production of IFN-γ (prevention: 0.53 ± 0.21 vs 1.84 ± 0.04 ng/ml, p < 0.05; treatment: 8.4 ± 0.4 vs 12.4 ± 0.7 ng/ml, p < 0.005) and TNF (prevention: 61.5 ± 13 vs 134 ± 19 pg/ml, p < 0.01; treatment: 333.5 ± 11 vs 496 ± 20 pg/ml, p < 0.001). The number of activated lamina propria lymphocytes was also reduced after antibiotic treatment. In conclusion, antibiotic therapy significantly ameliorates the severity of ileitis in SAMP1/YitFc mice by a mechanism involving down-regulation of activated gut lymphocytes and inhibition of intestinal Th1 cytokine production.
Delineation of Five Thyroglobulin T Cell Epitopes with Pathogenic Potential in Experimental Autoimmune Thyroiditis
Authors: Verginis Panayotis , Stanford Marianne M. , Carayanniotis George
Published: April 2014

Experimental autoimmune thyroiditis (EAT) is a T cell-mediated disease that can be induced in mice after challenge with thyroglobulin (Tg) or Tg peptides. To date, five pathogenic Tg peptides have been identified, four of which are clustered toward the C-terminal end. Because susceptibility to EAT is under control of H-2Ak genes, we have used an algorithm-based approach to identify Ak-binding peptides with pathogenic potential within mouse Tg. Eight candidate synthetic peptides, varying in size from 9 to 15 aa, were tested and five of those (p306, p1579, p1826, p2102, and p2596) were found to induce EAT in CBA/J (H-2k) mice either after direct challenge with peptide in adjuvant or by adoptive transfer of peptide-sensitized lymph node cells (LNCs) into naive hosts. These pathogenic peptides were immunogenic at the T cell level, eliciting specific LNC proliferative responses and IL-2 and/or IFN-γ secretion in recall assays in vitro, but contained nondominant epitopes. All immunogenic peptides were confirmed as Ak binders because peptide-specific LNC proliferation was blocked by an Ak-specific mAb, but not by a control mAb. Peptide-specific serum IgG was induced only by p2102 and p2596, but these Abs did not bind to intact mouse Tg. This study reaffirms the predictive value of Ak-binding motifs in epitope mapping and doubles the number of known pathogenic T cell determinants in Tg that are now found scattered throughout the length of this large autoantigen. This knowledge may contribute toward our understanding of the pathogenesis of autoimmune thyroiditis.
Cutting Edge: Major CD8 T Cell Response to Live Bacillus Calmette-Guérin Is Mediated by CD1 Molecules
Authors: Kawashima Tetsuo , Norose Yoshihiko , Watanabe Yoshiyuki , Enomoto Yutaka , Narazaki Hidehiko , Watari Eiji , Tanaka Shigeo , Takahashi Hidemi , Yano Ikuya , Brenner Michael B. , Sugita Masahiko
Published: April 2014

MHC class I-restricted CD8+ T cells are a crucial component of the host defense against mycobacterial infection in mice, but it has often proved very difficult to identify the CD8 T cell response in humans. Human group 1 CD1 molecules (CD1a, -b, -c) mediate MHC-independent presentation of mycobacteria-derived lipid and glycolipid Ags to CD8+ T cells, and their intracellular localization to the endocytic system may favor efficient monitoring of phagosome-resident mycobacteria. Here, we show that bacillus Calmette-Guérin (BCG)-immunized subjects contain a significant circulating pool of CD8+ T cells that recognize BCG-infected DCs in a CD1-dependent, but MHC-independent, manner. These CD1-restricted T cells efficiently detected live, rather than dead, BCG and produced IFN-γ, an important cytokine for protection against mycobacterial infection. These results emphasize that lipid-reactive CD8+ T cells may contribute to host defense against mycobacterial infection.
Cutting Edge: Bradykinin Induces IL-12 Production by Dendritic Cells: A Danger Signal That Drives Th1 Polarization
Authors: Aliberti Julio , Viola João P. B. , Vieira-de-Abreu Adriana , Bozza Patricia T. , Sher Alan , Scharfstein Julio
Published: April 2014

Dendritic cells play a major role in the induction of both innate and acquired immune responses against pathogenic invaders. These cells are also able to sense endogenous activation signals liberated by injured tissues even in the absence of infection. In the present work, we demonstrate that kinins mobilize dendritic cells to produce IL-12 through activation of the B2 bradykinin receptor subtype and that bradykinin-induced IL-12 responses are tightly regulated both by angiotensin-converting enzyme, a kinin-degrading peptidase, and by endogenous IL-10. Using a mouse model of allergic inflammation, we further show that addition of bradykinin to OVA during immunization results in decreased eosinophil infiltration on Ag challenge. The latter effect was demonstrated to be due to IL-12-driven skewing of Ag-specific T cell responses to a type 1 cytokine profile. Our data thus indicate that kinin peptides can serve as danger signals that trigger dendritic cells to produce IL-12 through activation of B2 bradykinin receptors.
Differential Regulation of Human Eosinophil IL-3, IL-5, and GM-CSF Receptor α-Chain Expression by Cytokines: IL-3, IL-5, and GM-CSF Down-Regulate IL-5 Receptor α Expression with Loss of IL-5 Responsiveness, but Up-Regulate IL-3 Receptor α Expression
Authors: Gregory Bernard , Kirchem Antje , Phipps Simon , Gevaert Phillipe , Pridgeon Carol , Rankin Sara M. , Robinson Douglas S.
Published: April 2014

Our recent data suggested that tissue eosinophils may be relatively insensitive to anti-IL-5 treatment. We examined cross-regulation and functional consequences of modulation of eosinophil cytokine receptor expression by IL-3, IL-5 GM-CSF, and eotaxin. Incubation of eosinophils with IL-3, IL-5, or GM-CSF led to reduced expression of IL-5Rα, which was sustained for up to 5 days. Eosinophils incubated with IL-5 or IL-3 showed diminished respiratory burst and mitogen-activated protein kinase kinase phosphorylation in response to further IL-5 stimulation. In contrast to these findings, eosinophil expression of IL-3Rα was increased by IL-3, IL-5, and GM-CSF, whereas GM-CSF receptor α was down-regulated by GM-CSF, but was not affected by IL-3 or IL-5. CCR3 expression was down-regulated by IL-3 and was transiently reduced by IL-5 and GM-CSF, but rapidly returned toward baseline. Eotaxin had no effect on receptor expression for IL-3, IL-5, or GM-CSF. Up-regulation of IL-3Rα by cytokines was prevented by a phosphoinositol 3-kinase inhibitor, whereas this and other signaling inhibitors had no effect on IL-5Rα down-regulation. These data suggest dynamic and differential regulation of eosinophil receptors for IL-3, IL-5, and GM-CSF by the cytokine ligands. Since these cytokines are thought to be involved in eosinophil development and mobilization from the bone marrow and are present at sites of allergic inflammation, tissue eosinophils may have reduced IL-5R expression and responsiveness, and this may explain the disappointing effect of anti-IL-5 therapy in reducing airway eosinophilia in asthma.
Stimulation of Primary Human Endothelial Cell Proliferation by IFN
Authors: Gomez Dolores , Reich Nancy C.
Published: April 2014

The IFN family of cytokines has pleiotropic roles in immunity and development. In this study, we provide evidence that IFN can stimulate the proliferation of primary human endothelial cells. This is in contrast to the growth-suppressive effects of IFN observed on transformed human cells, thereby underscoring the distinctive responses of primary human cells. The growth-stimulatory effect of IFN was determined by an increase in DNA synthesis assessed with [3H]thymidine incorporation, an increase in G2 and M cell cycle phases assessed with flow cytometric analysis, and an increase in cell number. Distinct cell types, including primary human fibroblast and smooth muscle cells, were also growth stimulated by IFN. Neutralizing Abs to IFN were used to demonstrate the growth response was mediated specifically by the IFN cytokine. The signaling pathway of type I IFNs activates STAT1 and STAT2. In primary endothelial cells, we demonstrate that STAT3 and STAT5 are also activated, and these STATs may contribute to cellular proliferation. To evaluate possible effectors of positive growth, DNA microarray analyses were performed to assess gene induction in response to IFN. These results reveal changes in the RNA levels of genes in endothelial cells that encode proteins involved in cellular proliferation.
NK Cell Tolerance in Mixed Allogeneic Chimeras
Authors: Zhao Yong , Ohdan Hideki , Manilay Jennifer O. , Sykes Megan
Published: April 2014

Alterations in inhibitory receptor expression on NK cells have been detected in mixed allogeneic chimeras and in mosaic MHC class I-expressing transgenic mice. However, it is not known whether or not NK cells are tolerant to host and donor Ags in mixed chimeras. In vitro studies have shown a lack of mutual tolerance of separated donor and host NK cells obtained from mixed chimeras. Using BALB/c→B6 fully MHC-mismatched mixed chimeras, we have now investigated this question in vivo. Neither donor nor host NK cells in mixed chimeras showed evidence for activation, as indicated by expression of B220 and Thy-1.2 on NK cells in chimeric mice at levels similar to those in nonchimeric control mice. Lethally irradiated, established mixed BALB/c→ B6 chimeras rejected a low dose of β2-microglobulin-deficient bone marrow cells (BMC) efficiently but did not reject BALB/c or B6 BMCs. In contrast, similarly conditioned B6 mice rejected both BALB/c and β2-microglobulin-deficient BMCs. Thus, NK cells were specifically tolerant to the donor and the host in mixed allogeneic chimeras. The similar growth of RMA lymphoma cells in both chimeric and control B6 mice further supports the conclusion that donor BALB/c NK cells are tolerant to B6 Ags in chimeras. Administration of a high dose of exogenous IL-2 could not break NK cell tolerance in chimeric mice, suggesting that NK cell tolerance in chimeras is not due to a lack of activating cytokine. No reduction in the level of expression of the activating receptor Ly-49D, recognizing a donor MHC molecule, was detected among recipient NK cells in mixed chimeras. Thus, the present studies demonstrate that NK cells in mixed chimeras are stably tolerant to both donor and host Ags, by mechanisms that are as yet unexplained.
IL-21 in Synergy with IL-15 or IL-18 Enhances IFN-γ Production in Human NK and T Cells
Authors: Strengell Mari , Matikainen Sampsa , Sirén Jukka , Lehtonen Anne , Foster Don , Julkunen Ilkka , Sareneva Timo
Published: April 2014

NK and T cell-derived IFN-γ is a key cytokine that stimulates innate immune responses and directs adaptive T cell response toward Th1 type. IL-15, IL-18, and IL-21 have significant roles as activators of NK and T cell functions. We have previously shown that IL-15 and IL-21 induce the expression of IFN-γ, T-bet, IL-12Rβ2, and IL-18R genes both in NK and T cells. Now we have studied the effect of IL-15, IL-18, and IL-21 on IFN-γ gene expression in more detail in human NK and T cells. IL-15 clearly activated IFN-γ mRNA expression and protein production in both cell types. IL-18 and IL-21 enhanced IL-15-induced IFN-γ gene expression. IL-18 or IL-21 alone induced a modest expression of the IFN-γ gene but a combination of IL-21 and IL-18 efficiently up-regulated IFN-γ production. We also show that IL-15 activated the binding of STAT1, STAT3, STAT4, and STAT5 to the regulatory sites of the IFN-γ gene. Similarly, IL-21 induced the binding of STAT1, STAT3, and STAT4 to these elements. IL-15- and IL-21-induced STAT1 and STAT4 activation was verified by immunoprecipitation with anti-phosphotyrosine Abs followed by Western blotting with anti-STAT1 and anti-STAT4 Abs. IL-18 was not able to induce the binding of STATs to IFN-γ gene regulatory sites. IL-18, however, activated the binding of NF-κB to the IFN-γ promoter NF-κB site. Our results suggest that both IL-15 and IL-21 have an important role in activating the NK cell-associated innate immune response.
Drug-Inducible, Dendritic Cell-Based Genetic Immunization
Authors: Timares Laura , Safer Karim Mahmoud , Qu Baoxi , Takashima Akira , Johnston Stephen Albert
Published: April 2014

Determining the mechanism of Ag loading of Langerhans cells (LC) for genetic immunization (GI) is complicated by the inability to distinguish between the response generated by direct transfection of LC from that due to exogenous uptake. To unravel this mechanism, we examined the impact of gene gun treatment on LC with respect to their activation and migration from skin, transgene expression, and ability to initiate humoral and cellular immune responses upon transfer to naive mice. To assess responses generated by direct LC transfection, an RU486-inducible expression system was used as a GI vector. In vitro skin organ cultures were developed from gene gun immunized mouse ear specimens to obtain LC. Gene gun treatment markedly augmented (3-fold) LC migration from ear skin, and these LC expressed the transgene at RNA and protein levels. Transfer of 2 × 105 migratory cells resulted in identical cellular responses to, but 10-fold lower humoral responses than, standard GI. Using an RU486-inducible system, we were able to measure responses generated by directly transfected LC. Our results indicate that direct transfection is a predominant pathway for LC Ag loading. The ability to regulate transgene expression with inducible DC-based vaccines demonstrates a new level of immunological control.
Thyroid-Specific Expression of IFN-γ Limits Experimental Autoimmune Thyroiditis by Suppressing Lymphocyte Activation in Cervical Lymph Nodes
Authors: Barin Jobert G. , Afanasyeva Marina , Talor Monica V. , Rose Noel R. , Burek C. Lynne , Caturegli Patrizio
Published: April 2014

The role of IFN-γ in the pathogenesis of autoimmune disease is controversial, being described as immunostimulatory in some studies and immunosuppressive in others. To determine the contribution of local expression of IFN-γ, we derived NOD.H-2h4 transgenic mice overexpressing IFN-γ in a thyroid-restricted manner. Transgenic mice, which had serum IFN-γ levels similar to wild-type littermates, showed up-regulation of MHC class II on thyrocytes, but did not develop spontaneous thyroiditis. Upon immunization with murine thyroglobulin, transgenic mice developed milder disease and reduced IgG1 responses compared with wild type. The milder disease was associated with decreased frequency of activated CD44+ lymphocytes in the cervical lymph nodes. This suppressive effect was confirmed by showing that blockade of systemic IFN-γ with mAb enhanced disease and increased IgG1 responses. The study supports a disease-limiting role of IFN-γ in autoimmune thyroiditis. Furthermore, it provides the first evidence that local IFN-γ activity in the thyroid is sufficient for disease suppression.
Novel Variants of the IL-10 Receptor 1 Affect Inhibition of Monocyte TNF-α Production
Authors: Gasche Christoph , Grundtner Paul , Zwirn Petra , Reinisch Walter , Shaw Sarah H. , Zdanov Alexander , Sarma Usha , Williams Lynn M. , Foxwell Brian M. , Gangl Alfred
Published: April 2014

IL-10-deficient mice exhibit spontaneous enterocolitis and other symptoms akin to Crohn’s disease, indicating that IL-10 might regulate normal physiology in the gut. However, clinical trials with IL-10 in Crohn’s disease were disappointing, although some patients showed healing of intestinal mucosa. This study searched for genetic polymorphisms within the IL-10 pathway. We decided to screen for mutations of the IL-10R1 cDNA in healthy volunteers and Crohn’s disease patients and identified two novel variants: a serine 138-to-glycine (S138G) and a glycine 330-to-arginine (G330R) substitution. The allelic frequency in a European cohort was relatively high (16% for the S138G and 33% for the G330R), and S138G was in strong linkage disequilibrium with G330R. A similar allele frequency was found in a group of Crohn’s patients. In IL-10R1 G330R-expressing monocytes, the inhibitory effect of IL-10 on TNF-α production was diminished, indicating that this variant may be a loss-of-function allele. No such difference was observed between haplotypes 4 (G330R only) and 7 (S138G and G330R). In addition, these IL-10R1 variants had no influence on the IL-10R1 expression density. Structural analysis of the S138G variant revealed that the substitution of S138G may interfere with binding of IL-10 to IL-10R1.
Wound Healing and Expression of Antimicrobial Peptides/Polypeptides in Human Keratinocytes, a Consequence of Common Growth Factors
Authors: Sørensen Ole E. , Cowland Jack B. , Theilgaard-Mönch Kim , Liu Lide , Ganz Tomas , Borregaard Niels
Published: April 2014

In addition to acting as a physical barrier against microorganisms, the skin produces antimicrobial peptides and proteins. After wounding, growth factors are produced to stimulate the regeneration of tissue. The growth factor response ceases after regeneration of the tissue, when the physical barrier protecting against microbial infections is re-established. We found that the growth factors important in wound healing, insulin-like growth factor I and TGF-α, induce the expression of the antimicrobial peptides/polypeptides human cationic antimicrobial protein hCAP-18/LL-37, human β-defensin 3, neutrophil gelatinase-associated lipocalin, and secretory leukocyte protease inhibitor in human keratinocytes. Both an individual and a synergistic effect of these growth factors were observed. These findings offer an explanation for the expression of these peptides/polypeptides in the skin disease psoriasis and in wound healing and define a host defense role for growth factors in wound healing.
IKKβ Plays an Essential Role in the Phosphorylation of RelA/p65 on Serine 536 Induced by Lipopolysaccharide
Authors: Yang Fan , Tang Eric , Guan Kunliang , Wang Cun-Yu
Published: April 2014

Activation of the IκB kinase (IKK) complex by LPS induces phosphorylation and degradation of IκBα, leading to the nuclear translocation of NF-κB. Although it is essential for NF-κB activation, emerging evidence has indicated that the nuclear translocation of NF-κB is not sufficient to activate NF-κB-dependent transcription. Here, we reported that LPS induced the phosphorylation of the p65 trans-activation domain on serine 536 in monocytes/macrophages. Using mouse embryonic fibroblasts lacking either IKKα or IKKβ, we found that IKKβ played an essential role in LPS-induced p65 phosphorylation on serine 536, while IKKα was partially required for the p65 phosphorylation. The LPS-induced p65 phosphorylation on serine 536 was independent of the phosphatidylinositol 3′-kinase/Akt signaling pathway. Furthermore, we found that the phosphorylation on serine 536 increased the p65 transcription activity. In summary, our results demonstrate that IKKβ plays an essential role in the LPS-induced p65 phosphorylation on serine 536, which may represent a mechanism to regulate the NF-κB transcription activity by LPS.
Effective Mucosal Immunity to Anthrax: Neutralizing Antibodies and Th Cell Responses Following Nasal Immunization with Protective Antigen
Authors: Boyaka Prosper N. , Tafaro Angela , Fischer Romy , Leppla Stephen H. , Fujihashi Kohtaro , McGhee Jerry R.
Published: April 2014

Mucosal, but not parenteral, immunization induces immune responses in both systemic and secretory immune compartments. Thus, despite the reports that Abs to the protective Ag of anthrax (PA) have both anti-toxin and anti-spore activities, a vaccine administered parenterally, such as the aluminum-adsorbed anthrax vaccine, will most likely not induce the needed mucosal immunity to efficiently protect the initial site of infection with inhaled anthrax spores. We therefore took a nasal anthrax vaccine approach to attempt to induce protective immunity both at mucosal surfaces and in the peripheral immune compartment. Mice nasally immunized with recombinant PA (rPA) and cholera toxin (CT) as mucosal adjuvant developed high plasma PA-specific IgG Ab responses. Plasma IgA Abs as well as secretory IgA anti-PA Abs in saliva, nasal washes, and fecal extracts were also induced when a higher dose of rPA was used. The anti-PA IgG subclass responses to nasal rPA plus CT consisted of IgG1 and IgG2b Abs. A more balanced profile of IgG subclasses with IgG1, IgG2a, and IgG2b Abs was seen when rPA was given with a CpG oligodeoxynucleotide as adjuvant, suggesting a role for the adjuvants in the nasal rPA-induced immunity. The PA-specific CD4+ T cells from mice nasally immunized with rPA and CT as adjuvant secreted low levels of CD4+ Th1-type cytokines in vitro, but exhibited elevated IL-4, IL-5, IL-6, and IL-10 responses. The functional significance of the anti-PA Ab responses was established in an in vitro macrophage toxicity assay in which both plasma and mucosal secretions neutralized the lethal effects of Bacillus anthracis toxin.
Urokinase-Type Plasminogen Activator Potentiates Lipopolysaccharide-Induced Neutrophil Activation
Authors: Abraham Edward , Gyetko Margaret R. , Kuhn Katherine , Arcaroli John , Strassheim Derek , Park Jong Sung , Shetty Sreerama , Idell Steven
Published: April 2014

Urokinase plasminogen activator (uPA) is a serine protease that catalyzes the conversion of plasminogen to plasmin. Although increased circulating levels of uPA are present in endotoxemia and sepsis, conditions in which activated neutrophils contribute to the development of acute organ dysfunction, the ability of uPA to participate directly in LPS-induced neutrophil activation has not been examined. In the present experiments, we show that uPA can enhance activation of neutrophils exposed to submaximal stimulatory doses of LPS. In particular, uPA increased LPS-induced activation of intracellular signaling pathways, including Akt and c-Jun N-terminal kinase, nuclear translocation of the transcriptional regulatory factor NF-κB, and expression of proinflammatory cytokines, including IL-1β, macrophage-inflammatory protein-2, and TNF-α. There was no effect of uPA on LPS-induced activation of p38 mitogen-activated protein kinase in neutrophils. Transgenic mice unable to produce uPA (uPA−/−) were protected from endotoxemia-induced lung injury, as determined by development of lung edema, pulmonary neutrophil accumulation, lung IL-1β, macrophage-inflammatory protein-2, and TNF-α cytokine levels. These results demonstrate that uPA can potentiate LPS-induced neutrophil responses and also suggest that such effects are sufficiently important in vivo to play a major contributory role in neutrophil-mediated inflammatory responses, such as the development of acute lung injury.
IFN Regulatory Factor-1 Is Required for the Up-Regulation of the CD40-NF-κB Activator 1 Axis During Airway Inflammation
Authors: Zhao Zhendong , Qian Youcun , Wald Dave , Xia Yi-Feng , Geng Jian-Guo , Li Xiaoxia
Published: April 2014

Recent studies show that NF-κB activator 1 (Act1) functions as an important adapter molecule for CD40-mediated signaling in epithelial cells. To explore the physiological function of the CD40-Act1 axis, we studied the regulation of gene expression of CD40 and Act1 both in vivo and in cell culture models. Although CD40 and Act1 are up-regulated in mouse lung upon LPS stimulation, IL-1 plus IFN-α, -β, or -γ synergistically up-regulate both CD40 and Act1 gene expression in human epithelial A549 cells. Cycloheximide superinduces the Act1 mRNA, whereas actinomycin D completely abolishes the Act1 mRNA, indicating that the induction of Act1 mRNA is at the transcriptional level and does not require protein synthesis. Promoter sequence analyses identified putative IFN regulatory factor (IRF)-1, C/EBP-β, and AP-1 transcription factor binding sites in the Act1 promoter. Although mutation of any of the three sites abolished the promoter activity, Abs against IRF-1 and C/EBP-β, but not AP-1, blocked the formation of the DNA-binding complex induced by IL-1 plus IFN-β stimulation, suggesting cooperative action between IRF-1 and C/EBP-β in mediating Act1 promoter activity. Importantly, LPS-induced gene expression of CD40 and Act1 in the mouse lung is abolished in IRF-1−/− mice, indicating an essential role of transcription factor IRF-1 in the coordinated regulation of these two genes during airway inflammation. The induced expression of the CD40-Act1 axis by inflammatory cytokines in epithelial cells probably plays an important role in priming these cells for their response to CD40 ligand during airway inflammation.
Regulation of Airway Epithelial Cell NF-κB-Dependent Gene Expression by Protein Kinase Cδ
Authors: Page Kristen , Li Jing , Zhou Limei , Iasvoyskaia Svetlana , Corbit Kevin C. , Soh Jae-Won , Weinstein I. Bernard , Brasier Allan R. , Lin Anning , Hershenson Marc B.
Published: April 2014

Airway epithelial cells synthesize proinflammatory molecules such as IL-8, GM-CSF, RANTES, and ICAM-1, the expression of which is increased in the airways of patients with asthma. We investigated the regulation of these NF-κB-dependent genes by the novel protein kinase C (PKC) isoform PKCδ in 16HBE14o- human airway epithelial cells, focusing on IL-8 expression. Transient transfection with the constitutively active catalytic subunit of PKCδ (PKCδ-CAT), and treatment with bryostatin 1, an activator of PKCδ, each increased transcription from the IL-8 promoter, whereas overexpression of PKCε had minor effects. Expression of a dominant negative PKCδ mutant (PKCδ-KR) or pretreatment of cells with rottlerin, a chemical PKCδ inhibitor, attenuated TNF-α- and phorbol ester-induced transcription from the IL-8 promoter. Bryostatin 1 treatment increased IL-8 protein abundance in primary airway epithelial cells. Selective activation of PKCδ by bryostatin also activated NF-κB, as evidenced by p65 RelA and p50 NF-κB1 binding to DNA, NF-κB trans-activation, and IκB degradation. The sufficiency of PKCδ to induce NF-κB nuclear translocation and binding to DNA was confirmed in a 16HBE14o- cell line inducibly expressing PKCδ-CAT under the tet-off system. Deletion of the NF-κB response element severely attenuated PKCδ-induced IL-8 promoter activity. Finally, PKCδ-CAT induced transcription from the GM-CSF, RANTES, and ICAM-1 promoters. Together these data suggest that PKCδ plays a key role in the regulation of airway epithelial cell NF-κB-dependent gene expression.
Localization of MHC Class II/Human Cartilage Glycoprotein-39 Complexes in Synovia of Rheumatoid Arthritis Patients Using Complex-Specific Monoclonal Antibodies
Authors: Steenbakkers Peter G. A. , Baeten Dominique , Rovers Eric , Veys Eric M. , Rijnders Antonius W. M. , Meijerink Jan , De Keyser Filip , Boots Annemieke M. H.
Published: April 2014

Recently human cartilage gp-39 (HC gp-39) was identified as a candidate autoantigen in rheumatoid arthritis (RA). To further investigate the relevance of this Ag in RA, we have generated a set of five mAbs to a combination epitope of complexes of HC gp-39263–275 and the RA-associated DRαβ1*0401 HLA class II molecules. FACS studies revealed that these mAb recognize specific complexes on homozygous DRαβ1*0401-positive B lymphoblastoid cells pulsed with HC gp-39263–275. The best mAb, 12A, was further characterized using a set of irrelevant DRαβ1*0401-binding peptides and truncated/elongated versions of HC gp-39263–275 itself. The minimal epitope recognized in combination with DRαβ1*0401 was HC gp-39263–273. Peptides not encompassing HC gp-39263–273 were not recognized. Three of five mAb were able to inhibit (up to 90%) the response of HC gp-39263–275-specific DRαβ1*0401-restricted T cell hybridomas to peptide-pulsed APC or purified complexes. Using mAb 12A, we have been able to identify and localize dendritic cells that present DRαβ1*0401/HC gp-39263–275 complexes in synovial tissue of DRαβ1*0401-positive RA patients, indicating local presentation of the HC gp-39263–275 epitope in the inflamed target tissue by professional APC. These data support a role of HC gp-39 in the local autoimmune response that leads to chronic inflammation and joint destruction.
P2X7 Receptor-Dependent Blebbing and the Activation of Rho-Effector Kinases, Caspases, and IL-1β Release
Authors: Verhoef Philip A. , Estacion Mark , Schilling William , Dubyak George R.
Published: April 2014

In response to ATP binding, the P2X7R facilitates cation channel activation, nonspecific pore formation, rapid changes in plasma membrane morphology, and secretion of IL-1β from LPS-primed macrophages. To investigate the relationship between the P2X7R-dependent changes in plasma membrane organization and the release of IL-1β, we generated time-lapse movies of ATP-stimulated BAC1 murine macrophages in conjunction with biochemical analyses of IL-1β release. Similar image analyses in human embryonic kidney 293 cells expressing recombinant P2X7R (HEK-P2X7) permitted comparison of P2X7R-dependent effects in macrophage vs nonmacrophage backgrounds. Whereas HEK-P2X7 cells exhibit zeiotic blebbing within 5 min of ATP treatment, BAC1 macrophages initiated a distinct “tethered” blebbing 10 min after ATP addition. This blebbing was comparably induced by the P2X7R-selective agonist BzATP and was blocked by P2X7R inhibitors KN-62 and oxidized ATP. Blebbing was initiated at ATP concentrations ≥3 mM, but optimal IL-1β release occurred at 1 mM ATP. P2X7R-dependent blebbing was abrogated in the presence of Rho-effector kinase inhibitors Fasudil and Y-27632, but ATP-induced IL-1β release was unaffected. ATP-induced activation of RhoA could be detected in both HEK-P2X7 cells and BAC1 murine macrophages. Thus, P2X7R activation signals distinct, novel membrane blebbing events (dependent on RhoA activation and Rho-effector kinase activity) and simultaneously initiates release of IL-1β. Our observations that blebbing and IL-1β release are dissociable suggest these events occur via parallel rather than convergent signaling pathways.
Marked Airway Eosinophilia Prevents Development of Airway Hyper-responsiveness During an Allergic Response in IL-5 Transgenic Mice
Authors: Kobayashi Takao , Iijima Koji , Kita Hirohito
Published: April 2014

Tissue eosinophilia probably plays important roles in the pathophysiology of bronchial asthma and allergic disorders; however, this concept was challenged recently by controversial results in mouse models of bronchial asthma treated with anti-IL-5 Ab and the failure of anti-IL-5 therapy in humans. We have now used a unique model, IL-5 transgenic (TG) mice, to address a fundamental question: is airway eosinophilia beneficial or detrimental in the allergic response? After sensitization and challenge with OVA, IL-5 TG mice showed a marked airway eosinophilia. Surprisingly, these IL-5 TG mice showed lower airway reactivity to methacholine. Immunohistochemical analysis of the lungs revealed a marked peribronchial infiltration of eosinophils, but no eosinophil degranulation. In vitro, mouse eosinophils from peritoneal lavage fluids did not produce superoxide anion, but did produce an anti-inflammatory and fibrotic cytokine, TGF-β1. Indeed, the TGF-β1 levels in bronchoalveolar lavage fluid specimens from IL-5 TG mice directly correlated with airway eosinophilia (r = 0.755). Furthermore, anti-IL-5 treatment of IL-5 TG mice decreased both airway eosinophilia and TGF-β1 levels in bronchoalveolar lavage fluids and increased airway reactivity. Thus, in mice, marked eosinophilia prevents the development of airway hyper-reactivity during an allergic response. Overall, the roles of eosinophils in asthma and in animal models need to be addressed carefully for their potentially detrimental and beneficial effects.
Allospecific T Cell Epitope Sharing Reveals Extensive Conservation of the Antigenic Features of Peptide Ligands Among HLA-B27 Subtypes Differentially Associated with Spondyloarthritis
Authors: Montserrat Veronica , Martí Mercè , de Castro José A. López
Published: April 2014

HLA-B*2702, B*2704, and B*2705 are strongly associated with spondyloarthritis, whereas B*2706 is not. Subtypes differ among each other by a few amino acid changes and bind overlapping peptide repertoires. In this study we asked whether differential subtype association with disease is related to differentially bound peptides or to altered antigenicity of shared ligands. Alloreactive CTL raised against B*2704 were analyzed for cross-reaction with B*2705, B*2702, B*2706, and mutants mimicking subtype changes. These CTL are directed against many alloantigen-bound peptides and can be used to analyze the antigenicity of HLA-B27 ligands on different subtypes. Cross-reaction of anti-B*2704 CTL with B*2705 and B*2702 correlated with overlap of their peptidic anchor motifs, suggesting that many shared ligands have similar antigenic features on these three subtypes. Moreover, the percent of anti-B*2704 CTL cross-reacting with B*2706 was only slightly lower than the overlap between the corresponding peptide repertoires, suggesting that most shared ligands have similar antigenic features on these two subtypes. Cross-reaction with B*2705 or mutants mimicking changes between B*2704 and B*2705 was donor-dependent. In contrast, cross-reaction with B*2702 or B*2706 was less variable among individuals. Conservation of antigenic properties among subtypes has implications for allorecognition, as it suggests that shared peptides may determine cross-reaction across exposed amino acid differences in the MHC molecules and that the antigenic distinctness of closely related allotypes may differ among donors. Our results also suggest that differential association of HLA-B27 subtypes with spondyloarthritis is more likely related to differentially bound peptides than to altered antigenicity of shared ligands.
Cutting Edge: B Cell Receptor Signals Regulate BLyS Receptor Levels in Mature B Cells and Their Immediate Progenitors
Authors: Smith Susan Harless , Cancro Michael P.
Published: April 2014

This study examines how B lymphocyte stimulator (BLyS) receptor expression and responsiveness are influenced by B cell receptor (BcR) signaling. Our results show that resting and BcR-stimulated B cells are dependent on BLyS for survival and that B cells remain BLyS responsive during BcR-induced activation. Further, BcR ligation up-regulates expression of the BLySR B cell maturation defect/BLySR3 (Bcmd/BR3), but not other known BLySRs. Finally, the coupling of BcR signaling with Bcmd/BR3 expression is limited to late transitional and mature B cells. Together, these findings establish the coupling of BcR signaling with Bcmd/BR3 expression as a fundamental aspect of follicular B cell selection, survival, and activation.
Notch-Regulated Ankyrin-Repeat Protein Inhibits Notch1 Signaling: Multiple Notch1 Signaling Pathways Involved In T Cell Development
Authors: Yun Theodore J. , Bevan Michael J.
Published: April 2014

We have characterized the function of Notch-regulated ankyrin-repeat protein (Nrarp) in mouse cell lines and in hematopoietic stem cells (HSCs). Nrarp overexpression is able to block Notch-induced activation of CBF-1. In AKR1010 thymoma cells, Nrarp overexpression blocks CBF-1-dependent transcriptional activation of Notch-responsive genes and inhibits phenotypic changes associated with Notch activation. Enforced expression of Nrarp in mouse HSCs results in a profound block in T lineage commitment and progression through early stages of thymocyte maturation. In contrast, Deltex-1 overexpression in HSCs can also block T lineage commitment but not progression through the early double negative stages of thymocyte maturation. The different effects of Deltex-1 and Nrarp overexpression suggest that alternate Notch signaling pathways mediate T vs B lineage commitment and thymocyte maturation.
CTLA-4 · FasL Induces Alloantigen-Specific Hyporesponsiveness
Authors: Elhalel Michal Dranitzki , Huang Jui-Han , Schmidt William , Rachmilewitz Jacob , Tykocinski Mark L.
Published: April 2014

The APC:T cell interface can be effectively targeted with immunotherapeutic proteins. We previously described a unique trans signal converter protein, CTLA-4 · Fas ligand (FasL), that has the inherent capacities to tether the T cell inhibitor FasL (CD95 ligand) to the surfaces of B7 (CD80 and CD86)-positive APC (via CTLA-4:B7 interaction), and in so doing, to simultaneously interfere with B7-to-CD28 T cell activation signals. Given the continuing need for agents capable of inducing allograft tolerance without generalized immunosuppression, we have explored in depth the functional activity of CTLA-4 · FasL in human allogeneic MLR. CTLA-4 · FasL inhibits 1° MLR and induces specific hyporesponsiveness in 2° MLR, with both effects only partially reversible with exogenous IL-2. Moreover, the presence of exogenous IL-2 during the 1° MLR does not affect the induction of hyporesponsiveness upon restimulation. Furthermore, CTLA-4 · FasL enables partial activation of allostimulated T cells, reduces the fraction of actively dividing cells, and increases the percentage of dead cells among dividing T cells. Taken together, these findings suggest that CTLA-4 · FasL-mediated inhibition of secondary alloantigenic responses involves both anergy induction and clonal deletion. Thus, CTLA-4 · FasL, a paradigmatic trans signal converter protein, manifests unique functional properties and emerges as a potentially useful immunotherapeutic for modulating alloresponsiveness.
Minor Histocompatibility Antigens on Canine Hemopoietic Progenitor Cells
Authors: Weber Martin , Lange Claudia , Günther Wolfgang , Franz Monika , Kremmer Elisabeth , Kolb Hans-Jochem
Published: April 2014

Adoptive immunotherapy with CTL against minor histocompatibility Ags (mHA) provides a promising way to treat leukemia relapse in allogeneic chimeras. Here we describe the in vitro generation of CTL against mHA in the dog. We tested their inhibitory effect on the growth of hemopoietic progenitor cells stimulated by hemopoietic growth factors in a 4-day suspension culture. CTL were produced by coculture of donor PBMC with bone marrow-derived dendritic cells (DCs). These DCs were characterized by morphology, high expression of MHC class II and CD1a, and the absence of the monocyte-specific marker CD14. Characteristically these cells stimulated allogeneic lymphocytes (MLR) and, after pulsing with a foreign Ag (keyhole limpet hemocyanin), autologous T cells. CTL were generated either ex vivo by coculture with DCs of DLA-identical littermates or in vivo by immunization of the responder with DCs obtained from a DLA-identical littermate. In suspension culture assays the growth of hemopoietic progenitor cells was inhibited in 53% of DLA-identical littermate combinations. In canine families mHA segregated with DLA as restriction elements. One-way reactivity against mHA was found in five littermate combinations. In two cases mHA might be Y chromosome associated, in three cases autosomally inherited alleles were detected. We conclude that CTL can be produced in vitro and in vivo against mHA on canine hemopoietic progenitor cells using bone marrow-derived DCs.
Atrial Natriuretic Peptide Polarizes Human Dendritic Cells Toward a Th2-Promoting Phenotype Through Its Receptor Guanylyl Cyclase-Coupled Receptor A
Authors: Morita Rimpei , Ukyo Naoya , Furuya Mayumi , Uchiyama Takashi , Hori Toshiyuki
Published: April 2014

Atrial natriuretic peptide (ANP) is a cardiovascular hormone secreted mainly by the cardiac atria and regulates the volume-pressure homeostasis. The action of ANP is mediated by its receptor, guanylyl cyclase-coupled receptor A (GC-A). In this study, we explored the possibility that ANP and GC-A may play a role in the dendritic cell (DC)-mediated immune regulation. We first examined the expression of GC-A in human monocyte-derived DCs in comparison with monocytes and found that DCs but not monocytes express GC-A at both the mRNA and protein levels. DCs responded to ANP with an increase in intracellular cGMP in a dose-dependent manner, indicating that GC-A expressed on DCs is functional. Furthermore, treatment of DCs with ANP decreased production of IL-12 and TNF-α and conversely increased that of IL-10 upon stimulation with LPS. In accordance with this change of cytokine production, DCs treated with ANP plus LPS promoted differentiation of naive CD4+ T cells into a Th2 phenotype. Finally, we presented evidence that ANP affected cytokine production of fresh whole blood stimulated with LPS in line with the above-mentioned results. These results indicate that ANP polarizes human DCs toward a Th2-promoting phenotype through GC-A and thus can regulate immune responses.