bluuebunny/crossref_metadata_2025 huggingface dataset:
Stress-Induced Inhibition of the NF-κB Signaling Pathway Results from the Insolubilization of the IκB Kinase Complex following Its Dissociation from Heat Shock Protein 90
Authors: Pittet Jean-Francois , Lee Hyon , Pespeni Melissa , O’Mahony Allison , Roux Jeremie , Welch William J.
Published: April 2014

Activation of the stress response attenuates proinflammatory responses by suppressing cytokine-stimulated activation of the NF-κB signaling pathway. In this study, we show that the activation of the cellular stress response, either by heat shock treatment or after exposure to sodium arsenite, leads to a transient inhibition of IκBα phosphorylation. Inhibition of IκBα phosphorylation after stress was associated with the detergent insolubilization of the upstream kinases, IκB kinase α (IKKα) and IκB kinase β, components involved in IκBα phosphorylation. Pretreatment of cells with glycerol, a chemical chaperone that reduces the extent of stress-induced protein denaturation, reduced the stress-dependent detergent insolubility of the IKK complex and restored the cytokine-stimulated phosphorylation of IκB. The stress-dependent insolubility of the IKK complex appeared reversible; as the cells recovered from the heat shock treatment, the IKK complex reappeared within the soluble fraction of cells and was again capable of mediating the phosphorylation of IκBα in response to added cytokines. Treatment of cells with geldanamycin, an inhibitor of heat shock protein 90 (Hsp90) function, also resulted in IKK detergent insolubility and proteasome-mediated degradation of the IKK complex. Furthermore, while IKKα coprecipitated with Hsp90 in control cells, coprecipitation of the two proteins was greatly reduced in those cells early after stress or following exposure to geldanamycin. Stress-induced transient insolubilization of the IκB kinase complex following its dissociation from Hsp90 represents a novel mechanism by which the activation of the stress response inhibits the NF-κB signaling pathway in response to proinflammatory stimuli.
Acetyl-Boswellic Acids Inhibit Lipopolysaccharide-Mediated TNF-α Induction in Monocytes by Direct Interaction with IκB Kinases
Authors: Syrovets Tatiana , Büchele Berthold , Krauss Christine , Laumonnier Yves , Simmet Thomas
Published: April 2014

Expression of proinflammatory cytokines by monocytes is tightly regulated by transcription factors such as NF-κB. In this study, we show that, in LPS-stimulated human peripheral monocytes, the pentacyclic triterpenes acetyl-α-boswellic acid (AαBA) and acetyl-11-keto-β-boswellic acid (AKβBA) down-regulate the TNF-α expression. AαBA and AKβBA inhibited NF-κB signaling both in LPS-stimulated monocytes as detected by EMSA, as well as in a NF-κB-dependent luciferase gene reporter assay. By contrast, the luciferase expression driven by the IFN-stimulated response element was unaffected, implying specificity of the inhibitory effect observed. Both AαBA and AKβBA did not affect binding of recombinant p50/p65 and p50/c-Rel dimers to DNA binding sites as analyzed by surface plasmon resonance. Instead, both pentacyclic triterpenes inhibited the LPS-induced degradation of IκBα, as well as phosphorylation of p65 at Ser536 and its nuclear translocation. AαBA and AKβBA inhibited specifically the phosphorylation of recombinant IκBα and p65 by IκBα kinases (IKKs) immunoprecipitated from LPS-stimulated monocytes. In line with this, AαBA and AKβBA also bound to and inhibited the activities of active human recombinant GST-IKKα and His-IKKβ. The LPS-triggered induction of TNF-α in monocytes is dependent on IKK activity, as confirmed by IKK-specific antisense oligodeoxynucleotides. Thus, via their direct inhibitory effects on IKK, AαBA and AKβBA convey inhibition of NF-κB and subsequent down-regulation of TNF-α expression in activated human monocytes. These findings provide a molecular basis for the anti-inflammatory properties ascribed to AαBA- and AKβBA-containing drugs and suggest acetyl-boswellic acids as tools for the development of novel therapeutic interventions.
Cyclooxygenase Inhibition Augments Allergic Inflammation through CD4-Dependent, STAT6-Independent Mechanisms
Authors: Hashimoto Koichi , Sheller James R. , Morrow Jason D. , Collins Robert D. , Goleniewska Kasia , O’Neal Jamye , Zhou Weisong , Ji Shaoquan , Mitchell Daphne B. , Graham Barney S. , Peebles R. Stokes
Published: April 2014

Nonselective cyclooxygenase (COX) inhibition during the development of allergic disease in a murine model causes an increase in type 2 cytokines and lung eosinophilia; however, the mechanisms responsible for this augmented allergen-induced inflammation have not been examined. Ab depletion of CD4 and CD8 cells revealed that the heightened allergic inflammation caused by COX inhibition was CD4, but not CD8, dependent. Allergen sensitization and airway challenge alone led to undetectable levels of IL-5 and IL-13 in the lungs of IL-4, IL-4Rα, and STAT6 knockout (KO) mice, but COX inhibition during the development of allergic inflammation resulted in wild-type levels of IL-5 and IL-13 and heightened airway eosinophilia in each of the three KO mice. These results indicate that the effect of COX inhibition was independent of signaling through IL-4, IL-4Rα, and STAT6. However, whereas COX inhibition increased IgE levels in allergic wild-type mice, IgE levels were undetectable in IL-4, IL-4Rα, and STAT6 KO mice, suggesting that IL-13 alone is not a switch factor for IgE synthesis in this model. These results illustrate the central role played by products derived from the COX pathway in the regulation of allergic immune responses.
Human Autoantibodies Modulate the T Cell Epitope Repertoire but Fail to Unmask a Pathogenic Cryptic Epitope
Authors: Quaratino Sonia , Ruf Jean , Osman Mohamed , Guo Jin , McLachlan Sandra , Rapoport Basil , Londei Marco
Published: April 2014

Abs can tune the responses of Ag-specific T cells by influencing the nature of the epitope repertoire displayed by APCs. We explored the interaction between human self-reactive T cells and human monoclonal autoantibodies from combinatorial Ig-gene libraries derived from autoimmune thyroiditis patients and specific for the main autoantigen thyroid peroxidase (TPO). All human mAbs extensively influenced the T cell epitope repertoire recognized by different TPO-specific T cell clones. The action of the human mAbs was complex, because sometimes the same Ab suppressed or enhanced the epitopes recognized by the 10 different TPO-specific T cell clones. The human mAbs could modulate the epitope repertoire when TPO was added exogenously and when expressed constitutively on the surface of APCs. However, they could not unmask an immunodominant cryptic TPO epitope. In this study, we show that human autoantibodies influence the activity of self-reactive T cells and prove their relevance in concealing or exposing epitopes recognized by self-reactive T cells. However, our results further stress the biological significance of the immunodominant cryptic epitope we have defined and its potential importance in the evolution of autoimmunity.
Bacterial Lipoprotein Induces Resistance to Gram-Negative Sepsis in TLR4-Deficient Mice via Enhanced Bacterial Clearance
Authors: O’Brien Gavin C. , Wang Jiang Huai , Redmond H. Paul
Published: April 2014

TLRs are highly conserved pathogen recognition receptors. As a result, TLR4-deficient C3H/HeJ mice are highly susceptible to Gram-negative sepsis. We have previously demonstrated that tolerance induced by bacterial lipoprotein (BLP) protects wild-type mice against polymicrobial sepsis-induced lethality. In this study, we assessed whether pretreatment of C3H/HeJ mice with BLP could induce resistance to a subsequent Gram-negative Salmonella typhimurium infection. Pretreatment with BLP resulted in a significant survival benefit in TLR4-deficient C3H/HeJ mice (p < 0.0002 vs control C3H/HeJ) after challenge with live S. typhimurium (0.25 × 106 CFU/mouse). This survival benefit was associated with enhanced bacterial clearance from the circulation and in the visceral organs (p < 0.05 vs control C3H/HeJ). Furthermore, pretreatment with BLP resulted in significant increases in complement receptor type 3 (CR3) and FcγIII/IIR expression on polymorphonuclear neutrophils (PMNs) and macrophages (p < 0.05 vs control C3H/HeJ). There was impaired bacterial recognition and phagocytosis in TLR4-deficient mice compared with wild-type mice. However, a significant augmented uptake, ingestion, and intracellular killing of S. typhimurium by PMNs and peritoneal macrophages was evident in BLP-pretreated C3H/HeJ mice (p < 0.05 vs control C3H/HeJ). An up-regulation of inducible NO synthase and increased production of intracellular NO were observed in peritoneal macrophages from BLP-pretreated C3H/HeJ mice (p < 0.05 vs control C3H/HeJ). Depletion of PMNs did not diminish the beneficial effects of BLP with regard to both animal survival and bacterial clearance. These results indicate that BLP, a TLR2 ligand, protects highly susceptible TLR4-deficient mice from Gram-negative sepsis via enhanced bacterial clearance.
Restricted IgA Repertoire in Both B-1 and B-2 Cell-Derived Gut Plasmablasts
Authors: Stoel Maaike , Jiang Han-Qing , van Diemen Cleo C. , Bun Judy C. A. M. , Dammers Peter M. , Thurnheer M. Christine , Kroese Frans G. M. , Cebra John J. , Bos Nicolaas A.
Published: April 2014

Mucosal IgA is the most abundantly produced Ig upon colonization of the intestinal tract with commensal organisms in the majority of mammals. The repertoire of these IgA molecules is still largely unknown; a large amount of the mucosal IgA cannot be shown to react with the inducing microorganisms. Analysis of the repertoire of used H chain Ig (VH) genes by H-CDR3 spectrotyping, cloning, and sequencing of VH genes from murine intestinal IgA-producing plasma cells reveals a very restricted usage of VH genes and multiple clonally related sequences. The restricted usage of VH genes is a very consistent observation, and is observed for IgA plasma cells derived from B-1 or conventional B-2 cells from different mouse strains. Clonal patterns from all analyzed VH gene sequences show mainly independently acquired somatic mutations in contrast to the clonal evolution patterns often observed as a consequence of affinity maturation in germinal center reactions in peripheral lymphoid organs and Peyer’s patches. Our data suggest a model of clonal expansion in which many mucosal IgA-producing B cells develop in the absence of affinity maturation. The affinity of most produced IgA might not be the most critical factor for its possible function to control the commensal organisms, but simply the abundance of large amounts of IgA that can bind with relatively unselected affinity to redundant epitopes on such organisms.
Mast Cells, Basophils, and Eosinophils Acquire Constitutive IL-4 and IL-13 Transcripts during Lineage Differentiation That Are Sufficient for Rapid Cytokine Production
Authors: Gessner André , Mohrs Katja , Mohrs Markus
Published: April 2014

Mast cells, basophils, and eosinophils are myeloid cells that are distinguished by their capability to produce IL-4 and IL-13. However, it is not clear how this potential is related to the lineage differentiation of these subsets. In the present study we used bicistronic IL-4 reporter (4get) mice to directly visualize IL-4 expression by nonlymphoid cells in vitro and in vivo at the single-cell level. Our data show that frequent expression of both Il4 alleles is initiated and maintained during ontogeny by an IL-4Rα- or Stat6-independent mechanism. Despite the constitutive presence of cytokine transcripts in differentiated cells under steady state conditions, cytokine production is not detectable in the absence of stimulation. Moreover, mature mast cells, basophils, and eosinophils also constitutively express IL-13. Both preformed IL-4 and IL-13 mRNAs are sufficient for rapid cytokine production upon stimulation. Our data show that mast cells, basophils, and eosinophils are programmed for IL-4 and IL-13 expression early in ontogeny. These novel findings have important implications for the prevention and therapeutic intervention of allergic and asthmatic diseases.
A2A Adenosine Receptor Induction Inhibits IFN-γ Production in Murine CD4+ T Cells
Authors: Lappas Courtney M. , Rieger Jayson M. , Linden Joel
Published: April 2014

Incubation of purified C57BL/6 murine CD4+ T lymphocytes with anti-CD3 mAb serves as a model of TCR-mediated activation and results in increased IFN-γ production and cell surface expression of CD25 and CD69. We demonstrate here that signaling through the TCR causes a rapid (4-h) 5-fold increase in A2A adenosine receptor (AR) mRNA, which is correlated with a significant increase in the efficacy of A2AAR-mediated cAMP accumulation in these cells. A2AAR activation reduces TCR-mediated production of IFN-γ by 98% with a potency order of 4-{3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxytetrahydrofuran-2-yl)-9H-purin-2-yl]prop-2-ynyl}cyclohexanecarboxylic acid methyl ester (ATL146e; EC50 = 0.19 ± 0.03 nM) > 4-{3-[6-amino-9-(5-cyclopropyl-carbamoyl-3,4-dihydroxytetrahydrofuran-2-yl)-9H-purin-2-yl]prop-2-ynyl}piperidine-1-carboxylic acid methyl ester (ATL313; 0.43 ± 0.06 nM) > 5′-N-ethylcarboxamidoadenosine (3.5 ± 0.77 nM) > 2-[4-(2-carboxyethyl)phenethylamino]-5′-N-ethylcarboxamidoadenosine (CGS21680; 7.2 ± 1.4 nM) ≫ N6-cyclohexyladenosine (110 ± 33 nM) > 2-chloro-N6-(3-iodobenzyl)-5′-N-methylcarboxamide (390 ± 160 nM), similar to the potency order to compete for radioligand binding to the recombinant murine A2AAR but not the A3AR. The selective A2AAR antagonist, 4-(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol (ZM241385), inhibits the effect of ATL146e with a pA2 of 0.34 nM and also inhibits the effects of N6-cyclohexyl-adenosine and 2-chloro-N6-(3-iodobenzyl)-5′-N-methylcarboxamide. In CD4+ T cells derived from A2AAR−/− and A2AAR+/− mice, the IFN-γ release response to ATL146e is reduced by 100 and 50%, respectively, indicative of a gene dose effect. The response of T cells to the phosphodiesterase inhibitor, 4-(3′-cyclopentyloxy-4′-methoxyphenyl)-2-pyrrolidone (rolipram), is not affected by A2AAR deletion. We conclude that the rapid induction of the A2AAR mRNA in T cells provides a mechanism for limiting T cell activation and secondary macrophage activation in inflamed tissues.
TNF Receptor-Associated Factor 6 Is an Essential Mediator of CD40-Activated Proinflammatory Pathways in Monocytes and Macrophages
Authors: Mukundan Lata , Bishop Gail A. , Head Kimberly Z. , Zhang Lihua , Wahl Larry M. , Suttles Jill
Published: April 2014

The interaction between CD40 and its ligand, CD154, has been shown to play a role in the onset and maintenance of inflammatory disease. Contributing to this process is the ability of CD40 to signal monocyte and macrophage inflammatory cytokine production. We have shown that this event is dependent on Src family tyrosine kinase activity and the subsequent activation of ERK1/2. To address the role of TNFR-associated factor (TRAF) family members in facilitating this signaling pathway, we transfected a CD40-deficient macrophage cell line with wild-type human CD40, or with CD40 containing disrupted TRAF binding sites. Ligation of either wild-type CD40, or a CD40 mutant unable to bind TRAF2/3/5, resulted in the stimulation of inflammatory cytokine production. However, ligation of a CD40 mutant lacking a functional TRAF6 binding site did not initiate inflammatory cytokine production, and this mutant was found to be defective in CD40-mediated activation of ERK1/2, as well as IκB kinase (IKK) and NF-κB. Likewise, introduction of a dominant-negative TRAF6 into a wild-type (CD40+) macrophage cell line resulted in abrogation of CD40-mediated induction of inflammatory cytokine synthesis. Finally, treatment of monocytes with a cell-permeable peptide corresponding to the TRAF6-binding motif of CD40 inhibited CD40 activation of ERK1/2, IKK, and inflammatory cytokine production. These data demonstrate that TRAF6 acts as a critical adapter of both the Src/ERK1/2 and IKK/NF-κB proinflammatory signaling pathways in monocytes and macrophages.
Lipoprotein I, a TLR2/4 Ligand Modulates Th2-Driven Allergic Immune Responses
Authors: Revets Hilde , Pynaert Gwenda , Grooten Johan , De Baetselier Patrick
Published: April 2014

Asthma is an inflammatory lung disease that is initiated and directed by Th2 and inhibited by Th1 cytokines. Microbial infections have been shown to prevent allergic responses by inducing the secretion of the Th1 cytokines IL-12 and IFN-γ. In this study, we examined whether administration of lipoprotein I (OprI) from Pseudomonas aeruginosa could prevent the inflammatory and physiological manifestations of asthma in a murine model of OVA-induced allergic asthma. OprI triggered dendritic cells to make IL-12 and TNF-α, with subsequent IFN-γ production from T cells. OprI stimulation of dendritic cells involved both TLR2 and TLR4. Intranasal coadministration of OprI with OVA allergen resulted in a significant decrease in airway eosinophilia and Th2 (IL-4 and IL-13) cytokines and this effect was sustained after repeated allergen challenge. The immediate suppressive effect of OprI (within 2 days of administration) was accompanied by an increase in Th1 cytokine IFN-γ production and a significant, but transient infiltration of neutrophils. OprI did not redirect the immune system toward a Th1 response since no increased activation of locally recruited Th1 cells could be observed upon repeated challenge with allergen. Our data show for the first time that a bacterial lipoprotein can modulate allergen-specific Th2 effector cells in an allergic response in vivo for a prolonged period via stimulation of the TLR2/4 signaling pathway.
Differential Role of Three Major New Zealand Black-Derived Loci Linked with Yaa-Induced Murine Lupus Nephritis
Authors: Kikuchi Shuichi , Fossati-Jimack Liliane , Moll Thomas , Amano Hirofumi , Amano Eri , Ida Akinori , Ibnou-Zekri Nabila , Laporte Catherine , Santiago-Raber Marie-Laure , Rozzo Stephen J. , Kotzin Brian L. , Izui Shozo
Published: April 2014

By assessing the development of Y-linked autoimmune acceleration (Yaa) gene-induced systemic lupus erythematosus in C57BL/6 (B6) × (New Zealand Black (NZB) × B6.Yaa)F1 backcross male mice, we mapped three major susceptibility loci derived from the NZB strain. These three quantitative trait loci (QTL) on NZB chromosomes 1, 7, and 13 differentially regulated three different autoimmune traits: anti-nuclear autoantibody production, gp70-anti-gp70 immune complex (gp70 IC) formation, and glomerulonephritis. Contributions to the disease traits were further confirmed by generating and analyzing three different B6.Yaa congenic mice, each carrying one individual NZB QTL. The chromosome 1 locus that overlapped with the previously identified Nba2 (NZB autoimmunity 2) locus regulated all three traits. A newly identified chromosome 7 locus, designated Nba5, selectively promoted anti-gp70 autoantibody production, hence the formation of gp70 IC and glomerulonephritis. B6.Yaa mice bearing the NZB chromosome 13 locus displayed increased serum gp70 production, but not gp70 IC formation and glomerulonephritis. This locus, called Sgp3 (serum gp70 production 3), selectively regulated the production of serum gp70, thereby contributing to the formation of nephritogenic gp70 IC and glomerulonephritis, in combination with Nba2 and Nba5 in NZB mice. Among these three loci, a major role of Nba2 was demonstrated, because B6.Yaa Nba2 congenic male mice developed the most severe disease. Finally, our analysis revealed the presence in B6 mice of an H2-linked QTL, which regulated autoantibody production. This locus had no apparent individual effect, but most likely modulated disease severity through interaction with NZB-derived susceptibility loci.
Cutting Edge: Bcl-3 Up-Regulation by Signal 3 Cytokine (IL-12) Prolongs Survival of Antigen-Activated CD8 T Cells
Authors: Valenzuela Javier O. , Hammerbeck Christopher D. , Mescher Matthew F.
Published: April 2014

Clonal expansion of T cells requires cell division and survival during the proliferative phase of the response. Naive murine CD8 T cells responding to Ag and costimulation undergo an abortive response characterized by impaired clonal expansion, failure to develop effector functions, and long-term tolerance. A third signal provided by IL-12 is required for full expansion, activation, and establishment of memory. The enhanced survival, and thus clonal expansion, supported by IL-12 is not due to increased Bcl-2 or Bcl-xL expression; both are maximally activated by signals 1 and 2. In contrast, Bcl-3, recently shown to enhance survival when ectopically expressed in T cells, is increased only when IL-12 is present. Furthermore, examination of Bcl-3-deficient CD8 T cells demonstrates that the increased survival caused by IL-12 depends upon Bcl-3. The time courses of expression suggest that Bcl-2 and Bcl-xL promote survival early in the response, whereas Bcl-3 acts later in the response.
LIGHT Is Constitutively Expressed on T and NK Cells in the Human Gut and Can Be Induced by CD2-Mediated Signaling
Authors: Cohavy Offer , Zhou Jaclyn , Ware Carl F. , Targan Stephan R.
Published: April 2014

The TNF superfamily cytokine, lymphotoxin-like inducible protein that competes with glycoprotein D for binding herpesvirus entry mediator on T cells (LIGHT; TNFSF14), can augment T cell responses inducing IFN-γ production and can drive pathological gut inflammation when expressed as a transgene in mouse T cells. LIGHT expression by human intestinal T cells suggests the possibility that LIGHT may play a key role in regulation of the mucosal immune system. A nonenzymatic method was developed for the isolation of T cells from the human lamina propria, permitting analysis of native cell surface protein expression. Cell surface LIGHT was constitutively expressed on mucosal T and NK cells and a subpopulation of gut-homing CD4+ T cells in the periphery. In addition, CD2-mediated stimulation induced efficient LIGHT expression on intestinal CD4+ T cells, but not on peripheral blood T cells, suggesting a gut-specific, Ag-independent mechanism for LIGHT induction. By contrast, herpesvirus entry mediator expression on gut T cells was unperturbed, implicating the transcriptional regulation of LIGHT as a mechanism modulating signaling activity in the gut. Quantitative analysis of LIGHT mRNA in a cohort of inflammatory bowel disease patients indicated elevated expression in biopsies from small bowel and from inflamed sites, implicating LIGHT as a mediator of mucosal inflammation.
Autocrine Regulation of T Cell Motility by Calreticulin-Thrombospondin-1 Interaction
Authors: Li Shu Shun , Forslöw Anna , Sundqvist Karl-Gösta
Published: April 2014

The mechanisms regulating T lymphocyte migration within the extracellular matrix are not understood. We show in this study that the thrombospondin-1 binding site of calreticulin, spanning aa 19–32, is a major triggering factor for T cell motility and migration within a three-dimensional collagen type 1 matrix, and that exogenous motogenic factors such as chemokines can stimulate migration via a calreticulin-thrombospondin-1 pathway. Endogenous calreticulin binding to the N-terminal domain of endogenous thrombospondin-1 elicited a motogenic signal to the T cells through the C-terminal domain of thrombospondin-1 and its cell surface receptor integrin-associated protein (CD47). Our data further revealed that thrombospondin-1 was expressed on the cell surface with a high turnover, and that PI3K and the Janus family of tyrosine kinases were required for T cell motility mediated through calreticulin, thrombospondin-1, and CD47. These results unveil an autocrine mechanism of calreticulin-thrombospondin-1-CD47 interaction for the control of T cell motility and migration within three-dimensional extracellular matrix substrata.
Phagocytosis-Induced Apoptosis in Macrophages Is Mediated by Up-Regulation and Activation of the Bcl-2 Homology Domain 3-Only Protein Bim
Authors: Kirschnek Susanne , Ying Songmin , Fischer Silke F. , Häcker Hans , Villunger Andreas , Hochrein Hubertus , Häcker Georg
Published: April 2014

Cell death by apoptosis is important in immune cell homeostasis and in the defense against infectious microorganisms. The physiological event of uptake and intracellular destruction of bacteria is a powerful apoptotic stimulus to macrophages and neutrophil granulocytes. In this study, we provide a molecular analysis of phagocytosis-induced apoptosis. Apoptosis was blocked by Bcl-2 in a mouse macrophage cell line and in primary mouse macrophages. Analysis of the upstream mechanisms revealed that apoptosis was triggered by the Bcl-2 homology domain 3-only protein Bim/Bod. Contact with bacteria or bacterial components induced a strong increase in Bim-expression through TLR and MyD88. Inhibition of the MAPK p38 and JNK reduced both up-regulation of Bim and apoptosis. Phosphorylation of Bim was further observed in mouse macrophages, which appeared to be the result of TLR-dependent phosphatase inhibition. Although TLR-induced Bim was, unlike Bim in resting cells, not bound to the microtubuli cytoskeleton, the up-regulation of Bim was not sufficient to cause apoptosis. A second signal was required that was generated in the process of phagocytosis. Phagocytosis-induced apoptosis was strongly reduced in Bim−/− macrophages. These data provide the molecular context of a form of apoptosis that may serve to dispose of terminally differentiated phagocytes.
Dysregulated B7-1 and B7-2 Expression on Nonobese Diabetic Mouse B Cells Is Associated with Increased T Cell Costimulation and the Development of Insulitis
Authors: Hussain Shabbir , Delovitch Terry L.
Published: April 2014

Little is known about the pathogenic role of B cell dysfunction in T cell-mediated autoimmune disease. We previously reported that B cell hyper-responsiveness, resistance to apoptosis, and accumulation in islets occur during the onset of insulitis, but not in type 1 diabetes (T1D), in NOD mice. In this study we extended these studies to further determine how islet-infiltrated B cells contribute to this inflammatory insulitis. We demonstrate the presence of an increased percentage of B7-1+ and a decreased percentage of B7-2+ B cells in the spleen of autoimmune disease-prone NOD and nonobese diabetes-resistant mice compared with the spleen of nonautoimmune disease-prone C57BL/6 and BALB/c mice. An age-dependent differential expression of B7-1 and B7-2 was associated with the development of insulitis and CD4+CD25+ T cell deficiency in autoimmune disease-prone mice. Whereas BCR and LPS stimulation increased B7-2 expression on B cells from autoimmune disease-prone and nonautoimmune disease-prone mice, LPS-induced B7-1 expression was higher on NOD than C57BL/6 B cells. Interestingly, increased expression of B7-1 and B7-2 was found on islet-infiltrated B cells, and this increase was associated with enhanced T cell costimulation. Islet-infiltrated B cells were shown to be a source of TNF-α production in islets. B7 blockade of BCR-stimulated NOD B cells by anti-B7-1 and anti-B7-2 mAbs during coadoptive transfer with diabetogenic T cells into NOD.scid mice protected these recipients from T1D. These results suggest that increased B7-1 and B7-2 expression on islet-infiltrated NOD B cells is associated with increased T cell costimulation and the development of inflammatory insulitis in NOD mice.
Vaccination with an Acidic Polymerase Epitope of Influenza Virus Elicits a Potent Antiviral T Cell Response but Delayed Clearance of an Influenza Virus Challenge
Authors: Crowe Sherry R. , Miller Shannon C. , Shenyo Rachael M. , Woodland David L.
Published: April 2014

The mechanisms underlying epitope selection and the potential impact of immunodominance hierarchies on peptide-based vaccines are not well understood. Recently, we have shown that two immunodominant MHC class I-restricted epitopes, NP366–374/Db (nucleoprotein (NP)) and PA224–233/Db (acidic polymerase (PA)), which drive the CD8+ T cell response to influenza virus infection in C57BL/6 mice, are differentially expressed on infected cells. Whereas NP appears to be strongly expressed on all infected cells, PA appears to be strongly expressed on dendritic cells but only weakly expressed on nondendritic cells. Thus, the immune response to influenza virus may involve T cells specific for epitopes, such as PA, that are poorly expressed at the site of infection. To examine the consequences of differential Ag presentation on peptide vaccination, we compared the kinetics of the T cell response and influenza virus clearance in mice vaccinated with the NP or PA peptide. Vaccination with either the NP or PA peptide resulted in accelerated and enhanced Ag-specific T cell responses at the site of infection following influenza virus challenge. These T cells were fully functional in terms of their ability to produce IFN-γ and TNF-α and to mediate cytolytic activity. Despite this enhancement of the Ag-specific T cell response, PA vaccination had a detrimental effect on the clearance of influenza virus compared with unvaccinated or NP-vaccinated mice. These data suggest that differential Ag presentation impacts the efficacy of T cell responses to specific epitopes and that this needs to be considered for the development of peptide-based vaccination strategies.
Identification of Antigen-Capturing Cells as Basophils
Authors: Mack Matthias , Schneider Martin A. , Moll Cordula , Cihak Josef , Brühl Hilke , Ellwart Joachim W. , Hogarth Mark P. , Stangassinger Manfred , Schlöndorff Detlef
Published: April 2014

Binding of intact Ag is a hallmark of Ag-specific B cells. Apart from B cells, a small number of non-B cells can bind Ag with comparable efficacy as B cells and are found in the peripheral blood, spleen, and bone marrow of mice. This population has been observed for a long time and recently named “Ag-capturing cells.” Their identity remained enigmatic. In this study, we show that these cells are basophilic granulocytes. Their ability to capture Ags is dependent on surface IgE receptors and on Ag-specific plasma IgE molecules appearing after immunization. Several surface markers including surface bound IgE, IL-3R, CD45, CD16/32, and the chemokine receptor CCR2 were used to clearly identify these cells. Cross-linkage of surface Igs results in the release of large amounts of IL-4 and IL-6. The data identify basophils as Ag-capturing cells and support the concept of basophils as important regulators of humoral immune responses.
Langerhans Cells Derived from Genetically Modified Human CD34+ Hemopoietic Progenitors Are More Potent Than Peptide-Pulsed Langerhans Cells for Inducing Antigen-Specific CD8+ Cytolytic T Lymphocyte Responses
Authors: Yuan Jianda , Latouche Jean-Baptiste , Reagan John L. , Heller Glenn , Riviere Isabelle , Sadelain Michel , Young James W.
Published: April 2014

Sustained Ag expression by human dendritic cells (DCs) is an attractive means of optimizing Ag presentation for stimulating durable cellular immunity. To establish proof of principle, we used Langerhans cell (LC) progeny of retrovirally transduced CD34+ hemopoietic progenitor cells to stimulate responses against the HLA-A*0201-restricted influenza matrix peptide (fluMP). Retroviral transduction of CD34+ hemopoietic progenitor cells, during pre-expansion by thrombopoietin, c-kit ligand, and FLT-3 ligand, on recombinant fibronectin, but in the absence of FCS, resulted in gene expression by 20–30% of the LCs. Expression persisted at least 28 days, with little decline (<30%) over that time. Retroviral transduction did not alter the phenotype or potent immunogenicity of normal mature DCs. FluMP-transduced LCs stimulated a 130-fold expansion of T cells reactive with HLA-A*0201-fluMP tetramers, even at LC:T cell ratios of 1:100–150 and lower, whereas fluMP-pulsed LCs stimulated only a 30-fold expansion. FluMP-transduced LCs also stimulated higher IFN-γ secretion (100–123 spot-forming cells/105 CD8+ T cells) than did fluMP-pulsed LCs (10–91 spot-forming cells/105 CD8+ T cells). CD8+ T cells stimulated by transduced LCs did not react preferentially with retrovirally transduced targets, indicating that the responses targeted only the immunizing influenza and not the retroviral vector Ags, even though these could have provided nonspecific helper epitopes presented by the transduced LCs. These data demonstrate that gene-transduced LCs maintain the activated phenotype as well potent immunogenicity typical of mature DCs. LCs genetically modified to express fluMP are also more potent stimulators of Ag-specific CD8+ T cell responses than are peptide-pulsed LCs.
CpG Oligodeoxynucleotides Enhance Neonatal Resistance to Listeria Infection
Authors: Ito Shuichi , Ishii Ken J. , Gursel Mayda , Shirotra Hidekazu , Ihata Atsushi , Klinman Dennis M.
Published: April 2014

Infection by Listeria monocytogenes causes serious morbidity and mortality during the neonatal period. Previous studies established that immunostimulatory CpG oligodeoxynucleotides (ODN) can increased the resistance of adult mice to many infectious pathogens, including Listeria. This work examines the capacity of CpG ODN to stimulate a protective immune response in newborns. Results indicate that dendritic cells, macrophages, and B cells from 3-day-old mice respond to CpG stimulation by secreting IFN-γ, IL-12, and/or TNF-α. Spleen cells from CpG-treated neonates produce large amounts of cytokine and NO when exposed to bacteria in vitro. Newborns treated with CpG ODN are protected from lethal Listeria challenge and generate Ag-specific CD4 and CD8 T cells that afford long-term protection against subsequent infection. These results demonstrate that cellular elements of the neonatal immune system respond to stimulation by CpG ODN, thereby reducing host susceptibility to infectious pathogens.
Targeted Disruption of the2B4Gene in Mice Reveals an In Vivo Role of 2B4 (CD244) in the Rejection of B16 Melanoma Cells
Authors: Vaidya Swapnil V. , Stepp Susan E. , McNerney Megan E. , Lee Jae-Kyung , Bennett Michael , Lee Kyung-Mi , Stewart Colin L. , Kumar Vinay , Mathew Porunelloor A.
Published: April 2014

Murine 2B4 (CD244) is a cell surface receptor expressed on all NK cells, γδ-T cells, a subset of CD8+ T cells, and all CD14+ monocytes. 2B4 binds to CD48 with high affinity, and cross-linking 2B4 with anti-2B4 Ab in vitro causes activation of NK cells. To study its physiological role, we have generated, by gene targeting, mice deficient in the expression of this cell surface molecule. The expression of lymphoid cell surface markers on PBMC and splenocytes of mice homozygous for the mutation in 2B4 (2B4−/−) is identical to that in wild-type mice. However, thymocytes from female 2B4−/− mice, but not male 2B4−/− mice, have an increase in the immature CD4−/CD8− population. To investigate the in vivo role of 2B4, wild-type and 2B4−/− mice were injected with CD48+ and CD48− metastatic B16 melanoma cells. Wild-type mice rejected CD48+ melanoma poorly compared with CD48− tumor cells, suggesting that ligation of 2B4 by CD48 on melanoma cells is inhibitory. In keeping with this, male 2B4−/− mice showed enhanced ability to reject CD48+ melanoma cells. However, female 2B4−/− mice poorly rejected both CD48+ and CD48− melanoma cells, revealing a gender-specific and CD48-independent defect in mice lacking 2B4. In vitro and in vivo experiments reveal a complex role of NK cells in gender specificity.
Human CD4+ Effector Memory T Cells Persisting in the Microenvironment of Lung Cancer Xenografts Are Activated by Local Delivery of IL-12 to Proliferate, Produce IFN-γ, and Eradicate Tumor Cells
Authors: Broderick Lori , Yokota Sandra J. , Reineke Joshua , Mathiowitz Edith , Stewart Carleton C. , Barcos Maurice , Kelleher Raymond J. , Bankert Richard B.
Published: April 2014

The implantation of small pieces of human primary lung tumor biopsy tissue into SCID mice results in a viable s.c. xenograft in which the tissue architecture, including tumor-associated leukocytes, tumor cells, and stromal cells, is preserved in a functional state. By monitoring changes in tumor volume, gene expression patterns, cell depletion analysis, and the use of function-blocking Abs, we previously established in this xenograft model that exogenous IL-12 mobilizes human tumor-associated leukocytes to kill tumor cells in situ by indirect mechanisms that are dependent upon IFN-γ. In this study immunohistochemistry and FACS characterize the early cellular events in the tumor microenvironment induced by IL-12. By 5 days post-IL-12 treatment, the constitutively present human CD45+ leukocytes have expanded and infiltrated into tumor-rich areas of the xenograft. Two weeks post-treatment, there is expansion of the human leukocytes and complete effacement of the tumor compared with tumor progression and gradual loss of most human leukocytes in control-treated xenografts. Immunohistochemical analyses reveal that the responding human leukocytes are primarily activated or memory T cells, with smaller populations of B cells, macrophages, plasma cells, and plasmacytoid dendritic cells capable of producing IFN-α. The predominant cell population was also characterized by FACS and was shown to have a phenotype consistent with a CD4+ effector memory T cell. We conclude that quiescent CD4+ effector memory T cells are present within the tumor microenvironment of human lung tumors and can be reactivated by the local and sustained release of IL-12 to proliferate and secrete IFN-γ, leading to tumor cell eradication.
Quality of Recombinant Protein Determines the Amount of Autoreactivity Detected against the Tumor-Associated Epithelial Cell Adhesion Molecule Antigen: Low Frequency of Antibodies against the Natural Protein
Authors: Schmetzer Oliver , Moldenhauer Gerhard , Riesenberg Rainer , Pires José Ricardo , Schlag Peter , Pezzutto Antonio
Published: April 2014

The human epithelial cell adhesion molecule (EpCAM) is expressed on normal epithelial cells and is overexpressed in most carcinomas. EpCAM-targeted immunotherapy has been tried in several clinical studies. High titers of autoantibodies against EpCAM have been reported by different authors. We have generated large amounts of purified protein in S2 Drosophila cells (S2-EpCAM) with a purity of >96%. In contrast, the protein produced in baculovirus-dependent systems (baculo-EpCAM) that has been used in previous studies shows a purity of 79%. 1H nuclear magnetic resonance spectrum of S2-EpCAM is typical of folded protein, whereas the baculo-EpCAM sample shows a spectrum corresponding to a partially unfolded protein. Using S2-EpCAM, denatured S2-EpCAM, and baculo-EpCAM, we measured EpCAM Abs of different isotypes in the serum of healthy controls and cancer patients. We found Ab titers against EpCAM in a much lower percentage of sera as published previously, and support the hypothesis that Ab reactivity in some published studies might be due to reactivity against denatured protein, to contaminating proteins in the baculovirus preparations, and to reactivity with BSA. Tetanus toxoid-reactive IgG Abs are present in 1000-fold higher titers compared with EpCAM-reactive Abs. Only IgA Abs were found in higher proportions and in higher concentrations than tetanus toxoid-specific Abs. Our study shows that EpCAM only rarely induces autoantibodies against native protein and emphasizes the importance of using extremely purified Ag preparations when evaluating Abs against tumor-associated Ags.
Tristetraprolin Down-Regulates IL-2 Gene Expression through AU-Rich Element-Mediated mRNA Decay
Authors: Ogilvie Rachel L. , Abelson Michelle , Hau Heidi H. , Vlasova Irina , Blackshear Perry J. , Bohjanen Paul R.
Published: April 2014

Posttranscriptional regulation of IL-2 gene expression at the level of mRNA decay is mediated by an AU-rich element (ARE) found in the 3′-untranslated region. We hypothesized that the ARE-binding protein tristetraprolin (TTP) regulates T lymphocyte IL-2 mRNA decay by interacting with the IL-2 ARE and targeting the transcript for decay. rTTP protein expressed in HeLa cells bound specifically to the IL-2 ARE with high affinity in a gel shift assay. In primary human T lymphocytes, TTP mRNA and protein expression were induced by TCR and CD28 coreceptor stimulation. Using a gel shift assay, we identified a cytoplasmic RNA-binding activity that was induced by TCR and CD28 coreceptor stimulation and bound specifically to the IL-2 ARE sequence. Using anti-TTP Abs, we showed by supershift that this inducible activity contained TTP. We also showed that insertion of the IL-2 ARE sequence into the 3′-untranslated region of a β-globin reporter construct conferred TTP-dependent mRNA destabilization on the β-globin reporter. To determine whether TTP also regulates IL-2 gene expression in vivo, we examined IL-2 expression in primary cells from wild-type and TTP knockout mice. Compared with their wild-type counterparts, TCR- and CD28-activated splenocytes and T cells from TTP knockout mice overexpressed IL-2 mRNA and protein. Also, IL-2 mRNA was more stable in activated splenocytes from TTP knockout mice compared with wild-type mice. Taken together, these data suggest that TTP functions to down-regulate IL-2 gene expression through ARE-mediated mRNA decay.
Reductions in IκBε and Changes in NF-κB Activity during B Lymphocyte Differentiation
Authors: Doerre Stefan , Mesires Kristin Perkins , Daley Kylle M. , McCarty Thomas , Knoetig Sonja , Corley Ronald B.
Published: April 2014

The levels and stability of IκBε have been examined in unstimulated and stimulated splenic B cells and compared with that of IκBα and IκBβ. Primary murine splenic B cells but not T cells were found to contain high levels of IκBε protein, equivalent to levels of the abundant IκBα. Most agents that activate IκBα and IκBβ degradation do not induce rapid degradation of IκBε. Interestingly, however, the levels of IκBε, but not of IκBα or IκBβ, are dramatically reduced upon the stimulation of B cells both in vivo and in vitro. Since IκBε exhibits substrate specificity for NF-κB Rel homodimers, this suggested the possibility that changes in NF-κB-responsive genes might also occur during this transition. Consistent with this hypothesis, we found that a NF-κB reporter construct sensitive to p65/RelA homodimers is activated at the time that IκBε levels decline following B cell stimulation. In IgG+ B cell lines, which contain low levels of IκBε, this same reporter construct was inactive, suggesting that the increases in Rel homodimer activity that accompany B cell stimulation are transient. However, there are differences in the level of expression of NF-κB-responsive genes in these IgG+ B cell lines compared with their IgM+ counterparts. From these data, we conclude that there are transient changes in NF-κB activity due to reductions in IκBε, which might contribute to long-term, persistent changes that accompany B cell differentiation. We propose an important role for IκBε in the differential regulation of nuclear NF-κB activity in stimulated B cells.
Innate Immunity in the Human Female Reproductive Tract: Antiviral Response of Uterine Epithelial Cells to the TLR3 Agonist Poly(I:C)
Authors: Schaefer Todd M. , Fahey John V. , Wright Jacqueline A. , Wira Charles R.
Published: April 2014

The objective of this study was to examine the expression of TLR by human primary uterine epithelial cells (UEC) and to determine whether exposure to the TLR agonist poly(I:C) would induce an antiviral response. The secretion of several cytokines and chemokines was examined as well as the mRNA expression of human β-defensin-1 and -2 (HBD1 and HBD2), IFN-β, and the IFN-β-stimulated genes myxovirus resistance gene 1 and 2′,5′ oligoadenylate synthetase. The expression of TLR1–9 by UEC was demonstrated by RT-PCR, with only TLR10 not expressed. Stimulation of UEC with the TLR3 agonist poly(I:C) induced the expression of the proinflammatory cytokines TNF-α, IL-6, GM-CSF, and G-CSF, as well as the chemokines CXCL8/IL-8, CCL2/MCP-1, and CCL4/MIP-1β. In addition, poly(I:C) exposure induced the mRNA expression of HBD1 and HBD2 by 6- and 4-fold, respectively. Furthermore, upon exposure to poly(I:C) UEC initiated a potent antiviral response resulting in the induction of IFN-β mRNA expression 70-fold and myxovirus resistance gene 1 and 2′,5′ oligoadenylate synthetase mRNA expression (107- and 96-fold), respectively. These results suggest that epithelial cells that line the uterine cavity are sensitive to viral infection and/or exposure to viral dsRNA released from killed epithelial cells. Not only do UEC release proinflammatory cytokines and chemokines that mediate the initiation of an inflammatory response and recruitment of immune cells to the site of infection, but they also express β-defensins, IFN-β, and IFN-β-stimulated genes that can have a direct inhibiting effect on viral replication.
Sphingolipids and the Balancing of Immune Cell Function: Lessons from the Mast Cell
Authors: Olivera Ana , Rivera Juan
Published: April 2014

Recent studies reveal that metabolites of sphingomyelin are critically important for initiation and maintenance of diverse aspects of immune cell activation and function. The conversion of sphingomyelin to ceramide, sphingosine, or sphingosine-1-phosphate (S1P) provides interconvertible metabolites with distinct biological activities. Whereas ceramide and sphingosine function to induce apoptosis and to dampen mast cell responsiveness, S1P functions as a chemoattractant and can up-regulate some effector responses. Many of the S1P effects are mediated through S1P receptor family members (S1P1–5). S1P1, which is required for thymocyte emigration and lymphocyte recirculation, is also essential for Ag-induced mast cell chemotaxis, whereas S1P2 is important for mast cell degranulation. S1P is released to the extracellular milieu by Ag-stimulated mast cells, enhancing inflammatory cell functions. Modulation of S1P receptor expression profiles, and of enzymes involved in sphingolipid metabolism, particularly sphingosine kinases, are key in balancing mast cell and immune cell responses. Current efforts are unraveling the complex underlying mechanisms regulating the sphingolipid pathway. Pharmacological intervention of these key processes may hold promise for controlling unwanted immune responses.
Cutting Edge: TLR Ligands Are Not Sufficient to Break Cross-Tolerance to Self-Antigens
Authors: Hamilton-Williams Emma E. , Lang Andreas , Benke Dirk , Davey Gayle M. , Wiesmüller Karl-Heinz , Kurts Christian
Published: April 2014

Cross-presentation of peripheral self-Ags by dendritic cells (DC) can induce deletion of autoreactive CTL by a mechanism termed cross-tolerance. Activation of DC by microbial TLR ligands is thought to result in adaptive immunity. However, activation of tolerogenic DC may cause autoimmunity by stimulating instead of deleting autoreactive CTL. To investigate this scenario, we have monitored the response of autoreactive CTL in specific for the transgenic self Ag, OVA, expressed in pancreatic islets of RIP-mOVA mice injected with ligands of TLR2, 3, 4, and 9. This somewhat enhanced proliferation and cytokine production, and moderately reduced the CTL number able to induce autoimmunity. Nevertheless, physiological CTL numbers were deleted before disease ensued, unless specific CD4 T cell help was provided. In conclusion, DC activation by TLR ligands was insufficient to break peripheral cross-tolerance in the absence of specific CD4 T cell help, and triggered autoimmunity by stimulating the early effector phase of autoreactive CTL only when their precursor frequency was extremely high.