bluuebunny/crossref_metadata_2025 huggingface dataset:
IL-4 Is a Mediator of IL-12p70 Induction by Human Th2 Cells: Reversal of Polarized Th2 Phenotype by Dendritic Cells
Authors: Kaliński Paweł , Smits Hermelijn H. , Schuitemaker Joost H. N. , Vieira Pedro L. , van Eijk Marco , de Jong Esther C. , Wierenga Eddy A. , Kapsenberg Martien L.
Published: April 2014

IL-12 is a key inducer of Th1-associated inflammatory responses, protective against intracellular infections and cancer, but also involved in autoimmune tissue destruction. We report that human Th2 cells interacting with monocyte-derived dendritic cells (DC) effectively induce bioactive IL-12p70 and revert to Th0/Th1 phenotype. In contrast, the interaction with B cells preserves polarized Th2 phenotype. The induction of IL-12p70 in Th2 cell-DC cocultures is prevented by IL-4-neutralizing mAb, indicating that IL-4 acts as a Th2 cell-specific cofactor of IL-12p70 induction. Like IFN-γ, IL-4 strongly enhances the production of bioactive IL-12p70 heterodimer in CD40 ligand-stimulated DC and macrophages and synergizes with IFN-γ at low concentrations of both cytokines. However, in contrast to IFN-γ, IL-4 inhibits the CD40 ligand-induced production of inactive IL-12p40 and the production of either form of IL-12 induced by LPS, which may explain the view of IL-4 as an IL-12 inhibitor. The presently described ability of IL-4 to act as a cofactor of Th cell-mediated IL-12p70 induction may allow Th2 cells to support cell-mediated immunity in chronic inflammatory states, including cancer, autoimmunity, and atopic dermatitis.
Loss of IL-6 Receptor Expression in Cervical Carcinoma Cells Inhibits Autocrine IL-6 Stimulation: Abrogation of Constitutive Monocyte Chemoattractant Protein-1 Production
Authors: Hess Sigrun , Smola Hans , Sandaradura de Silva Ute , Hadaschik Dirk , Kube Dieter , Baldus Stephen E. , Flucke Uta , Pfister Herbert
Published: April 2014

IL-6 is synthesized in human pampilloma virus (HPV)-transformed cervical carcinoma cell lines and is supposed to stimulate these cells in an autocrine manner. We studied IL-6 production and responsiveness in nonmalignant HPV-transformed keratinocytes and cervical carcinoma cells in detail. IL-6 was detected in cervical carcinomas in situ. Correspondingly, HPV-positive carcinoma cell lines expressed high IL-6 levels. However, these carcinoma cell lines showed low responsiveness to IL-6 as revealed by low constitutive STAT3 binding activity, which was not further enhanced by exogenous IL-6. In contrast, in vitro-transformed nonmalignant keratinocytes without endogenous IL-6 production strongly responded to exogenous IL-6 with activation of STAT3. STAT3 protein expression levels were comparable in both responsive and nonresponsive cell lines. Also, gp130, the upstream signal-transducing receptor subunit conveying IL-6 signals into the cell, was expressed in all tested cell lines. However, the IL-6 binding subunit gp80 was lost in the malignant cells. Addition of soluble gp80 was sufficient to restore IL-6 responsiveness in carcinoma cells as shown by enhanced activation of STAT3 binding activity. As a consequence of the restored IL-6 responsiveness, carcinoma cells strongly produced the chemokine monocyte chemoattractant protein-1 (MCP-1). Our data demonstrate that cervical carcinoma cells producing high amounts of IL-6 only weakly respond to IL-6 in an autocrine manner due to limited gp80 expression. While production of IL-6 might contribute to a local immunosuppressive effect, silencing an autocrine IL-6 response prevents constitutive production of the mononuclear cell-attracting chemokine MCP-1. Both mechanisms might help the tumor to escape the immune system.
Macrophages Present Pinocytosed Exogenous Antigen Via MHC Class I Whereas Antigen Ingested by Receptor-Mediated Endocytosis Is Presented Via MHC Class II
Authors: Peppelenbosch Maikel P. , DeSmedt Marjory , Pynaert Gwenda , van Deventer Sander J. H. , Grooten Johan
Published: April 2014

Macrophages present exogenous Ag either via MHC class I or MHC class II molecules. We investigated whether the mode of hemagglutinin (HA) uptake influences the class of MHC molecule by which this Ag is presented. Normally, HA is ingested by receptor-mediated endocytosis, but this may be switched to macropinocytosis and pinocytosis by adding phorbol esters to the cells. This switch resulted in altered intracellular routing of ingested Ag and a transition from Ag presentation via MHC class II molecules to presentation via MHC class I molecules. Similarly, inhibition of receptor-mediated HA endocytosis, by treating the cells with the HA receptor destroying enzyme neuraminidase, abrogated Ag presentation via MHC class II molecules and induced presentation via MHC class I molecules. If, however, under these conditions, receptor-mediated uptake of HA was restored, by virtue of HA/anti-HA Ab interaction and subsequent uptake of HA via the Fc receptor, presentation via MHC class II was restored as well, whereas presentation of HA via MHC class I molecules was no longer detectable. We conclude that in macrophages the mode of Ag uptake is decisive in determining via which class of MHC molecules Ag is presented: pinocytosis and macropinocytosis produce exclusive presentation of exogenous Ag via MHC class I molecules whereas receptor-mediated endocytosis leads exclusively to presentation via class II molecules.
Frequent Contribution of T Cell Clonotypes with Public TCR Features to the Chronic Response Against a Dominant EBV-Derived Epitope: Application to Direct Detection of Their Molecular Imprint on the Human Peripheral T Cell Repertoire
Authors: Lim Annick , Trautmann Lydie , Peyrat Marie-Alix , Couedel Chrystelle , Davodeau François , Romagné François , Kourilsky Philippe , Bonneville Marc
Published: April 2014

In an attempt to provide a global picture of the TCR repertoire diversity of a chronic T cell response against a common Ag, we performed an extensive TCR analysis of cells reactive against a dominant HLA-A2-restricted EBV epitope (hereafter referred to as GLC/A2), obtained after sorting PBL or synovial fluid lymphocytes from EBV-seropositive individuals using MHC/peptide multimers. Although TCR β-chain diversity of GLC/A2+ T cells was extensive and varied greatly from one donor to another, we identified in most cell lines several recurrent Vβ subsets (Vβ2, Vβ4, and Vβ16 positive) with highly conserved TCRβ complementarity-determining region 3 (CDR3) length and junctional motifs, which represented from 11 to 98% (mean, 50%) of GLC/A2-reactive cells. While TCR β-chains expressed by these subsets showed limited CDR1, CDR2, and CDR3 homology among themselves, their TCR α-chains comprised the same TCRAV region, thus suggesting hierarchical contribution of TCR α-chain vs TCR β-chain CDR to recognition of this particular MHC/peptide complex. The common occurrence of T cell clonotypes with public TCR features within GLC/A2-specific T cells allowed their direct detection within unsorted PBL using ad hoc clonotypic primers. These results, which suggest an unexpectedly high contribution of public clonotypes to the TCR repertoire against a dominant epitope, have several implications for the follow-up and modulation of T cell-mediated immunity.
Antigen Triggering Selectively Increases TCRBV Gene Transcription
Authors: Lennon Greig P. , Sillibourne James E. , Furrie Elizabeth , Doherty Martin J. , Kay Richard A.
Published: April 2014

When the TCR binds Ag it is phosphorylated, internalized, and degraded. We wished to examine whether this process was accompanied by a specific concomitant increase in TCR mRNA levels. To do this, PBMC and a T cell clone were cultured with a series of superantigens and an alloantigen. Only T cells specifically responding to an antigenic stimulus had increased levels of TCR β-chain variable (TCRBV)-specific mRNA. This response was apparent after 48 h, peaked around 72 h, and was still elevated after 7 days. Increased gene transcription appeared to be driven solely by Ag as specific Ag depletion prevented culture supernatants transferring this effect. The level of TCRBV mRNA elevation was not influenced by the stimulating Ag, but appeared dependent on the gene encoding the stimulated TCR. Reporter gene assays, using cloned TCRBV gene promoters, confirmed both that TCR gene transcription rises after stimulation and that basal and stimulated levels of TCR transcription vary between different TCRBV genes. These data conclusively demonstrate that there is no direct relationship between TCRBV mRNA and T cell number, and that future repertoire studies must take both factors into account.
The Cleavage of Two C1s Subunits by a Single Active C1r Reveals Substantial Flexibility of the C1s-C1r-C1r-C1s Tetramer in the C1 Complex
Authors: Lörincz Zsolt , Gál Péter , Dobó József , Cseh Sándor , Szilágyi Katalin , Ambrus Géza , Závodszky Péter
Published: April 2014

The activation of the C1s-C1r-C1r-C1s tetramer in the C1 complex, which involves the cleavage of an Arg-Ile bond in the catalytic domains of the subcomponents, is a two-step process. First, the autolytic activation of C1r takes place, then activated C1r cleaves zymogen C1s. The Arg463Gln mutant of C1r (C1rQI) is stabilized in the zymogen form. This mutant was used to form a C1q-(C1s-C1rQI-C1r-C1s) heteropentamer to study the relative position of the C1r and C1s subunits in the C1 complex. After triggering the C1 by IgG-Sepharose, both C1s subunits are cleaved by the single proteolytically active C1r subunit in the C1s-C1rQI-C1r-C1s tetramer. This finding indicates that the tetramer is flexible enough to adopt different conformations within the C1 complex during the activation process, enabling the single active C1r to cleave both C1s, the neighboring and the sequentially distant one.
Human Lymphocyte-Specific Protein 1, the Protein Overexpressed in Neutrophil Actin Dysfunction with 47-kDa and 89-kDa Protein Abnormalities (NAD 47/89), Has Multiple F-Actin Binding Domains
Authors: Zhang Qihong , Li Yao , Howard Thomas H.
Published: April 2014

Human lymphocyte-specific protein 1 (LSP1) is an F-actin binding protein, which has an acidic N-terminal half and a basic C-terminal half. In the basic C-terminal half, there are amino acid sequences highly homologous to the actin-binding domains of two known F-actin binding proteins: caldesmon and the villin headpieces (CI, CII, VI, VII). However, the exact numbers and locations of the F-actin binding domains within LSP1 are not clearly defined. In this report, we utilized 125I-labeled F-actin ligand blotting and high-speed F-actin cosedimentation assays to analyze the F-actin binding properties of truncated LSP1 peptides and to define the F-actin binding domains. Results show that LSP1 has at least three and potentially a fourth F-actin binding domain. All F-actin binding domains are located in the basic C-terminal half and correspond to the caldesmon and villin headpiece homologous regions. LSP1 181–245 and LSP1 246–295, containing sequences homologous to caldesmon F-actin binding site I and II, respectively (CI, CII), binds F-actin; similarly, LSP1 306–339 can bind F-actin and contains two inseparable villin headpiece-like F-actin binding domains (VI, VII). Although LSP1 1–305, which does not contain VI and VII regions, retains F-actin binding activity, its binding affinity for F-actin is much weaker than that of full-length LSP1. Site-directed mutagenesis of the basic amino acids in the KRYK (VI) or KYEK (VII) sequences to acidic amino acids create mutants that bind F-actin with lower affinity than full-length wild-type LSP1. High KCl concentrations decrease full-length LSP1 binding to F-actin, suggesting the affinity between LSP1 and F-actin is mainly through electrostatic interaction.
Deficient In Vitro and In Vivo Phagocytosis of Apoptotic T Cells by Resident Murine Alveolar Macrophages
Authors: Hu Bin , Sonstein Joanne , Christensen Paul J. , Punturieri Antonello , Curtis Jeffrey L.
Published: April 2014

Apoptotic lymphocytes are readily identified in murine lungs, both during the response to particulate Ag and in normal mice. Because apoptotic lymphocytes are seldom detected in other organs, we hypothesized that alveolar macrophages (AMφ) clear apoptotic lymphocytes poorly. To test this hypothesis, we compared in vitro phagocytosis of apoptotic thymocytes by resident AMφ and peritoneal macrophages (PMφ) from normal C57BL/6 mice. AMφ were deficient relative to PMφ both in percentage containing apoptotic thymocytes (19.1 ± 1% vs 96 ± 2.6% positive) and in phagocytic index (0.23 ± 0.02 vs 4.2 ± 0.67). This deficiency was not due to kinetic differences, was seen with six other inbred mouse strains, and was not observed using carboxylate-modified polystyrene microbeads. Annexin V blockade indicated that both Mφ types cleared apoptotic T cells by a mechanism involving phosphatidylserine expression. By contrast, neither mAb blockade of a variety of receptors (CD11b, CD29, CD51, and CD61) known to be involved in clearance of apoptotic cells, nor the tetrapeptide RGDS (arginine-glycine-aspartic acid-serine) blocked ingestion by either type of macrophage. To confirm these studies, apoptotic thymocytes were given intratracheally or i.p. to normal mice, and then AMφ or PMφ were recovered 30–240 min later. Ingestion of apoptotic thymocytes by AMφ in vivo was significantly decreased at all times. Defective ingestion of apoptotic lymphocytes may preserve AMφ capacity to produce proinflammatory cytokines in host defense, but could contribute to development of autoimmunity by failing to eliminate nucleosomes.
E-Selectin-Dependent Signaling Via the Mitogen-Activated Protein Kinase Pathway in Vascular Endothelial Cells
Authors: Hu Yenya , Kiely Jeanne-Marie , Szente Brian E. , Rosenzweig Anthony , Gimbrone Michael A.
Published: April 2014

E-selectin, a cytokine-inducible adhesion molecule, supports rolling and stable arrest of leukocytes on activated vascular endothelium. Previous studies have suggested that this transmembrane protein can also transduce signals into the endothelial cell. We now demonstrate activation of the mitogen-activated protein kinase (MAPK) signaling cascade in cultured HUVEC in response to E-selectin-dependent leukocyte adhesion and Ab-mediated cross-linking of cell surface E-selectin. Adhesion of increasing numbers of HL60 cells to IL-1β-activated HUVEC stimulated robust increases in MAPK activity that were abrogated by an E-selectin blocking Ab. Cross-linking of cell surface E-selectin with Abs, as a mimic of multivalent ligand engagement, strongly stimulated MAPK/extracellular signal-related kinase (ERK) kinase (MEK)-dependent MAPK activation and concomitant up-regulation of mRNA for c-fos, an immediate early response gene, whereas Ab cross-linking of HLA class I molecules (present at comparable density) failed to do so. Coimmunoprecipitation documented Ras, Raf-1 and, phospho-MEK complex formation. Unactivated HUVEC transduced with a full-length adenoviral E-selectin construct also exhibited cross-link-induced MAPK activation, macromolecular complex formation, and c-fos up-regulation, whereas HUVEC transduced with a cytoplasmic domain deletion mutant failed to respond. These observations indicate that E-selectin can transduce an activating stimulus via the MAPK cascade into the endothelial cell during leukocyte adhesion.
Oncostatin M Stimulates the Growth of Dermal Fibroblasts Via a Mitogen-Activated Protein Kinase-Dependent Pathway
Authors: Ihn Hironobu , Tamaki Kunihiko
Published: April 2014

Oncostatin M (OSM), a member of the hemopoietic cytokine family, has been implicated in the process of fibrosis and dermal wound healing. As a part of an ongoing study of the mechanisms of fibrosis and dermal wound healing, we have investigated the mechanism of the growth regulation of dermal fibroblasts by OSM. OSM stimulates the mitogenesis of dermal fibroblasts in a dose-dependent manner. This effect was completely blocked by anti-OSM IgG, but not by anti-IL-6 IgG. Furthermore, OSM induction was abolished by genistein, a tyrosine kinase inhibitor, or by PD98059, a specific mitogen-activated protein (MAP) kinase pathway inhibitor, but not by calphostin C, a protein kinase C inhibitor. Immunoblotting analysis using a specific Ab against phosphorylated MAP kinase (Thr202/Tyr204) showed that OSM induces phosphorylation of MAP kinase in dermal fibroblasts. Furthermore, transient transfection of the dominant-negative mutant MAP kinase into dermal fibroblasts abolished the OSM induction. These results strongly suggest that OSM stimulates the growth of dermal fibroblasts via a MAP kinase-dependent pathway.
Human Galectin-3 Is a Novel Chemoattractant for Monocytes and Macrophages
Authors: Sano Hideki , Hsu Daniel K. , Yu Lan , Apgar John R. , Kuwabara Ichiro , Yamanaka Tohru , Hirashima Mitsuomi , Liu Fu-Tong
Published: April 2014

Galectin-3 is a β-galactoside-binding protein implicated in diverse biological processes. We found that galectin-3 induced human monocyte migration in vitro in a dose-dependent manner, and it was chemotactic at high concentrations (1.0 μM) but chemokinetic at low concentrations (10–100 nM). Galectin-3-induced monocyte migration was inhibited by its specific mAb and was blocked by lactose and a C-terminal domain fragment of the protein, indicating that both the N-terminal and C-terminal domains of galectin-3 are involved in this activity. Pertussis toxin (PTX) almost completely blocked monocyte migration induced by high concentrations of galectin-3. Galectin-3 caused a Ca2+ influx in monocytes at high, but not low, concentrations, and both lactose and PTX inhibited this response. There was no cross-desensitization between galectin-3 and any of the monocyte-reactive chemokines examined, including monocyte chemotactic protein-1, macrophage inflammatory protein-1α, and stromal cell-derived factor-1α. Cultured human macrophages and alveolar macrophages also migrated toward galectin-3, but not monocyte chemotactic protein-1. Finally, galectin-3 was found to cause monocyte accumulation in vivo in mouse air pouches. These results indicate that galectin-3 is a novel chemoattractant for monocytes and macrophages and suggest that the effect is mediated at least in part through a PTX-sensitive (G protein-coupled) pathway.
Inducible Expression of a Th2-Type CC Chemokine Thymus- and Activation-Regulated Chemokine by Human Bronchial Epithelial Cells
Authors: Sekiya Takashi , Miyamasu Misato , Imanishi Masako , Yamada Hirokazu , Nakajima Toshiharu , Yamaguchi Masao , Fujisawa Takao , Pawankar Ruby , Sano Yasuyuki , Ohta Ken , Ishii Akira , Morita Yutaka , Yamamoto Kazuhiko , Matsushima Kouji , Yoshie Osamu , Hirai Koichi
Published: April 2014

CCR4 is now known to be selectively expressed in Th2 cells. Since the bronchial epithelium is recognized as an important source of mediators fundamental to the manifestation of respiratory allergic inflammation, we studied the expression of two functional ligands for CCR4, i.e., macrophage-derived chemokine (MDC) and thymus- and activation-regulated chemokine (TARC), in bronchial epithelial cells. The bronchial epithelium of asthmatics and normal subjects expressed TARC protein, and the asthmatics showed more intense expression than the normal subjects. On the other hand, MDC expression was only weakly detected in the asthmatics, but the intensity was not significantly different from that of normal subjects. Combination of TNF-α and IL-4 induced expression of TARC protein and mRNA in bronchial epithelial A549 cells, which was slightly up-regulated by IFN-γ. The enhancement by IFN-γ was more pronounced in bronchial epithelial BEAS-2B cells, and a maximum production occurred with combination of TNF-α, IL-4, and IFN-γ. On the other hand, MDC was essentially not expressed in any of the cultures. Furthermore, expressions of TARC protein and mRNA were almost completely inhibited by glucocorticoids. These results indicate that the airway epithelium represents an important source of TARC, which potentially plays a role via a paracrine mechanism in the development of allergic respiratory diseases. Furthermore, the beneficial effect of inhaled glucocorticoids on asthma may be at least in part due to their direct inhibitory effect on TARC generation by the bronchial epithelium.
Autoantigen-Specific CD4+CD28low T Cell Subset Prevents Autoimmune Exocrinopathy in Murine Sjögren’s Syndrome
Authors: Saegusa Kaoru , Ishimaru Naozumi , Yanagi Kumiko , Haneji Norio , Nishino Mizuho , Azuma Miyuki , Saito Ichiro , Hayashi Yoshio
Published: April 2014

Organ-specific autoimmune exocrinopathy resembling Sjögren’s syndrome (SS) that spontaneously develops in NFS/sld mutant mice thymectomized 3 day after birth is dependent on Th1-type CD4+ T cells. We previously reported that a cleavage product of 120-kDa α-fodrin may be an important autoantigen in the pathogenesis of SS in both an animal model and the patients. We demonstrate that in an animal model of SS with overt exocrinopathy, a unique CD4+ T cell subset expressing CD28low is dramatically increased in spleen cells before the disease onset, but that the CD4+ T cells of diseased mice were virtually all CD28high. We found that the spleen cells in these mice before the disease onset showed a significant increase in autoantigen-specific T cell proliferation. Analysis of in vitro cytokine production by spleen cells indicated, before the disease onset, severely impaired production of IL-2 and IFN-γ in the animal model, whereas high levels of IL-4 were observed. Expression of cytokine genes, including IL-4, IL-10, and TGF-β, was detected in FACS-sorted CD4+CD28low T cells by RT-PCR analysis. Transfer of CD4+CD28low T cells into the animal model actually prevented the development of autoimmune lesions including autoantibody production. These results suggest that a CD4+CD28low T cell subset that is continuously activated by an organ-specific autoantigen may play a regulatory role in the development of organ-specific autoimmune disease in an animal model of SS.
NF-κB as a Central Mediator in the Induction of TGF-β in Monocytes from Patients with Idiopathic Myelofibrosis: An Inflammatory Response Beyond the Realm of Homeostasis
Authors: Rameshwar Pranela , Narayanan Ramaswamy , Qian Jing , Denny Thomas N. , Colon Cristina , Gascon Pedro
Published: April 2014

Immune-mediated mechanisms have been implicated in the etiology of idiopathic bone marrow fibrosis (IMF). However, the mechanism remains poorly defined. Compared with healthy controls, IMF monocytes are overactivated, with increased production of TGF-β and IL-1. TGF-β is central to the progression of fibrosis in different organs. In the lung, fibrosis is associated with up-regulation of TGF-β-inducible genes. Because IL-1 and TGF-β have pro- and antiinflammatory properties and neither appears to regulate the high levels of each other in IMF, we studied the mechanism of this paradigm. We focused on the role of RelA, a subunit of the transcription factor, NF-κB that is associated with inflammatory responses. We transiently knocked out RelA from IMF monocytes with antisense oligonucleotides and showed that RelA is central to IL-1 and TGF-β production and to the adhesion of IMF monocytes. Because the NF-κB family comprises subunits other than RelA, we used aspirin and sodium salicylate to inhibit kinases that activate NF-κB and showed effects similar to those of the RelA knockout system. It is unlikely that RelA could be interacting directly with the TGF-β gene. Therefore, we determined its role in TGF-β production and showed that exogenous IL-1 could induce TGF-β and adherence of IMF monocytes despite the depletion of NF-κB. The results indicate that IL-1 is necessary for TGF-β production in IMF monocytes, but NF-κB activation is required for the production of endogenous IL-1. Initial adhesion activates NF-κB, which led to IL-1 production. Through autocrine means, IL-1 induces TGF-β production. In total, these reactions maintain overactivation of IMF monocytes.
Development of Streptococcal Pyrogenic Exotoxin C Vaccine Toxoids That Are Protective in the Rabbit Model of Toxic Shock Syndrome
Authors: McCormick John K. , Tripp Timothy J. , Olmsted Stephen B. , Matsuka Yury V. , Gahr Pamala J. , Ohlendorf Douglas H. , Schlievert Patrick M.
Published: April 2014

Streptococcal pyrogenic exotoxin C (SPE C) is a superantigen produced by many strains of Streptococcus pyogenes that (along with streptococcal pyrogenic exotoxin A) is highly associated with streptococcal toxic shock syndrome (STSS) and other invasive streptococcal diseases. Based on the three-dimensional structure of SPE C, solvent-exposed residues predicted to be important for binding to the TCR or the MHC class II molecule, or important for dimerization, were generated. Based on decreased mitogenic activity of various single-site mutants, the double-site mutant Y15A/N38D and the triple-site mutant Y15A/H35A/N38D were constructed and analyzed for superantigenicity, toxicity (lethality), immunogenicity, and the ability to protect against wild-type SPE C-induced STSS. The Y15A/N38D and Y15A/H35A/N38D mutants were nonmitogenic for rabbit splenocytes and human PBMCs and nonlethal in two rabbit models of STSS, yet both mutants were highly immunogenic. Animals vaccinated with the Y15A/N38D or Y15A/H35A/N38D toxoids were protected from challenge with wild-type SPE C. Collectively, these data indicate that the Y15A/N38D and Y15A/H35A/N38D mutants may be useful as toxoid vaccine candidates.
Cutting Edge: Functional Requirement for SAP in 2B4-Mediated Activation of Human Natural Killer Cells as Revealed by the X-Linked Lymphoproliferative Syndrome
Authors: Tangye Stuart G. , Phillips Joseph H. , Lanier Lewis L. , Nichols Kim E.
Published: April 2014

X-linked lymphoproliferative syndrome (XLP) is an immunodeficiency characterized by life-threatening infectious mononucleosis and EBV-induced B cell lymphoma. The gene mutated in XLP encodes SLAM (signaling lymphocytic activation molecule-associated protein)-associated protein (SAP), a small SH2 domain-containing protein. SAP associates with 2B4 and SLAM, activating receptors expressed by NK and T cells, and prevents recruitment of SH2 domain-containing protein tyrosine phosphatase-2 SHP-2) to the cytoplasmic domains of these receptors. The phenotype of XLP may therefore result from perturbed signaling through SAP-associating receptors. We have addressed the functional consequence of SAP deficiency on 2B4-mediated NK cell activation. Ligating 2B4 on normal human NK cells with anti-2B4 mAb or interaction with transfectants bearing the 2B4 ligand CD48 induced NK cell cytotoxicity. In contrast, ligation of 2B4 on NK cells from a SAP-deficient XLP patient failed to initiate cytotoxicity. Despite this, CD2 or CD16-induced cytotoxicity of SAP-deficient NK cells was similar to that of normal NK cells. Thus, selective impairment of 2B4-mediated NK cell activation may contribute to the immunopathology of XLP.
Cutting Edge: HMG-1 as a Mediator of Acute Lung Inflammation
Authors: Abraham Edward , Arcaroli John , Carmody Aaron , Wang Haichao , Tracey Kevin J.
Published: April 2014

Acute inflammatory lung injury is often a delayed complication of critical illness and is associated with increased mortality. High mobility group-1 (HMG-1) protein, in addition to its role as a transcriptional regulatory factor, has recently been identified as a late mediator of endotoxin lethality. In the present studies, HMG-1 given intratracheally produced acute inflammatory injury to the lungs, with neutrophil accumulation, the development of lung edema, and increased pulmonary production of IL-1β, TNF-α, and macrophage-inflammatory protein-2. In endotoxin-induced acute lung inflammation, administration of anti-HMG-1 Abs either before or after endotoxin exposure decreased the migration of neutrophils to the lungs as well as lung edema. These protective effects of anti-HMG-1 were specific, because pulmonary levels of IL-1β, TNF-α, or macrophage-inflammatory protein-2 were not decreased after therapy with anti-HMG-1. Together, these findings indicate that HMG-1 is a distal mediator of acute inflammatory lung injury.
Somatostatin Through Its Specific Receptor Inhibits Spontaneous and TNF-α- and Bacteria-Induced IL-8 and IL-1β Secretion from Intestinal Epithelial Cells
Authors: Chowers Yehuda , Cahalon Liora , Lahav Maor , Schor Hagai , Tal Ruth , Bar-Meir Simon , Levite Mia
Published: April 2014

Intestinal epithelial cells secrete proinflammatory cytokines and chemokines that are crucial in mucosal defense. However, this secretion must be tightly regulated, because uncontrolled secretion of proinflammatory mediators may lead to chronic inflammation and mucosal damage. The aim of this study was to determine whether somatostatin, secreted within the intestinal mucosa, regulates secretion of cytokines from intestinal epithelial cells. The spontaneous as well as TNF-α- and Salmonella-induced secretion of IL-8 and IL-1β derived from intestinal cell lines Caco-2 and HT-29 was measured after treatment with somatostatin or its synthetic analogue, octreotide. Somatostatin, at physiological nanomolar concentrations, markedly inhibited the spontaneous and TNF-α-induced secretion of IL-8 and IL-1β. This inhibition was dose dependent, reaching >90% blockage at 3 nM. Furthermore, somatostatin completely abrogated the increased secretion of IL-8 and IL-1β after invasion by Salmonella. Octreotide, which mainly stimulates somatostatin receptor subtypes 2 and 5, affected the secretion of IL-8 and IL-1β similarly, and the somatostatin antagonist cyclo-somatostatin completely blocked the somatostatin- and octreotide-induced inhibitory effects. This inhibition was correlated to a reduction of the mRNA concentrations of IL-8 and IL-1β. No effect was noted regarding cell viability. These results indicate that somatostatin, by directly interacting with its specific receptors that are expressed on intestinal epithelial cells, down-regulates proinflammatory mediator secretion by a mechanism involving the regulation of transcription. These findings suggest that somatostatin plays an active role in regulating the mucosal inflammatory response of intestinal epithelial cells after physiological and pathophysiological stimulations such as bacterial invasion.
Cd22aPRE-mRNA Dysregulated Expression of theCd22Gene as a Result of a Short Interspersed Nucleotide Element Insertion inCd22aLupus-Prone Mice
Authors: Mary Charles , Laporte Catherine , Parzy Daniel , Santiago Marie-Laure , Stefani Franck , Lajaunias Frédéric , Parkhouse R. Michael E. , O’Keefe Theresa L. , Neuberger Michael S. , Izui Shozo , Reininger Luc
Published: April 2014

The Cd22 gene encodes a B cell-specific adhesion molecule that modulates B cell Ag receptor-mediated signal transduction, and is allelic to a lupus-susceptibility locus in New Zealand White (NZW) mice. In this study, we show that, in addition to the wild-type transcripts, NZW (Cd22a) mice synthesize aberrant CD22 mRNAs that contain ∼20–120 nucleotide insertions upstream of the coding region between exons 2 and 3, and/or ∼100–190 nucleotide deletions of exon 4. Sequence analysis revealed that these aberrant mRNA species arose by alternative splicing due to the presence in the NZW strain of a 794-bp sequence insertion in the second intron, containing a cluster of short interspersed nucleotide elements. Both the presence of sequence insertion and aberrantly spliced mRNAs were specific to mice bearing the Cd22a and Cd22c alleles. Up-regulation of CD22 expression after LPS activation appeared impaired in Cd22a spleen cells (twice lower than in Cd22b B cells). Furthermore, we show that partial CD22 deficiency, i.e., heterozygous level of CD22 expression, markedly promotes the production of IgG anti-DNA autoantibodies in C57BL/6 (Cd22b) mice bearing the Y chromosome-linked autoimmune acceleration gene, Yaa. Taken together, these results suggest that a lower up-regulation of CD22 on activated B cells (resulting from Cd22 gene anomaly in Cd22a mice or from CD22 heterozygosity in mutants obtained by gene targeting) is implicated in autoantibody production, providing support for Cd22a as a possible candidate allele contributing to lupus susceptibility.
Regulation of the Fas Death Pathway by FLICE-Inhibitory Protein in Primary Human B Cells
Authors: Hennino Ana , Berard Marion , Casamayor-Pallejà Montserrat , Krammer Peter H. , Defrance Thierry
Published: April 2014

The Fas/Fas ligand (L) system plays an important role in the maintenance of peripheral B cell tolerance and the prevention of misguided T cell help. CD40-derived signals are required to induce Fas expression on virgin B cells and to promote their susceptibility to Fas-mediated apoptosis. In the current study, we have analyzed the early biochemical events occurring upon Fas ligation in CD40L-activated primary human tonsillar B cells with respect to Fas-associated death domain protein (FADD), caspase-8/FADD-like IL-1β-converting enzyme (FLICE), and c-FLICE inhibitory protein (FLIP). We report here that Fas-induced apoptosis in B cells does not require integrity of the mitochondrial Apaf-1 pathway and that caspase-8 is activated by association with the death-inducing signaling complex (DISC), i.e., upstream of the mitochondria. We show that both FADD and the zymogen form of caspase-8 are constitutively expressed at high levels in virgin B cells, whereas c-FLIP expression is marginal. In contrast, c-FLIP, but neither FADD nor procaspase-8, is strongly up-regulated upon ligation of CD40 or the B cell receptor on virgin B cells. Finally, we have found that c-FLIP is also recruited and cleaved at the level of the DISC in CD40L-activated virgin B cells. We propose that c-FLIP expression delays the onset of apoptosis in Fas-sensitive B cells. The transient protection afforded by c-FLIP could offer an ultimate safeguard mechanism against inappropriate cell death or allow recruitment of phagocytes to ensure efficient removal of apoptotic cells.
The Dual Role of IL-2 in the Generation and Maintenance of CD8+ Memory T Cells
Authors: Dai Zhenhua , Konieczny Bogumila T. , Lakkis Fadi G.
Published: April 2014

The mechanisms responsible for the generation and maintenance of T cell memory are unclear. In this study, we tested the role of IL-2 in allospecific CD8+ T cell memory by analyzing the long-term survival, phenotype, and functional characteristics of IL-2-replete (IL-2+/+) and IL-2-deficient (IL-2−/−) CD8+ TCR-transgenic lymphocytes in an adoptive transfer model. We found that IL-2 is not essential for the in vivo generation, maintenance, or recall response of CD8+ memory T cells. However, IL-2 increased the size of the CD8+ memory pool if present at the time of initial T cell activation but reduced the size of the pool if present during memory maintenance by inhibiting the proliferation of CD8+ memory T cells. Thus, IL-2-based vaccine strategies or immunosuppressive regimens that target IL-2 should take into account the divergent roles of IL-2 in CD8+ T cell immunity.
Disparate Cytotoxic Activity of Nickel-Specific CD8+ and CD4+ T Cell Subsets Against Keratinocytes
Authors: Traidl Claudia , Sebastiani Silvia , Albanesi Cristina , Merk Hans F. , Puddu Pietro , Girolomoni Giampiero , Cavani Andrea
Published: April 2014

Allergic contact dermatitis (ACD) is the result of an exaggerated immune reaction to haptens mediated by skin-homing T cells, but the effector mechanisms responsible for the tissue damage are poorly understood. Here we studied the capacity of distinct subsets of hapten-specific T cells to induce apoptosis in autologous keratinocytes. Skin- and blood-derived nickel-specific CD8+ T cytotoxic 1 (Tc1) and Tc2 clones as well as CD4+ Th1 and Th2 expressed the cutaneous lymphocyte-associated Ag and exhibited strong MHC-restricted cytotoxicity against nickel-coupled B lymphoblasts, as detected by the [3H]TdR release assay. Both Tc1 and Tc2 clones, but not CD4+ T cells, displayed a significant cytotoxic activity against resting nickel-modified keratinocytes. Following IFN-γ treatment, keratinocytes expressed MHC class II and ICAM-1 and became susceptible to Th1-mediated, but not Th2-mediated, cytotoxicity. The molecules of the two major cytotoxic pathways, Fas ligand (FasL) and perforin, were expressed by Tc1, Tc2, and Th1 cells, whereas Th2 cells expressed only FasL. Experiments performed in the presence of specific inhibitors of the perforin (concanamycin A) and FasL (brefeldin A) pathway indicated that perforin-mediated killing dominated in Tc1 and Tc2, and FasL-mediated cytotoxicity prevailed in Th2 clones, with a more heterogeneous behavior in the case of Th1 cells. Finally, perforin mRNA was expressed in ACD lesional skin, as assessed by RT-PCR analysis. In aggregate, our results indicate that keratinocytes can be target of multiple hapten-specific CTL responses, that may have distinct roles in the epidermal injury during ACD.
Precursor Thymocyte Proliferation and Differentiation Are Controlled by Signals Unrelated to the Pre-TCR
Authors: Petrie Howard T. , Tourigny Michelle , Burtrum Douglas B. , Livak Ferenc
Published: April 2014

In-frame rearrangement of the TCR-β locus and expression of the pre-TCR are compulsory for the production of CD4+8+ thymocytes from CD4−8− precursors. Signals delivered via the pre-TCR are thought to induce the differentiation process as well as the extensive proliferation that accompanies this transition. However, it is equally possible that pre-TCR expression is required for the success of this transition, but does not play a direct role in the inductive process. In the present manuscript we examine this possibility using a variety of normal and genetically modified mouse models. Our evidence shows that differentiation and mitogenesis can both occur independently of pre-TCR expression. However, these processes are absolutely dependent on the presence of normal thymic architecture and cellular composition. These findings are consistent with a checkpoint role for the pre-TCR in regulating the divergence of survival and cell death fates at the CD4−8− to CD4+8+ transition. Further, our data suggest that precursor thymocyte differentiation is induced by other, probably ubiquitous, mechanisms that require the presence of normal thymic cellularity, composition, and architecture.
CD25 Is a Marker for CD4+ Thymocytes That Prevent Autoimmune Diabetes in Rats, But Peripheral T Cells with This Function Are Found in Both CD25+ and CD25− Subpopulations
Authors: Stephens Leigh Andrea , Mason Don
Published: April 2014

Previously we have shown that autoimmune diabetes, induced in rats by a protocol of adult thymectomy and split-dose gamma irradiation, can be prevented by the transfer of a subset of CD4+ T cells with a memory phenotype (CD45RC−), as well as by CD4+CD8− thymocytes, from syngeneic donors. Further studies now reveal that in the thymus the regulatory cells are observed in the CD25+ subset of CD4+CD8− cells, whereas transfer of the corresponding CD25− thymocyte subset leads to acceleration of disease onset in prediabetic recipients. However, in the periphery, not all regulatory T cells were found to be CD25+. In thoracic duct lymph, cells that could prevent diabetes were found in both CD25− and CD25+ subsets of CD4+CD45RC− cells. Further, CD25− regulatory T cells were also present within the CD4+CD45RC− cell subset from spleen and lymph nodes, but were effective in preventing diabetes only after the removal of CD25− recent thymic emigrants. Phenotypic analysis of human thymocytes showed the presence of CD25+ cells in the same proportions as in rat thymus. The possible developmental relationship between CD25+ and CD25− regulatory T cells is discussed.
Impaired Affinity Maturation inCr2−/− Mice Is Rescued by Adjuvants Without Improvement in Germinal Center Development
Authors: Wu Xiaobo , Jiang Ning , Fang Yi-Fu , Xu Chenguang , Mao Dailing , Singh Jasvinder , Fu Yang-Xin , Molina Hector
Published: April 2014

Cr2 −/− mice have an impairment in humoral immunity, as shown by the decrease in the Ab titers against T cell-dependent Ags and abnormalities in germinal center formation. Germinal centers are present, but they are decreased in size and number, indicating problems in their development. In this study, we investigated whether this abnormality in germinal center development is associated with problems in the establishment of optimal affinity maturation and the generation of memory B cells, processes closely related to the germinal center reaction. We immunized the Cr2−/− animals with different Ags with or without adjuvants. We showed that, when immunized without adjuvants, complement receptors are absolutely required for optimal affinity maturation. Although limited affinity maturation is elicited in the Cr2−/− Ab response, it is decreased as compared with normal animals. Memory B cell generation is also impaired. In the presence of adjuvants, germinal center development in the Cr2−/− mice is still abnormal, as demonstrated by their decreased size and number. Surprisingly, adjuvants establish optimal affinity maturation and partially restore the amount of Ab produced during the primary response and memory B cell generation. However, adjuvants cannot improve the ability of follicular dendritic cells to retain Ags in the form of immune complexes. These observations indicate that immunization with inflammatory Ags offset some of the immunological abnormalities found in the Cr2−/− mice and show that optimal affinity maturation in the Cr2−/− mice can be achieved in the absence of normal germinal centers.
The Brain Parenchyma Is Permissive for Full Antitumor CTL Effector Function, Even in the Absence of CD4 T Cells
Authors: Walker Paul R. , Calzascia Thomas , Schnuriger Valérie , Scamuffa Nathalie , Saas Philippe , de Tribolet Nicolas , Dietrich Pierre-Yves
Published: April 2014

Effective antitumor immune responses against cerebral malignancies have been demonstrated in several models, but precise cellular function of specific effector cells is poorly understood. We have explored this topic by analyzing the MHC class I-restricted T cell response elicited after implantation of HLA-CW3-transfected P815 mastocytoma cells (P815-CW3) in syngeneic mice. In this model, tumor-specific CTLs use a distinctive repertoire of TCRs that allows ex vivo assessment of the response by immunophenotyping and TCR spectratyping. Thus, for the first time in a brain tumor model, we are able to directly visualize ex vivo CTLs specific for a tumor-expressed Ag. Tumor-specific CTLs are detected in the CNS after intracerebral implantation of P815-CW3, together with other inflammatory cells. Moreover, despite observations in other models suggesting that CTLs infiltrating the brain may be functionally compromised and highly dependent upon CD4 T cells, in this syngeneic P815-CW3 model, intracerebral tumors were efficiently rejected, whether or not CD4 T cells were present. This observation correlated with potent ex vivo cytotoxicity of brain-infiltrating CTLs, specific for the immunodominant epitope CW3170–179 expressed on P815-CW3 tumor cells.
Production of Plasminogen Activator Inhibitor-1 by Human Mast Cells and Its Possible Role in Asthma
Authors: Cho Seong H. , Tam Sun W. , Demissie-Sanders Sossiena , Filler Scott A. , Oh Chad K.
Published: April 2014

The plasminogen activator inhibitor type 1 (PAI-1) has an essential role in tissue remodeling. The PAI-1 gene was induced by a combination of phorbol ester and calcium ionophore at the highest level among the inducible human mast cell genes that we have analyzed on a DNA microarray. PAI-1 was secreted by both a human mast cell line (HMC)-1 and primary cultured human mast cells upon stimulation, whereas PAI-1 was undetectable in either group of unstimulated cells. The secretion of PAI-1 was due to de novo synthesis of PAI-1 rather than secretion of preformed PAI-1. The functional significance of PAI-1 secretion was demonstrated by complete inhibition of tissue-type plasminogen activator activity with supernatants of stimulated HMC-1 cells. Furthermore, we were able to regulate PAI-1 gene expression in HMC-1 cells by known therapeutic agents. High-dose (1 μM) dexamethasone induced PAI-1 mRNA expression. Cyclosporin down-regulated the expression of the PAI-1 gene. Cycloheximide abrogated PAI-1 mRNA expression, suggesting that transcription of the PAI-1 gene requires de novo synthesis of early gene products, including transcription factors. Finally, we demonstrated PAI-1 in lung mast cells from a patient with asthmatic attack by double-immunofluorescence study. This is the first report demonstrating that activated human mast cells release a striking amount of functionally active PAI-1. These results suggest that PAI-1 could play an important role in airway remodeling of asthma, and inhibition of PAI-1 activity could represent a novel therapeutic approach in the management of airway remodeling.
IFN-γ Up-Regulates IL-18 Gene Expression Via IFN Consensus Sequence-Binding Protein and Activator Protein-1 Elements in Macrophages
Authors: Kim Yong-Man , Im Joo Young , Han Seung Hyun , Kang Hyung Sik , Choi Inpyo
Published: April 2014

Constitutive IL-18 expression is detected from many different cells, including macrophages, keratinocytes, and osteoblasts. It has been known that IL-18 gene expression is regulated by two different promoters (p1 promoter and p2 promoter). When RAW 264.7 macrophages were treated with IFN-γ, IL-18 gene expression was increased in a dose- and time-dependent manner. IFN-γ activated the inducible promoter 1, but not the constitutive promoter 2. Mutagenesis studies indicated that an IFN consensus sequence-binding protein (ICSBP) binding site between −39 and −22 was critical for the IFN-γ inducibility. EMSA using an ICSBP oligonucleotide probe showed that IFN-γ treatment increased the formation of DNA-binding complex, which was supershifted with anti-IFN regulatory factor-1 Ab and anti-ICSBP Ab. Another element, an AP-1 site between −1120 and −1083, was important. EMSA using an AP-1-specific oligonucleotide demonstrated that IFN-γ or LPS treatment increased the AP-1-binding activity. The addition of anti-c-Jun Ab or anti-c-Fos Ab to IFN-γ- or LPS-treated nuclear extracts resulted in the reduction of AP-1 complex or the formation of a supershifted complex. Taken together, these results indicate that IFN-γ increased IL-18 gene expression via ICSBP and AP-1 elements.
The I-Ag7 MHC Class II Molecule Linked to Murine Diabetes Is a Promiscuous Peptide Binder
Authors: Stratmann Thomas , Apostolopoulos Vasso , Mallet-Designe Valérie , Corper Adam L. , Scott Christopher A. , Wilson Ian A. , Kang Angray S. , Teyton Luc
Published: April 2014

Susceptibility to insulin-dependent diabetes mellitus is linked to MHC class II genes. The only MHC class II molecule expressed by nonobese diabetic (NOD) mice, I-Ag7, shares a common α-chain with I-Ad but has a peculiar β-chain. As with most β-chain alleles linked to diabetes susceptibility, I-Ag7 contains a nonaspartic residue at position β57. We have produced large amounts of empty I-Ag7 molecules using a fly expression system to characterize its biochemical properties and peptide binding by phage-displayed peptide libraries. The identification of a specific binding peptide derived from glutamic acid decarboxylase (GAD65) has allowed us to crystallize and obtain the three-dimensional structure of I-Ag7. Structural information was critical in evaluating the binding studies. I-Ag7, like I-Ad, appears to be very promiscuous in terms of peptide binding. Their binding motifs are degenerate and contain small and/or small hydrophobic residues at P4 and P6 of the peptide, a motif frequently found in most globular proteins. The degree of promiscuity is increased for I-Ag7 over I-Ad as a consequence of a larger P9 pocket that can specifically accommodate negatively charged residues, as well as possibly residues with bulky side chains. So, although I-Ad and I-Ag7 are structurally closely related, stable molecules and good peptide binders, they differ functionally in their ability to bind significantly different peptide repertoires that are heavily influenced by the presence or the absence of a negatively charged residue at position 57 of the β-chain. These characteristics link I-Ag7 with autoimmune diseases, such as insulin-dependent diabetes mellitus.