bluuebunny/crossref_metadata_2025 huggingface dataset:
Prostaglandin D2 and Its Metabolites Induce Caspase-Dependent Granulocyte Apoptosis That Is Mediated Via Inhibition of IκBα Degradation Using a Peroxisome Proliferator-Activated Receptor-γ-Independent Mechanism
Authors: Ward Carol , Dransfield Ian , Murray Joanna , Farrow Stuart N. , Haslett Christopher , Rossi Adriano G.
Published: April 2014

Many inflammatory mediators retard granulocyte apoptosis. Most natural PGs studied herein (e.g., PGE2, PGA2, PGA1, PGF2α) either delayed apoptosis or had no effect, whereas PGD2 and its metabolite PGJ2 selectively induced eosinophil, but not neutrophil apoptosis. This novel proapoptotic effect does not appear to be mediated via classical PG receptor ligation or by elevation of intracellular cAMP or Ca2+. Intriguingly, the sequential metabolites Δ12PGJ2 and 15-deoxy-Δ12, Δ14-PGJ2 (15dPGJ2) induced caspase-dependent apoptosis in both granulocytes, an effect that did not involve de novo protein synthesis. Despite the fact that Δ12PGJ2 and 15dPGJ2 are peroxisome proliferator-activated receptor-γ (PPAR-γ) activators, apoptosis was not mimicked by synthetic PPAR-γ and PPAR-α ligands or blocked by an irreversible PPAR-γ antagonist. Furthermore, Δ12PGJ2 and 15dPGJ2 inhibited LPS-induced IκBα degradation and subsequent inhibition of neutrophil apoptosis, suggesting that apoptosis is mediated via PPAR-γ-independent inhibition of NF-κB activation. In addition, we show that TNF-α-mediated loss of cytoplasmic IκBα in eosinophils is inhibited by 15dPGJ2 in a concentration-dependent manner. The selective induction of eosinophil apoptosis by PGD2 and PGJ2 may help define novel therapeutic pathways in diseases in which it would be desirable to specifically remove eosinophils but retain neutrophils for antibacterial host defense. The powerful proapoptotic effects of Δ12PGJ2 and 15dPGJ2 in both granulocyte types suggest that these natural products control the longevity of key inflammatory cells and may be relevant to understanding the control and resolution of inflammation.
Expression of Functional CCR and CXCR Chemokine Receptors in Podocytes
Authors: Huber Tobias Bruno , Reinhardt Hans Christian , Exner Markus , Burger Jan Andreas , Kerjaschki Dontscho , Saleem Moin A. , Pavenstädt Hermann
Published: April 2014

Chemokines and their receptors play an important role in the pathogenesis of acute and chronic glomerular inflammation. However, their expression pattern and function in glomerular podocytes, the primary target cells in a variety of glomerulopathies, have not been investigated as of yet. Using RT-PCR, we now demonstrate the expression of CCR4, CCR8, CCR9, CCR10, CXCR1, CXCR3, CXCR4, and CXCR5 in cultured human podocytes. Stimulation of these receptors induced a concentration-dependent biphasic increase of the free cytosolic calcium concentration in podocytes in culture. In addition, we demonstrate that podocytes release IL-8 in the presence of FCS and that IL-8 down-regulates cell surface CXCR1. Chemokine stimulation of the detected CCRs and CXCRs increased activity of NADPH-oxidase, the primary source of superoxide anions in podocytes. Immunohistochemistry studies revealed only diffuse and weak CXCR expression in healthy human glomerula. In contrast, in membranous nephropathy, a characteristic podocyte disorder, the expression of CXCR1, CXCR3, and CXCR5 is up-regulated in podocytes. In conclusion, podocytes in culture and podocytes in human kidney sections express a set of chemokine receptors. The release of oxygen radicals that accompanies the activation of CCRs and CXCRs may contribute to podocyte injury and the development of proteinuria during membranous nephropathy.
Peptide Mimotopes of Pneumococcal Capsular Polysaccharide of 6B Serotype: A Peptide Mimotope Can Bind to Two Unrelated Antibodies
Authors: Shin Jeon-Soo , Yu Jigui , Lin Jisheng , Zhong Linghao , Bren Kara L. , Nahm Moon H.
Published: April 2014

Two groups of bacteriophage clones displaying the antigenic properties of serotype 6B pneumococcal capsular polysaccharide (PS) were obtained from different phage libraries expressing random heptameric peptides. One group, biopanned with a mouse mAb (Hyp6BM1), is comprised of 17 phage clones expressing 10 unique sequences of linear peptides. The other group, selected with another mAb (Hyp6BM8), contained six clones, all of which expressed the identical circular peptide. Phage clones expressing the linear peptides (e.g., PhaM1L3) bound only to Hyp6BM1, but not other 6B PS-specific mAb, and their binding could be inhibited with pneumococcal capsular type 6B PS only. In contrast, a phage clone expressing the circular peptide (PhaM8C1) cross-reacted with several other 6B PS-specific mAbs, and their binding could be inhibited with pneumococcal capsular PS of 6A and 6B serotypes. Two short peptides, PepM1L3 and PepM8C1, reflecting the peptide inserts of the corresponding phage clones, could inhibit the binding of the two clones to their respective mAb. Interestingly, the peptide insert in PhaM8C1 was identical to that in PhaB3C4, a previously reported mimotope of α(2→8) polysialic acid, Neisseria meningitidis group B PS. Indeed, PhaM8C1 bound to HmenB3 (a meningococcal Ab), and their association could be inhibited with α(2–8) polysialic acid, but not with 6B PS. Conversely, α(2–8) polysialic acid could not inhibit the binding of PhaM8C1 to Hyp6BM8. The two-dimensional nuclear magnetic resonance studies indicate that PepM8C1 peptide can assume several conformations in solution. The ability of this peptide to assume multiple conformations might account for its ability to mimic more than one Ag type.
Role of Membrane Cofactor Protein (CD46) in Regulation of C4b and C3b Deposited on Cells
Authors: Barilla-LaBarca Maria L. , Liszewski M. Kathryn , Lambris John D. , Hourcade Dennis , Atkinson John P.
Published: April 2014

C4b and C3b deposited on host cells undergo limited proteolytic cleavage by regulatory proteins. Membrane cofactor protein (MCP; CD46), factor H, and C4b binding protein mediate this reaction, known as cofactor activity, that also requires the plasma serine protease factor I. To explore the roles of the fluid phase regulators vs those expressed on host cells, a model system was used examining complement fragments deposited on cells transfected with human MCP as assessed by FACS and Western blotting. Following incubation with Ab and complement on MCP+ cells, C4b was progressively cleaved over the first hour to C4d and C4c. There was no detectable cleavage of C4b on MCP− cells, indicating that MCP (and not C4BP in the serum) primarily mediates this cofactor activity. C3b deposition was not blocked on MCP+ cells because classical pathway activation occurred before substantial C4b cleavage. Cleavage, though, of deposited C3b was rapid (<5 min) and iC3b was the dominant fragment on MCP− and MCP+ cells. Studies using a function-blocking mAb further established factor H as the responsible cofactor. If the level of Ab sensitization was reduced 8-fold or if Mg2+-EGTA was used to block the classical pathway, MCP efficiently inhibited C3b deposition mediated by the alternative pathway. Thus, for the classical pathway, MCP is the cofactor for C4b cleavage and factor H for C3b cleavage. However, if the alternative pathway mediates C3b deposition, then MCP’s cofactor activity is sufficient to restrict complement activation.
Analysis of IgE Antibodies from a Patient with Atopic Dermatitis: Biased V Gene Usage and Evidence for Polyreactive IgE Heavy Chain Complementarity-Determining Region 3
Authors: Edwards Michael R. , Brouwer Wandy , Choi Caroline H. Y. , Ruhno John , Ward Robyn L. , Collins Andrew M.
Published: April 2014

To better understand V gene usage, specificity, and clonal origins of IgE Abs in allergic reactions, we have constructed a combinatorial Ab library from the mRNA of an adult patient with atopic dermatitis. Sequence analysis of random clones revealed that 33% of clones used the IGHV6-1 H chain V gene segment, the only member of the VH6 gene family. IGHV6-1 is rarely used in the expressed adult repertoire; however, it is associated with fetal derived Abs. Features of the VH6 rearrangements included short complementarity-determining region 3, frequent use of IGHD7-27 D gene, and little nucleotide addition at the D-J junction. There was also a low level of mutation compared with VH1, VH3, and VH4 rearrangements. The library was expressed as phage-Fab fusions, and specific phage selected by panning on the egg allergen ovomucoid. Upon expression as soluble IgE Fabs, 12 clones demonstrated binding to ovomucoid, skim milk, and BSA by ELISA. Nucleotide sequencing demonstrated that the IGHV6-1 V gene segment encoded each of the 12 multiply reactive IgE Fabs. A cyclic peptide was designed from the complementarity-determining region 3 of several of these clones. The cyclic peptide bound both self and nonself Ags, including ovomucoid, human IgG, tetanus toxoid, and human and bovine von Willebrand factor. These results suggest that some IgE Abs may bind more than one Ag, which would have important implications for understanding the multiple sensitivities seen in conditions such as atopic dermatitis.
Memory Th2 Effector Cells Can Develop in the Absence of B7-1/B7-2, CD28 Interactions, and Effector Th Cells After Priming with an Intestinal Nematode Parasite
Authors: Ekkens Melinda J. , Liu Zhugong , Liu Qian , Foster Anthony , Whitmire Jeannette , Pesce John , Sharpe Arlene H. , Urban Joseph F. , Gause William C.
Published: April 2014

B7-1/B7-2 interactions are required for many Th2-cell mediated primary immune responses including the response that follows infection with the intestinal nematode parasite, Heligmosomoides polygyrus. However, few studies have examined the role of B7-1/B7-2/CD28 interactions in the development of a Th2 memory immune response. We examined the development of the memory Th2 response to H. polygyrus in BALB/c mice deficient in both B7-1 and B7-2 (B7-1/B7-2−/−) and in BALB/c mice deficient in CD28 (CD28−/−). Following primary inoculation with H. polygyrus, adult worms in the gut were cleared with an anti-helminthic drug and mice were subsequently challenge-inoculated with H. polygyrus larvae. The memory Th2 response is readily distinguished by its inhibitory effect on adult worm maturation, resulting in marked reductions in adult worm egg production that are not observed during the primary immune response. Following H. polygyrus challenge inoculation, comparable decreases in egg production and similar increases in mesenteric lymph node cell IL-4 production were observed in B7-1/B7-2−/− and B7-1/B7-2+/+ mice. However, elevations in total serum IgG1 and IgE were reduced, while increases in serum Ag-specific IgG1 and IgE and germinal center formation were blocked in H. polygyrus-challenged B7-1/B7-2−/− mice. In contrast, in H. polygyrus-challenged CD28−/− mice, marked elevations in Ag-specific IgG1 and IgE and increased germinal center formation were observed. The results of these studies demonstrate that effector Th2 memory cells that produce IL-4 and mediate host defense can develop when B7-1/B7-2 interactions, and associated effector Th2 cell development, are blocked during priming. However, humoral immunity is impaired and differentially affected in B7-1/B7-2−/− mice and CD28−/− mice following H. polygyrus challenge.
Murine Lyme Arthritis Development Mediated by p38 Mitogen-Activated Protein Kinase Activity
Authors: Anguita Juan , Barthold Stephen W. , Persinski Rafal , Hedrick Michael N. , Huy Christy A. , Davis Roger J. , Flavell Richard A. , Fikrig Erol
Published: April 2014

Borrelia burgdorferi, the Lyme disease agent, causes joint inflammation in an experimental murine model. Inflammation occurs, in part, due to the ability of B. burgdorferi to induce the production of proinflammatory cytokines and a strong CD4+ T helper type 1 response. The mechanisms by which spirochetes induce these responses are not completely known, although transcription factors, such as NF-κB in phagocytic cells, initiate the proinflammatory cytokine burst. We show here that the mitogen-activated protein (MAP) kinase of 38 kDa (p38 MAP kinase) is involved in the proinflammatory cytokine production elicited by B. burgdorferi Ags in phagocytic cells and the development of murine Lyme arthritis. B. burgdorferi Ags activated p38 MAP kinase in vitro, and the use of a specific inhibitor repressed the spirochete-induced production of TNF-α. The infection of mice that are deficient for a specific upstream activator of the kinase, MAP kinase kinase 3, resulted in diminished proinflammatory cytokine production and the development of arthritis, without compromising the ability of CD4+ T cells to respond to borrelial Ags or the production of specific Abs. Overall, these data indicated that the p38 MAP kinase pathway plays an important role in B. burgdorferi-elicited inflammation and point to potential new therapeutic approaches to the treatment of inflammation induced by the spirochete.
TheTrypanosoma cruziTc52-Released Protein Induces Human Dendritic Cell Maturation, Signals Via Toll-Like Receptor 2, and Confers Protection Against Lethal Infection
Authors: Ouaissi Ali , Guilvard Eliane , Delneste Yves , Caron Gersende , Magistrelli Giovanni , Herbault Nathalie , Thieblemont Nathalie , Jeannin Pascale
Published: April 2014

The intracellular protozoan parasite Trypanosoma cruzi is the etiological agent of Chagas disease. We have recently identified a T. cruzi-released protein related to thiol-disulfide oxidoreductase family, called Tc52, which is crucial for parasite survival and virulence. In vitro, Tc52 in combination with IFN-γ activates human macrophages. In vivo, active immunization with Tc52 relieves the immunosuppression associated to acute infection and elicits a specific immune response. As dendritic cells (DC) have a central role in the initiation of immune responses, we investigated whether Tc52 may modulate DC activity. We show that Tc52 induces human DC maturation. Tc52-treated immature DC acquire CD83 and CD86 expression, produce inflammatory chemokines (IL-8, monocyte chemoattractant protein-1, and macrophage-inflammatory protein-1α), and present potent costimulatory properties. Tc52 binds to DC by a mechanism with the characteristics of a saturable receptor system and signals via Toll-like receptor 2. While Tc52-mediated signaling involves its reduced glutathione-binding site, another portion of the molecule is involved in Tc52 binding to DC. Finally, we report that immunization with Tc52 protects mice in vivo against lethal infection with T. cruzi. Together these data evidence complex molecular interactions between the T. cruzi-derived molecule, Tc52, and DC, and suggest that Tc52 and related class of proteins might represent a new type of pathogen-associated molecular patterns. Moreover, the immune protection data suggest that Tc52 is among candidate molecules that may be used to design an optimal multicomponent vaccine to control T. cruzi infection.
Inhibition of CXCR4-Tropic HIV-1 Infection by Lipopolysaccharide: Evidence of Different Mechanisms in Macrophages and T Lymphocytes
Authors: Verani Alessia , Sironi Francesca , Siccardi Antonio G. , Lusso Paolo , Vercelli Donata
Published: April 2014

Bacterial LPS protects primary human macrophages from infection by CCR5-tropic HIV-1 isolates through the release of the CC chemokines RANTES and macrophage inflammatory protein-1α and -1β. Here, we show that LPS also suppresses infection of macrophages by CXCR4-tropic HIV-1 isolates. A marked down-regulation of both CD4 and CXCR4 expression was associated with this effect. Furthermore, a soluble factor(s) released by macrophages upon LPS treatment inhibited infection with CXCR4-tropic HIV-1 isolate viruses in both macrophages and T lymphocytes. Infection of both cell types appeared to be blocked at the level of viral entry and was independent of stromal cell-derived factor-1, the only known natural ligand of CXCR4. Moreover, the suppressive effect of LPS was unrelated to the release of IFN-α and -β, macrophage-derived chemokine, leukemia inhibitory factor, or TNF-α. These results suggest the existence of potent HIV-1 inhibitory factor(s), uncharacterized to date, released by activated cells of the mononuclear phagocytic system.
The Role of Lipopolysaccharide Binding Protein in Resistance to Salmonella Infections in Mice
Authors: Fierer Joshua , Swancutt Mark A. , Heumann Didier , Golenbock Douglas
Published: April 2014

Polymorphonuclear leukocytes (PMN) and LPS-binding protein (LBP) are both components of the innate immune system. LBP is a plasma protein that binds to lipid A and enhances the biological activity of LPS 100- to 1000-fold. Recently it was reported that LBP-deficient mice are more susceptible to Salmonella typhimurium infection. Here we report that LBP KO mice are more susceptible to Salmonella peritonitis, but not to oral or i.v. infection. LBP knockout (KO) mice responded normally to i.p. injections of Staphylococcus aureus and casein, but not to i.p. injection of S. typhimurium or Salmonella LPS. Mice with a mutation in Toll-like receptor 4 (C3H/HeJ) have a similar defect in PMN chemotaxis. In normal mice S. typhimurium stimulated production of the CXC chemokines macrophage inflammatory protein-2 and cytokine-induced neutrophil chemoattractant, but levels of cytokine-induced neutrophil chemoattractant and macrophage inflammatory protein-2 were greatly reduced in the LBP KO mice. LBP KO mice pretreated with casein to attract PMN in an LBP-independent manner were more resistant to Salmonella infection, but neutropenic mice were not protected by casein. Splenic TNF-α mRNA levels were also lower in LBP KO than in control mice infected with Salmonella. Since TNF-α can activate PMN, LBP KO mice may have both fewer and less active PMN in the first few hours after Salmonella are injected, making LBP KO mice more susceptible. This work confirms the importance of PMN in resistance to Salmonella infections and shows that this is facilitated by LBP.
Protein Kinase R Regulates Double-Stranded RNA Induction of TNF-α But Not IL-1β mRNA in Human Epithelial Cells
Authors: Meusel Tiffany R. , Kehoe Kelly E. , Imani Farhad
Published: April 2014

Epithelial cells represent the initial site of respiratory viral entry and the first line of defense against such infections. This early antiviral response is characterized by an increase in the production of proinflammatory cytokines such as TNF-α and IL-1β. dsRNA, which is a common factor present during the life cycle of both DNA and RNA viruses, is known to induce TNF-α and IL-1β in a variety of cells. In this work we provide data showing that dsRNA treatment induces TNF-α and IL-1β in human lung epithelial cells via two different mechanisms. Our data show that dsRNA activation of dsRNA-activated protein kinase (PKR) is associated with induction of TNF-α but not IL-1β expression. An inhibitor of PKR activation blocked the dsRNA-induced elevations in TNF-α but not IL-1β mRNA in epithelial cells. Data obtained from infection of epithelial cells with a vaccinia virus lacking the PKR inhibitory polypeptide, E3L, revealed that PKR activation was essential for TNF-α but not for IL-1β expression. In this report, we provide experimental support for the differential regulation of proinflammatory cytokine expression by dsRNA and viral infections in human airway epithelial cells.
Fgr Deficiency Results in Defective Eosinophil Recruitment to the Lung During Allergic Airway Inflammation
Authors: Vicentini Lucia , Mazzi Paola , Caveggion Elena , Continolo Silvia , Fumagalli Laura , Lapinet-Vera José A. , Lowell Clifford A. , Berton Giorgio
Published: April 2014

Using a mouse model of allergic lung inflammation, we found that mice deficient of Fgr, a Src family tyrosine kinase highly expressed in myelomonocytic cells, fail to develop lung eosinophilia in response to repeated challenge with aerosolized OVA. Both tissue and airway eosinophilia were markedly reduced in fgr−/− mice, whereas mice with the sole deficiency of Hck, another Src family member, responded normally. Release of allergic mediators, such as histamine, IL-4, RANTES/CCL5, and eotaxin/CCL11, in the airways of OVA-treated animals was equal in wild-type and fgr−/− mice. However, lung eosinophilia in Fgr-deficient mice correlated with a defective accumulation of GM-CSF and IL-5 in the airways, whereas secretion of these cytokines by spleen cells in response to OVA was normal. Examination of mRNA expression in whole lung tissue allowed us to detect comparable expression of transcripts for eotaxin/CCL11, macrophage-inflammatory protein-1α/CCL3, macrophage-inflammatory protein-1β/CCL4, monocyte chemoattractant protein-1/CCL2, TCA-3/CCL1, IL-4, IL-10, IL-2, IL-3, IL-9, IL-15, and IFN-γ in OVA-sensitized wild-type and fgr−/− mice. In contrast, the increase in IL-5 and IL-13 mRNA expression was lower in fgr−/− compared with wild-type mice. These findings suggest that deficiency of Fgr results in a marked reduction of lung eosinophilia and the establishment of a positive feedback loop based on autocrine secretion of eosinophil-active cytokines. These results identify Fgr as a novel pharmacological target to control allergic inflammation.
Absence of Platelet Endothelial Cell Adhesion Molecule-1 (CD31) Leads to Increased Severity of Local and Systemic IgE-Mediated Anaphylaxis and Modulation of Mast Cell Activation
Authors: Wong Mae-Xhum , Roberts Donna , Bartley Paul A. , Jackson Denise E.
Published: April 2014

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a newly assigned member of the Ig-immunoreceptor tyrosine-based inhibitory motif superfamily, and its functional role is suggested to be an inhibitory receptor that modulates immunoreceptor tyrosine-based activation motif-dependent signaling cascades. In this study, we hypothesized that PECAM-1 plays an essential in vivo role as a counterregulator of immediate hypersensitivity reactions. We found that PECAM-1 was highly expressed on the surface of immature bone marrow mast cells and at a lower density on mature peritoneal mast cells. Examination of skin biopsies from PECAM-1+/+ and PECAM-1−/− mice revealed that absence of PECAM-1 did not affect mast cell development or the capacity of mast cells to populate tissues. To examine whether the absence of PECAM-1 would influence immediate hypersensitivity reactions, PECAM-1+/+ and PECAM-1−/− mice were presensitized with anti-DNP mouse IgE and then challenged 20 h later with DNP-BSA or PBS. PECAM-1−/− mice exhibited elevated serum histamine concentrations after Ag stimulation compared with PECAM-1+/+ mice, indicating an increased severity of systemic IgE-mediated anaphylaxis. PECAM-1−/− mice have increased sensitivity to local cutaneous IgE-dependent anaphylaxis compared with PECAM-1+/+ mice, as assessed by greater tissue swelling of their ears and mast cell degranulation in situ. PECAM-1−/− bone marrow mast cells showed enhanced dense granule serotonin release after FcεRI cross-linking in vitro. These results suggest that PECAM-1 acts as a counterregulator in allergic disease susceptibility and severity and negatively modulates mast cell activation.
TGF-β1 as an Enhancer of Fas-Mediated Apoptosis of Lung Epithelial Cells
Authors: Hagimoto Naoki , Kuwano Kazuyoshi , Inoshima Ichiro , Yoshimi Michihiro , Nakamura Norio , Fujita Masaki , Maeyama Takashige , Hara Nobuyuki
Published: April 2014

Transforming growth factor-β1 (TGF-β1) has important roles in lung fibrosis and the potential to induce apoptosis in several types of cells. We previously demonstrated that apoptosis of lung epithelial cells induced by Fas ligation may be involved in the development of pulmonary fibrosis. In this study, we show that TGF-β1 induces apoptosis of primary cultured bronchiolar epithelial cells via caspase-3 activation and down-regulation of cyclin-dependent kinase inhibitor p21. Concentrations of TGF-β1 that were not sufficient to induce apoptosis alone could enhance agonistic anti-Fas Ab or rFas ligand-mediated apoptosis of cultured bronchiolar epithelial cells. Soluble Fas ligand in the bronchoalveolar lavage fluid (BALF) from patients with idiopathic pulmonary fibrosis (IPF) also induced apoptosis of cultured bronchiolar epithelial cells that was significantly attenuated by anti-TGF-β Ab. Otherwise, BALF from patients with hypersensitivity pneumonitis (HP) could not induce apoptosis on bronchiolar epithelial cells, despite its comparable amounts of soluble Fas ligand. The concentrations of TGF-β1 in BALF from patients with IPF were significantly higher compared with those in BALF from patients with HP or controls. Furthermore, coincubation with the low concentration of TGF-β1 and HP BALF created proapoptotic effects comparable with the IPF BALF. In vivo, the administration of TGF-β1 could enhance Fas-mediated epithelial cell apoptosis and lung injury via caspase-3 activation in mice. Our results demonstrate a novel role of TGF-β1 in the pathophysiology of pulmonary fibrosis as an enhancer of Fas-mediated apoptosis of lung epithelial cells.
Developmental and Functional Defects of Thymic and Epidermal Vγ3 Cells in IL-15-Deficient and IFN Regulatory Factor-1-Deficient Mice
Authors: De Creus An , Van Beneden Katrien , Stevenaert Frederik , Debacker Veronique , Plum Jean , Leclercq Georges
Published: April 2014

In this study, the role of IL-15 and its regulation by the transcription factor IFN regulatory factor-1 (IRF-1) in murine Vγ3 T cell development and activity is assessed. Compared with wild-type (WT) mice, reduced numbers of mature Vγ3 cells were found in the fetal thymus of IL-15−/− mice, while IRF-1−/− mice displayed normal frequencies. Vγ3+ dendritic epidermal T cells (DETCs) were absent in IL-15−/− mice but present in IRF-1−/− mice. DETCs from IRF-1−/− mice displayed morphologically a less mature phenotype and showed different emergence kinetics during ontogeny. This corresponded with lower IL-15 mRNA levels in the skin epidermis. Comparable levels of IL-7 were found in the skin of WT and IL-15−/− mice. Adoptive transfer experiments of WT fetal thymocytes into IL-15−/− mice did not result in the development of Vγ3+ DETCs, confirming the nonredundant role of IL-15 in the skin during DETC development. In vitro, cytolytic activity of IL-15−/− Vγ3 cells was normal after stimulation with IL-15 and was further enhanced by addition of IL-12. In contrast, cytolytic activity of IRF-1−/− Vγ3 cells remained defective after stimulation with IL-15 in combination with IL-12. These data suggest that IL-15 is redundant for the development and/or survival of mature Vγ3 cells in the fetal thymus, whereas it is essential for the localization of Vγ3 cells in the skin. Furthermore, a possible role for IRF-1 in inducing morphological maturation of DETCs and cytolytic capacity of Vγ3 cells is suggested.
Impaired Proliferative Response of Vα24 NKT Cells from Cancer Patients Against α-Galactosylceramide
Authors: Yanagisawa Kazuhiko , Seino Ken-ichiro , Ishikawa Yuriko , Nozue Mutsumi , Todoroki Takeshi , Fukao Katashi
Published: April 2014

Human invariant Vα24+ NKT cells are a relatively new subpopulation of lymphocytes. It has been reported that Vα24 NKT cells are significantly involved in some human diseases. We have evaluated the number and function of Vα24 NKT cells in both healthy volunteers and cancer patients. In this study we found that Vα24 NKT cells in unfractionated PBMCs obtained from cancer patients did not respond efficiently to α-galactosylceramide (α-GalCer) in vitro. Thus, their proportion after stimulation with α-GalCer was smaller than that found in healthy volunteers. However, the cancer patients’ Vα24 NKT cells retained cytotoxic activity against malignant target cells, and they could efficiently proliferate to α-GalCer when fractionated by sorting. Furthermore, we found that addition of G-CSF to the culture could restore the low proliferative response of Vα24 NKT cells from cancer patients. These results suggest that some functions of NKT cells in cancer patients are impaired, and this observation carries significant implications for immunotherapy-based cancer treatments.
Cutting Edge: In Vitro-Generated Tolerogenic APC Induce CD8+ T Regulatory Cells That Can Suppress Ongoing Experimental Autoimmune Encephalomyelitis
Authors: Faunce Douglas E. , Terajewicz Ania , Stein-Streilein Joan
Published: April 2014

APC exposed to TGFβ2 and Ag (tolerogenic APC) promote peripheral Ag-specific tolerance via the induction of CD8+ T regulatory cells capable of suppressing Th1 and Th2 immunity. We postulated that tolerogenic APC might reinstate tolerance toward self-neuronal Ags and ameliorate ongoing experimental autoimmune encephalomyelitis (EAE). Seven days after immunization with myelin basic protein (MBP), mice received MBP-specific tolerogenic APC, and EAE was evaluated clinically. To test for the presence and the phenotype of T regulatory cells, CD4 and/or CD8 T cells from tolerogenic APC-treated mice were transferred to naive mice before their immunization with MBP. The MBP-specific tolerogenic APC decreased both the severity and incidence of ongoing EAE. Tolerance to self-neuronal Ags was induced in naive recipient mice via adoptive transfer of CD8+, but not CD4+ T cells. Rational use of in vitro-generated tolerogenic APC may lead to novel therapy for autoimmune disease.
Cutting Edge: IL-26 Signals through a Novel Receptor Complex Composed of IL-20 Receptor 1 and IL-10 Receptor 2
Authors: Sheikh Faruk , Baurin Vitaliy V. , Lewis-Antes Anita , Shah Nital K. , Smirnov Sergey V. , Anantha Shubha , Dickensheets Harold , Dumoutier Laure , Renauld Jean-Christophe , Zdanov Alexander , Donnelly Raymond P. , Kotenko Sergei V.
Published: April 2014

The receptor for IL-26 (AK155), a cytokine of the IL-10 family, has not previously been defined. We demonstrate that the active receptor complex for IL-26 is a heterodimer composed of two receptor proteins: IL-20R1 and IL-10R2. Signaling through the IL-26R results in activation of STAT1 and STAT3 which can be blocked by neutralizing Abs against IL-20R1 or IL-10R2. IL-10R2 is broadly expressed on a wide variety of tissues, whereas only a limited number of tissues express IL-20R1. Therefore, the ability to respond to IL-26 is restricted by the expression of IL-20R1. IL-10, IL-19, IL-20, IL-22, and IL-24 fail to signal through the combination of IL-10R2 and IL-20R1 proteins, demonstrating that this receptor combination is unique and specific for IL-26.
Cutting Edge: T Cells Trigger CD40-Dependent Platelet Activation and Granular RANTES Release: A Novel Pathway for Immune Response Amplification
Authors: Danese Silvio , de la Motte Carol , Reyes Brenda M. Rivera , Sans Miquel , Levine Alan D. , Fiocchi Claudio
Published: April 2014

Platelets, in addition to exerting hemostatic activity, contribute to immunity and inflammation. The recent report that platelets express CD40 led us to hypothesize that CD40 ligand (CD40L)-positive T cells could bind to platelets, cause their activation, and trigger granular RANTES release, creating a T cell recruitment feedback loop. Platelets were cocultured with resting or activated autologous T cells and their activation was assessed by P-selectin expression. RANTES binding to endothelial cells was assessed by confocal microscopy, and its biological activity was demonstrated by a T cell adhesion assay. CD40L-positive T cells induced platelet activation through a contact-mediated, CD40-dependent pathway resulting in RANTES release, which bound to endothelial cells and mediated T cell recruitment. Soluble CD40L induced the same events via p38, but not extracellular signal-regulated kinase, phosphorylation. These results show the existence of a novel platelet-dependent pathway of immune response amplification which brings these nonimmune cells close to the level of pathogenic relevance traditionally attributed to classical immune cells.
Visualizing the Site and Dynamics of IgG Salvage by the MHC Class I-Related Receptor, FcRn
Authors: Ober Raimund J. , Martinez Cruz , Vaccaro Carlos , Zhou Jinchun , Ward E. Sally
Published: April 2014

The MHC class I-related receptor, FcRn, plays a central role in regulating the serum levels of IgG. FcRn is expressed in endothelial cells, suggesting that these cells may be involved in maintaining IgG levels. We have used live cell imaging of FcRn-green fluorescent protein transfected human endothelial cells to analyze the intracellular events that control IgG homeostasis. We show that segregation of FcRn-IgG complexes from unbound IgG occurs in the sorting endosome. FcRn or FcRn-IgG complexes are gradually depleted from sorting endosomes to ultimately generate multivesicular bodies whose contents are destined for lysosomal degradation. In addition, the pathways taken by FcRn and the transferrin receptor overlap, despite distinct mechanisms of ligand uptake. The studies provide a dynamic view of the trafficking of FcRn and its ligand and have relevance to understanding how FcRn functions to maintain IgG homeostasis.
Visualization of Antigen Presentation by Actin-Mediated Targeting of Glycolipid-Enriched Membrane Domains to the Immune Synapse of B Cell APCs
Authors: Gordy Claire , Mishra Sudha , Rodgers William
Published: April 2014

Glycolipid-enriched membrane (GEM) domains, or lipid rafts, function in signaling in immune cells, but their properties during Ag presentation are less clear. To address this question, GEM domains were studied using fluorescence cell imaging of mouse CH27 B cells presenting Ag to D10 T cells. Our experiments showed that APCs were enriched with GEM domains in the immune synapse, and this occurred in an actin-dependent manner. This enrichment was specific to GEM domains, because a marker for non-GEM regions of the membrane was excluded from the immune synapse. Furthermore, fluorescence photobleaching experiments showed that protein in the immune synapse was dynamic and rapidly exchanged with that in other compartments of CH27 cells. To identify the signals for targeting GEM domains to the immune synapse in APCs, capping of the domains was measured in cells after cross-linking surface molecules. This showed that co-cross-linking CD48 with MHC class II was required for efficient capping and intracellular signaling. Capping of GEM domains by co-cross-linking CD48 and MHC class II occurred with co-capping of filamentous actin, and both domain capping and T cell-CH27 cell conjugation were inhibited by pretreating CH27 cells with latrunculin B. Furthermore, disruption of the actin cytoskeleton of the CH27 cells also inhibited formation of a mature immune synapse in those T cells that did conjugate to APCs. Thus, Ag presentation and efficient T cell stimulation occur by an actin-dependent targeting of GEM domains in the APC to the site of T cell engagement.
Inhibitory Effect of Toll-Like Receptor 4 on Fusion between Phagosomes and Endosomes/Lysosomes in Macrophages
Authors: Shiratsuchi Akiko , Watanabe Ikuko , Takeuchi Osamu , Akira Shizuo , Nakanishi Yoshinobu
Published: April 2014

Toll-like receptor 4 (TLR4) of macrophages recognizes LPS of Gram-negative bacteria in cooperation with CD14, which is also involved in the recognition of apoptotic cells. In this study we asked whether TLR4 plays a role in the phagocytic clearance of apoptotic cells by macrophages. Macrophages were prepared from peritoneal fluid of thioglycolate-treated mice carrying either a wild-type or a disrupted TLR4-encoding gene and were examined for their ability to phagocytose apoptotic mouse thymocytes, apoptotic Jurkat T cells, Ig-opsonized mouse thymocytes, Ig-opsonized zymosan particles, and latex beads. Both populations of macrophages equally expressed CD14 on their surfaces and showed almost equal activities of binding to and engulfing all these targets. However, apoptotic thymocytes, apoptotic Jurkat cells, and opsonized thymocytes disappeared more rapidly in TLR4-deficient macrophages than in wild-type macrophages, and the fusion between endosomes/lysosomes and phagosomes containing any target cells or particles was accelerated in mutant macrophages. Activation of the transcription factor NF-κB appeared not to occur in wild-type macrophages after engulfment, and the rate of apoptotic cell degradation in wild-type macrophages remained the same regardless of the activation of NF-κB. Finally, immunohistochemical analyses showed that ectopically expressed TLR4 was associated with phagosomes in a macrophage-derived cell line. All these results collectively indicate that TLR4 negatively regulates the degradation of engulfed cells in macrophages via a pathway independent of NF-κB.
IL-21 Induces the Functional Maturation of Murine NK Cells
Authors: Brady Jason , Hayakawa Yoshihiro , Smyth Mark J. , Nutt Stephen L.
Published: April 2014

IL-21 is a recently identified cytokine that stimulates mouse NK cell effector functions in vitro. In this study we demonstrate that IL-21 achieves its stimulatory effect by inducing the development of mature NK cells into a large granular lymphocyte phenotype with heightened effector function. IL-21 treatment results in increased cell size and granularity and a corresponding decrease in cell viability and proliferative potential. These cells up-regulate the expression of the inhibitory CD94-NKG2A receptor complex and the activation markers CD154 and killer cell, lectin-like-receptor G1. Surprisingly, IL-21 treatment also results in down-regulation of the pan-NK marker, NK1.1. Coinciding with these cellular changes IL-21 enhances cytolytic capacity across a spectrum of target sensitivities and induces IL-10 and IFN-γ production. In vivo treatment with IL-21 results in a very similar activation and phenotypic maturation of NK cells as well as a potent increase in NK cell-mediated anti-tumor immunity that is perforin dependent. These developmental changes suggested that IL-21 functions to induce the terminal differentiation of mouse NK cells, resulting in heightened NK cell-mediated cytotoxicity and immune surveillance.
NF-κB-Inducing Kinase Establishes Self-Tolerance in a Thymic Stroma-Dependent Manner
Authors: Kajiura Fumiko , Sun Shijie , Nomura Takashi , Izumi Keisuke , Ueno Tomoo , Bando Yoshimi , Kuroda Noriyuki , Han Hongwei , Li Yi , Matsushima Akemi , Takahama Yousuke , Sakaguchi Shimon , Mitani Tasuku , Matsumoto Mitsuru
Published: April 2014

Physical contact between thymocytes and the thymic stroma is essential for T cell maturation and shapes the T cell repertoire in the periphery. Stromal elements that control these processes still remain elusive. We used a mouse strain with mutant NF-κB-inducing kinase (NIK) to examine the mechanisms underlying the breakdown of self-tolerance. This NIK-mutant strain manifests autoimmunity and disorganized thymic structure with abnormal expression of Rel proteins in the stroma. Production of immunoregulatory T cells that control autoreactive T cells was impaired in NIK-mutant mice. The autoimmune disease seen in NIK-mutant mice was reproduced in athymic nude mice by grafting embryonic thymus from NIK-mutant mice, and this was rescued by supply of exogenous immunoregulatory T cells. Impaired production of immunoregulatory T cells by thymic stroma without normal NIK was associated with altered expression of peripheral tissue-restricted Ags, suggesting an essential role of NIK in the thymic microenvironment in the establishment of central tolerance.
Mechanisms Governing B Cell Developmental Defects in Invariant Chain-Deficient Mice
Authors: Benlagha Kamel , Park Se-Ho , Guinamard Rodolphe , Forestier Claire , Karlsson Lars , Chang Cheong-Hee , Bendelac Albert
Published: April 2014

Invariant chain (Ii)-deficient mice exhibit profound B cell defects that have remained poorly understood, because they could not be simply explained by impaired Ag presentation. We found that Ii deficiency induced cell autonomous defects of two distinct B cell lineages. The life span of mature follicular (FO) B cells was reduced, accounting for their markedly decreased frequency, whereas, in contrast, marginal zone (MZ) B cells accumulated. Other Ii-expressing lineages such as B1 B cells and dendritic cells were unaffected. Surprisingly, the life span of FO B cells was fully corrected in Ii/I-Aβ doubly deficient mice, revealing that Ii-free I-Aβ chains alter FO B cell survival. In contrast, the accumulation of MZ B cells was controlled by a separate mechanism independent of I-Aβ. Interestingly, in Ii-deficient mice lacking FO B cells, the MZ B cells invaded the FO zone, suggesting that intact follicules contribute to the retention of B cells in the MZ. These findings reveal unexpected consequences of Ii deficiency on the development and organization of B cell follicles.
Complete Loss of Fas Ligand Gene Causes Massive Lymphoproliferation and Early Death, Indicating a Residual Activity of gld Allele
Authors: Karray Saoussen , Kress Chantal , Cuvellier Sylvain , Hue-Beauvais Catherine , Damotte Diane , Babinet Charles , Lévi-Strauss Matthieu
Published: April 2014

To investigate the in vivo function of Fas ligand (FasL), we produced a mouse strain with a FasL gene flanked by loxP sequences. Mice with homozygous floxed FasL gene showed no obvious abnormalities. However, germline deletion of the FasL gene, obtained after mating with mice expressing ubiquitous Cre recombinase, resulted in an unexpectedly severe phenotype. FasL−/− mice exhibited an extreme splenomegaly and lymphadenopathy associated with lymphocytic infiltration into multiple organs and autoimmune disease. This severe phenotype led to the premature death at 4 mo of age of >50% of the homozygous mice. It stands in sharp contrast with the milder disease observed in gld (generalized lymphoproliferative disease) mice, indicating that the FasL allele of these mice encodes a protein still able to bind, albeit at a very low level, the Fas receptor.
Exosomes as Potent Cell-Free Peptide-Based Vaccine. I. Dendritic Cell-Derived Exosomes Transfer Functional MHC Class I/Peptide Complexes to Dendritic Cells
Authors: André Fabrice , Chaput Nathalie , Schartz Nöel E. C. , Flament Caroline , Aubert Nathalie , Bernard Jacky , Lemonnier François , Raposo Graça , Escudier Bernard , Hsu Di-Hwei , Tursz Thomas , Amigorena Sebastian , Angevin Eric , Zitvogel Laurence
Published: April 2014

Current immunization protocols in cancer patients involve CTL-defined tumor peptides. Mature dendritic cells (DC) are the most potent APCs for the priming of naive CD8+ T cells, eventually leading to tumor eradication. Because DC can secrete MHC class I-bearing exosomes, we addressed whether exosomes pulsed with synthetic peptides could subserve the DC function consisting in MHC class I-restricted, peptide-specific CTL priming in vitro and in vivo. The priming of CTL restricted by HLA-A2 molecules and specific for melanoma peptides was performed: 1) using in vitro stimulations of total blood lymphocytes with autologous DC pulsed with GMP-manufactured autologous exosomes in a series of normal volunteers; 2) in HLA-A2 transgenic mice (HHD2) using exosomes harboring functional HLA-A2/Mart1 peptide complexes. In this study, we show that: 1) DC release abundant MHC class I/peptide complexes transferred within exosomes to other naive DC for efficient CD8+ T cell priming in vitro; 2) exosomes require nature’s adjuvants (mature DC) to efficiently promote the differentiation of melanoma-specific effector T lymphocytes producing IFN-γ (Tc1) effector lymphocytes in HLA-A2 transgenic mice (HHD2). These data imply that exosomes might be a transfer mechanism of functional MHC class I/peptide complexes to DC for efficient CTL activation in vivo.
Exosomes as Potent Cell-Free Peptide-Based Vaccine. II. Exosomes in CpG Adjuvants Efficiently Prime Naive Tc1 Lymphocytes Leading to Tumor Rejection
Authors: Chaput Nathalie , Schartz Nöel E. C. , André Fabrice , Taïeb Julien , Novault Sophie , Bonnaventure Pierre , Aubert Nathalie , Bernard Jacky , Lemonnier François , Merad Miriam , Adema Gosse , Adams Malcolm , Ferrantini Maria , Carpentier Antoine F. , Escudier Bernard , Tursz Thomas , Angevin Eric , Zitvogel Laurence
Published: April 2014

Ideal vaccines should be stable, safe, molecularly defined, and out-of-shelf reagents efficient at triggering effector and memory Ag-specific T cell-based immune responses. Dendritic cell-derived exosomes could be considered as novel peptide-based vaccines because exosomes harbor a discrete set of proteins, bear functional MHC class I and II molecules that can be loaded with synthetic peptides of choice, and are stable reagents that were safely used in pioneering phase I studies. However, we showed in part I that exosomes are efficient to promote primary MHC class I-restricted effector CD8+ T cell responses only when transferred onto mature DC in vivo. In this work, we bring evidence that among the clinically available reagents, Toll-like receptor 3 and 9 ligands are elective adjuvants capable of triggering efficient MHC-restricted CD8+ T cell responses when combined to exosomes. Exosome immunogenicity across species allowed to verify the efficacy of good manufactory procedures-manufactured human exosomes admixed with CpG oligonucleotides in prophylactic and therapeutic settings of melanoma in HLA-A2 transgenic mice. CpG adjuvants appear to be ideal adjuvants for exosome-based cancer vaccines.
Novel Regulatory Mechanisms of CD40-Induced Prostanoid Synthesis by IL-4 and IL-10 in Human Monocytes
Authors: Inoue Yasushi , Otsuka Takeshi , Niiro Hiroaki , Nagano Shuji , Arinobu Yojirou , Ogami Eiichi , Akahoshi Mitsuteru , Miyake Katsuhisa , Ninomiya Ichiro , Shimizu Sakiko , Nakashima Hitoshi , Harada Mine
Published: April 2014

Interleukins IL-4 and IL-10 are considered to be central regulators for the limitation and eventual termination of inflammatory responses in vivo, based on their potent anti-inflammatory effects toward LPS-stimulated monocytes/macrophages and neutrophils. However, their role in T cell-dependent inflammatory responses has not been fully elucidated. In this study, we investigated the effects of both cytokines on the production of PGE2, a key molecule of various inflammatory conditions, in CD40-stimulated human peripheral blood monocytes. CD40 ligation of monocytes induced the synthesis of a significant amount of PGE2 via inducible expression of the cyclooxygenase (COX)-2 gene. Both IL-10 and IL-4 significantly inhibited PGE2 production and COX-2 expression in CD40-stimulated monocytes. Using specific inhibitors for extracellular signal-related kinase (ERK) and p38 mitogen-activated protein kinase (MAPK), we found that both kinase pathways are involved in CD40-induced COX-2 expression. CD40 ligation also resulted in the activation of NF-κB. Additional experiments exhibited that CD40 clearly induced the activation of the upstream kinases MAPK/ERK kinase 1/2, MAPK kinase 3/6, and I-κB in monocytes. IL-10 significantly inhibited CD40-induced activation of the ERK, p38 MAPK, and NF-κB pathways; however, inhibition by IL-4 was limited to the ERK pathway in monocytes. Neither IL-10 nor IL-4 affected the recruitment of TNFR-associated factors 2 and 3 to CD40 in monocytes. Collectively, IL-10 and IL-4 use novel regulatory mechanisms for CD40-induced prostanoid synthesis in monocytes, thus suggesting a potential role for these cytokines in regulating T cell-induced inflammatory responses, including autoimmune diseases.
Reduced Ig Class Switch in Aged Mice Correlates with Decreased E47 and Activation-Induced Cytidine Deaminase
Authors: Frasca Daniela , Van der Put Elaine , Riley Richard L. , Blomberg Bonnie B.
Published: April 2014

The capacity to class switch the IgH chain is critical to the effectiveness of humoral immune responses. We show that in vitro-stimulated splenic B cells from senescent mice are deficient in production of multiple class switch isotypes (IgG1, G2a, G3, and E), class switch recombination (CSR), and induction of the E2A-encoded transcription factor E47. E47 has previously been shown to be required for CSR, at least in part via expression of the activation-induced cytidine deaminase. Our studies show that impaired induction of E47, and subsequently activation-induced cytidine deaminase, contribute to poor CSR and production of secondary isotypes in senescence.