bluuebunny/crossref_metadata_2025 huggingface dataset:
Linker for Activation of T Cells, ζ-Associated Protein-70, and Src Homology 2 Domain-Containing Leukocyte Protein-76 are Required for TCR-Induced Microtubule-Organizing Center Polarization
Authors: Kuhné Michelle R. , Lin Joseph , Yablonski Deborah , Mollenauer Marianne N. , Ehrlich Lauren I. Richie , Huppa Johannes , Davis Mark M. , Weiss Arthur
Published: April 2014

Engagement of the T cell with Ag on an APC results in a series of immediate signaling events emanating from the stimulation of the TCR. These events include the induced phosphorylation of a number of cellular proteins with a subsequent increase in intracellular calcium and the restructuring of the microtubule and actin cytoskeleton within the T cell. This restructuring of the cytoskeleton culminates in the polarization of the T cell’s secretory apparatus toward the engaging APC, allowing the T cell to direct secretion of cytokines toward the appropriate APC. This polarization can be monitored by analyzing the position of the microtubule-organizing center (MTOC), as it moves toward the interface of the T cell and APC. The requirements for MTOC polarization were examined at a single-cell level by studying the interaction of a Jurkat cell line expressing a fluorescently labeled MTOC with Staphylococcal enterotoxin superantigen-bound Raji B cell line, which served as the APC. We found that repolarization of the MTOC substantially followed fluxes in calcium. We also used immobilized anti-TCR mAb and Jurkat signaling mutants, defective in TCR-induced calcium increases, to determine whether signaling components that are necessary for a calcium response also play a role in MTOC polarization. We found that ζ-associated protein-70 as well as its substrate adaptor proteins linker for activation of T cells and Src homology 2 domain-containing leukocyte protein-76 are required for MTOC polarization. Moreover, our studies revealed that a calcium-dependent event not requiring calcineurin or calcium/calmodulin-dependent kinase is required for TCR-induced polarization of the MTOC.
Stalk Region of β-Chain Enhances the Coreceptor Function of CD8
Authors: Wong Jenny S. , Wang Xiaosong , Witte Torsten , Nie Linghu , Carvou Nicolas , Kern Petra , Chang Hsiu-Ching
Published: April 2014

CD8 glycoproteins are expressed as either αα homodimers or αβ heterodimers on the surface of T cells. CD8αβ is a more efficient coreceptor than the CD8αα for peptide Ag recognition by TCR. Each CD8 subunit is composed of four structural domains, namely, Ig-like domain, stalk region, transmembrane region, and cytoplasmic domain. In an attempt to understand why CD8αβ is a better coreceptor than CD8αα, we engineered, expressed, and functionally tested a chimeric CD8α protein whose stalk region is replaced with that of CD8β. We found that the β stalk region enhances the coreceptor function of chimeric CD8αα to a level similar to that of CD8αβ. Surprisingly, the β stalk region also restored functional activity to an inactive CD8α variant, carrying an Ala mutation at Arg8 (R8A), to a level similar to that of wild-type CD8αβ. Using the R8A variant of CD8α, a panel of anti-CD8α Abs, and three MHC class I (MHCI) variants differing in key residues known to be involved in CD8α interaction, we show that the introduction of the CD8β stalk leads to a different topology of the CD8α-MHCI complex without altering the overall structure of the Ig-like domain of CD8α or causing the MHCI to employ different residues to interact with the CD8α Ig domain. Our results show that the stalk region of CD8β is capable of fine-tuning the coreceptor function of CD8 proteins as a coreceptor, possibly due to its distinct protein structure, smaller physical size and the unique glycan adducts associated with this region.
Characterization of Pig-Tailed Macaque Classical MHC Class I Genes: Implications for MHC Evolution and Antigen Presentation in Macaques
Authors: Lafont Bernard A. P. , Buckler-White Alicia , Plishka Ron , Buckler Charles , Martin Malcolm A.
Published: April 2014

MHC-dependent CD8+ T cell responses have been associated with control of viral replication and slower disease progression during lentiviral infections. Pig-tailed macaques (Macaca nemestrina) and rhesus monkeys (Macaca mulatta), two nonhuman primate species commonly used to model HIV infection, can exhibit distinct clinical courses after infection with different primate lentiviruses. As an initial step in assessing the role of MHC class I restricted immune responses to these infections, we have cloned and characterized classical MHC class I genes of pig-tailed macaques and have identified 19 MHC class I alleles (Mane) orthologous to rhesus macaque MHC-A, -B, and -I genes. Both Mane-A and Mane-B loci were found to be duplicated, and no MHC-C locus was detected. Pig-tailed and rhesus macaque MHC-A alleles form two groups, as defined by 14 polymorphisms affecting mainly their B peptide-binding pockets. Furthermore, an analysis of multiple pig-tailed monkeys revealed the existence of three MHC-A haplotypes. The distribution of these haplotypes in various Old World monkeys provides new insights about MHC-A evolution in nonhuman primates. An examination of B and F peptide-binding pockets in rhesus and pig-tailed macaques suggests that their MHC-B molecules present few common peptides to their respective CTLs.
Maintenance of Long Term γ-Herpesvirus B Cell Latency Is Dependent on CD40-Mediated Development of Memory B Cells
Authors: Kim In-Jeong , Flaño Emilio , Woodland David L. , Lund Frances E. , Randall Troy D. , Blackman Marcia A.
Published: April 2014

It has been proposed that the γ-herpesviruses maintain lifelong latency in B cells by gaining entry into the memory B cell pool and taking advantage of host mechanisms for maintaining these cells. We directly tested this hypothesis by kinetically monitoring viral latency in CD40+ and CD40− B cells from CD40+CD40− mixed bone marrow chimera mice after infection with a murine γ-herpesvirus, MHV-68. CD40+ B cells selectively entered germinal centers and differentiated into memory B cells. Importantly, latency was progressively lost in the CD40− B cells and preferentially maintained in the long-lived, isotype-switched CD40+ B cells. These data directly demonstrate viral exploitation of the normal B cell differentiation pathway to maintain latency.
Susceptibility to Experimental Lyme Arthritis Correlates with KC and Monocyte Chemoattractant Protein-1 Production in Joints and Requires Neutrophil Recruitment Via CXCR2
Authors: Brown Charles R. , Blaho Victoria A. , Loiacono Christie M.
Published: April 2014

The development of experimental Lyme arthritis has been correlated with the expression of a number of chemokines and cytokines, however, none of these have been measured directly from the arthritic joint. We examined the temporal expression of IL-1β, IL-4, IL-6, IL-10, IL-12p70, GM-CSF, IFN-γ, TNF-α, macrophage inflammatory protein-2, KC, macrophage inflammatory protein-1α, and monocyte chemoattractant protein-1 directly from the tibiotarsal joint in arthritis-resistant C57BL/6 (B6) and -susceptible C3H/He (C3H) mice. Only the chemokines KC and monocyte chemoattractant protein-1 were differentially expressed in joints of B6 and C3H mice and correlated with the development of Lyme arthritis. Infection of CXCR2−/− mice on either genetic background resulted in a significant decrease in the development of pathology, although infection of CCR2−/− mice had little or no effect. Neutrophils in CXCR2−/− mice were marginalized within blood vessels and could not enter the joint tissue. These results suggest that chemokine-mediated recruitment of neutrophils into the infected joint is a key requirement for the development of experimental Lyme arthritis.
Effects of Human Cytomegalovirus Infection on Ligands for the Activating NKG2D Receptor of NK Cells: Up-Regulation of UL16-Binding Protein (ULBP)1 and ULBP2 Is Counteracted by the Viral UL16 Protein
Authors: Rölle Alexander , Mousavi-Jazi Mehrdad , Eriksson Mikael , Odeberg Jenny , Söderberg-Nauclér Cecilia , Cosman David , Kärre Klas , Cerboni Cristina
Published: April 2014

Human CMV (HCMV) interferes with NK cell functions at various levels. The HCMV glycoprotein UL16 binds some of the ligands recognized by the NK-activating receptor NKG2D, namely UL16-binding proteins (ULBP) 1 and 2 and MHC class I-related chain B, possibly representing another mechanism of viral immune escape. This study addressed the expression and function of these proteins in infected cells. HCMV induced the expression of all three ULBPs, which were predominantly localized in the endoplasmic reticulum of infected fibroblasts together with UL16. However, while at a lower viral dose ULBP1 and 2 surface expression was completely inhibited compared to ULBP3, at a higher viral dose cell surface expression of ULBP1 and ULBP2 was delayed. The induction of ULBPs correlated with an increased dependency on NKG2D for recognition; however, the overall NK sensitivity did not change (suggesting that additional viral mechanisms interfere with NKG2D-independent pathways for recognition). Infection with a UL16 deletion mutant virus resulted in a different pattern compared to the wild type: all three ULBP molecules were induced with similar kinetics at the cell surface, accompanied by a pronounced, entirely NKG2D-dependent increase in NK sensitivity. Together our findings show that upon infection with HCMV, the host cell responds by expression of ULBPs and increased susceptibility to the NKG2D-mediated component of NK cell recognition, but UL16 limits these effects by interfering with the surface expression of ULBP1 and ULBP2.
Sepsis Induces Apoptosis and Profound Depletion of Splenic Interdigitating and Follicular Dendritic Cells
Authors: Tinsley Kevin W. , Grayson Mitchell H. , Swanson Paul E. , Drewry Anne M. , Chang Katherine C. , Karl Irene E. , Hotchkiss Richard S.
Published: April 2014

Dendritic cells are a phenotypically diverse group of APC that have unique capabilities to regulate the activity and survival of B and T cells. Although proper function of dendritic cells is essential to host control of invading pathogens, few studies have examined the impact of sepsis on dendritic cells. The purpose of this study was to determine the effect of sepsis on splenic interdigitating dendritic cells (IDCs) and follicular dendritic cells (FDCs) using a clinically relevant animal model. Immunohistochemical staining for FDCs showed that sepsis induced an initial marked expansion in FDCs that peaked at 36 h after onset. The FDCs expanded to fill the entire lymphoid zone otherwise occupied by B cells. Between 36 and 48 h after sepsis, there was a profound caspase 3 mediated apoptosis induced depletion of FDCs such that only a small contingent of cells remained. In contrast to the initial increase in FDCs, IDC numbers were decreased to ∼50% of control by 12 h after onset of sepsis. IDC death occurred by caspase 3-mediated apoptosis. Such profound apoptosis induced loss of FDCs and IDCs may significantly compromise B and T cell function and impair the ability of the host to survive sepsis.
Enhanced Recognition of Human NK Receptors After Influenza Virus Infection
Authors: Achdout Hagit , Arnon Tal I. , Markel Gal , Gonen-Gross Tsufit , Katz Gil , Lieberman Niva , Gazit Roi , Joseph Aviva , Kedar Eli , Mandelboim Ofer
Published: April 2014

The NK cell cytotoxic activity is regulated by both inhibitory and activating NK receptors. Thus, changes in the expression levels and in the affinity or avidity of those receptors will have a major effect on the killing of target cells. In this study, we demonstrate that the binding of NK-inhibitory receptors is enhanced after influenza virus infection. Surprisingly, however, no change in the level of class I MHC protein expression was observed on the surface of the infected cells. The increased binding was general, because it was observed in both the killer cell Ig-like receptor 2 domain long tail 1 and leukocyte Ig-like receptor-1. The increased binding was functional, was not dependent on the interaction with viral hemagglutinin-neuraminidase, was not dependent on the glycosylation site, and was not abolished after mutating the transmembrane or cytosolic portions of the class I MHC proteins. Confocal microscopy experiments showed increased binding of NK receptor-coated beads to infected cells expressing the appropriate class I MHC proteins. In addition, specific cell-free bead aggregates covered with class I MHC proteins were observed only in infected cells. We therefore suggest that the influenza virus use a novel mechanism for the inhibition of NK cell activity. This mechanism probably involves the generation of class I MHC complexes in infected cells that cause increased recognition of NK receptors.
Identification of Uteroglobin-Related Protein 1 and Macrophage Scavenger Receptor with Collagenous Structure as a Lung-Specific Ligand-Receptor Pair
Authors: Bin Liang-Hua , Nielson Larry D. , Liu Xinqi , Mason Robert J. , Shu Hong-Bing
Published: April 2014

High in normal (HIN)-1 is a secreted protein highly expressed in normal breast epithelium and down-regulated in breast carcinomas. By searching GenBank expressed sequence tag databases, we identified HIN-2, a protein homologous to HIN-1. HIN-2 is identical with a recently identified protein called uteroglobin-related protein 1 (UGRP1). Northern blot analysis demonstrated that UGRP1 is specifically expressed by lung, but not by the other tissues examined. By in situ hybridization experiments, UGRP1 was shown to be expressed by lung Clara-like cells in the bronchial epithelium and to be up-regulated in cystic fibrosis. In a mammalian expression system, secreted recombinant UGRP1 was copurified with apolipoprotein A-I. Using a retroviral vector-mediated expression cloning approach, we identified macrophage scavenger receptor with collagenous structure (MARCO) as a receptor for UGRP1. Northern blot and in situ hybridization experiments indicated that MARCO is expressed by alveolar macrophages in the lung. UGRP1 also bound to bacteria and yeast. LPS, a previously identified MARCO ligand, competed with UGRP1 for binding to MARCO and bacteria. Our findings suggest that UGRP1-MARCO is a ligand-receptor pair that is probably involved in inflammation and pathogen clearance in the lung.
Inactivation of Human β-Defensins 2 and 3 by Elastolytic Cathepsins
Authors: Taggart Clifford C. , Greene Catherine M. , Smith Stephen G. , Levine Rodney L. , McCray Paul B. , O’Neill Shane , McElvaney Noel G.
Published: April 2014

β-Defensins are antimicrobial peptides that contribute to the innate immune responses of eukaryotes. At least three defensins, human β-defensins 1, 2, and 3 (HBD-1, -2, and -3), are produced by epithelial cells lining the respiratory tract and are active toward Gram-positive (HBD-3) and Gram-negative (HBD-1, -2, and -3) bacteria. It has been postulated that the antimicrobial activity of defensins is compromised by changes in airway surface liquid composition in lungs of patients with cystic fibrosis (CF), therefore contributing to the bacterial colonization of the lung by Pseudomonas and other bacteria in CF. In this report we demonstrate that HBD-2 and HBD-3 are susceptible to degradation and inactivation by the cysteine proteases cathepsins B, L, and S. In addition, we show that all three cathepsins are present and active in CF bronchoalveolar lavage. Incubation of HBD-2 and -3 with CF bronchoalveolar lavage leads to their degradation, which can be completely (HBD-2) or partially (HBD-3) inhibited by a cathepsin inhibitor. These results suggest that β-defensins are susceptible to degradation and inactivation by host proteases, which may be important in the regulation of β-defensin activity. In chronic lung diseases associated with infection, overexpression of cathepsins may lead to increased degradation of HBD-2 and -3, thereby favoring bacterial infection and colonization.
GM-CSF Restores Innate, But Not Adaptive, Immune Responses in Glucocorticoid-Immunosuppressed Human Blood In Vitro
Authors: Xu Jian , Lucas Rudolf , Schuchmann Marcus , Kühnle Simone , Meergans Thomas , Barreiros Ana P. , Lohse Ansgar W. , Otto Gerd , Wendel Albrecht
Published: April 2014

Infection remains the major complication of immunosuppressive therapy in organ transplantation. Therefore, reconstitution of the innate immunity against infections, without activation of the adaptive immune responses, to prevent graft rejection is a clinically desirable status in transplant recipients. We found that GM-CSF restored TNF mRNA and protein expression without inducing IL-2 production and T cell proliferation in glucocorticoid-immunosuppressed blood from either healthy donors or liver transplant patients. Gene array experiments indicated that GM-CSF selectively restored a variety of dexamethasone-suppressed, LPS-inducible genes relevant for innate immunity. A possible explanation for the lack of GM-CSF to restore T cell proliferation is its enhancement of the release of IL-1βR antagonist, rather than of IL-1β itself, since exogenously added IL-1β induced an IL-2-independent Con A-stimulated proliferation of glucocorticoid-immunosuppressed lymphocytes. Finally, to test the in vivo relevance of our findings, we showed that GM-CSF restored the survival of dexamethasone- or cyclosporine A-immunosuppressed mice from an otherwise lethal infection with Salmonella typhimurium. In addition to this increased resistance to infection, GM-CSF did not induce graft rejection of a skin allotransplant in cyclosporine A-immunosuppressed mice. The selective restoration potential of GM-CSF suggests its therapeutic use in improving the resistance against infections upon organ transplantation.
Dependence of IL-4, IL-13, and Nematode-Induced Alterations in Murine Small Intestinal Smooth Muscle Contractility on Stat6 and Enteric Nerves
Authors: Zhao Aiping , McDermott Joseph , Urban Joseph F. , Gause William , Madden Kathleen B. , Yeung Karla Au , Morris Suzanne C. , Finkelman Fred D. , Shea-Donohue Terez
Published: April 2014

IL-4 and IL-13 promote gastrointestinal worm expulsion in part through effects on nonlymphoid cells, such as intestinal smooth muscle cells. The roles of Stat6 in IL-4-, IL-13-, and parasitic nematode-induced effects on small intestinal smooth muscle contractility were investigated in BALB/c wild-type and Stat6-deficient mice treated with a long-lasting formulation of recombinant mouse IL-4 (IL-4C) or IL-13 for 7 days. Separate groups of BALB/c mice were infected with Nippostrongylus brasiliensis or were drug-cured of an initial Heligmosomoides polygyrus infection and later reinfected. Infected mice were studied 9 and 12 days after inoculation, respectively. Segments of jejunum were suspended in an organ bath, and responses to nerve stimulation and to acetylcholine and substance P in the presence and absence of tetradotoxin, a neurotoxin, were determined. Both IL-4 and IL-13 increased smooth muscle responses to nerve stimulation in wild-type mice, but the effects were greater in IL-13-treated mice and were absent in IL-13-treated Stat6-deficient mice. Similarly, hypercontractile responses to nerve stimulation in H. polygyrus- and N. brasiliensis-infected mice were dependent in part on Stat6. IL-13, H. polygyrus, and N. brasiliensis, but not IL-4, also increased contractility to acetylcholine by mechanisms that involved Stat6 and enteric nerves. These studies demonstrate that both IL-4 and IL-13 promote intestinal smooth muscle contractility, but by different mechanisms. Differences in these effects correlate with differences in the relative importance of these cytokines in the expulsion of enteric nematode parasites.
Sublethal Hyperoxia Impairs Pulmonary Innate Immunity
Authors: Baleeiro Carlos E. O. , Wilcoxen Steven E. , Morris Susan B. , Standiford Theodore J. , Paine Robert
Published: April 2014

Supplemental oxygen is often required in the treatment of critically ill patients. The impact of hyperoxia on pulmonary host defense is not well-established. We hypothesized that hyperoxia directly impairs pulmonary host defense, beyond effects on alveolar wall barrier function. C57BL/6 mice were kept in an atmosphere of >95% O2 for 4 days followed by return to room air. This exposure does not lead to mortality in mice subsequently returned to room air. Mice kept in room air served as controls. Mice were intratracheally inoculated with Klebsiella pneumoniae and followed for survival. Alveolar macrophages (AM) were harvested by bronchoalveolar lavage after 4 days of in vivo hyperoxia for ex vivo experiments. Mortality from pneumonia increased significantly in mice exposed to hyperoxia compared with infected mice in room air. Burden of organisms in the lung and dissemination of infection were increased in the hyperoxia group whereas accumulation of inflammatory cells in the lung was impaired. Hyperoxia alone had no impact on AM numbers, viability, or ability to phagocytize latex microbeads. However, following in vivo hyperoxia, AM phagocytosis and killing of Gram-negative bacteria and production of TNF-α and IL-6 in response to LPS were significantly reduced. AM surface expression of Toll-like receptor-4 was significantly decreased following in vivo hyperoxia. Thus sublethal hyperoxia increases Gram-negative bacterial pneumonia mortality and has a significant adverse effect on AM host defense function. Impaired AM function due to high concentrations of supplemental oxygen may contribute to the high rate of ventilator-associated pneumonia seen in critically ill patients.
Early Self-Regulatory Mechanisms Control the Magnitude of CD8+ T Cell Responses Against Liver Stages of Murine Malaria
Authors: Hafalla Julius C. R. , Morrot Alexandre , Sano Gen-ichiro , Milon Geneviève , Lafaille Juan J. , Zavala Fidel
Published: April 2014

Following immunization with Plasmodium yoelii sporozoites, the CD8+ T cell population specific for the SYVPSAEQI epitope expressed in sporozoite and liver stages of this malaria parasite revealed the existence of a short term Ag presentation process that translated into a single clonal burst. Further expansion of this CD8+ T cell population in conditions of sustained Ag exposure and additional supply of naive cells was inhibited by regulatory mechanisms that were developed as early as 24–48 h after priming. Studies using mouse models for Plasmodium or influenza virus infections revealed that this mechanism is Ag specific and is mediated by activated CD8+ T cells that inhibit the priming of naive cells. This interference of the priming of naive cells appeared to result from limited access to Ag-presenting dendritic cells, which become disabled or are eliminated after contact with activated cells. Thus, concomitantly with the development of their effector antimicrobial capacity, CD8+ T cells also acquire a self-regulatory role that is likely to represent one of the earliest mechanisms induced in the course of an immune response and that limits the magnitude of the early expansion of CD8+ T lymphocytes reactive to microorganisms.
Colitogenic Th1 Cells Are Present in the Antigen-Experienced T Cell Pool in Normal Mice: Control by CD4+ Regulatory T Cells and IL-10
Authors: Asseman Chrystelle , Read Simon , Powrie Fiona
Published: April 2014

CD4+ regulatory T cells have been shown to prevent intestinal inflammation; however, it is not known whether they act to prevent the priming of colitogenic T cells or actively control these cells as part of the memory T cell pool. In this study, we describe the presence of colitogenic Th1 cells within the CD4+CD45RBlow population. These pathogenic cells enrich within the CD25− subset and are not recent thymic emigrants. CD4+CD45RBlow cells from germfree mice were significantly reduced in their ability to transfer colitis to immune deficient recipients, suggesting the presence of commensal bacteria in the donor mice drives colitogenic T cells into the Ag-experienced/memory T cell pool. This potentially pathogenic population of Ag-experienced T cells is subject to T cell-mediated regulation in vivo by both CD4+CD25+ and CD4+CD25− cells in an IL-10-dependent manner. Furthermore, administration of an anti-IL-10R mAb to unmanipulated adult mice was sufficient to induce the development of colitis. Taken together, these data indicate that colitogenic Th1 cells enter into the Ag-experienced pool in normal mice, but that their function is controlled by regulatory T cells and IL-10. Interestingly, IL-10 was not absolutely required for CD4+CD25+ T cell-mediated inhibition of colitis induced by transfer of naive CD4+CD45RBhigh cells, suggesting a differential requirement for IL-10 in the regulation of naive and Ag-experienced T cells.
Inhibition of IFN-γ-Mediated Inducible Nitric Oxide Synthase Induction by the Peroxisome Proliferator-Activated Receptor γ Agonist, 15-Deoxy-Δ12,14-Prostaglandin J2, Involves Inhibition of the Upstream Janus Kinase/STAT1 Signaling Pathway
Authors: Chen Ching-Wen , Chang Ying-Hsin , Tsi Chin-Ju , Lin Wan-Wan
Published: April 2014

Peroxisome proliferator-activated receptor γ (PPARγ) ligands have been reported to exert anti-inflammatory activities in macrophages by competition for transcriptional coactivators with some transcriptional factors, including NF-κB. In the present study the influence of PPARγ activators on IFN-γ-elicited macrophage stimulation and signaling cascades was investigated. The results show that IFN-γ-induced inducible NO synthase (iNOS) gene transcription, iNOS protein induction, and NO production are more sensitive to inhibition by 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2) than by the other two PPARγ agonists, GW1929 and ciglitazone. Delayed addition of 15dPGJ2 for 2 h resulted in reduced inhibition, suggesting action by 15dPGJ2 on the upstream signaling cascades. Immunoblotting, DNA binding, and reporter gene assays consistently revealed the inhibitory ability of 15dPGJ2, but not GW1929 or ciglitazone, on IFN-γ-elicited signaling cascades, including tyrosine phosphorylation of Janus tyrosine protein kinase 2 and STAT1, DNA binding, and IFN regulatory factor-1 trans-activation of STAT1. These effects of 15dPGJ2 were not abrogated by the PPARγ antagonist, bisphenol A diglycidyl ether, indicating the PPARγ-independent actions. 15dPGJ2 also attenuated IL-6-induced tyrosine phosphorylation of STAT1 and STAT3 in Hep3B hepatoma cells. Consistent with the inhibitory effect of reactive oxygen species on STAT1 signaling, STAT1 inhibition by 15dPGJ2 was abrogated by N-acetylcysteine, glutathione, superoxide dismutase, and catalase. Furthermore, 15dPGJ2-induced inhibition of STAT1 phosphorylation and NO production still occurred in the presence of peroxovanadate, ruling out the action mechanism of 15dPGJ2 on tyrosine phosphatase. Taken together, for the first time in this study we demonstrate that 15dPGJ2 can inhibit cytokine-stimulated Janus kinase 2-STAT signaling through a PPARγ-independent, reactive oxygen species-dependent mechanism. These data provide a novel molecular mechanism of iNOS inhibition by 15dPGJ2 and confirm its physiological role in anti-inflammation.
Amplification Mechanisms of Inflammation: Paracrine Stimulation of Arachidonic Acid Mobilization by Secreted Phospholipase A2 Is Regulated by Cytosolic Phospholipase A2-Derived Hydroperoxyeicosatetraenoic Acid
Authors: Balboa María A. , Pérez Rebeca , Balsinde Jesús
Published: April 2014

In macrophages and other major immunoinflammatory cells, two phospholipase A2 (PLA2) enzymes act in concert to mobilize arachidonic acid (AA) for immediate PG synthesis, namely group IV cytosolic phospholipase A2 (cPLA2) and a secreted phospholipase A2 (sPLA2). In this study, the molecular mechanism underlying cross-talk between the two PLA2s during paracrine signaling has been investigated. U937 macrophage-like cells respond to Con A by releasing AA in a cPLA2-dependent manner, and addition of exogenous group V sPLA2 to the activated cells increases the release. This sPLA2 effect is abolished if the cells are pretreated with cPLA2 inhibitors, but is restored by adding exogenous free AA. Inhibitors of cyclooxygenase and 5-lipoxygenase have no effect on the response to sPLA2. In contrast, ebselen strongly blocks it. Reconstitution experiments conducted in pyrrophenone-treated cells to abolish cPLA2 activity reveal that 12- and 15-hydroperoxyeicosatetraenoic acid (HPETE) are able to restore the sPLA2 response to levels found in cells displaying normal cPLA2 activity. Moreover, 12- and 15-HPETE are able to enhance sPLA2 activity in vitro, using a natural membrane assay. Neither of these effects is mimicked by 12- or 15-hydroxyeicosatetraenoic acid, indicating that the hydroperoxy group of HPETE is responsible for its biological activity. Collectively, these results establish a role for 12/15-HPETE as an endogenous activator of sPLA2-mediated phospholipolysis during paracrine stimulation of macrophages and identify the mechanism that connects sPLA2 with cPLA2 for a full AA mobilization response.
Inhibitory Effects of Surfactant Protein A on Surfactant Phospholipid Hydrolysis by Secreted Phospholipases A2
Authors: Chabot Sophie , Koumanov Kamen , Lambeau Gérard , Gelb Michael H. , Balloy Viviane , Chignard Michel , Whitsett Jeffrey A. , Touqui Lhousseine
Published: April 2014

Hydrolysis of surfactant phospholipids by secreted phospholipases A2 (sPLA2) contributes to surfactant dysfunction in acute respiratory distress syndrome. The present study demonstrates that sPLA2-IIA, sPLA2-V, and sPLA2-X efficiently hydrolyze surfactant phospholipids in vitro. In contrast, sPLA2-IIC, -IID, -IIE, and -IIF have no effect. Since purified surfactant protein A (SP-A) has been shown to inhibit sPLA2-IIA activity, we investigated the in vitro effect of SP-A on the other active sPLA2 and the consequences of sPLA2-IIA inhibition by SP-A on surfactant phospholipid hydrolysis. SP-A inhibits sPLA2-X activity, but fails to interfere with that of sPLA2-V. Moreover, in vitro inhibition of sPLA2-IIA-induces surfactant phospholipid hydrolysis correlates with the concentration of SP-A in surfactant. Intratracheal administration of sPLA2-IIA to mice causes hydrolysis of surfactant phosphatidylglycerol. Interestingly, such hydrolysis is significantly higher for SP-A gene-targeted mice, showing the in vivo inhibitory effect of SP-A on sPLA2-IIA activity. Administration of sPLA2-IIA also induces respiratory distress, which is more pronounced in SP-A gene-targeted mice than in wild-type mice. We conclude that SP-A inhibits sPLA2 activity, which may play a protective role by maintaining surfactant integrity during lung injury.
Cutting Edge: Invariant Vα14 NKT Cells Are Required for Allergen-Induced Airway Inflammation and Hyperreactivity in an Experimental Asthma Model
Authors: Lisbonne Mariette , Diem Séverine , de Castro Keller Alexandre , Lefort Jean , Araujo Luiza M. , Hachem Patricia , Fourneau Jean-Marie , Sidobre Stéphane , Kronenberg Mitchell , Taniguchi Masuru , Van Endert Peter , Dy Michel , Askenase Philip , Russo Momtchilo , Vargaftig B. Boris , Herbelin André , Leite-de-Moraes Maria C.
Published: April 2014

Airway hyperreactivity (AHR), eosinophilic inflammation with a Th2-type cytokine profile, and specific Th2-mediated IgE production characterize allergic asthma. In this paper, we show that OVA-immunized Jα18−/− mice, which are exclusively deficient in the invariant Vα14+ (iVα14), CD1d-restricted NKT cells, exhibit impaired AHR and airway eosinophilia, decreased IL-4 and IL-5 production in bronchoalveolar lavage fluid, and reduced OVA-specific IgE compared with wild-type (WT) littermates. Adoptive transfer of WT iVα14 NKT cells fully reconstitutes the capacity of Jα18−/− mice to develop allergic asthma. Also, specific tetramer staining shows that OVA-immunized WT mice have activated (CD69+) iVα14 NKT cells. Importantly, anti-CD1d mAb treatment blocked the ability of iVα14 T cells to amplify eosinophil recruitment to airways, and both Th2 cytokine and IgE production following OVA challenge. In conclusion, these findings clearly demonstrate that iVα14 NKT cells are required to participate in allergen-induced Th2 airway inflammation through a CD1d-dependent mechanism.
Cutting Edge: The B Cell Chemokine CXC Chemokine Ligand 13/B Lymphocyte Chemoattractant Is Expressed in the High Endothelial Venules of Lymph Nodes and Peyer’s Patches and Affects B Cell Trafficking Across High Endothelial Venules
Authors: Ebisuno Yukihiko , Tanaka Toshiyuki , Kanemitsu Naotoshi , Kanda Hidenobu , Yamaguchi Kazuhito , Kaisho Tsuneyasu , Akira Shizuo , Miyasaka Masayuki
Published: April 2014

While CCR7 ligands direct T cell trafficking into lymph nodes (LNs) and Peyer’s patches (PPs), chemokines that regulate B cell trafficking across high endothelial venules (HEVs) remain to be fully elucidated. Here we report that CXC chemokine ligand (CXCL)13 (B lymphocyte chemoattractant) is detected immunohistologically in the majority of HEVs in LNs and PPs of nonimmunized mice. Systemically administered anti-CXCL13 Ab bound to the surface of ∼50% of HEVs in LNs and PPs, but not to other types of blood vessels, indicating that CXCL13 is expressed in the HEV lumen. In CXCL13-null mice, B cells rarely adhered to PP HEVs, whereas T cells did efficiently. Superfusion of CXCL13-null PPs with CXCL13 restored the luminal presentation of CXCL13 and also B cell arrest in PP HEVs at least partially. Collectively, these results indicate that CXCL13 expressed in the HEV lumen plays a crucial role in B cell trafficking into secondary lymphoid tissues such as PPs.
Cutting Edge: SR-PSOX/CXC Chemokine Ligand 16 Mediates Bacterial Phagocytosis by APCs Through its Chemokine Domain
Authors: Shimaoka Takeshi , Nakayama Takashi , Kume Noriaki , Takahashi Shu , Yamaguchi Junko , Minami Manabu , Hayashida Kazutaka , Kita Toru , Ohsumi Jun , Yoshie Osamu , Yonehara Shin
Published: April 2014

SR-PSOX and CXC chemokine ligand (CXCL)16, which were originally identified as a scavenger receptor and a transmembrane-type chemokine, respectively, are indicated to be identical. In this study, we demonstrate that membrane-bound SR-PSOX/CXCL16 mediates adhesion and phagocytosis of both Gram-negative and Gram-positive bacteria. Importantly, our prepared anti-SR-PSOX mAb, which suppressed chemotactic activity of SR-PSOX, significantly inhibited bacterial phagocytosis by human APCs including dendritic cells. Various scavenger receptor ligands inhibited the bacterial phagocytosis of SR-PSOX. In addition, the recognition specificity for bacteria was determined by only the chemokine domain of SR-PSOX/CXCL16. Thus, SR-PSOX/CXCL16 may play an important role in facilitating uptake of various pathogens and chemotaxis of T and NKT cells by APCs through its chemokine domain.
Cutting Edge: Induced Indoleamine 2,3 Dioxygenase Expression in Dendritic Cell Subsets Suppresses T Cell Clonal Expansion
Authors: Mellor Andrew L. , Baban Babak , Chandler Phillip , Marshall Brendan , Jhaver Kanchan , Hansen Anna , Koni Pandelakis A. , Iwashima Makio , Munn David H.
Published: April 2014

In mice, immunoregulatory APCs express the dendritic cell (DC) marker CD11c, and one or more distinctive markers (CD8α, B220, DX5). In this study, we show that expression of the tryptophan-degrading enzyme indoleamine 2,3 dioxygenase (IDO) is selectively induced in specific splenic DC subsets when mice were exposed to the synthetic immunomodulatory reagent CTLA4-Ig. CTLA4-Ig did not induce IDO expression in macrophages or lymphoid cells. Induction of IDO completely blocked clonal expansion of T cells from TCR transgenic mice following adoptive transfer, whereas CTLA4-Ig treatment did not block T cell clonal expansion in IDO-deficient recipients. Thus, IDO expression is an inducible feature of specific subsets of DCs, and provides a potential mechanistic explanation for their T cell regulatory properties.
Germinal Centers Regulate Human Th2 Development
Authors: Johansson-Lindbom Bengt , Ingvarsson Sigurdur , Borrebaeck Carl A. K.
Published: April 2014

In the present study we demonstrate that all CD4+ T cells in human tonsil expressing the Th2-selective receptor chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) also 1) express high levels of CXCR5, and 2) display a transitional CD45RA/RO phenotype and consistently do not produce significant amounts of cytokines when immediately analyzed ex vivo. Hence, they represent precursors of Th2 effector cells, a conclusion confirmed by their robust production of IL-4, IL-5, and IL-13, but not IFN-γ, after in vitro activation. CD4+ T cells, which express only intermediate levels of CXCR5, instead develop into IFN-γ-producing cells under identical culture conditions, thus establishing a correlation between relative levels of CXCR5 expression and the acquired cytokine profile. Because CXCR5 is critically involved in follicular localization, the results suggest that these CRTH2+ Th2 cells preferentially develop their cytokine-producing phenotype within germinal centers (GCs), whereas extrafollicular differentiation instead promotes Th1 development. In support for this proposal, we show that T cells with an intermediate expression of CXCR5 can be forced to also produce IL-4 and IL-13 if cultured with allogenic GC B cells. Finally, we demonstrate that the previously described CD57+ GC T cells also express high levels of CXCR5 but instead of comprising a Th2 precursor, they represent anergized T cells. Taken together, these data suggest that GCs and B cells regulate CD4+ T cell differentiation in a finely tuned fashion, either by promoting differentiation of Th2 cells, which apparently leave the lymphoid tissue before evolving a cytokine-producing phenotype, or by furnishing T cell unresponsiveness.
Lymphocyte Accumulation in the Spleen of Retinoic Acid Receptor-Related Orphan Receptor γ-Deficient Mice
Authors: Zhang Nu , Guo Jian , He You-Wen
Published: April 2014

The hormone nuclear receptor retinoic acid receptor-related orphan receptor γ (RORγ) plays important roles in thymocyte development and lymphoid organogenesis. RORγ and its thymus-specific isoform RORγt are expressed in the thymus, but not in the spleen and bone marrow (BM). However, RORγ−/− mice have 2- to 3-fold more splenocytes than wild-type controls due to an accumulation of conventional resting B lymphocytes. The increase in B lymphocytes in RORγ−/− mice is caused neither by abnormal B cell development in the BM nor by an obvious defect in the peripheral T cell compartment. Furthermore, analyses of BM chimeras using either RORγ−/− or recombinase-activating gene-2−/− mice as recipients and wild-type or RORγ−/− mice as donors, respectively, demonstrate that the splenic microenvironment of RORγ−/− mice is defective, since wild-type T and B lymphocytes accumulated in these chimeric mice. In addition, T lymphocyte homeostasis was altered due to a lowered thymic output in RORγ−/− mice. Collectively, these results suggest that RORγ regulates lymphocyte homeostasis at multiple levels.
Mechanisms of Ganglioside Inhibition of APC Function
Authors: Caldwell Sheila , Heitger Andreas , Shen Weiping , Liu Yihui , Taylor Barbara , Ladisch Stephan
Published: April 2014

Gangliosides shed by tumor cells exert potent inhibitory effects on cellular immune responses. Here we have studied ganglioside inhibition of APC function. When human monocytes were preincubated in 50 μM highly purified ganglioside GD1a, pulsed with tetanus toxoid (TT), and washed, the expected Ag-induced proliferative response of autologous normal T cells added to these monocytes was inhibited by 81%. Strikingly, there was also almost complete (92%) and selective inhibition of the up-regulation of the monocyte costimulatory molecule CD80, while I-CAM-1, LFA-3, HLA-DR, and CD86 expression were unaffected. Purified LPS-stimulated monocytes that had been preincubated in GD1a likewise showed inhibition of CD80 up-regulation (59%) as well as down-regulation of CD40 (54%) and impaired release of IL-12 and TNF-α (reduced by 59 and 51%). GD1a-preincubated human dendritic cells (DC) were also affected. They had reduced constitutive expression of CD40 (33%) and CD80 (61%), but not CD86, and marked inhibition of release of IL-6 (72%), IL-12 (70%), and TNF-α (46%). Even when pulsed with TT, these ganglioside-preincubated DC remained deficient in costimulatory molecule expression and cytokine secretion and were unable to induce a normal T cell proliferative response to TT. Finally, significant inhibition of nuclear localization of NF-κB proteins in activated DC suggests that disruption of NF-κB activation may be one mechanism contributing to ganglioside interference with APC expression of costimulatory molecules and cytokine secretion, which, in turn, may diminish antitumor immune responses.
Plasma Cell Survival Is Mediated by Synergistic Effects of Cytokines and Adhesion-Dependent Signals
Authors: Cassese Giuliana , Arce Sergio , Hauser Anja E. , Lehnert Katja , Moewes Beate , Mostarac Miro , Muehlinghaus Gwendolin , Szyska Martin , Radbruch Andreas , Manz Rudolf A.
Published: April 2014

Recent results suggest that plasma cell longevity is not an intrinsic capacity, but depends on yet unknown factors produced in their environment. In this study, we show that the cytokines IL-5, IL-6, TNF-α, and stromal cell-derived factor-1α as well as signaling via CD44 support the survival of isolated bone marrow plasma cells. The cytokines IL-7 and stem cell factor, crucially important for early B cell development, do not mediate plasma cell survival, indicating that plasma cells and early B cells have different survival requirements. As shown in IL-6-deficient mice, IL-6 is required for a normal induction, but not for the maintenance of plasma cell responses in vivo, indicating that the effects of individual survival factors are redundant. Optimal survival of isolated plasma cells requires stimulation by a combination of factors acting synergistically. These results strongly support the concept that plasma cell survival depends on niches in which a combination of specific signals, including IL-5, IL-6, stromal cell-derived factor-1α, TNF-α, and ligands for CD44, provides an environment required to mediate plasma cell longevity.
Differential Localization and Function of ADP-Ribosylation Factor-6 in Anergic Human T Cells: A Potential Marker for Their Identification
Authors: Tzachanis Dimitrios , Appleman Leonard J. , van Puijenbroek Andre A. F. L. , Berezovskaya Alla , Nadler Lee M. , Boussiotis Vassiliki A.
Published: April 2014

Anergy is a state of immunologic tolerance in which T cells are viable but incapable of responding to antigenic stimulation. Recent data indicate that anergic cells have a distinct gene expression program that determines their unique function. In this study we show that anergic human T cells selectively express the small GTPase ADP-ribosylation factor-6 (ARF6), which is involved in membrane traffic and regulation of the cortical actin cytoskeleton. ARF6 was expressed in the GTP-bound form that localizes at the plasma membrane, resulting in a distinct morphologic appearance of anergic cells. Forced expression of ARF6-GTP in Jurkat T cells prevented TCR-mediated reorganization of cortical actin, extracellular signal-regulated kinase1/2 activation, and IL-2 transcription. Forced expression of ARF6-GTP in primary human T cells inhibited extracellular signal-regulated kinase1/2 activation and proliferative responses. Importantly, T cells with the distribution pattern of ARF6-GTP were detected in peripheral blood, suggesting that anergic T cells may constitutively exist in vivo.
Functional Dissection of Lupus Susceptibility Loci on the New Zealand Black Mouse Chromosome 1: Evidence for Independent Genetic Loci Affecting T and B Cell Activation
Authors: Wither Joan E. , Lajoie Ginette , Heinrichs Svinda , Cai Yong-Chun , Chang Nan , Ciofani Amelia , Cheung Yui-Ho , MacLeod Ralph
Published: April 2014

In previous work, we demonstrated linkage between a broad region on New Zealand Black (NZB) chromosome 1 and increased costimulatory molecule expression on B cells and autoantibody production. In this study, we produced C57BL/6 congenic mice with homozygous NZB chromosome 1 intervals of differing lengths. We show that both B6.NZBc1(35–106) (numbers denote chromosomal interval length) and B6.NZBc1(85–106) mice produce IgG anti-nuclear autoantibodies, but B6.NZBc1(35–106) mice develop significantly higher titers of autoantibodies and more severe renal disease than B6.NZBc1(85–106) mice. Cellular analysis of B6.NZBc1(85–106) mice revealed splenomegaly and increased numbers of memory T cells. In addition to these features, B6.NZBc1(35–106) mice had altered B and T cell activation with increased expression of CD69, and for B cells, costimulatory molecules and MHC. Introduction of an anti-hen egg white lysozyme Ig transgene, as a representative nonself-reactive Ig receptor, onto the B6.NZBc1(35–106) background corrected the B cell activation phenotype and led to dramatic normalization of splenomegaly and T cell activation, but had little impact on the increased proportion of memory T cells. These findings indicate that there are multiple lupus susceptibility genes on NZB chromosome 1, and that although B cell defects play an important role in lupus pathogenesis in these mice, they act in concert with T cell activation defects.
Selective Inhibition of T Cell Activation Via CD147 Through Novel Modulation of Lipid Rafts
Authors: Staffler Günther , Szekeres Andreas , Schütz Gerhard J. , Säemann Marcus D. , Prager Elisabeth , Zeyda Maximilian , Drbal Karel , Zlabinger Gerhard J. , Stulnig Thomas M. , Stockinger Hannes
Published: April 2014

The plasma membrane is compartmentalized into microdomains and the association/dissociation of receptors and signaling molecules with/from these membrane domains is a major principle for regulation of signal transduction. By following the reorganization of microdomains on living cells and performing biochemical studies, we show that Ab targeting of the T cell activation-associated Ag CD147 prevents TCR stimulation-dependent reorganization and clustering of microdomains. Triggering CD147 induces a displacement of the GPI-anchored coreceptors CD48 and CD59 from microdomains in human T lymphocytes. This perturbation of microdomains is accompanied by a selective inhibition of TCR-mediated T cell proliferation. The CD147-inhibited cells secret normal levels of IL-2 but acquire reduced amounts of the IL-2 receptor α-chain CD25. These results indicate that negative regulating signals can modulate microdomains and suggest a general mechanism for inhibition of receptor signaling.
Blastocyst MHC, a Putative Murine Homologue of HLA-G, Protects TAP-Deficient Tumor Cells from Natural Killer Cell-Mediated Rejection In Vivo
Authors: Tajima Atsushi , Tanaka Toshitaka , Ebata Tomohiko , Takeda Kazuyoshi , Kawasaki Akemi , Kelly Janice M. , Darcy Phillip K. , Vance Russell E. , Raulet David H. , Kinoshita Katsuyuki , Okumura Ko , Smyth Mark J. , Yagita Hideo
Published: April 2014

Blastocyst MHC is a recently identified mouse MHC class Ib gene, which is selectively expressed in blastocyst and placenta, and may be the mouse homolog of HLA-G gene the products of which have been implicated in protection of fetal trophoblasts from maternal NK cells and evasion of some tumor cells from NK cell attack. In this study, we identified two blastocyst MHC gene transcripts encoding a full-length α-chain (bc1) and an alternatively spliced form lacking the α2 domain (bc2), which may be homologous to HLA-G1 and HLA-G2, respectively. Both placenta and a teratocarcinoma cell line predominantly expressed the bc2 transcript. When these cDNAs were expressed in TAP-deficient RMA-S or TAP-sufficient RMA cells, only bc1 protein was expressed on the surface of RMA cells, but both bc1 and bc2 proteins were retained in the cytoplasm of RMA-S cells. Significantly, the RMA-S cells expressing either bc1 or bc2 were protected from lysis by NK cells in vitro. This protection was at least partly mediated by up-regulation of Qa-1b expression on the surface of RMA-S cells, which engaged the CD94/NKG2A inhibitory receptor on NK cells. More importantly, the bc1- or bc2-expressing RMA-S cells were significantly protected from NK cell-mediated rejection in vivo. These results suggested a role for blastocyst MHC in protecting TAP-deficient trophoblasts and tumor cells from NK cell attack in vivo.