bluuebunny/crossref_metadata_2025 huggingface dataset:
The Inhibitory Receptor NKG2A Determines Lysis of Vaccinia Virus-Infected Autologous Targets by NK Cells
Authors: Brooks Collin R. , Elliott Tim , Parham Peter , Khakoo Salim I.
Published: April 2014

Signals transduced by inhibitory receptors that recognize self-MHC class I molecules prevent NK cells from being activated by autologous healthy target cells. In order for NK cells to be activated upon contact with an infected cell, the balance between the activating and inhibitory signals that regulate NK cell function must be altered in favor of activation. By studying liver-derived NK cells, we show that only a subpopulation of NK cells expressing high levels of the inhibitory receptor NKG2A are able to lyse autologous vaccinia-infected targets, and that this is due to selective down-regulation of HLA-E. These data demonstrate that release from an inhibitory receptor:ligand interaction is one mechanism that permits NK cell recognition of a virally infected target, and that the variegated expression of inhibitory receptors in humans generates a repertoire of NK cells with different antiviral potentials.
Polyoma Virus-Like Particles Elicit Polarized Cytokine Responses in APCs from Tumor-Susceptible and -Resistant Mice
Authors: Velupillai Palanivel , Garcea Robert L. , Benjamin Thomas L.
Published: April 2014

PERA/Ei (PE) mice are highly susceptible to tumor induction by polyoma virus, whereas C57BR/cdj (BR) mice are highly resistant. PE mice respond to viral infection with a type 2 (IL-10) and BR mice with a type 1 (IL-12) cytokine response, underlining the importance of a sustained T cell response for effective antitumor immunity. PE and BR mice showed comparable Ab responses to the virus, indicating that a Th1 response is fully compatible with strong humoral immunity. Tumor susceptibility is dominant, and a type 2 response prevails in F1 mice derived from these strains. In this study, we show that the different cytokine responses of virus-infected hosts are recapitulated in vitro by exposure of APCs from uninfected PE, BR, and F1 animals to the virus. Importantly, virus-like particles formed from recombinant VP1, the major viral capsid protein, elicited the same host-specific cytokine responses as infectious virus. Assembly of VP1 pentamers into capsid shells is required because unassembled VP1 pentamers were ineffective. Binding of virus-like particles to sialic acid is required because pretreatment of APCs with neuraminidase prevented the response. Expression of TLR2 and TLR4 differed among different subpopulations of APCs and also between resistant and susceptible mice. Evidence is presented indicating that these TLRs play a role in mediating the host-specific cytokine responses to the virus.
Bovine Peptidoglycan Recognition Protein-S: Antimicrobial Activity, Localization, Secretion, and Binding Properties
Authors: Tydell C. Chace , Yuan Jun , Tran Patti , Selsted Michael E.
Published: April 2014

Peptidoglycan (PGN) recognition proteins (PGRPs) are pattern recognition molecules of innate immunity that are conserved from insects to humans. Various PGRPs are reported to have diverse functions: they bind bacterial molecules, digest PGN, and are essential to the Toll pathway in Drosophila. One family member, bovine PGN recognition protein-S (bPGRP-S), has been found to bind and kill microorganisms in a PGN-independent manner, raising questions about the identity of the bPGRP-S ligand. Addressing this, we have determined the binding and microbicidal properties of bPGRP-S in a range of solutions approximating physiologic conditions. In this study we show that bPGRP-S interacts with other bacterial components, including LPS and lipoteichoic acid, with higher affinities than for PCP, as determined by their abilities to inhibit bPGRP-S-mediated killing of bacteria. Where and how PGRPs act in vivo is not yet clear. Using Immunogold electron microscopy, PGRP-S was localized to the dense/large granules of naive neutrophils, which contain the oxygen-independent bactericidal proteins of these cells, and to the neutrophil phagolysosome. In addition, Immunogold staining and secretion studies demonstrate that neutrophils secrete PGRP-S when exposed to bacteria. Bovine PGRP-S can mediate direct lysis of heat-killed bacteria; however, PGRP-S-mediated killing of bacteria is independent of this activity. Evidence that bPGRP-S has multiple activities and affinity to several bacterial molecules challenges the assumption that the PGRP family of proteins recapitulates the evolution of TLRs. Mammalian PGRPs do not have a single antimicrobial activity against a narrow range of target organisms; rather, they are generalists in their affinity and activity.
Extracellular Acidosis Induces Neutrophil Activation by a Mechanism Dependent on Activation of Phosphatidylinositol 3-Kinase/Akt and ERK Pathways
Authors: Martínez Diego , Vermeulen Mónica , Trevani Analía , Ceballos Ana , Sabatté Juan , Gamberale Romina , Álvarez María Eugenia , Salamone Gabriela , Tanos Tamara , Coso Omar A. , Geffner Jorge
Published: April 2014

Inflammation in peripheral tissues is usually associated with the development of local acidosis; however, there are few studies aimed at analyzing the influence of acidosis on immune cells. We have shown previously that extracellular acidosis triggers human neutrophil activation, inducing a transient increase in intracellular Ca2+ concentration, a shape change response, the up-regulation of CD18 expression, and a delay of apoptosis. In this study, we analyzed the signaling pathways responsible for neutrophil activation. We found that acidosis triggers the phosphorylation of Akt (the main downstream target of PI3K) and ERK MAPK, but not that of p38 and JNK MAPK. No degradation of IκB was observed, supporting the hypothesis that NF-κB is not activated under acidosis. Inhibition of PI3K by wortmannin or LY294002 markedly decreased the shape change response and the induction of Ca2+ transients triggered by acidosis, whereas the inhibition of MEK by PD98059 or U0126 significantly inhibited the shape change response without affecting the induction of Ca2+ transients. We also found that acidosis not only induces a shape change response and the induction of Ca2+ transients in human neutrophils but also stimulates the endocytosis of FITC-OVA and FITC-dextran. Stimulation of endocytosis was partially prevented by inhibitors of PI3K and MEK. Together, our results support the notion that the stimulation of human neutrophils by extracellular acidosis is dependent on the activation of PI3K/Akt and ERK pathways. Of note, using mouse peritoneal neutrophils we observed that the enhancement of endocytosis induced by acidosis was associated with an improved ability to present extracellular Ags through a MHC class I-restricted pathway.
Pristane, a Non-Antigenic Adjuvant, Induces MHC Class II-Restricted, Arthritogenic T Cells in the Rat
Authors: Holmberg Jens , Tuncel Jonatan , Yamada Hisakata , Lu Shemin , Olofsson Peter , Holmdahl Rikard
Published: April 2014

Pristane-induced arthritis (PIA) in rats, a model for rheumatoid arthritis (RA), is a T cell-dependent disease. However, pristane itself is a lipid and unable to form a stable complex with a MHC class II molecule. Therefore, the specificity and function of the T cells in PIA are as unclear as in rheumatoid arthritis. In this study, we show that activated CD4+ αβT cells, which target peripheral joints, transfer PIA. The pristane-primed T cells are of oligo or polyclonal origin as determined by their arthritogenicity after stimulation with several mitogenic anti-TCRVβ and anti-TCRVα mAbs. Arthritogenic cells secreted IFN-γ and TNF-α (but not IL-4) when stimulated with Con A in vitro, and pretreatments of recipient rats with either anti-IFN-γ or a recombinant TNF-α receptor before transfer ameliorated arthritis development. Most importantly, we show that these T cells are MHC class II restricted, because treatment with Abs against either DQ or DR molecules ameliorates arthritis development. The MHC class II restriction was confirmed by transferring donor T cells to irradiated recipients that were syngenic, semiallogenic, or allogenic to MHC class II molecules, in which only syngenic and semiallogenic recipients developed arthritis. These data suggest that the in vivo administration of a non-antigenic adjuvant, like pristane, activates CD4+ αβT cells that are MHC class II restricted and arthritogenic.
TGF-β1 Released from Activated Platelets Can Induce TNF-Stimulated Human Brain Endothelium Apoptosis: A New Mechanism for Microvascular Lesion during Cerebral Malaria
Authors: Wassmer Samuel C. , de Souza J. Brian , Frère Corinne , Candal Francisco J. , Juhan-Vague Irène , Grau Georges E.
Published: April 2014

Platelets have recently been shown to accumulate in brain microvessels of patients with cerebral malaria and to modulate the binding of Plasmodium falciparum-infected red cells to human brain endothelium in vitro. In the present study we used a platelet-endothelial cell coculture model to investigate the mechanisms by which platelets modify the function of human brain microvascular endothelial cells (HBEC). Platelets were found to have a proapoptotic effect on TNF-activated HBEC, and this was contact-dependent, as inhibiting platelet binding prevented endothelial cell killing. We also showed that the supernatants of thrombin-activated platelets killed TNF-stimulated HBEC and that TGF-β1 was the main molecule involved in endothelial cell death, because its inhibition completely abrogated the activated-platelet supernatant effect. Our data illustrate another aspect of the duality of TGF-β1 in malaria and may provide new insights into the pathogenesis of cerebral malaria.
Abrupt Expression of TLR4 in TLR4-Deficient Macrophages Imposes a Selective Disadvantage: Genetic Evidence for TLR4-Dependent Responses to Endogenous, Nonmicrobial Stimuli
Authors: Cohn Ellen F. , Nathan Carl , Radzioch Danuta , Yu Hongwei , Xiang Zhaoying , Ding Aihao
Published: April 2014

TLR4 is crucial for macrophage responses to LPS. It is less clear whether TLR4 may also transduce signals from host factors, and if so, with what consequences. Immortalized bone marrow-derived macrophage cell lines, termed T4Cr and T4ko, were established from TLR4null strains, C57BL/10ScNCr and TLR4 knockout mice, respectively. Multiple transfections and selections were conducted to stably introduce TLR4 into these cell lines. Among 196 individual clones isolated, 48 expressed TLR4 on the cell surface but did not respond to LPS due to a deletion in the MyD88 gene. The remaining clones integrated TLR4 DNA into the genome but expressed neither detectable TLR4 mRNA nor TLR4 protein. To test the possibility that TLR4null cells lack modulating factors to protect against a harmful effect of TLR4, 15 stably transfected clones were generated in the presence of conditioned media from wild-type macrophages. Some of these cells expressed a small amount of TLR4 and regained responsiveness to LPS. Because no microbial ligands were available to the cell lines during their generation, signaling via endogenous ligands is likely to have occurred in TLR4-expressing, signal-competent macrophages and imposed a proliferative or other selective disadvantage. These studies support the existence of constitutive signaling via TLR4 during in vitro culture of macrophages without microbial products, and help account for the lack of reports of restoration of TLR4 expression in normally TLR4-expressing types of cells in vitro whose TLR4 genes are deleted or disrupted.
Activation of TLR-9 Induces IL-8 Secretion through Peroxynitrite Signaling in Human Neutrophils
Authors: József Levente , Khreiss Tarek , El Kebir Driss , Filep János G.
Published: April 2014

Bacterial DNA containing unmethylated CpG motifs is emerging as an important regulator of functions of human neutrophil granulocytes (polymorphonuclear leukocytes (PMN)). These motifs are recognized by TLR-9. Recent studies indicate that peroxynitrite (ONOO−) may function as an intracellular signal for the production of IL-8, one of the key regulators of leukocyte trafficking in inflammation. In this study we investigated whether bacterial DNA (CpG-DNA) could induce ONOO− signaling in human PMN. Human whole blood, isolated PMN (purity, >95%), and high purity (>99%) PMN respond to CpG-DNA, but not to calf thymus DNA, with secretion of IL-8 and, to a lesser extent, IL-6 and TNF. Methylation of cytosines in CpG-DNA resulted in a complete loss of activity. The endosomal acidification inhibitors, bafilomycin A and chloroquine, inhibited CpG-DNA-induced cytokine release from PMN. CpG-DNA-induced IL-8 mRNA expression and release was also blocked by the NO synthase inhibitor Nω-nitro-l-arginine methyl ester. CpG-DNA evoked concomitant increases in intracellular superoxide and NO levels, leading to enhanced ONOO− formation and, consequently, nuclear accumulation of c-Fos and NF-κB. Pharmacological inhibition of NF-κB activation attenuated ∼75% of CpG-DNA-evoked IL-8 release. These results identify ONOO−-dependent activation of NF-κB and c-Fos as an important mechanism that mediates PMN responses, including IL-8 gene expression and release, to bacterial DNA and unmethylated CpG motifs in particular. Enhanced ONOO− formation represents a mechanism by which bacterial DNA may contribute to prolongation and amplification of the inflammatory response.
A Potent Adjuvant Monophosphoryl Lipid A Triggers Various Immune Responses, but Not Secretion of IL-1β or Activation of Caspase-1
Authors: Okemoto Kazuo , Kawasaki Kiyoshi , Hanada Kentaro , Miura Masami , Nishijima Masahiro
Published: April 2014

Lipid A, the membrane anchor portion of LPS, is responsible for the endotoxin activity of LPS and induces many inflammatory responses in macrophages. Monophosphoryl lipid A (MPL), a lipid A derivative lacking a phosphate residue, induces potent immune responses with low toxicity. To elucidate the mechanism underlying the low toxicity of MPL, we examined the effects of MPL on the secretion of proinflammatory cytokines by mouse peritoneal macrophages, a murine macrophage-like cell line (RAW 264.7), and a human macrophage-like cell line (THP-1). MPL enhanced the secretion of TNF-α, but not that of IL-1β, whereas Escherichia coli-type lipid A (natural source-derived and chemically synthesized lipid A) enhanced the secretion of both cytokines. Although MPL enhanced the levels of IL-1β mRNA and IL-1β precursor protein to levels similar to those induced by lipid A, IL-1β precursor processing in MPL-treated cells was much lower than that in E. coli-type lipid A-treated ones. Moreover, MPL, unlike E. coli-type lipid A, failed to induce activation of caspase-1, which catalyzes IL-1β precursor processing. These results suggest that an immune response without activation of caspase-1 or secretion of IL-1β results in the low toxicity of this adjuvant.
Oxidants Selectively Reverse TGF-β Suppression of Proinflammatory Mediator Production
Authors: Xiao Yi Qun , Freire-de-Lima Celio G. , Janssen William J. , Morimoto Konosuke , Lyu Dennis , Bratton Donna L. , Henson Peter M.
Published: April 2014

Although TGF-β inhibits the production of proinflammatory mediators in vitro and in vivo, its anti-inflammatory activities may be ineffective in early or severe acute inflammatory circumstances. In this study, we suggest a role for oxidative stress on TGF-β signaling, leading to prevention of its normal anti-inflammatory effects but leaving its Smad-driven effects on cellular differentiation or matrix production unaffected. Stimulation of the RAW 264.7 macrophage cells, human or mouse alveolar macrophages with LPS led to NF-κB-driven production of proinflammatory mediators, which were inhibited by TGF-β. This inhibition was prevented in the presence of hydrogen peroxide. We found that hydrogen peroxide acted by inducing p38 MAPK activation, which then prevented the ERK activation and MAPK phosphatase-1 up-regulation normally induced by TGF-β. This was mediated through Src tyrosine kinases and protein phosphatase-1/2A. By contrast, hydrogen peroxide had no effects on TGF-β-induced Smad2 phosphorylation and SBE-luc reporter gene transcription.
Ezrin/Radixin/Moesin Proteins Are Phosphorylated by TNF-α and Modulate Permeability Increases in Human Pulmonary Microvascular Endothelial Cells
Authors: Koss McKenzie , Pfeiffer Gordon R. , Wang Ying , Thomas Sharon T. , Yerukhimovich Michael , Gaarde William A. , Doerschuk Claire M. , Wang Qin
Published: April 2014

Endothelial cells (ECs) respond to TNF-α by altering their F-actin cytoskeleton and junctional permeability through mechanisms that include protein kinase C (PKC) and p38 MAPK. Ezrin, radixin, and moesin (ERM) regulate many cell processes that often require a conformational change of these proteins as a result of phosphorylation on a conserved threonine residue near the C terminus. This study tested the hypothesis that ERM proteins are phosphorylated on this critical threonine residue through TNF-α-induced activation of PKC and p38 and modulate permeability increases in pulmonary microvascular ECs. TNF-α induced ERM phosphorylation on the threonine residue that required activation of p38, PKC isoforms, and phosphatidylinositol-4-phosphate 5-kinase Iα, a major enzyme generating phosphatidylinositol 4,5-bisphosphate, and phosphorylated ERM were prominently localized at the EC periphery. TNF-α-induced ERM phosphorylation was accompanied by cytoskeletal changes, paracellular gap formation, and increased permeability to fluxes of dextran and albumin. These changes required activation of p38 and PKC and were completely prevented by inhibition of ERM protein expression using small interfering RNA. Thus, ERM proteins are phosphorylated through p38 and PKC-dependent mechanisms and modulate TNF-α-induced increases in endothelial permeability. Phosphorylation of ERM likely plays important roles in EC responses to TNF-α by modulating the F-actin cytoskeleton, adhesion molecules, and signaling events.
Anti-Inflammatory Effect of Lactobacillus casei on Shigella-Infected Human Intestinal Epithelial Cells
Authors: Tien Meng-Tsung , Girardin Stephen E. , Regnault Béatrice , Le Bourhis Lionel , Dillies Marie-Agnès , Coppée Jean-Yves , Bourdet-Sicard Raphaëlle , Sansonetti Philippe J. , Pédron Thierry
Published: April 2014

Shigella invades the human intestinal mucosa, thus causing bacillary dysentery, an acute recto-colitis responsible for lethal complications, mostly in infants and toddlers. Conversely, commensal bacteria live in a mutualistic relationship with the intestinal mucosa that is characterized by homeostatic control of innate responses, thereby contributing to tolerance to the flora. Cross-talk established between commensals and the intestinal epithelium mediate this active process, the mechanisms of which remain largely uncharacterized. Probiotics such as Lactobacillus casei belong to a subclass of these commensals that modulate mucosal innate responses and possibly display anti-inflammatory properties. We analyzed whether L. casei could attenuate the pro-inflammatory signaling induced by Shigella flexneri after invasion of the epithelial lining. Cultured epithelial cells were infected with L. casei, followed by a challenge with S. flexneri. Using macroarray DNA chips, we observed that L. casei down-regulated the transcription of a number of genes encoding pro-inflammatory effectors such as cytokines and chemokines and adherence molecules induced by invasive S. flexneri. This resulted in an anti-inflammatory effect that appeared mediated by the inhibition of the NF-κB pathway, particularly through stabilization of I-κBα. In a time-course experiment using GeneChip hybridization analysis, the expression of many genes involved in ubiquitination and proteasome processes were modulated during L. casei treatment. Thus, L. casei has developed a sophisticated means to maintain intestinal homeostasis through a process that involves manipulation of the ubiquitin/proteasome pathway upstream of I-κBα.
Human Lung Mast Cells Adhere to Human Airway Smooth Muscle, in Part, via Tumor Suppressor in Lung Cancer-1
Authors: Yang Weidong , Kaur Davinder , Okayama Yoshimichi , Ito Akihiko , Wardlaw Andrew J. , Brightling Christopher E. , Bradding Peter
Published: April 2014

Mast cells infiltrate the airway smooth muscle (ASM) of patients with asthma, an event which is likely to be a key factor in the development of this disease. Adhesion is a fundamental mechanism facilitating cellular cross-talk. We have examined whether human lung mast cells (HLMC) and ASM adhere, and have also examined the mechanism involved. Primary cultures of HLMC and confluent human ASM were cocultured for 30 min, then nonadherent HLMC were removed by centrifugation. HLMC adhered avidly to ASM monolayers (mean ± SEM adhesion 43.2 ± 1.2%, n = 41). Adhesion was increased to 58.8 ± 2.7% by 1 mM Mn2+ (p = 0.015), and was reduced by EDTA and EGTA to 20.5 ± 1.5% and 21.0 ± 1.3%, respectively (p < 0.0001). Adhesion-blocking Abs for ICAM-1, VCAM-1, CD18, and the α4 and β1 integrins had no effect on HLMC adhesion. HLMC expressed tumor suppressor in lung cancer-1 (TSLC-1) and blocking this reduced adhesion from 38.5 ± 4.8% to 28.3 ± 3.7% (p = 0.004, n = 7). ASM did not express TSLC-1, indicating that TSLC-1 acts as a heterophilic adhesion molecule. In summary, HLMC adhere avidly to ASM in part via TSLC-1 and in part via an as-yet-undefined Ca2+-dependent pathway. This supports the hypothesis that adhesion is important in the recruitment and retention of HLMC by the ASM in asthma, and for the functional interaction of these cells.
Defective Activation of ERK in Macrophages Lacking the p50/p105 Subunit of NF-κB Is Responsible for Elevated Expression of IL-12 p40 Observed after Challenge with Helicobacter hepaticus
Authors: Tomczak Michal F. , Gadjeva Mihaela , Wang Yan Yan , Brown Ketorah , Maroulakou Ioanna , Tsichlis Philip N. , Erdman Susan E. , Fox James G. , Horwitz Bruce H.
Published: April 2014

Helicobacter hepaticus is an enterohepatic Helicobacter species that induces lower bowel inflammation in susceptible mouse strains, including those lacking the p50/p105 subunit of NF-κB. H. hepaticus-induced colitis is associated with elevated levels of IL-12 p40 expression, and p50/p105-deficient macrophages express higher levels of IL-12 p40 than wild-type macrophages after challenge with H. hepaticus. However, the molecular mechanisms by which the p50/p105 subunit of NF-κB suppresses IL-12 p40 expression have not yet been elucidated. In this study we have demonstrated that H. hepaticus challenge of macrophages induces ERK activation, and this event plays a critical role in inhibiting the ability of H. hepaticus to induce IL-12 p40. Activation of ERK requires both p50/p105 and the MAPK kinase kinase, Tpl-2. Inhibition of the induction of IL-12 p40 by ERK was independent of c-Rel, a known positive regulator of IL-12 p40. Instead, it was linked to the induction of c-Fos, a known inhibitor of IL-12 p40 expression. These results suggest that H. hepaticus induces ERK activation by a pathway dependent upon Tpl-2 and p105, and that activation of ERK inhibits the expression of IL-12 p40 by inducing c-Fos. Thus, a defect in ERK activation could play a pivotal role in the superinduction of IL-12 p40 observed after challenge of macrophages lacking the p50/p105 subunit of NF-κB with H. hepaticus.
T Cell Epitope Mimicry in Antiglomerular Basement Membrane Disease
Authors: Arends Jon , Wu Jean , Borillo Jason , Troung Luan , Zhou Cindy , Vigneswaran Nadarajah , Lou Ya-Huan
Published: April 2014

Antiglomerular basement membrane (GBM) disease or Goodpasture’s syndrome is among the earliest recognized human autoimmune diseases. Although collagen 4α3 NC1 (Col4α3NC1) has been identified as the responsible autoantigen, it remains unknown how autoimmunity to this autoantigen is provoked. We have demonstrated in our rat model that a single nephritogenic T cell epitope pCol28–40 of Col4α3NC1 induces glomerulonephritis. We hypothesized that microbial peptides that mimic this T cell epitope could induce the disease. Based on the critical residue motif (xxtTxNPsxx) of pCol28–40, seven peptides derived from human infection-related microbes were chosen through GenBank search and synthesized. All peptides showed cross-reactivity with pCol28–40-specific T cells at various levels. Only four peptides induced transient proteinuria and minor glomerular injury. However, the other three peptides induced severe proteinuria and modest to severe glomerulonephritis in 16–25% of the immunized rats. Unexpectedly, the most nephritogenic peptide, pCB, derived from Clostridium botulinum, also induced modest (25%) to severe (25%) pulmonary hemorrhage, another important feature of anti-GBM disease; this was not correlated with the severity of glomerulonephritis. This finding suggests that subtle variations in T cell epitope specificity may lead to different clinical manifestations of anti-GBM disease. In summary, our study raises the possibility that a single T cell epitope mimicry by microbial Ag may be sufficient to induce the anti-GBM disease.
Thyrocytes Responding to IFN-γ Are Essential for Development of Lymphocytic Spontaneous Autoimmune Thyroiditis and Inhibition of Thyrocyte Hyperplasia
Authors: Yu Shiguang , Sharp Gordon C. , Braley-Mullen Helen
Published: April 2014

IFN-γ promotes the development of lymphocytic spontaneous autoimmune thyroiditis (L-SAT) in NOD.H-2h4 mice and inhibits the development of thyrocyte hyperplasia and proliferation (TEC H/P). The precise mechanisms by which IFN-γ promotes L-SAT and inhibits TEC H/P are unknown. To determine whether responsiveness of lymphocytes or thyrocytes to IFN-γ is important for the development of these lesions, IFN-γR−/− mice, which develop TEC H/P similar to IFN-γ−/− mice, were used as recipients for adoptive cell transfer. Wild-type (WT) splenocytes or bone marrow induced L-SAT and inhibited TEC H/P in IFN-γ−/−, but not IFN-γR−/− recipients. IFN-γR−/− recipients of WT cells developed severe TEC H/P, but did not develop L-SAT, suggesting that thyrocytes responding to IFN-γ are important for inhibition of TEC H/P. Unexpectedly, IFN-γR−/− splenocytes or bone marrow did not induce L-SAT in IFN-γ−/− or WT mice even though IFN-γR−/− lymphocyte donors produced as much IFN-γ as lymphocytes from WT donors, and thyrocytes could respond to IFN-γ. Real-time PCR indicated that recipients of IFN-γR−/− bone marrow expressed less mRNA for IFN-γ-inducible chemokines compared with recipients of WT bone marrow. This might limit the migration of IFN-γR−/− lymphocytes to thyroids. Few IFN-γR−/− lymphocytes infiltrated thyroids even in the presence of WT lymphocytes, suggesting that lymphocytes unable to respond to IFN-γ are not induced to migrate to thyroids. These results suggest that thyrocytes must be able to respond to IFN-γ for the development of L-SAT and inhibition of TEC H/P, and lymphocytes must be able to respond to IFN-γ to induce L-SAT.
Ex Vivo-Expanded CD4+CD25+ Immunoregulatory T Cells Prevent Graft-versus-Host-Disease by Inhibiting Activation/Differentiation of Pathogenic T Cells
Authors: Trenado Aurélie , Sudres Muriel , Tang Qizhi , Maury Sébastien , Charlotte Frédéric , Grégoire Sylvie , Bonyhadi Mark , Klatzmann David , Salomon Benoît L. , Cohen José L.
Published: April 2014

CD4+CD25+ immunoregulatory T cells (Tregs) can be administered to inhibit graft-vs-host disease (GVHD) while preserving graft-vs-leukemia activity after allogeneic bone marrow transplantation in mice. Preclinical studies suggest that it is necessary to infuse as many Tregs as conventional donor T cells to achieve a clinical effect on GVHD. Thus, it would be necessary to expand Tregs ex vivo before transplantation. Two strategies have been proposed: expansion of Tregs stimulated by anti-CD3/CD28-coated microbeads for polyclonal activation or by host-type allogeneic APCs for selecting Tregs specific for host Ags. In this study, we describe the mechanisms by which ex vivo-expanded Tregs act on donor T cells to prevent GVHD in mice. We demonstrate that expanded Tregs strongly inhibited the division, expansion, and differentiation of donor T cells, with a more pronounced effect with Tregs specific for host Ags. These latter cells permit the efficient and durable control of GVHD and favor immune reconstitution.
HIV-Specific IL-10-Positive CD8+ T Cells Are Increased in Advanced Disease and Are Associated with Decreased HIV-Specific Cytolysis
Authors: Elrefaei Mohamed , Barugahare Banson , Ssali Francis , Mugyenyi Peter , Cao Huyen
Published: April 2014

IL-10-producing T cells have been shown to inhibit Ag-specific CD8+ T cell responses, and may play a role in the immune dysregulation observed in HIV-1 infection. We characterized the Gag-specific IL-10 responses by CD8+ T cells in HIV-1-positive volunteers from Uganda. HIV-specific IL-10 responses were detected in 32 of 61 (52.4%) antiretroviral naive and 2 of 15 (13.3%) volunteers with a complete virologic response on antiretroviral therapy (< 400 copies/ml). The frequency of HIV-specific IL-10-positive cells was significantly higher in volunteers with advanced disease (CD4+ T cell count <200 cells/mm3; p = 0.0004), and correlated positively with plasma HIV RNA (r = 0.43, p = 0.0004). Interestingly, the frequency of Gag-specific CD107a/b-, but not IFN-γ-, positive cells was significantly lower in individuals with detectable IL-10-positive CD8+ T cells (p = 0.004). Gag-specific IL-10-positive CD8+ T cells demonstrated a pattern of surface memory marker expression that is distinct compared with CD107a/b- and IFN-γ-positive CD8+ T cell populations (p < 0.0001). Our study describes a distinct population of IL-10-positive CD8+ T cells that may play a role in HIV-associated immune dysfunction.
A Case for Regulatory B Cells
Authors: Mizoguchi Atsushi , Bhan Atul K.
Published: April 2014

B cells are typically characterized by their ability to produce Abs, including autoantibodies. However, B cells possess additional immune functions, including the production of cytokines and the ability to function as a secondary APC. As with T cells, the B cell population contains functionally distinct subsets capable of performing both pathogenic and regulatory functions. Recent studies indicate that regulatory B cells develop in several murine models of chronic inflammation, including inflammatory bowel disease, rheumatoid arthritis, and experimental autoimmune encephalomyelitis. The regulatory function may be directly accomplished by the production of regulatory cytokines IL-10 and TGF-β and/or by the ability of B cells to interact with pathogenic T cells to dampen harmful immune responses. In this review, we make a case for the existence of regulatory B cells and discuss the possible developmental pathways and functional mechanisms of these B cells.
Cutting Edge: Evidence for Ligand-Independent Multimerization of the IL-17 Receptor
Authors: Kramer Jill M. , Yi Ling , Shen Fang , Maitra Amarnath , Jiao Xuanmao , Jin Tian , Gaffen Sarah L.
Published: April 2014

IL-17 and its receptor are founding members of a novel inflammatory cytokine family. To date, only one IL-17 receptor subunit has been identified, termed IL-17RA. All known cytokine receptors consist of a complex of multiple subunits. Although IL-17-family cytokines exist as homodimers, the configuration and stoichiometry of the IL-17R complex remain unknown. We used fluorescence resonance energy transfer (FRET) to determine whether IL-17RA subunits multimerize, and, if so, whether they are preassembled in the plasma membrane. HEK293 cells coexpressing IL-17RA fused to cyan or yellow fluorescent proteins (CFP or YFP) were used to evaluate FRET before and after IL-17A or IL-17F treatment. In the absence of ligand, IL-17RA molecules exhibited significant specific FRET efficiency, demonstrating that they exist in a multimeric, preformed receptor complex. Strikingly, treatment with IL-17A or IL-17F markedly reduced FRET efficiency, suggesting that IL-17RA subunits within the IL-17R complex undergo a conformational change upon ligand binding.
Cutting Edge: Modulation of Fas-Mediated Apoptosis by Lipid Rafts in T Lymphocytes
Authors: Legembre Patrick , Daburon Sophie , Moreau Patrick , Moreau Jean-François , Taupin Jean-Luc
Published: April 2014

In type I cells, Fas-mediated cell death requires cytoplasmic membrane subdomains called microdomains or lipid rafts. On the contrary, Fas signaling is independent of these structures in type II cells. We report that in human T cells, CD28, CD59, and CD55 are all localized into lipid rafts and that CD28 is concentrated into microdomains enriched in ganglioside GM1, whereas CD59 and CD55 are not. Moreover, CD28 cross-linking leads to the formation of lipid raft clusters which exclude CD59 and CD55, and reciprocally. Coligation of Fas with CD55 or CD59 inhibits the apoptotic signal, whereas CD28 recruitment amplifies the Fas signaling pathway. Therefore, we conclude that 1) different types of microdomains exist on the cell surface, with distinct functional properties and 2) the recruitment of these distinct structures may differentially modulate the Fas pathway. Moreover, our results demonstrate that Fas-induced apoptosis can be controlled at the level of the cytoplasmic membrane.
Cutting Edge: Tissue Inhibitor of Metalloproteinase 3 Regulates TNF-Dependent Systemic Inflammation
Authors: Smookler David S. , Mohammed Fazilat F. , Kassiri Zamaneh , Duncan Gordon S. , Mak Tak W. , Khokha Rama
Published: April 2014

Host response to infectious agents must be rapid and powerful. One mechanism is the release of presynthesized membrane-bound TNF. TNF shedding is mediated by TNF-α converting enzyme, which is selectively inhibited by the tissue inhibitor of metalloproteinase 3 (TIMP3). We show that loss of TIMP3 impacts innate immunity by dysregulating cleavage of TNF and its receptors. Cultured timp3−/− macrophages release more TNF in response to LPS than wild-type macrophages. In timp3−/− mice, LPS causes serum levels of TNF and its receptors to rise more rapidly and remain higher compared with wild-type mice. The altered kinetics of ligand and receptor shedding enhances TNF signaling in timp3−/− mice, indicated by elevated serum IL-6. Physiologically, timp3−/− mice are more susceptible to LPS-induced mortality. Ablation of the TNF receptor gene p55 (Tnfrsf1a) or treatment with a synthetic metalloproteinase inhibitor rescues timp3−/− mice. Thus, TIMP3 is essential for normal innate immune function.
Cutting Edge: Heligmosomoides polygyrus Induces TLR4 on Murine Mucosal T Cells That Produce TGFβ after Lipopolysaccharide Stimulation
Authors: Ince M. Nedim , Elliott David E. , Setiawan Tommy , Blum Arthur , Metwali Ahmed , Wang Ying , Urban Joseph F. , Weinstock Joel V.
Published: April 2014

Helminths are immune modulators that down-regulate colitis in inflammatory bowel disease. In animal models, intestinal bacteria drive colitis and in humans certain alleles of the LPS receptor protein TLR4 increase inflammatory bowel disease susceptibility. To understand helminthic immune modulation in the gut, we studied the influence of intestinal Heligmosomoides polygyrus colonization on LPS-induced lamina propria mononuclear cell (LPMC) cytokine responses in mice. LPS did not stimulate TGFβ production from LPMC of uninfected mice. LPS strongly induced LPMC from worm-infected animals to secrete TGFβ, but not TNF-α or IL-12. The TGFβ derived from mucosal T cells. Helminth infection up-regulated TLR4 expression only in lamina propria T cells. LPMC from worm-infected TLR4 mutant animals did not respond to LPS, suggesting that LPS required TLR4 to stimulate TGFβ secretion. Thus, during helminth infection, LPS challenge induces mucosal T cells to make TGFβ through a TLR4-dependent process without promoting synthesis of proinflammatory cytokines.
Cutting Edge: Three-Dimensional Architecture of the Thymus Is Required to Maintain Delta-Like Expression Necessary for Inducing T Cell Development
Authors: Mohtashami Mahmood , Zúñiga-Pflücker Juan Carlos
Published: April 2014

The three-dimensional microarchitecture of the thymus plays a unique role in directing T cell lineage commitment and development. This is supported by the fact that, in contrast to fetal thymic organ cultures, thymic stromal cell monolayer cultures (TSMC) fail to support T lymphopoiesis. Nevertheless, OP9-DL1 cell monolayer cultures induce T lineage commitment and differentiation. Thus, the inability of TSMC to support T lymphopoiesis may be due to a loss of Notch ligand expression and/or function during culture. In this study, we report that, in contrast to fetal thymic organ cultures, TSMC fail to maintain expression of the Notch ligands, Delta-like (Dll) 1 and Dll4, and concomitantly lose the ability to support T lymphopoiesis. Importantly, ectopic re-expression of Dll1 or Dll4 is sufficient to restore the ability of TSMC to support T lymphopoiesis. These findings demonstrate that maintenance of endogenous Dll1 or Dll4 expression by thymic stromal cells is required for the commitment and differentiation of T cells in the absence of a three-dimensional microenvironment.
Cutting Edge: Innate Immune Cells Contribute to the IFN-γ-Dependent Regulation of Antigen-Specific CD8+ T Cell Homeostasis
Authors: Sercan Özen , Hämmerling Günter J. , Arnold Bernd , Schüler Thomas
Published: April 2014

IFN-γ has a dual function in the regulation of T cell homeostasis. It promotes the expansion of effector T cells and simultaneously programs their contraction. The cellular mechanisms leading to this functional dichotomy of IFN-γ have not been identified to date. In this study we show: 1) that expansion of wild-type CD8+ T cells is defective in IFN-γ-deficient mice but increased in IFN-γR-deficient mice; and 2) that contraction of the effector CD8+ T cell pool is impaired in both mouse strains. Furthermore, we show that CD11b+ cells responding to IFN-γ are sufficient to limit CD8+ T cell expansion and promote contraction. The data presented here reveal that IFN-γ directly promotes CD8+ T cell expansion and simultaneously induces suppressive functions in CD11b+ cells that counter-regulate CD8+ T cell expansion, promote contraction, and limit memory formation. Thus, innate immune cells contribute to the IFN-γ-dependent regulation of Ag-specific CD8+ T cell homeostasis.
T Cell Zones of Lymphoid Organs Constitutively Express Th1 Cytokine mRNA: Specific Changes during the Early Phase of an Immune Response
Authors: Kalies Kathrin , Blessenohl Maike , Nietsch Julia , Westermann Jürgen
Published: April 2014

The cytokine milieu of the T cell zones in lymphoid organs is involved in the activation of naive T cells. Quantitative data regarding the local expression of cytokines are lacking. Therefore, the expression of Th1 (IL-2, IL-12p40, IFN-γ), Th2 (IL-4, IL-10), as well as TGFβ1 and IL-15 mRNA was studied after laser microdissection in the steady state and during an immune response in rats. Our results show that Th1 cytokines are preferentially found in lymphoid tissues and in the T cell zones, whereas Th2 cytokines are expressed throughout the organs and especially in the B cell zones. After injection of sheep RBC, IL-2 and IFN-γ mRNA are significantly increased in the T cell zone only, a change not seen by analyzing the whole spleen. Studying the spatial and temporal expression of genes will reveal new insights into the regulation of immune responses.
Human Monocytes as Intermediaries between Allogeneic Endothelial Cells and Allospecific T Cells: A Role for Direct Scavenger Receptor-Mediated Endothelial Membrane Uptake in the Initiation of Alloimmunity
Authors: Xu He , Dhanireddy Kiran K. , Kirk Allan D.
Published: April 2014

Recipient monocytes, T cells, and donor endothelial cells (ECs) are recognized as critical components of allograft rejection. We have recently shown that human monocytes infiltrate vascularized allografts before clinical rejection and have thus hypothesized that monocytes, rather than costimulation-poor ECs, initiate an alloimmune response. However, the nature of the interactions between ECs, monocytes, and T cells has been incompletely defined. Specifically, it is not clear whether these cells interact in a hierarchical manner, nor is it apparent what constitutes an interaction. We therefore studied human ECs, monocytes, and T cells in various isolated in vitro combinations to define the salient features of their contact and to determine whether their interactions were sequential in nature. We find that T cells proliferate poorly to allogeneic ECs and autologous monocytes but well to autologous monocytes following allogeneic EC contact. We show that monocytes gain their stimulatory capacity by phagocytizing allogeneic but not autologous EC membranes in a process governed by scavenger receptors. This process facilitates the subsequent presentation of intact donor HLA molecules to T cells (semidirect presentation). Moreover, monocytes are receptive to T cell help only after exposure to ECs and require CD4+ T cells to optimally express costimulatory molecules and foster Ag presentation. Our results indicate that monocytes engage allogeneic ECs through scavenger receptors and are then primed to facilitate T cell activation in a codependent manner. This reciprocal codependence allows for monocytes to serve as a regulated bridge between the allograft and T cells.
Role of Breast Milk in a Mouse Model of Maternal Transmission of Asthma Susceptibility
Authors: Leme Adriana S. , Hubeau Cedric , Xiang Yuhong , Goldman Alejandra , Hamada Kaoru , Suzaki Yasue , Kobzik Lester
Published: April 2014

Epidemiologic data suggest a link between nursing by asthmatic mothers and increased risk of allergy in babies. We sought to experimentally test the potential contribution of breast milk mediator(s) in a mouse model of maternal transmission of asthma risk by evaluating the effect of adoptive nursing on asthma susceptibility in the offspring. We measured airway hyperresponsiveness (AHR) and allergic airway inflammation (AI) after an intentionally suboptimal OVA Ag sensitization, tested the allergen independence of the maternal effect by using a second allergen, casein, for sensitization of the baby mice, and tested the potential role of cytokines by measuring their levels in breast milk. Offspring of asthmatic, but not normal, mothers showed AHR and AI, indicating a maternal transfer of asthma risk. After adoptive nursing, both groups (litters born to asthmatic mothers and nursed by normal mothers, and normal babies nursed by asthmatic mothers) showed AHR (enhanced pause after methacholine aerosol, 50 mg/ml, 3.7 ± 0.7, 4.2 ± 0.5, respectively, vs 1.1 ± 0.1 normal controls, n = 25, p < 0.01) and AI, seen as eosinophilia on histology and bronchoalveolar lavage (40.7 ± 4.5%, 28.7 ± 3.7%, vs 1.0 ± 0.5% normals, n = 25, p < 0.01) after OVA sensitization. Similar results using casein allergen were observed. Multiplex assays for cytokines (IFN-γ, IL-2, IL-4, IL-5, TNF-α, and IL-13) in breast milk were negative. Breast milk is sufficient, but not necessary, to mediate allergen-independent maternal transmission of asthma risk to offspring.
Lymphoid Sequestration of Alloreactive Memory CD4 T Cells Promotes Cardiac Allograft Survival
Authors: Zhang Qiwei , Chen Yifa , Fairchild Robert L. , Heeger Peter S. , Valujskikh Anna
Published: April 2014

Memory T cells specific for donor Ags present a unique challenge in transplantation. In addition to expressing robust immune responses to a transplanted organ, memory T cells may be resistant to the effects of immunosuppressive therapies used to prolong graft survival. In this study, we explore the possibility of controlling deleterious donor-reactive memory CD4 T cells through lymphoid sequestration. We showed that sphingosine 1-phosphate receptor-1 agonist FTY720 induces relocation of circulating memory CD4 T cells into secondary lymphoid organs. Lymphoid sequestration of these donor-reactive memory CD4 T cells prolonged survival of murine heterotopic cardiac allografts and synergizes with conventional costimulatory blockade to further increase graft survival. Despite limited trafficking, memory CD4 T cells remain capable of providing help for the induction of anti-donor CD8 T cell and alloantibody responses. Elimination of antidonor humoral immunity resulted in indefinite allograft survival proving the pathogenicity of alloantibody under these conditions. Overall, this is the first demonstration that FTY720 influences memory CD4 T cell trafficking and attenuates their contribution to allograft rejection. The data have important implications for guiding FTY720 therapy and for designing combinatorial strategies aimed at prolonging allograft survival in sensitized transplant patients with donor-specific memory T cells.
Galectin-3 and Galectin-1 Bind Distinct Cell Surface Glycoprotein Receptors to Induce T Cell Death
Authors: Stillman Brianna N. , Hsu Daniel K. , Pang Mabel , Brewer C. Fred , Johnson Pauline , Liu Fu-Tong , Baum Linda G.
Published: April 2014

Galectins are a family of mammalian β-galactoside-binding proteins that positively and negatively regulate T cell death. Extracellular galectin-1 directly induces death of T cells and thymocytes, while intracellular galectin-3 blocks T cell death. In contrast to the antiapoptotic function of intracellular galectin-3, we demonstrate that extracellular galectin-3 directly induces death of human thymocytes and T cells. However, events in galectin-3- and galectin-1-induced cell death differ in a number of ways. Thymocyte subsets demonstrate different susceptibility to the two galectins: whereas galectin-1 kills double-negative and double-positive human thymocytes with equal efficiency, galectin-3 preferentially kills double-negative thymocytes. Galectin-3 binds to a complement of T cell surface glycoprotein receptors distinct from that recognized by galectin-1. Of these glycoprotein receptors, CD45 and CD71, but not CD29 and CD43, appear to be involved in galectin-3-induced T cell death. In addition, CD7 that is required for galectin-1-induced death is not required for death triggered by galectin-3. Following galectin-3 binding, CD45 remains uniformly distributed on the cell surface, in contrast to the CD45 clustering induced by galectin-1. Thus, extracellular galectin-3 and galectin-1 induce death of T cells through distinct cell surface events. However, as galectin-3 and galectin-1 cell death are neither additive nor synergistic, the two death pathways may converge inside the cell.
Early Preplasma Cells Define a Tolerance Checkpoint for Autoreactive B Cells
Authors: Culton Donna A. , O’Conner Brian P. , Conway Kara L. , Diz Ramiro , Rutan Jennifer , Vilen Barbara J. , Clarke Stephen H.
Published: April 2014

Ab-secreting plasma cells (PCs) are the effectors of humoral immunity. In this study, we describe regulation of autoreactive B cells specific for the ribonucleoprotein Smith (Sm) at an early pre-PC stage. These cells are defined by the expression of the PC marker CD138 and normal levels of CD19 and B220. They are present at a high frequency in normal mouse spleen and bone marrow, are Ag dependent, and are located predominantly along the T cell-B cell border and near bridging channels. Anti-Sm pre-PCs also occur at a high frequency in nonautoimmune mice and show additional phenotypic characteristics of PC differentiation. However, while some of these pre-PCs are Ab-secreting cells, those specific for Sm are not, indicating regulation. Consistent with this, anti-Sm pre-PCs have a higher turnover rate and higher frequency of cell death than those that do not bind Sm. Regulation of anti-Sm pre-PCs occurs upstream of the transcriptional repressor, B lymphocyte-induced maturation protein-1, expression. Regulation at this stage is overcome in autoimmune MRL/lpr mice and is accompanied by an altered B lymphocyte stimulator receptor profile. These data reveal a new B cell tolerance checkpoint that is overcome in autoimmunity.