bluuebunny/crossref_metadata_2025 huggingface dataset:
The Novel Cytokine p43 Induces IL-12 Production in Macrophages via NF-κB Activation, Leading to Enhanced IFN-γ Production in CD4+ T Cells
Authors: Kim Eugene , Kim Seung Hyun , Kim Sunghoon , Kim Tae Sung
Published: April 2014

Recently, we determined that p43, an auxiliary factor of mammalian multiaminoacyl-tRNA synthetases, is secreted, and functions as a novel pleiotropic cytokine. In this study, we have attempted to characterize the effects of p43 on the generation of IL-12 in mouse macrophages. p43 was determined to induce significant IL-12 production from mouse macrophages in a dose-dependent manner. The stimulatory effect of p43 on the activation of IL-12p40 promoter was mapped to a region harboring an NF-κB binding site. The nuclear extracts from the p43-stimulated macrophages exhibited profound NF-κB DNA-binding activity, as determined by the EMSA. In addition, the p43-stimulated IL-12 induction and NF-κB DNA-binding activity were significantly suppressed by caffeic acid phenethyl ester and BAY11-7082, both inhibitors of NF-κB activation, indicating that p43 induced the production of IL-12 in macrophages mainly via the activation of NF-κB. Importantly, p43 increased the level of IFN-γ production in the Ag-primed lymph node cells, but had no effect on IL-4 levels. The addition of a neutralizing anti-IL-12p40 mAb to the cell cultures resulted in a decrease of the production of p43-enhanced IFN-γ by the keyhole limpet hemocyanin-primed lymph node cells. Furthermore, coincubation with p43-pretreated macrophages enhanced the production of IFN-γ by the keyhole limpet hemocyanin-primed CD4+ T cells, thereby indicating that p43 may enhance IFN-γ expression in CD4+ T cells via the induction of IL-12 production in macrophages. These results indicate that p43 may play an essential role in the development of the Th1 immune responses associated with cancer immunotherapy and protective immunity against intracellular pathogens.
Hydrolytic and Nonenzymatic Functions of Acetylcholinesterase Comodulate Hemopoietic Stress Responses
Authors: Grisaru Dan , Pick Marjorie , Perry Chava , Sklan Ella H. , Almog Ronit , Goldberg Ilan , Naparstek Elizabeth , Lessing Joseph B. , Soreq Hermona , Deutsch Varda
Published: April 2014

Glucocorticoid-initiated granulocytosis, excessive proliferation of granulocytes, persists after cortisol levels are lowered, suggesting the involvement of additional stress mediator(s). In this study, we report that the stress-induced acetylcholinesterase variant, AChE-R, and its cleavable, cell-penetrating C-terminal peptide, ARP, facilitate granulocytosis. In postdelivery patients, AChE-R-expressing granulocyte counts increased concomitantly with serum cortisol and AChE activity levels, yet persisted after cortisol had declined. Ex vivo, mononuclear cells of adult peripheral blood responded to synthetic ARP26 by overproduction of hemopoietically active proinflammatory cytokines (e.g., IL-6, IL-10, and TNF-α). Physiologically relevant ARP26 levels promoted AChE gene expression and induced the expansion of cultured CD34+ progenitors and granulocyte maturation more effectively than cortisol, suggesting autoregulatory prolongation of ARP effects. In vivo, transgenic mice overexpressing human AChE-R, unlike matched controls, showed enhanced expression of the myelopoietic transcription factor PU.1 and maintained a stable granulocytic state following bacterial LPS exposure. AChE-R accumulation and the consequent inflammatory consequences can thus modulate immune responses to stress stimuli.
Synergistic Activation of Macrophages via CD40 and TLR9 Results in T Cell Independent Antitumor Effects
Authors: Buhtoiarov Ilia N. , Lum Hillary D. , Berke Gideon , Sondel Paul M. , Rakhmilevich Alexander L.
Published: April 2014

We have previously shown that macrophages (Mφ) can be activated by CD40 ligation to become cytotoxic against tumor cells in vitro. Here we show that treatment of mice with agonistic anti-CD40 mAb (anti-CD40) induced up-regulation of intracellular TLR9 in Mφ and primed them to respond to CpG-containing oligodeoxynucleotides (CpG), resulting in synergistic activation. The synergy between anti-CD40 and CpG was evidenced by increased production of IFN-γ, IL-12, TNF-α, and NO by Mφ, as well as by augmented apoptogenic effects of Mφ against tumor cells in vitro. The activation of cytotoxic Mφ after anti-CD40 plus CpG treatment was dependent on IFN-γ but not TNF-α or NO, and did not require T cells and NK cells. Anti-CD40 and CpG also synergized in vivo in retardation of tumor growth in both immunocompetent and immunodeficient mice. Inactivation of Mφ in SCID/beige mice by silica treatment abrogated the antitumor effect. Taken together, our results show that Mφ can be activated via CD40/TLR9 ligation to kill tumor cells in vitro and inhibit tumor growth in vivo even in immunocompromised tumor-bearing hosts, indicating that this Mφ-based immunotherapeutic strategy may be appropriate for clinical testing.
Immunodomination in the Evolution of Dominant Epitope-Specific CD8+ T Lymphocyte Responses in Simian Immunodeficiency Virus-Infected Rhesus Monkeys
Authors: Newberg Michael H. , McEvers Kimberly J. , Gorgone Darci A. , Lifton Michelle A. , Baumeister Susanne H. C. , Veazey Ronald S. , Schmitz Jörn E. , Letvin Norman L.
Published: April 2014

Because the control of HIV-1 replication is largely dependent on CD8+ T lymphocyte responses specific for immunodominant viral epitopes, vaccine strategies that increase the breadth of dominant epitope-specific responses should contribute to containing HIV-1 spread. Developing strategies to elicit such broad immune responses will require an understanding of the mechanisms responsible for focusing CD8+ T lymphocyte recognition on a limited number of epitopes. To explore this biology, we identified cohorts of rhesus monkeys that expressed the MHC class I molecules Mamu-A*01, Mamu-A*02, or both, and assessed the evolution of their dominant epitope-specific CD8+ T lymphocyte responses (Gag p11C- and Tat TL8-specific in the Mamu-A*01+ and Nef p199RY-specific in the Mamu-A*02+ monkeys) following acute SIV infection. The Mamu-A*02+ monkeys that also expressed Mamu-A*01 exhibited a significant delay in the evolution of the CD8+ T lymphocyte responses specific for the dominant Mamu-A*02-restricted SIV epitope, Nef p199RY. This delay in kinetics was not due to differences in viral load kinetics or magnitude or in viral escape mutations, but was associated with the evolution of the Mamu-A*01-restricted CD8+ T lymphocyte responses to the highly dominant SIV epitopes Gag p11C and Tat TL8. Thus, the evolution of dominant epitope-specific CD8+ T lymphocyte responses can be suppressed by other dominant epitope-specific responses, and this immunodomination is important in determining the kinetics of dominant epitope-specific responses.
Specificity of CD4+CD25+ Regulatory T Cell Function in Alloimmunity
Authors: Sánchez-Fueyo Alberto , Sandner Sigrid , Habicht Antje , Mariat Christophe , Kenny James , Degauque Nicolas , Zheng Xin Xiao , Strom Terry B. , Turka Laurence A. , Sayegh Mohamed H.
Published: April 2014

CD4+CD25+ regulatory T cells (TRegs) are critical for the acquisition of peripheral allograft tolerance. However, it is unclear whether TRegs are capable of mediating alloantigen-specific suppressive effects and, hence, contributing to the specificity of the tolerant state. In the current report we have used the ABM TCR transgenic (Tg) system, a C57BL/6-derived strain in which CD4+ T cells directly recognize the allogeneic MHC-II molecule I-Abm12, to assess the capacity of TRegs to mediate allospecific effects. In these mice, 5–6% of Tg CD4+ T cells exhibit conventional markers of the TReg phenotype. ABM TRegs are more effective than wild-type polyclonal TRegs at suppressing effector immune responses directed against I-Abm12 alloantigen both in vitro and in vivo. In contrast, they are incapable of suppressing responses directed against third-party alloantigens unless these are expressed in the same allograft as I-Abm12. Taken together, our results indicate that in transplantation, TReg function is dependent on TCR stimulation, providing definitive evidence for their specificity in the regulation of alloimmune responses.
Src-Like Adaptor Protein Regulates B Cell Development and Function
Authors: Dragone Leonard L. , Myers Margaret D. , White Carmen , Sosinowski Tomasz , Weiss Arthur
Published: April 2014

The avidity of BCRs and TCRs influences signal strength during processes of lymphocyte development. Avidity is determined by both the intrinsic affinity for Ag and surface levels of the Ag receptor. The Src-like adaptor protein (SLAP) is a regulator of TCR levels on thymocytes, and its deficiency alters thymocyte development. We hypothesized that SLAP, which is expressed in B cells, also is important in regulating BCR levels, signal strength, and B cell development. To test this hypothesis, we analyzed the B cell compartment in SLAP-deficient mice. We found increased splenic B cell numbers and decreased surface IgM levels on mature, splenic B cells deficient in SLAP. Immature bone marrow and splenic B cells from BCR-transgenic, SLAP-deficient mice were found to express higher surface levels of IgM. In contrast, mature splenic B cells from BCR-transgenic mice expressed decreased levels of surface BCR associated with decreased calcium flux and activation-induced markers, compared with controls. These data suggest that SLAP regulates BCR levels and signal strength during lymphocyte development.
Neonatally Primed Lymph Node, but Not Splenic T Cells, Display a Gly-Gly Motif within the TCR β-Chain Complementarity-Determining Region 3 That Controls Affinity and May Affect Lymphoid Organ Retention
Authors: Caprio-Young Jacque C. , Bell J. Jeremiah , Lee Hyun-Hee , Ellis Jason , Nast Danielle , Sayler Gary , Min Booki , Zaghouani Habib
Published: April 2014

Ig-proteolipid protein 1 (Ig-PLP1) is an Ig chimera expressing the encephalitogenic PLP1 peptide corresponding to amino acid residues 139–151 of PLP. Newborn mice given Ig-PLP1 in saline on the day of birth and challenged 7 wk later with PLP1 peptide in CFA develop an organ-specific neonatal immunity that confers resistance against experimental allergic encephalomyelitis. The T cell responses in these animals are comprised of Th2 cells in the lymph node and anergic Th1 lymphocytes in the spleen. Intriguingly, the anergic splenic T cells, although nonproliferative and unable to produce IFN-γ or IL-4, secrete significant amounts of IL-2. Studies were performed to determine whether the two populations display any structural differences in the TCR H chain variable region that could contribute to the differential affinity and retention in different organs. Responsive Th2 lymph node T cells and anergic splenic lymphocytes were immortalized, and the structures of their TCR Vβ were determined. The results show that Vβ and Jβ usage was random, but the CDR3 regions of the lymph node cells had a conserved Gly-Gly motif. Analysis of TCR affinity/avidity correlated the Gly-Gly motif with lower affinity and retention of the Th2 cells in the lymph node. Also, it is suggested that a higher TCR affinity may be a contributing factor for the development of the neonatal Th1 response in the spleen.
Novel Insights into the Mechanism of Action of FTY720 in a Transgenic Model of Allograft Rejection: Implications for Therapy of Chronic Rejection
Authors: Habicht Antje , Clarkson Michael R. , Yang Jun , Henderson Joel , Brinkmann Volker , Fernandes Stacey , Jurewicz Mollie , Yuan Xueli , Sayegh Mohamed H.
Published: April 2014

FTY720 is a high-affinity agonist at the sphingosine 1-phosphate receptor 1 that prevents lymphocyte egress from lymphoid tissue and prolongs allograft survival in several animal models of solid organ transplantation. In this study we used a recently developed adoptive transfer model of TCR transgenic T cells to track allospecific CD4+ T cell expansion and trafficking characteristics, cytokine secretion profiles, and surface phenotype in vivo in the setting of FTY720 administration. We report that FTY720 administration had no effect on alloantigen-driven T cell activation, proliferation, acquisition of effector-memory function, or T cell apoptosis. However, FTY720 caused a reversible sequestration of alloantigen-specific effector-memory T cells in regional lymphoid tissue associated with a decrease in T cell infiltration within the allograft and a subsequent prolongation in allograft survival. Furthermore, delayed administration of FTY720 in a cardiac model of chronic allograft rejection attenuated the progression of vasculopathy and tissue fibrosis consistent with the hypothesis that FTY720 interrupts the trafficking of activated effector-memory T cells. These data have important implications for targeting the sphingosine 1-phosphate receptor 1 in solid organ transplantation.
Altered Self-Erythrocyte Recognition and Destruction in an Inbred Line of Tilapia (Oreochromis aureus)
Authors: Shirak Andrey , Bendersky Anna , Hulata Gideon , Ron Micha , Avtalion Ramy R.
Published: April 2014

Carboxyfluorescein diacetate (cFDA)-stained autologous and syngeneic tilapia (Oreochromis aureus) erythrocytes are recognized by effector peripheral blood leukocytes and lysed after a short culture period of 4 h. The hemolysis level was evaluated by measuring the fluorescence of the released cFDA. The degree of lysis of stained target erythrocytes of 60 individuals revealed a trimodal distribution statistically stratified into three groups of low (LR), intermediate (IR), and high (HR) responders. Depletion of the majority of phagocytes from leukocytes lowered the lysis level of HR to that of LR. A highly significant increase of LR cytotoxicity was obtained after the addition of conditioned medium from HR but only in the presence of phagocytes. Genetic analysis of offspring from four crosses (IR × HR, IR × LR, HR × LR, and LR × LR) revealed a quantitative trait locus (QTL) segregating for the level of response linked to markers UNH207 and UNH231 on linkage group 6 of tilapia. Based on segregation analysis of 58 gynogenetic BIU-1 offspring, the distances from the centromere were estimated as 21.5, 11.5, and 9.0 cM for UNH207, UNH231, and the QTL, respectively. It is suggested that 1) self-target recognition and destruction requires both cFDA-altered self-erythrocyte membrane and membrane structures normally present in autologous, syngeneic, and xenogeneic targets; 2) natural cytotoxic cells and/or macrophages are involved in erythrocyte lysis; and 3) the lysis level is codominantly inherited by a QTL segregating on tilapia linkage group 6.
A Signal Adaptor SLAM-Associated Protein Regulates Spontaneous Autoimmunity and Fas-Dependent Lymphoproliferation in MRL-Fas
lpr Lupus Mice
Authors: Komori Hiroaki , Furukawa Hiroshi , Mori Shiro , Ito Mitsuko R. , Terada Miho , Zhang Ming-Cai , Ishii Naoto , Sakuma Nobuhiro , Nose Masato , Ono Masao
Published: April 2014

Autoantibody production and lymphadenopathy are common features of systemic autoimmune disease. Targeted or spontaneous mutations in the mouse germline have generated many autoimmune models with these features. Importantly, the models have provided evidence for the gene function in prevention of autoimmunity, suggesting an indispensable role for the gene in normal immune response and homeostasis. We describe here pathological and genetic characterizations of a new mutant strain of mice, the mutation of which spontaneously occurred in the Fas-deficient strain, MRL/Mp.Faslpr (MRL/lpr). MRL/lpr is known to stably exhibit systemic lupus erythematosus-like diseases. However, the mutant mice barely displayed autoimmune phenotypes, though the original defect in Fas expression was unchanged. Linkage analysis using (mutant MRL/lpr × C3H/lpr)F2 mice demonstrated a nucleotide insertion that caused loss of expression of small adaptor protein, signaling lymphocyte activation molecule (SLAM)-associated protein (SAP). SAP is known to be a downstream molecule of SLAM family receptors and to mediate the activation signal for tyrosine kinase Fyn. Recent studies have shown pleiotropic roles of SAP in T, B, and NK cell activations and NKT cell development. The present study will provide evidence for an essential role for SAP in the development of autoimmune diseases, autoantibodies, and lymphadenopathy in MRL/lpr lupus mice.
Defining the Origins and Evolution of the Chemokine/Chemokine Receptor System
Authors: DeVries Mark E. , Kelvin Alyson A. , Xu Luoling , Ran Longsi , Robinson John , Kelvin David J.
Published: April 2014

The chemokine system has a critical role in mammalian immunity, but the evolutionary history of chemokines and chemokine receptors are ill-defined. We used comparative whole genome analysis of fruit fly, sea urchin, sea squirt, pufferfish, zebrafish, frog, and chicken to identify chemokines and chemokine receptors in each species. We report 127 chemokine and 70 chemokine receptor genes in the 7 species, with zebrafish having the most chemokines, 63, and chemokine receptors, 24. Fruit fly, sea urchin, and sea squirt have no identifiable chemokines or chemokine receptors. This study represents the most comprehensive analysis of the chemokine system to date and the only complete characterization of chemokine systems outside of mouse and human. We establish a clear evolutionary model of the chemokine system and trace the origin of the chemokine system to ∼650 million years ago, identifying critical steps in their evolution and demonstrating a more extensive chemokine system in fish than previously thought.
The Viral Protein A238L Inhibits TNF-α Expression through a CBP/p300 Transcriptional Coactivators Pathway
Authors: Granja Aitor G. , Nogal Maria L. , Hurtado Carolina , del Aguila Carmen , Carrascosa Angel L. , Salas María L. , Fresno Manuel , Revilla Yolanda
Published: April 2014

African swine fever virus (ASFV) is able to inhibit TNF-α-induced gene expression through the synthesis of A238L protein. This was shown by the use of deletion mutants lacking the A238L gene from the Vero cell-adapted Ba71V ASFV strain and from the virulent isolate E70. To further analyze the molecular mechanism by which the viral gene controls TNF-α, we have used Jurkat cells stably transfected with the viral gene to identify the TNF-α regulatory elements involved in the induction of the gene after stimulation with PMA and calcium ionophore. We have thus identified the cAMP-responsive element and κ3 sites on the TNF-α promoter as the responsible of the gene activation, and demonstrate that A238L inhibits TNF-α expression through these DNA binding sites. This inhibition was partially reverted by overexpression of the transcriptional factors NF-AT, NF-κB, and c-Jun. Furthermore, we present evidence that A238L inhibits the activation of TNF-α by modulating NF-κB, NF-AT, and c-Jun trans activation through a mechanism that involves CREB binding protein/p300 function, because overexpression of these transcriptional coactivators recovers TNF-α promoter activity. In addition, we show that A238L is a nuclear protein that binds to the cyclic AMP-responsive element/κ3 complex, thus displacing the CREB binding protein/p300 coactivators. Taken together, these results establish a novel mechanism in the control of TNF-α gene expression by a viral protein that could represent an efficient strategy used by ASFV to evade the innate immune response.
Helminth-Primed Dendritic Cells Alter the Host Response to Enteric Bacterial Infection
Authors: Chen Chien-Chang , Louie Steve , McCormick Beth A. , Walker W. Allan , Shi Hai Ning
Published: April 2014

To examine whether intestinal helminth infection may be a risk factor for enteric bacterial infection, a murine model was established using the intestinal helminth Heligomosomoides polygyrus and a murine pathogen Citrobacter rodentium, which causes infectious colitis. Using this model we recently have shown that coinfection with the Th2-inducing H. polygyrus and C. rodentium promotes bacterial-associated disease and colitis. In this study, we expand our previous observations and examine the hypothesis that dendritic cells (DC) stimulated by helminth infection may play an important role in the regulation of the intestinal immune response to concurrent C. rodentium infection as well as in the modulation of the bacterial pathogenesis. We show that H. polygyrus infection induces DC activation and IL-10 expression, and that adoptive transfer of parasite-primed DC significantly impairs host protection to C. rodentium infection, resulting in an enhanced bacterial infection and in the development of a more severe colonic injury. Furthermore, we demonstrate that adoptive transfer of parasite-primed IL-10-deficient DCs fails to result in the development of a significantly enhanced C. rodentium-mediated colitis. Similarly, when the DC IL-10 response was neutralized by anti-IL-10 mAb treatment in mice that received parasite-primed DC, no deleterious effect of the parasite-primed DC on the host intestinal response to C. rodentium was detected. Thus, our results provide evidence to indicate that the H. polygyrus-dependent modulation of the host response to concurrent C. rodentium infection involves IL-10-producing DCs.
Listeria monocytogenes Activated p38 MAPK and Induced IL-8 Secretion in a Nucleotide-Binding Oligomerization Domain 1-Dependent Manner in Endothelial Cells
Authors: Opitz Bastian , Püschel Anja , Beermann Wiebke , Hocke Andreas C. , Förster Stefanie , Schmeck Bernd , van Laak Vincent , Chakraborty Trinad , Suttorp Norbert , Hippenstiel Stefan
Published: April 2014

Nucleotide-binding oligomerization domain (Nod) proteins serve as intracellular pattern recognition molecules recognizing peptidoglycans. To further examine intracellular immune recognition, we used Listeria monocytogenes as an organism particularly amenable for studying innate immunity to intracellular pathogens. In contrast to wild-type L. monocytogenes, the nonpathogenic Listeria innocua, or L. monocytogenes mutants lacking internalin B or listeriolysin O, poorly invaded host cells and escaped into host cell cytoplasm, respectively, and were therefore used as controls. In this study, we show that only the invasive wild-type L. monocytogenes, but not the listeriolysin O- or internalin B-negative L. monocytogenes mutants or L. innocua, substantially induced IL-8 production in HUVEC. RNA interference and Nod1-overexpression experiments demonstrated that Nod1 is critically involved in chemokine secretion and NF-κB activation initiated by L. monocytogenes in human endothelial cells. Moreover, we show for the first time that Nod1 mediated activation of p38 MAPK signaling induced by L. monocytogenes. Finally, L. monocytogenes- and Nod1-induced IL-8 production was blocked by a specific p38 inhibitor. In conclusion, L. monocytogenes induced a Nod1-dependent activation of p38 MAPK signaling and NF-κB which resulted in IL-8 production in endothelial cells. Thus, Nod1 is an important component of a cytoplasmic surveillance pathway.
Functional Importance of Regional Differences in Localized Gene Expression of Receptors for IL-13 in Murine Gut
Authors: Morimoto Motoko , Morimoto Masahiro , Zhao Aiping , Madden Kathleen B. , Dawson Harry , Finkelman Fred D. , Mentink-Kane Margaret , Urban Joseph F. , Wynn Thomas A. , Shea-Donohue Terez
Published: April 2014

IL-13 induces a STAT6-dependent hypercontractility of intestinal smooth muscle that is mediated by binding to the IL-13Rα1 component of the type 2 IL-4R that is linked to STAT6. IL-13 also binds to the IL-13Rα2 that is not linked to STAT6 and functions to limit the effects of IL-13 in vivo. In this study we assessed the contributions of regional and cellular differences in the distribution of the IL-13R components to the physiological regulation of smooth muscle function in wild-type mice and mice deficient in STAT6 or IL-13Rα2. The expression of IL-13 and IL-13Rα2 was higher in colon than in small intestine. Laser capture microdissection of specific cell types revealed that the expression of IL-13Rα2 was higher in the smooth muscle layer compared with levels in the epithelial cells of the mucosa. In contrast, there was a uniform distribution of IL-13α1 in smooth muscle, epithelia, and myenteric neurons. The significant hypercontractility of smooth muscle in mice deficient in IL-13Rα2, but not in STAT6, shows the physiological importance of IL-13 binding to IL-13Rα2. The pronounced differences in the expression of IL-13Rα2 suggest that the gut has developed sophisticated mechanisms for controlling the physiological and pathophysiological activities of IL-13.
Integrin αIIbβ3 Induces the Adhesion and Activation of Mast Cells through Interaction with Fibrinogen
Authors: Oki Toshihiko , Kitaura Jiro , Eto Koji , Lu Yang , Maeda-Yamamoto Mari , Inagaki Naoki , Nagai Hiroichi , Yamanishi Yoshinori , Nakajina Hideaki , Kumagai Hidetoshi , Kitamura Toshio
Published: April 2014

Integrin αIIb, a well-known marker of megakaryocyte-platelet lineage, has been recently recognized on hemopoietic progenitors. We now demonstrate that integrin αIIbβ3 is highly expressed on mouse and human mast cells including mouse bone marrow-derived mast cells, peritoneal mast cells, and human cord blood-derived mast cells, and that its binding to extracellular matrix proteins leads to enhancement of biological functions of mast cells in concert with various stimuli. With exposure to various stimuli, including cross-linking of FcεRI and stem cell factor, mast cells adhered to extracellular matrix proteins such as fibrinogen and von Willebrand factor in an integrin αIIbβ3-dependent manner. In addition, the binding of mast cells to fibrinogen enhanced proliferation, cytokine production, and migration and induced uptake of soluble fibrinogen in response to stem cell factor stimulation, implicating integrin αIIbβ3 in a variety of mast cell functions. In conclusion, mouse and human mast cells express functional integrin αIIbβ3.
Endotoxin-Induced Expression of Murine Bactericidal Permeability/Increasing Protein Is Mediated Exclusively by Toll/IL-1 Receptor Domain-Containing Adaptor Inducing IFN-β-Dependent Pathways
Authors: Eckert Melanie , Wittmann Irene , Röllinghoff Martin , Gessner André , Schnare Markus
Published: April 2014

Antimicrobial effector proteins are a key mechanism for the innate immune system to combat pathogens once they infect the host. We report the identification and cloning of the mouse homologue of human bactericidal permeability/increasing protein (BPI). Mouse BPI is constitutively expressed in lymphatic organs and tissues as well as in mouse testis. Upon stimulation with different TLR ligands, mouse BPI is strongly expressed in granulocytes and, surprisingly, in bone marrow-derived dendritic cells. Mouse BPI is most strongly induced by bacterial LPS through a signaling pathway that is completely dependent on TLR4-Toll/IL-1R domain-containing adaptor inducing IFN-β. Functional studies revealed that mouse BPI does have the potential to neutralize LPS and inhibits bacterial growth. Mouse BPI is expressed in granulocytes and bone marrow-derived dendritic cells, and the transcriptional activation is controlled by TLRs.
CD4+ T Lymphocytes Expressing CD40 Ligand Help the IgM Antibody Response to Soluble Pneumococcal Polysaccharides via an Intermediate Cell Type
Authors: Jeurissen Axel , Billiau An D. , Moens Leen , Shengqiao Li , Landuyt Willy , Wuyts Greet , Boon Louis , Waer Mark , Ceuppens Jan L. , Bossuyt Xavier
Published: April 2014

Streptococcus pneumoniae causes serious infections in children, the elderly, and immunocompromised patients. Protection against infections with S. pneumoniae is mediated through Abs against the capsular polysaccharides (caps-PS). We previously showed that the murine Ab response to caps-PS is dependent on CD40-CD40L interaction. In the present paper, we addressed the question of whether the CD40-CD40L-mediated modulation of the anti-caps-PS immune reaction is the result of a direct interaction between B lymphocytes and T lymphocytes or of an indirect interaction. SCID/SCID mice reconstituted with B lymphocytes from wild-type mice did not mount anti-caps-PS Abs. SCID/SCID mice reconstituted with B lymphocytes from wild-type mice and CD4+ T lymphocytes from wild-type mice but not CD4+ T lymphocytes from CD40L knockout mice stimulated the anti-caps-PS Ab response. This indicated that CD4+ T lymphocytes stimulated the anti-caps-PS Ab response in a CD40L-dependent manner. SCID/SCID mice reconstituted with B lymphocytes from CD40 knockout mice and CD4+ T lymphocytes from wild-type mice generated an anti-caps-PS Ab response that could be inhibited by MR1, a blocking anti-CD40L Ab. These data indicated that CD4+ T lymphocytes stimulated the anti-caps-PS Ab response in an indirect way. Finally, lethally irradiated CD40 knockout mice reconstituted with bone marrow from wild-type mice mounted an anti-caps-PS Ab response that was comparable to the Ab response in wild-type mice, revealing that the required CD40 was on hemopoietic cells. In conclusion, we provide evidence that CD4+ T lymphocytes expressing CD40L stimulate the Ab response to soluble caps-PS by interacting with CD40-expressing non-B cells.
Memory T Cell Populations in the Lung Airways Are Maintained by Continual Recruitment
Authors: Ely Kenneth H. , Cookenham Tres , Roberts Alan D. , Woodland David L.
Published: April 2014

Effector memory T cell populations in the periphery play a key role in cellular immune responses to secondary infections. However, it is unclear how these populations are maintained under steady-state conditions in nonlymphoid peripheral sites, such as the lung airways. In this study, we show that LFA-1 expression is selectively down-regulated following entry of memory T cells into the lung airways. Using Sendai virus as a mouse model of respiratory virus infection, we use LFA-1 expression levels to demonstrate that effector memory T cell populations in the lung airways are maintained by continual recruitment of new cells from the circulation. The rate of memory cell recruitment is surprisingly rapid, resulting in replacement of 90% of the population every 10 days, and is maintained for well over 1 year following viral clearance. These data indicate that peripheral T cell memory is dynamic and depends on a systemic source of T cells.
Doxycycline Modulates Nitric Oxide Production in Murine Lung Epithelial Cells
Authors: Hoyt Jeffrey C. , Ballering Janelle , Numanami Hiroki , Hayden John M. , Robbins Richard A.
Published: April 2014

Many effective therapeutic agents exhibit effects that are different from their intended primary mode of action. Antibiotics such as doxycycline and erythromycin A are no exception. They also display anti-inflammatory activity. Using LA4 murine lung alveolar epithelial cells, effects of doxycycline and erythromycin A on inducible NO synthase (iNOS) NO production as well as iNOS protein and mRNA production were investigated. Induction of iNOS was accomplished by treatment with cytomix (TNF-α, IL-1β, and IFN-γ each at 5 ng/ml). Production of NO or iNOS was not detected in controls with or without erythromycin A. In the presence of cytomix, erythromycin A did not decrease NO, nitrite, iNOS protein, or mRNA production. In contrast, doxycycline caused a dose-dependent decrease in NO, nitrite, iNOS protein, and mRNA production in cytomix-treated cells. Doxycycline at 30 μg/ml produced a 90% decrease in nitrite and NO production and a 52% decrease in iNOS mRNA transcription compared with cytomix treatment alone. Actinomycin D treatment suggests that doxycycline decreases stability of iNOS mRNA in cytomix-treated cells. To determine a mechanism for the decrease in iNOS expression, NF-κB and AP-1 transcription regulatory systems and p38 MAPK were examined. Doxycycline treatment gave no statistically significant change in NF-κB activation but did decrease p38 MAPK protein in cytomix-treated cells by 50%, suggesting that p38 MAPK may be responsible for stabilization of iNOS mRNA. These results demonstrate that doxycycline decreases NO production from iNOS by destabilization of iNOS mRNA via decreased expression of p38 MAPK.
Lipopolysaccharide Induces Cyclooxygenase-2 in Intestinal Epithelium via a Noncanonical p38 MAPK Pathway
Authors: Grishin Anatoly V. , Wang Jin , Potoka Douglas A. , Hackam David J. , Upperman Jeffrey S. , Boyle Patricia , Zamora Ruben , Ford Henri R.
Published: April 2014

Necrotizing enterocolitis (NEC), a severe intestinal inflammation in neonates, occurs following bacterial colonization of the gut. LPS-induced production of inflammatory factors in immature enterocytes may be a factor in NEC. Previously, we described LPS-induced p38 MAPK-dependent expression of cyclooxygenase-2 (COX-2) in rat IEC-6 cells. In this study, we examine COX-2 expression in newborn rat intestinal epithelium and further characterize the mechanisms of COX-2 regulation in enterocytes. Induction of NEC by formula feeding/hypoxia increased phospho-p38 and COX-2 levels in the intestinal mucosa. Celecoxib, a selective COX-2 inhibitor, exacerbated the disease, suggesting a protective role for COX-2. COX-2 was induced in the intestinal epithelium by LPS in vivo and ex vivo. The latter response was attenuated by the p38 inhibitor SB202190, but not by inhibitors of ERK, JNK, or NF-κB. In IEC-6 enterocytes, COX-2 was induced by the expression of MAPK kinase 3 EE (MKK3EE), a constitutive activator of p38, but not of activators of ERK or JNK pathways. However, neither MKK3/6 nor MKK4, the known p38 upstream kinases, were activated by LPS. Dominant-negative MKK3 or MKK4 or SB202190 failed to prevent LPS-induced, p38-activating phosphorylation, ruling out important roles of these kinases or p38 autophosphorylation. LPS increased COX-2 and activating phosphorylation of p38 with similar dose-response. Blockade of LPS-induced expression of COX-2-luciferase reporter and destabilization of COX-2 message by SB202190 indicate that p38 regulates COX-2 at transcription and mRNA stability levels. Our data indicate that p38-mediated expression of COX-2 proceeds through a novel upstream pathway and support the role of the neonate’s enterocytes as bacterial sensors.
Class IB-Phosphatidylinositol 3-Kinase (PI3K) Deficiency Ameliorates IA-PI3K-Induced Systemic Lupus but Not T Cell Invasion
Authors: Barber Domingo F. , Bartolomé Almira , Hernandez Carmen , Flores Juana M. , Fernandez-Arias Cristina , Rodríguez-Borlado Luis , Hirsch Emilio , Wymann Matthias , Balomenos Dimitrios , Carrera Ana C.
Published: April 2014

Class I PI3K catalyzes formation of 3-poly-phosphoinositides. The family is divided into IA isoforms, activated by Tyr kinases and the IB isoform (PI3Kγ), activated by G protein-coupled receptors. Mutations that affect PI3K are implicated in chronic inflammation, although the differential contribution of each isoform to pathology has not been elucidated. Enhanced activation of class IA-PI3K in T cells extends CD4+ memory cell survival, triggering an invasive lymphoproliferative disorder and systemic lupus. As both IA- and IB-PI3K isoforms regulate T cell activation, and activated pathogenic CD4+ memory cells are involved in triggering systemic lupus, we examined whether deletion of IB could reduce the pathological consequences of increased IA-PI3K activity. IB-PI3Kγ deficiency did not abolish invasion or lymphoproliferation, but reduced CD4+ memory cell survival, autoantibody production, glomerulonephritis, and systemic lupus. Deletion of the IB-PI3Kγ isoform thus decreased survival of pathogenic CD4+ memory cells, selectively inhibiting systemic lupus development. These results validate the PI3Kγ isoform as a target for systemic lupus erythematosus treatment.
Ligation of CD28 Stimulates the Formation of a Multimeric Signaling Complex Involving Grb-2-Associated Binder 2 (Gab2), Src Homology Phosphatase-2, and Phosphatidylinositol 3-Kinase: Evidence That Negative Regulation of CD28 Signaling Requires the Gab2 Pleckstrin Homology Domain
Authors: Parry Richard V. , Whittaker Gillian C. , Sims Martin , Edmead Christine E. , Welham Melanie J. , Ward Stephen G.
Published: April 2014

Grb-2-associated binder (Gab)2 is a scaffolding adaptor protein that has been reported to promote growth factor and cytokine receptor signal transduction, but inhibit TCR-mediated signaling events. In this study, we show that ligation of CD28 by its natural ligand B7-1/CD80, induces tyrosine phosphorylation of Gab2 and its coassociation with Src homology phosphatase (SHP)-2 and class IA PI3K in Jurkat cells. Overexpression of wild-type Gab2 revealed a negative role in regulation of CD3/CD28 induction of the transcription factors NF-κB and AP-1. To characterize this inhibitory function further, we used Gab2 mutants unable to bind either PI3K or SHP-2 and a PH domain deletion mutant. Although PI3K has previously been implicated as necessary for Gab2-mediated inhibition of TCR signaling, Gab2 mutants defective in their ability to bind PI3K or SHP-2 retained their inhibitory function, whereas deletion of the PH domain ablated the inhibitory effect of Gab2. Together, these data demonstrate that CD28 stimulation of T cells is sufficient to induce an inhibitory multimeric signaling complex involving Gab2, SHP-2, and PI3K. Furthermore, the inhibitory capacity of Gab2 is strictly dependent upon the integrity of its PH domain, suggesting phosphoinositide-mediated membrane recruitment is important to Gab2 function in T cells.
Tumor-Infiltrating Dendritic Cells Are Potent Antigen-Presenting Cells Able to Activate T Cells and Mediate Tumor Rejection
Authors: Preynat-Seauve Olivier , Schuler Prisca , Contassot Emmanuel , Beermann Friedrich , Huard Bertrand , French Lars E.
Published: April 2014

Dendritic cells (DC) are potent inducers of immune responses. DC have been shown to infiltrate tumors, but very little is known about the functional status of these naturally occurring tumor-infiltrating DC (TIDC). In this study, the status and function of TIDC from several types of mouse melanoma were investigated in detail. CD11c+/MHC II+ cells, consistent with a DC phenotype, were found in all of transplantable or spontaneous melanomas studied. These TIDC were predominantly myeloid (CD11c+/CD8α−/B220−) in nature with small numbers of plasmacytoid (CD11c+/B220+). TIDC had an intermediate maturation phenotype with some expression of costimulatory molecules and the capacity to take up particles. Upon culture overnight ex vivo, the TIDC markedly up-regulated the expression of costimulatory molecules and also increased IL-12 production. Importantly, such ex vivo-matured TIDC pulsed with OVA were able to migrate to lymph nodes, to activate naive OVA-specific CD4+ and CD8+ T cells, and to confer protection against a challenge with OVA-expressing tumor cells. In conclusion, melanomas are infiltrated by functional DC that can act as fully competent APC. These APC have the potential to be manipulated and may therefore represent a promising target for cancer immunotherapy.
Molecular Characterization of Rat Leukocyte P-Selectin Glycoprotein Ligand-1 and Effect of Its Blockade: Protection from Ischemia-Reperfusion Injury in Liver Transplantation
Authors: Tsuchihashi Sei-ichiro , Fondevila Constantino , Shaw Gray D. , Lorenz Meike , Marquette Kimberly , Benard Susan , Shen Xiu-Da , Ke Bibo , Busuttil Ronald W. , Kupiec-Weglinski Jerzy W.
Published: April 2014

P-selectin glycoprotein ligand-1 (PSGL-1) mediates the initial tethering of leukocytes to activated platelets and endothelium. We report molecular cloning and characterization of the rat PSGL-1 gene. A neutralizing Ab was generated, and its binding epitope was mapped to the N-terminal binding region of rat PSGL-1. We examined the effects of early PSGL-1 blockade in rat liver models of cold ischemia, followed by ex vivo reperfusion or transplantation (orthotopic liver transplantation (OLT)) using an anti-PSGL-1 Ab with diminished Fc-mediated effector function. In the ex vivo hepatic cold ischemia and reperfusion model, pretreatment with anti-PSGL-1 Ab improved portal venous flow, increased bile production, and decreased hepatocellular damage. Rat pretreatment with anti-PSGL-1 Ab prevented hepatic insult in a model of cold ischemia, followed by OLT, as assessed by 1) decreased hepatocellular damage (serum glutamic oxaloacetic transaminase/glutamic-pyruvic transaminase levels), and ameliorated histological features of ischemia/reperfusion injury, consistent with extended OLT survival; 2) reduced intrahepatic leukocyte infiltration, as evidenced by decreased expression of P-selectin, ED-1, CD3, and OX-62 cells; 3) inhibited expression of proinflammatory cytokine genes (TNF-α, IL-1β, IL-6, IFN-γ, and IL-2); and 4) prevented hepatic apoptosis accompanied by up-regulation of antiapoptotic Bcl-2/Bcl-xL protective genes. Thus, targeting PSGL-1 with a blocking Ab that has diminished Fc-mediated effector function is a simple and effective strategy that provides the rationale for novel therapeutic approaches to maximize the organ donor pool through the safer use of liver transplants despite prolonged periods of cold ischemia.
Bacterial-Responsive B Lymphocytes Induce Periodontal Bone Resorption
Authors: Han Xiaozhe , Kawai Toshihisa , Eastcott Jean W. , Taubman Martin A.
Published: April 2014

Host immune responses play a key role in periodontal diseases. We have found that B lymphocytes in human periodontal lesions bear abundant receptor activator of NF-κB ligand (RANKL), a major factor in the regulation of osteoclast differentiation. The purpose of this study was to evaluate Actinobacillus actinomycetemcomitans-responsive B lymphocytes in their level of RANKL expression and their effects on periodontal bone resorption. Congenitally athymic Rowett rats received injections of formalin-fixed A. actinomycetemcomitans into the gingival papillae, and donor B cells from normal rats immunized with A. actinomycetemcomitans were transferred via tail vein injection. We demonstrated that B cells from A. actinomycetemcomitans-immunized animals had greater levels of RANKL expression and induced a significantly higher level of osteoclast differentiation from RAW 264.7 cells than did nonimmune B cells that were not Ag specific. This activity was eliminated by incubation with the RANKL decoy receptor osteoprotegerin fusion protein. A. actinomycetemcomitans-binding B cell (ABB) and RANKL-expressing B cells were recovered from the gingival tissues of recipient rats transferred with ABB, but not from recipients of PBS nonimmune B cells or A. actinomycetemcomitans nonbinding B cells. Also, recipients of ABB exhibited increased osteoclast formation on the alveolar bone surface and significant periodontal bone resorption. This effect was antagonized by injection of osteoprotegerin fusion protein into the local gingival tissues. In summary, this study suggests that B lymphocytes can contribute to increased periodontal bone resorption in the absence of T lymphocytes. This effect is associated with the up-regulation of RANKL expression.
SC5 mAb Represents a Unique Tool for the Detection of Extracellular Vimentin as a Specific Marker of Sézary Cells
Authors: Huet Delphine , Bagot Martine , Loyaux Denis , Capdevielle Joël , Conraux Laurence , Ferrara Pascual , Bensussan Armand , Marie-Cardine Anne
Published: April 2014

Circulating malignant Sézary lymphocytes result from a clonal proliferation of memory/activated CD4+CD45RO+ T lymphocytes primarily involving the skin. Recently, the CD158k/KIR3DL2 cell surface receptor has been identified to phenotypically characterize these cells. We previously described a mAb termed SC5 that identifies an unknown early activation cell membrane molecule. It is expressed selectively by T lymphocytes isolated from healthy individuals upon activation, and by circulating Sézary syndrome lymphocytes. In addition, we found that SC5 mAb was reactive with all resting T lymphocytes once permeabilized, indicating that SC5 mAb-reactive molecule might present distinct cellular localization according to the T cell activation status. In this study, we show for the first time that SC5 mAb recognizes the intermediate filament protein vimentin when exported to the extracellular side of the plasma membrane of viable Sézary malignant cells. We demonstrate that SC5 mAb is unique as it reacts with both viable malignant lymphocytes and apoptotic T cells. As vimentin is also detected rapidly at the cell membrane surface after normal T lymphocyte activation, it suggests that its extracellular detection on Sézary cells could be a consequence of their constitutive activation status. Finally, as a probable outcome of vimentin cell surface expression, autoantibodies against vimentin were found in the sera of Sézary syndrome patients.
Models of Systemic Lupus Erythematosus: Development of Autoimmunity Following Peptide Immunizations of Noninbred Pedigreed Rabbits
Authors: Rai Geeta , Ray Satyajit , Shaw Robyn E. , DeGrange Paula F. , Mage Rose G. , Newman Barbara A.
Published: April 2014

Reported in this study are the initial results from studies to develop rabbit models of systemic lupus erythematosus (SLE) by immunizations using two distinct peptides on branched polylysine backbones (multiple Ag peptide)-peptides. Eleven rabbits received a peptide from the Sm B/B′ spliceosomal complex previously shown to be immunogenic in rabbits, and 13 rabbits received a peptide from the rabbit N-methyl-d-aspartate receptor NR2b. All 24 animals in different generations of pedigreed, noninbred rabbits produced peptide-specific responses. Anti-nuclear autoantibody responses, including anti-dsDNA, were seen in 17 of 24 rabbits. To date, two rabbits have been observed to have seizure-like events and a third nystagmus. A model for eliciting development of SLE in genetically related yet heterogeneous rabbits may more closely resemble development of human SLE than do some models in inbred mice. Through selective breeding, it may also ultimately provide additional information about the genetics and etiology of SLE and serve as a model for assessing new treatment options.
Premature Expression of Chemokine Receptor CCR9 Impairs T Cell Development
Authors: Uehara Shoji , Hayes Sandra M. , Li LiQi , El-Khoury Dalal , Canelles Matilde , Fowlkes B. J. , Love Paul E.
Published: April 2014

During thymocyte development, CCR9 is expressed on late CD4−CD8− (double-negative (DN)) and CD4+CD8+ (double-positive) cells, but is subsequently down-regulated as cells transition to the mature CD4+ or CD8+ (single-positive (SP)) stage. This pattern of expression has led to speculation that CCR9 may regulate thymocyte trafficking and/or export. In this study, we generated transgenic mice in which CCR9 surface expression was maintained throughout T cell development. Significantly, forced expression of CCR9 on mature SP thymocytes did not inhibit their export from the thymus, indicating that CCR9 down-regulation is not essential for thymocyte emigration. CCR9 was also expressed prematurely on immature DN thymocytes in CCR9 transgenic mice. Early expression of CCR9 resulted in a partial block of development at the DN stage and a marked reduction in the numbers of double-positive and SP thymocytes. Moreover, in CCR9-transgenic mice, CD25high DN cells were scattered throughout the cortex rather than confined to the subcapsular region of the thymus. Together, these results suggest that regulated expression of CCR9 is critical for normal development of immature thymocytes, but that down-regulation of CCR9 is not a prerequisite for thymocyte emigration.