bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of Mitogen-Activated Protein Kinase Signaling by Chloroquine
Authors: Weber Stephen M. , Chen Jian-Min , Levitz Stuart M.
Published: April 2014

Previously, we demonstrated that the anti-inflammatory drug chloroquine (CQ) inhibited LPS-induced TNF-α transcription. To define further the mechanism of CQ, we studied the effect of this drug on mitogen-activated protein kinase signaling pathways involved in regulation of TNF production. CQ interfered with phosphorylation of extracellular signal-regulated kinases (ERK)1/2 and the ERK-activating kinases mitogen-activating protein/ERK kinase (MEK)1/2. Both CQ and PD98059, a MEK1 inhibitor, reduced luciferase reporter activity driven by human TNF promoter sequences. However, CQ appeared to mediate these effects by deactivating Raf, the upstream activator of MEK. These findings were supported by functional data demonstrating that CQ and PD98059 interfered with TNF expression in several human and murine cell types while neither inhibitor blocked TNF production in murine RAW264.7 macrophages, a cell line that does not require MEK-ERK signaling for TNF production. Finally, we evaluated whether CQ could sensitize HeLa cells to undergo anti-Fas-mediated apoptosis, an effect observed when ERK activation is interrupted in this cell line. CQ rendered HeLa cells sensitive to anti-Fas treatment in a manner similar to PD98059. Taken together, these data argue that therapeutic concentrations of CQ interfere with ERK activation by a novel mechanism, an effect that could be responsible, at least in part, for the potent anti-inflammatory effects of this drug.
Inflammatory Microcrystals Alter the Functional Phenotype of Human Osteoblast-Like Cells In Vitro: Synergism with IL-1 to Overexpress Cyclooxygenase-2
Authors: Bouchard Line , de Médicis Rinaldo , Lussier André , Naccache Paul H. , Poubelle Patrice E.
Published: April 2014

Chronic crystal-associated arthropathies such as gout and pseudogout can lead to local bone destruction. Because osteoblasts, which orchestrate bone remodeling via soluble factors and cell-to-cell interactions, have been described in contact with microcrystals, particularly in uratic foci of gout, we hypothesized that microcrystals of monosodium urate monohydrate (MSUM) and of calcium pyrophosphate dihydrate (CPPD) could alter osteoblastic functions. MSUM and CPPD adhered to human osteoblastic cells (hOB) in vitro and were partly phagocytized as shown by scanning electron microscopy. MSUM and CPPD dose-dependently stimulated the production of PGE2 in hOB as assessed by enzyme immunoassay, a response that was synergistically enhanced in the presence of IL-1. The mechanism of this synergism was, at least in part, at the level of the expression of cyclooxygenase-2 as evaluated by immunoblot analysis. MSUM and CPPD also stimulated the expression of IL-6 and IL-8 and reduced the 1,25-dihydroxyvitamin D3-induced activity of alkaline phosphatase and osteocalcin in hOB (with no synergism with IL-1). MSUM- or CPPD-stimulated expression of IL-6 in hOB pretreated with the selective cyclooxygenase-2 inhibitor NS-398 was increased, unlike that induced by IL-1 alone which was partially reduced. MSUM-, CPPD- or IL-1-induced expression of IL-8 was unchanged by pretreating hOB with NS-398. These results suggest that inflammatory microcrystals alter the normal phenotype of hOB, redirecting them toward reduced bone formation and amplified osteoblast-mediated bone resorption, abnormalities that could play a role in the bone destruction associated with chronic crystal-induced arthritis.
A Proinflammatory Role for the Cyclopentenone Prostaglandins at Low Micromolar Concentrations: Oxidative Stress-Induced Extracellular Signal-Regulated Kinase Activation Without NF-κB Inhibition
Authors: Bureau Fabrice , Desmet Christophe , Mélotte Dorothée , Jaspar Fabrice , Volanti Cédric , Vanderplasschen Alain , Pastoret Paul-Pierre , Piette Jacques , Lekeux Pierre
Published: April 2014

An anti-inflammatory role and therapeutic potential for cyclopentenone PGs (cyPGs) has been suggested, based on observations that levels of cyPGs in exudates increase during the resolution phase of inflammation, and that exogenous cyPGs may attenuate the inflammatory response in vivo and in vitro mainly through inhibition of NF-κB, a critical activator of inflammatory gene expression. However, exogenous cyPGs inhibit NF-κB only at concentrations substantially higher than those of endogenous cyPGs present in inflammatory fluids, thus challenging the hypothesis that cyPGs are naturally occurring inhibitors of inflammation and suggesting that cyPGs at low concentrations might have previously unappreciated effects. In this study, using various cell types, we report that cyPGs, when used at concentrations substantially lower than required for NF-κB inhibition (viz, low micromolar concentrations), significantly potentiate the inflammatory response to TNF-α. At these concentrations, cyPGs induce production of reactive oxygen species, thereby synergizing with TNF-α to activate the extracellular signal-regulated kinase 1/2, an activation which in turn potentiates proinflammatory cytokine expression at both transcriptional and posttranscriptional levels. Our study establishes a proinflammatory role for cyPGs at low micromolar concentrations, raises the possibility that cyPGs do not act as physiologic anti-inflammatory mediators, and questions the therapeutic potential of these compounds.
Immune Responses to Small Nuclear Ribonucleoproteins: Antigen-Dependent Distinct B Cell Epitope Spreading Patterns in Mice Immunized with Recombinant Polypeptides of Small Nuclear Ribonucleoproteins
Authors: Deshmukh Umesh S. , Kannapell Carol C. , Fu Shu Man
Published: April 2014

Complex patterns of autoantibody reactivities with the small nuclear ribonucleoproteins (snRNPs) are observed in systemic lupus erythematosus. To investigate the role of individual snRNP components in the initiation and diversification of anti-snRNP Ab responses, we immunized A/J mice with recombinant Smith D (SmD), Smith B (SmB), and A ribonucleoprotein (A-RNP) with alum as adjuvant. Sera at different time points after initial immunizations were analyzed by Western blot and immunoprecipitation assays. In SmD-immunized mice, specific Abs to A-RNP and SmB were generated by 2 mo postimmunization, in addition to the detection of cross-reactive Abs between the immunogen and other snRNPs. Whereas Abs reactive with the immunogen decreased by 5 mo, Abs capable of immunoprecipitating A-RNP and SmB increased. In SmB-immunized mice, specific Abs to A-RNP were readily detectable, in addition to cross-reactive Abs. In contrast, A-RNP-immunized mice had only cross-reactive Abs to SmB without detectable Abs to SmD. However, in these mice, specific Abs to the 70-kDa protein were generated. Abs, which precipitated the native snRNP particle, were generated in all three groups of the immunized mice. Our results show that different initiating Ags from the same multiprotein antigenic complex induce distinct patterns of epitope spreading to proteins within that complex. These data have significant implications for the mechanisms of autoantibody diversification in systemic lupus erythematosus.
Anatomic Localization of Immature and Mature Dendritic Cells in an Ectopic Lymphoid Organ: Correlation with Selective Chemokine Expression in Rheumatoid Synovium
Authors: Page Guillaume , Lebecque Serge , Miossec Pierre
Published: April 2014

It remains to be clarified whether dendritic cells (DC) reach the rheumatoid arthritis (RA) synovium, considered an ectopic lymphoid organ, as mature cells or undergo local maturation. We characterized by immunohistochemistry the DC subsets and used tonsils as a control. Immature and mature DC were defined by CD1a and DC-lysosome-associated membrane protein/CD83 expression, respectively. Immature DC were mainly detected in the lining layer in RA synovium. Mature DC were exclusively detected in the lymphocytic infiltrates. The DC-lysosome-associated membrane protein/CD1a ratio was 1.1 in RA synovium and 5.3 in tonsils, suggesting the relative accumulation of immature DC in RA synovium. We then focused on the expression of CCL20/CCR6 and CCL19/CCR7, CCL21/CCR7 chemokine/receptor complex, which control immature and mature DC migration respectively. A close association was observed between CCL20-producing cells and CD1a+ cells, suggesting the contribution of CCL20 to CCR6+ cell homing. Conversely, CCL21 and CCL19 expression was only detected in perivascular infiltrates. The association among CCL19/21-producing cells, CCR7 expression, and mature DC accumulation is in line with the roles of these chemokines in mature CCR7+ DC homing to lymphocytic infiltrates. The role of DC in disease initiation and perpetuation makes chemokines involved in DC migration a potential therapeutic target.
p38 Mitogen-Activated Protein Kinase Is Activated and Linked to TNF-α Signaling in Inflammatory Bowel Disease
Authors: Waetzig Georg H. , Seegert Dirk , Rosenstiel Philip , Nikolaus Susanna , Schreiber Stefan
Published: April 2014

Inflammatory bowel diseases (IBD)—Crohn’s disease and ulcerative colitis—are relapsing chronic inflammatory disorders which involve genetic, immunological, and environmental factors. The regulation of TNF-α, a key mediator in the inflammatory process in IBD, is interconnected with mitogen-activated protein kinase pathways. The aim of this study was to characterize the activity and expression of the four p38 subtypes (p38α–δ), c-Jun N-terminal kinases (JNKs), and the extracellular signal-regulated kinases (ERK)1/2 in the inflamed intestinal mucosa. Western blot analysis revealed that p38α, JNKs, and ERK1/2 were significantly activated in IBD, with p38α showing the most pronounced increase in kinase activity. Protein expression of p38 and JNK was only moderately altered in IBD patients compared with normal controls, whereas ERK1/2 protein was significantly down-regulated. Immunohistochemical analysis of inflamed mucosal biopsies localized the main expression of p38α to lamina propria macrophages and neutrophils. ELISA screening of the supernatants of Crohn’s disease mucosal biopsy cultures showed that incubation with the p38 inhibitor SB 203580 significantly reduced secretion of TNF-α. In vivo inhibition of TNF-α by a single infusion of anti-TNF-α Ab (infliximab) resulted in a highly significant transient increase of p38α activity during the first 48 h after infusion. A significant infliximab-dependent p38α activation was also observed in THP-1 myelomonocytic cells. In human monocytes, infliximab enhanced TNF-α gene expression, which could be inhibited by SB 203580. In conclusion, p38α signaling is involved in the pathophysiology of IBD.
Intact Active Bone Transplantation Synergizes with Anti-CD40 Ligand Therapy to Induce B Cell Tolerance
Authors: Yin Dengping , Ma LianLi , Varghese Anncy , Shen JiKun , Chong Anita S.-F.
Published: April 2014

Blockade of T cell costimulatory pathways can result in the prolongation of allograft survival through the suppression of Th1 responses; however, late allograft rejection is usually accompanied by an emerging allograft-specific humoral response. We have recently determined that intact active bone (IAB) fragments transplanted under the kidney capsule can synergize with transient anti-CD40 ligand (CD40L) treatment to induce robust donor-specific allograft tolerance and suppress the alloantibody response. In this study, we take advantage of the ability of galactosyltransferase-deficient knockout (GT-Ko) mice to respond to the carbohydrate epitope, galactose-α1,3-galactose (Gal), to investigate whether IAB plus transient anti-CD40L therapy directly tolerize B cell responses. GT-Ko mice tolerized to Gal-expressing C3H hearts and IAB plus transient anti-CD40L therapy were challenged with pig kidney membranes that express high levels of Gal. The anti-Gal IgM and IgG responses were significantly suppressed in IAB-tolerant mice compared with controls, while the non-Gal anti-pig Ab responses were comparable. The anti-pig T cell cytokine response (IFN-γ and IL-4) was comparable in IAB-tolerant and control mice. The tolerant state for the anti-Gal IgM response could be reversed with repeated immunization, whereas the tolerant state for the IgG response was robust and resisted repeated immunization. These observations provide an important proof-of-concept that adjunct therapies can synergize with anti-CD40L Abs to tolerize B cell responses independent of their effects on T cells. This model, which does not require mixed chimerism, provides a unique opportunity for investigating the mechanism of peripheral tolerance in a clinically relevant population of carbohydrate-specific B cells.
Characterization of Drug-Specific T Cells in Phenobarbital- Induced Eruption
Authors: Hashizume Hideo , Takigawa Masahiro , Tokura Yoshiki
Published: April 2014

Phenobarbital has a high potential to elicit adverse reactions including severe skin eruptions and systemic involvements among the worldwide-prescribed drugs. Although phenobarbital hypersensitivity is thought to be mediated by T cells specific to the drug, its precise mechanism remains not fully elucidated. To characterize T cells reactive with phenobarbital, we generated drug-specific T cell clones and lines from PBMCs of patients with phenobarbital hypersensitivity showing various degrees of cutaneous and extracutaneous involvements. Although the TCR Vβ repertoire and phenotype in the T cell clones/T cell lines were heterogeneous among the patients, Vβ13.1+ and Vβ5.1+ clones or lines were raised from the individuals examined who possessed different HLA haplotypes. Histopathological examination suggested that Vβ5.1+CD8+ T cells and Vβ13.1+ T cells played a role in cutaneous and extracutaneous involvements, respectively. A Vβ13.1+CD4+ clone was found to proliferate in response to the Ag with processing-impaired, fixed APCs. Most of the clones and lines belonged to the Th2 phenotype, producing IL-4 and IL-5 but not IFN-γ upon phenobarbital stimulation. Clones/lines with Th1 or Th0 phenotypes also constituted minor populations. These observations clearly indicate the heterogeneity and a marked individual deviation of reactive T cell subsets among the patients in terms of CD4/8 phenotype, Vβ repertoire, Ag recognition pattern, and cytokine production; and thus provide evidence whereby each pathogenic T cell subset contributes to special elements of clinical presentation.
Molecular Mechanisms for Gender Differences in Susceptibility to T Cell-Mediated Autoimmune Diabetes in Nonobese Diabetic Mice
Authors: Bao Min , Yang Yang , Jun Hee-Sook , Yoon Ji-Won
Published: April 2014

Nonobese diabetic (NOD) mice spontaneously develop diabetes with a strong female prevalence; however, the mechanisms for this gender difference in susceptibility to T cell-mediated autoimmune diabetes are poorly understood. This investigation was initiated to find mechanisms by which sex hormones might affect the development of autoimmune diabetes in NOD mice. We examined the expression of IFN-γ, a characteristic Th1 cytokine, and IL-4, a characteristic Th2 cytokine, in islet infiltrates of female and male NOD mice at various ages. We found that the most significant difference in cytokine production between sexes was during the early stages of insulitis at 4 wk of age. IFN-γ was significantly higher in young females, whereas IL-4 was higher in young males. CD4+ T cells isolated from lymph nodes of female mice and activated with anti-CD3 and anti-CD28 Abs produced more IFN-γ, but less IL-4, as compared with males. Treatment of CD4+ T cells with estrogen significantly increased, whereas testosterone treatment decreased the IL-12-induced production of IFN-γ. We then examined whether the change in IL-12-induced IFN-γ production by treatment with sex hormones was due to the regulation of STAT4 activation. We found that estrogen treatment increased the phosphorylation of STAT4 in IL-12-stimulated T cells. We conclude that the increased susceptibility of female NOD mice to the development of autoimmune diabetes could be due to the enhancement of the Th1 immune response through the increase of IL-12-induced STAT4 activation by estrogen.
Cutting Edge: A-Kinase Anchor Proteins Are Involved in Maintaining Resting T Cells in an Inactive State
Authors: Williams Richard O.
Published: April 2014

A-kinase anchor proteins (AKAPs) target protein kinase A (PKA) to different subcellular locations and are thought to play important roles in the cAMP signaling pathway. The aims of this study were to determine whether T cells express AKAPs and, if so, to establish their physiological significance. CD4+ T cells were found to express eight AKAPs. Disruption of the AKAP-PKA interaction caused high levels of IL-2, IL-4, IL-5, and IFN-γ production in the absence of stimulation via CD3ε and CD28 molecules. Disruption of the AKAP-PKA interaction acted synergistically with suboptimal doses of Ag in boosting proliferative responses of T cells. Finally, disruption of the AKAP-PKA interaction rendered T cells insensitive to cAMP-elevating agents. It was concluded that AKAPs, through their association with PKA, are involved in maintaining T cell homeostasis and in regulating the sensitivity of T cells to incoming cAMP signals.
Ligation of HLA Class I Molecules on Endothelial Cells Induces Phosphorylation of Src, Paxillin, and Focal Adhesion Kinase in an Actin-Dependent Manner
Authors: Jin Yi-Ping , Singh Ram Pyare , Du Ze-Ying , Rajasekaran Ayyappan K. , Rozengurt Enrique , Reed Elaine F.
Published: April 2014

The development of chronic rejection is the major limitation to long-term allograft survival. HLA class I Ags have been implicated to play a role in this process because ligation of class I molecules by anti-HLA Abs stimulates smooth muscle cell and endothelial cell proliferation. In this study, we show that ligation of HLA class I molecules on the surface of human aortic endothelial cells stimulates phosphorylation of Src, focal adhesion kinase, and paxillin. Signaling through class I stimulated Src phosphorylation and mediated fibroblast growth factor receptor (FGFR) translocation to the nucleus. In contrast, Src kinase activity was not involved in class I-mediated transfer of FGFR from cytoplasmic stores to the cell surface. Inhibition of Src protein kinase activity blocked HLA class I-stimulated tyrosine phosphorylation of paxillin and focal adhesion kinase. Furthermore, HLA class I-mediated phosphorylation of the focal adhesion proteins and FGFR expression was inhibited by cytochalasin D and latrunculin A, suggesting a role for the actin cytoskeleton in the signaling process. These findings indicate that anti-HLA Abs have the capacity to transduce activation signals in endothelial cells that may promote the development of chronic rejection.
IL-23 and IL-12 Have Overlapping, but Distinct, Effects on Murine Dendritic Cells
Authors: Belladonna Maria Laura , Renauld Jean-Christophe , Bianchi Roberta , Vacca Carmine , Fallarino Francesca , Orabona Ciriana , Fioretti Maria Cristina , Grohmann Ursula , Puccetti Paolo
Published: April 2014

IL-23 is a recently discovered heterodimeric cytokine that shares biological properties with proinflammatory cytokines. The biologically active heterodimer consists of p19 and the p40 subunit of IL-12. IL-23 has been shown to possess biological activities on T cells that are similar as well distinct from those of IL-12. We have constructed single-chain IL-23 and IL-12 fusion proteins (IL-23-Ig and IL-12-Ig) and have compared the two recombinant proteins for effects on murine dendritic cells (DC). Here we show that the IL-23-Ig can bind a significant proportion of splenic DC of both the CD8α− and CD8α+ subtypes. Furthermore, IL-23and IL-12-Ig exert biological activities on DC that are only in part overlapping. While both proteins induce IL-12 production from DC, only IL-23-Ig can act directly on CD8α+ DC to promote immunogenic presentation of an otherwise tolerogenic tumor peptide. In addition, the in vitro effects of IL-23-Ig did not appear to require IL-12Rβ2 or to be mediated by the production of IL-12. These data may establish IL-23 as a novel cytokine with major effects on APC.
Complementary Roles for CD19 and Bruton’s Tyrosine Kinase in B Lymphocyte Signal Transduction
Authors: Fujimoto Manabu , Poe Jonathan C. , Satterthwaite Anne B. , Wahl Matthew I. , Witte Owen N. , Tedder Thomas F.
Published: April 2014

CD19 and Bruton’s tyrosine kinase (Btk) may function along common signaling pathways in regulating intrinsic and B cell Ag receptor (BCR)-induced signals. To identify physical and functional interactions between CD19 and Btk, a CD19-negative variant of the A20 B cell line was isolated, and CD19-deficient (CD19−/−) and CD19-overexpressing mice with the X-linked immunodeficient (Xid; Btk) mutation were generated. In A20 cells, Btk physically associated with CD19 following BCR engagement. CD19 and Btk interactions were not required for initial Btk phosphorylation, but CD19 expression maintained Btk in an activated state following BCR engagement. In primary B cells, CD19 signaling also required downstream Btk function since CD19-induced intracellular Ca2+ ([Ca2+]i) responses were modest in Xid B cells. In addition, CD19 overexpression did not normalize the Xid phenotype and most phenotypic and functional hallmarks of CD19 overexpression were not evident in these mice. However, CD19 and Btk also regulate independent signaling pathways since their combined loss had additive inhibitory effects on BCR-induced [Ca2+]i responses and CD19 deficiency induced a severe immunodeficiency in Xid mice. Thus, CD19 expression amplifies or prolongs Btk-mediated signaling, rather than serving as a required agent for Btk activation. Consistent with this, phosphatidylinositol 3-monophosphate kinase and Akt activation were normal in CD19−/− B cells following IgM engagement, although their kinetics of activation was altered. Thus, these biochemical and compound gene dosage studies indicate that Btk activation and [Ca2+]i responses following BCR engagement are regulated through multiple pathways, including a CD19/Src family kinase-dependent pathway that promotes the longevity of Btk signaling.
Stimulation Through Intercellular Adhesion Molecule-1 Provides a Second Signal for T Cell Activation
Authors: Chirathaworn Chintana , Kohlmeier Jacob E. , Tibbetts Scott A. , Rumsey Lisa M. , Chan Marcia A. , Benedict Stephen H.
Published: April 2014

Regulation of T cell activation requires two signals. First, appropriately presented Ag in the context of MHC interacts with the T cell Ag receptor-CD3 complex. The best-studied second signal is CD28, which resides on the T cell and responds to its counter receptor, B7. A second signal also can be delivered through LFA-1 residing on the T cell, responding to its counter receptor ICAM-1 residing on a different cell. Characterization of a second signal is tied to its ability to costimulate (along with stimulation through the TCR) proliferation, IL-2 secretion, and coactivation of phosphatidylinositol 3-kinase. We examined whether ICAM-1, residing on the T cell surface, could deliver a second signal into that T cell. Costimulation through CD3 plus ICAM-1 caused increased T cell proliferation, increased expression of the activation marker CD69, increased transcription through the IL-2 regulatory region, and increased secretion of selected Th1 but not Th2 cytokines. Costimulation through CD3 plus ICAM-1 caused synergistic activation of phosphatidylinositol 3-kinase. Finally, the combination of anti-CD3 plus anti-ICAM-1 (but not anti-CD3 alone) caused prolonged proliferation of naive T cells in a manner similar to costimulation through LFA-1 or CD28. Thus, we demonstrate for the first time that ICAM-1 resident on a T cell can deliver a costimulatory signal into that T cell.
Essential Role for IL-2 in the Regulation of Antiviral Extralymphoid CD8 T Cell Responses
Authors: D’Souza Warren N. , Schluns Kimberly S. , Masopust David , Lefrançois Leo
Published: April 2014

IL-2 is a cytokine produced primarily by activated T cells and is thought to be the quintessential T cell growth factor. The precise role of IL-2 in the regulation of CD8 T cell responses to foreign Ag in vivo however remains enigmatic. Using an adoptive transfer system with IL-2- or IL-2R-deficient TCR transgenic CD8 T cells and MHC class I tetramers, we demonstrated that the expansion of antiviral CD8 T cells in secondary lymphoid tissues was IL-2 independent, whereas IL-2 played a more significant role in supporting the continued expansion of these cells within nonlymphoid tissues. Paradoxically, autocrine IL-2 negatively regulated the overall magnitude of the CD8 T cell response in nonlymphoid tissues via a Fas-independent mechanism. Furthermore, autocrine IL-2 did not regulate the contraction or memory phase of the response. These experiments identified a novel role for IL-2 in regulation of antiviral CD8 T cell responses and homeostasis in nonlymphoid tissues.
Induction of Macrophage-Inflammatory Protein-3α Gene Expression by TNF-Dependent NF-κB Activation
Authors: Sugita Shinichi , Kohno Tomoko , Yamamoto Kazuo , Imaizumi Yoshitaka , Nakajima Hisayoshi , Ishimaru Tadayuki , Matsuyama Toshifumi
Published: April 2014

Macrophage-inflammatory protein-3α (MIP-3α), also designated as liver and activation-regulated chemokine (LARC), Exodus, or CCL20, is a recently identified CC chemokine that is expected to play a crucial role in the initiation of immune responses. In this study, we describe that MIP-3α expression is under the direct control of NF-κB, a key transcription factor of immune and inflammatory responses. Overexpression of the p65/RelA subunit of NF-κB significantly increased the MIP-3α mRNA level. MIP-3α transcription was stimulated by TNF, and this stimulation was inhibited by an NF-κB inhibitor, I-κBα superrepressor. Analysis of the human MIP-3α promoter demonstrated a functional NF-κB site responsible for its expression. We also show that MIP-3α expression is induced in LPS-treated mouse livers that were primed with Propionibacterium acnes, which developed massive liver injury with infiltration of inflammatory cells. This induction was fully dependent on the TNF signaling cascade, because it was not observed in the livers of TNFR1-deficient mice. Furthermore, pretreatment with gliotoxin, an inhibitor of NF-κB activity, abrogated the P. acnes/LPS-induced MIP-3α expression of wild-type mice. These results clearly demonstrate that MIP-3α gene expression is dependent on NF-κB activity in vitro, and indicate that the TNFR1-mediated TNF signaling cascade that leads to NF-κB activation plays an essential role in MIP-3α expression in the murine liver injury model.
Early Response to Rotavirus Infection Involves Massive B Cell Activation
Authors: Blutt Sarah E. , Warfield Kelly L. , Lewis Dorothy E. , Conner Margaret E.
Published: April 2014

Rotavirus is an acute enteric pathogen which induces severe diarrhea in infants and children. To determine the immune response to rotavirus in vivo, we used a mouse model of rotavirus infection. We observed dramatic increases in the sizes of both Peyer’s patches and mesenteric lymph nodes, but not spleen, between 1 and 6 days after infection with a homologous strain of murine rotavirus, EC wild type. Histological analysis showed large increases in the numbers of lymphocytes in these same tissues in rotavirus-infected mice. Flow cytometric analysis confirmed the increase in numbers of lymphocytes and revealed a large increase in the percentage of activated B, but not T, lymphocytes in both Peyer’s patches and mesenteric lymph nodes of rotavirus-infected mice compared with control mice. Fragment cultures from these tissues established at 3–4 days postinfection contain rotavirus-specific IgM but not IgA Ab. A similar degree of lymphoid hyperplasia and percentage of activated B cells were observed in rotavirus-infected TCR knockout mice. Taken together, our findings show that rotavirus infection, in the context of a normal immune response, induces a large increase in the percentages of activated B cells in the absence of any detectable increase in the percentage of activated T cells, implicating a T cell-independent B cell response as the primary mechanism for initial rotavirus clearance.
CCR3 Is Required for Tissue Eosinophilia and Larval Cytotoxicity After Infection withTrichinella spiralis
Authors: Gurish Michael F. , Humbles Alison , Tao Hong , Finkelstein Stella , Boyce Joshua A. , Gerard Craig , Friend Daniel S. , Austen K. Frank
Published: April 2014

The CCR3 binds at least seven different CC chemokines and is expressed on eosinophils, mast cells (MC), and a subset of Th cells (Th2) that generate cytokines implicated in mucosal immune responses. Using mice with a targeted disruption of CCR3 (CCR3−/−) and their +/+ littermates, we investigated the role of CCR3 in the amplification of tissue eosinophilia and MC hyperplasia in the mouse after infection with Trichinella spiralis. In CCR3−/− mice, eosinophils are not recruited to the jejunal mucosa after infection and are not present in the skeletal muscle adjacent to encysting larvae. In addition, the number of cysts in the skeletal muscle is increased and the frequency of encysted larvae exhibiting necrosis is reduced. The CCR3−/− mice exhibit the expected MC hyperplasia in the jejunum and caecum and reject the adult worms from the small intestine at a normal rate. This study is consistent with distinct functions for MC (adult worm expulsion) and eosinophils (toxicity to larvae) in immunity to a helminth, T. spiralis, and defines the essential requirement for CCR3 in eosinophil, but not MC recruitment to tissues.
Multiple Mechanisms Compensate to Enhance Tumor-Protective CD8+ T Cell Response in the Long-Term Despite Poor CD8+ T Cell Priming Initially: Comparison Between an Acute Versus a Chronic Intracellular Bacterium Expressing a Model Antigen
Authors: Dudani Renu , Chapdelaine Yvan , Faassen Henk van , Smith Dean K. , Shen Hao , Krishnan Lakshmi , Sad Subash
Published: April 2014

We evaluated CD8+ T cell responses against the dominant CTL epitope, OVA257–264, expressed by an acute (Listeria monocytogenes (LM) OVA) vs a chronic pathogen (Mycobacterium bovis bacillus Calmette-Guérin (BCG) OVA) to reveal the influence on CD8+ T cell memory and consequent protection against a challenge with OVA-expressing tumor cells. Infection with lower doses of both pathogens resulted in stronger bacterial growth but weaker T cell memory indicating that memory correlates with pathogen dose but not with bacterial expansion. The CD8+ T cell response induced by LM-OVA was helper T cell-independent and was characterized by a rapid effector response followed by a rapid, but massive, attrition. In contrast, BCG-OVA induced a delayed and weak response that was compensated for by a longer effector phase and reduced attrition. This response was partly dependent on CD4+ T cells. CD8+ T cell response induced by BCG-OVA, but not LM-OVA, was highly dependent on pathogen persistence to compensate for the weak initial CD8+ T cell priming. Despite a stronger initial T cell response with LM-OVA, BCG-OVA provided more effective tumor (B16OVA) control at both local and distal sites due to the induction of a persistently activated acquired, and a more potent innate, immunity.
Prothrombin Kringle-2 Activates Cultured Rat Brain Microglia
Authors: Ryu Jooyoung , Min Kyoung-jin , Rhim Tai Youn , Kim Tae Hyong , Pyo Hankyoung , Jin Byungkwan , Kim Seung-Up , Jou Ilo , Kim Soung Soo , Joe Eun-hye
Published: April 2014

Microglia, the major immune effector cells in the CNS, become activated when the brain suffers injury. In this study, we observed that prothrombin, a zymogen of thrombin, induced NO release and mRNA expression of inducible NO synthase, IL-1β, and TNF-α in rat brain microglia. The effect of prothrombin was independent of the protease activity of thrombin since hirudin, a specific inhibitor of thrombin, did not inhibit prothrombin-induced NO release. Furthermore, factor Xa enhanced the effect of prothrombin on microglial NO release. Kringle-2, a domain of prothrombin distinct from thrombin, mimicked the effect of prothrombin in inducing NO release and mRNA expression of inducible NO synthase, IL-1β, and TNF-α. Prothrombin and kringle-2 both triggered the same intracellular signaling pathways. They both activated mitogen-activated protein kinases and NF-κB in a similar pattern. NO release stimulated by either was similarly reduced by inhibitors of the extracellular signal-regulated kinase pathway (PD98059), p38 (SB203580), NF-κB (N-acetylcysteine), protein kinase C (Go6976, bisindolylmaleimide, and Ro31-8220), and phospholipase C (D609 and U73122). These results suggest that prothrombin can activate microglia, and that, in addition to thrombin, kringle-2 is a domain of prothrombin independently capable of activating microglia.
HER-2/neuand hTERT Cryptic Epitopes as Novel Targets for Broad Spectrum Tumor Immunotherapy
Authors: Scardino Antonio , Gross David-Alexandre , Alves Pedro , Schultze Joachim L. , Graff-Dubois Stéphanie , Faure Olivier , Tourdot Sophie , Chouaib Salem , Nadler Lee M. , Lemonnier François A. , Vonderheide Robert H. , Cardoso Angelo A. , Kosmatopoulos Kostas
Published: April 2014

Tolerance to tumor-nonmutated self proteins represents a major obstacle for successful cancer immunotherapy. Since this tolerance primarily concerns dominant epitopes, we hypothesized that targeting cryptic epitopes that have a low affinity for HLA could be an efficient strategy to breach the tolerance to tumor Ags. Using the P1Y heteroclitic peptide approach, we identified low affinity cryptic HLA-A*0201-restricted epitopes derived from two widely expressed tumor Ags, HER-2/neu and hTERT. The P1Y variants of four HER-2/neu (neu391, neu402, neu466, neu650)- and two hTERT (hTERT572 and hTERT988)-derived low affinity peptides exhibited strong affinity for HLA-A*0201 and stimulated specific CTL from healthy donor PBMCs. These CTL specifically recognized HER-2/neu- and hTERT-expressing tumor cells of various histological origins. In vivo studies showed that HLA-A*0201 transgenic HHD mice vaccinated with the P1Y variant peptides generated CTL that specifically lysed Ag-expressing tumor cells, thus recognizing the cognate endogenous Ags. These results suggest that heteroclitic variants of low affinity, cryptic epitopes of widely expressed tumor Ags may serve as valid tools for tumor immunotherapy.
Functional Heterogeneity of Vaccine-Induced CD8+ T Cells
Authors: Monsurrò Vladia , Nagorsen Dirk , Wang Ena , Provenzano Maurizio , Dudley Mark E. , Rosenberg Steven A. , Marincola Francesco M.
Published: April 2014

The functional status of circulating vaccine-induced, tumor-specific T cells has been questioned to explain their paradoxical inability to inhibit tumor growth. We enumerated with HLA-A*0201/peptide tetramers (tHLA) vaccine-elicited CD8+ T cell precursor frequency among PBMC in 13 patients with melanoma undergoing vaccination with the HLA-A*0201-associated gp100:209–217(210 M) epitope. T cell precursor frequency increased from undetectable to 12,400 ± 3,600 × 106 CD8+ T cells after vaccination and appeared heterogeneous according to previously described functional subtypes: CD45RA+CD27+ (14 ± 2.6% of tHLA-staining T cells), naive; CD45RA−CD27+ (14 ± 3.2%), memory; CD45RA+CD27− (43 ± 6%), effector; and CD45RA−CD27− (30 ± 4.1%), memory/effector. The majority of tHLA+CD8+ T cells displayed an effector, CD27− phenotype (73%). However, few expressed perforin (17%). Epitope-specific in vitro stimulation (IVS) followed by 10-day expansion in IL-2 reversed this phenotype by increasing the number of perforin+ (84 ± 3.6%; by paired t test, p < 0.001) and CD27+ (from 28 to 67%; by paired t test, p = 0.01) tHLA+ T cells. This conversion probably represented a change in the functional status of tHLA+ T cells rather than a preferential expansion of a CD27+ (naive and/or memory) PBMC, because it was reproduced after IVS of a T cell clone bearing a classic effector phenotype (CD45RA+CD27−). These findings suggest that circulating vaccine-elicited T cells are not as functionally active as inferred by characterization of IVS-induced CTL. In addition, CD45RA/CD27 expression may be more informative about the status of activation of circulating T cells than their status of differentiation.
Humoral Autoimmunity to Basement Membrane Antigens Is Regulated in C57BL/6 and MRL/MpJ Mice Transgenic for Anti-Laminin Ig Receptors
Authors: Rudolph Earl H. , Congdon Kendra L. , Sackey Faustina N. A. , Fitzsimons Muriel M. , Foster Mary H.
Published: April 2014

Basement membrane proteins are targeted in organ-limited and systemic autoimmune nephritis, yet little is known about the origin or regulation of immunity to these complex extracellular matrices. We used mice transgenic for a nephrotropic systemic lupus erythematosus (SLE) Ig H chain to test the hypothesis that humoral immunity to basement membrane is actively regulated. The LamH-Cμ Ig H chain transgene combines with diverse L chains to produce nephrotropic Ig reactive with murine laminin α1. To determine the fate of transgene-bearing B cells in vivo, transgenic mice were outcrossed onto nonautoimmune B6 and SLE-prone MRL backgrounds and exposed to potent mitogen or Ag in adjuvant. In this work we demonstrate that transgenic autoantibodies are absent in serum from M6 and M29 lineage transgenic mice and transgenic B cells hypoproliferate and fail to increase Ig production upon exposure to endotoxin or when subjected to B cell receptor cross-linking. Administration of LPS or immunization with autologous or heterologous laminin, maneuvers that induce nonoverlapping endogenous anti-laminin IgG responses, fails to induce a transgenic anti-laminin response. The marked reduction in splenic B cell number suggests that selected LamH-Cμ H chain and endogenous L chain combinations generate autospecificities that lead to B cell deletion. It thus appears that SLE-like anti-laminin B cells have access to and engage a tolerizing self-Ag in vivo. Failure to induce autoimmunity by global perturbations in immune regulation introduced by the MRL autoimmune background and exposure to potent environmental challenge suggests that humoral immunity to nephritogenic basement membrane epitopes targeted in systemic autoimmunity is tightly regulated.
Cutting Edge: Th2 Response Induction by Dendritic Cells: A Role for CD40
Authors: MacDonald Andrew S. , Straw Amy D. , Dalton Nicole M. , Pearce Edward J.
Published: April 2014

We investigated the influence of dendritic cell (DC) CD40 expression on Th2 and Th1 development by in vivo transfer of Ag-pulsed bone marrow-derived DC generated from wild-type (WT) or CD40−/− mice. Contrary to expectation, CD40−/− DC primed with Ag that inherently induce a Th2 response (soluble egg Ag from Schistosoma mansoni) failed to induce a Th2 response or any compensatory Th1 response, whereas CD40−/−DC primed with Ag that inherently induce a Th1 response (Propionibacterium acnes) generated a competent Th1 response. Thus, DC expression of CD40 is a prerequisite for initiation of Th2, but not Th1, responses by these Ag. Consistent with this, CD154−/− mice, unlike WT mice, failed to mount a Th2 response when directly injected with schistosome eggs but mounted a normal Th1 response after challenge with P. acnes. CD40-CD154 interaction can therefore play a major role in Th2 response induction.
Cutting Edge: Negative Regulation of Immune Synapse Formation by Anchoring Lipid Raft to Cytoskeleton Through Cbp-EBP50-ERM Assembly
Authors: Itoh Katsuhiko , Sakakibara Masahiro , Yamasaki Sho , Takeuchi Arata , Arase Hisashi , Miyazaki Masaru , Nakajima Nobuyuki , Okada Masato , Saito Takashi
Published: April 2014

Ag recognition by T lymphocytes induces immune synapse formation and recruitment of signaling molecules into a lipid raft. Cbp/PAG is a Csk-associated membrane adapter protein exclusively localized in a lipid raft. We identified NHERF/EBP50 as a Cbp-binding molecule, which connects the membrane raft and cytoskeleton by binding to both Cbp through its PDZ domain and ezrin-radixin-moesin through the C terminus. Overexpression of Cbp reduced the mobility of the raft on the cell surface of unstimulated T cells and prevented synapse formation and subsequent T cell activation, whereas a mutant incapable of EBP50 binding restored both synapse formation and activation. These results suggest that anchoring of lipid raft to the cytoskeleton through Cbp-EBP50-ezrin-radixin-moesin assembly regulates membrane dynamism for synapse formation and T cell activation.
Indirect IL-4 Pathway in Type 1 Immunity
Authors: Karulin Alexey Y. , Hesse Maike D. , Yip Hualin C. , Lehmann Paul V.
Published: April 2014

Recall Ag-specific IL-4 was detected in the spleen and in the blood, but not in lymph nodes of mice in which polarized type 1 immunity was induced. This IL-4 was not produced by T cells, but soluble factors secreted by the recall Ag-activated T cells, including IL-3, triggered cells of the innate immune system, primarily mast cells, to secrete IL-4. This notion has profound implications for immunodiagnostics: the detection of apparently recall Ag-specific IL-4 does not necessarily reflect the presence of Th2 or Th0 memory T cells with long-term cytokine commitment as is of interest for assessing adoptive immunity. We found that in vivo the indirect IL-4 pathway did not suffice to trigger IgE isotype switching, but promoted IgG1 production and inhibited type 1 T cell differentiation. Therefore, the indirect IL-4 pathway can explain partial type 2 immune response phenotypes in vivo in face of unipolar Th1 T cell immunity. The representation of mast cells in different tissues may explain why immune responses in certain organs are more type 2 biased. Therefore, the indirect pathway of IL-4 production represents a novel type of interaction between the innate and the adoptive immune system that can contribute to the outcome of host defense and immune pathology.
Tissue Expression of Human Toll-Like Receptors and Differential Regulation of Toll-Like Receptor mRNAs in Leukocytes in Response to Microbes, Their Products, and Cytokines
Authors: Zarember Kol A. , Godowski Paul J.
Published: April 2014

Members of the Toll-like receptor (TLR) family mediate dorsoventral patterning and cellular adhesion in insects as well as immune responses to microbial products in both insects and mammals. TLRs are characterized by extracellular leucine-rich repeat domains and an intracellular signaling domain that shares homology with cytoplasmic sequences of the mammalian IL-1 receptor and plant disease resistance genes. Ten human TLRs have been cloned as well as RP105, a protein similar to TLR4 but lacking the intracellular signaling domain. However, only five TLRs have described functions as receptors for bacterial products (e.g., LPS, lipoproteins). To identify potential sites of action, we used quantitative real-time RT-PCR to examine systematically the expression of mRNAs encoding all known human TLRs, RP105, and several other proteins important in TLR functions (e.g., MD-1, MD-2, CD14, MyD88). Most tissues tested expressed at least one TLR, and several expressed all (spleen, peripheral blood leukocytes). Analysis of TLR expression in fractionated primary human leukocytes (CD4+, CD8+, CD19+, monocytes, and granulocytes) indicates that professional phagocytes express the greatest variety of TLR mRNAs although several TLRs appear more restricted to B cells, suggesting additional roles for TLRs in adaptive immunity. Monocyte-like THP-1 cells regulate TLR mRNA levels in response to a variety of stimuli including phorbol esters, LPS, bacterial lipoproteins, live bacteria, and cytokines. Furthermore, addition of Escherichia coli to human blood ex vivo caused distinct changes in TLR expression, suggesting that important roles exist for these receptors in the establishment and resolution of infections and inflammation.
Potent CD4+ T Cell Responses Elicited by a Bicistronic HIV-1 DNA Vaccine Expressing gp120 and GM-CSF
Authors: Barouch Dan H. , Santra Sampa , Tenner-Racz Klara , Racz Paul , Kuroda Marcelo J. , Schmitz Joern E. , Jackson Shawn S. , Lifton Michelle A. , Freed Dan C. , Perry Helen C. , Davies Mary-Ellen , Shiver John W. , Letvin Norman L.
Published: April 2014

Virus-specific CD4+ T cell responses have been shown to play a critical role in controlling HIV-1 replication. Candidate HIV-1 vaccines should therefore elicit potent CD4+ as well as CD8+ T cell responses. In this report we investigate the ability of plasmid GM-CSF to augment CD4+ T cell responses elicited by an HIV-1 gp120 DNA vaccine in mice. Coadministration of a plasmid expressing GM-CSF with the gp120 DNA vaccine led to only a marginal increase in gp120-specific splenocyte CD4+ T cell responses. However, immunization with a bicistronic plasmid that coexpressed gp120 and GM-CSF under control of a single promoter led to a dramatic augmentation of vaccine-elicited CD4+ T cell responses, as measured by both cellular proliferation and ELISPOT assays. This augmentation of CD4+ T cell responses was selective, since vaccine-elicited Ab and CD8+ T cell responses were not significantly changed by the addition of GM-CSF. A 100-fold lower dose of the gp120/GM-CSF bicistronic DNA vaccine was required to elicit detectable gp120-specific splenocyte proliferative responses compared with the monocistronic gp120 DNA vaccine. Consistent with these findings, i.m. injection of the gp120/GM-CSF bicistronic DNA vaccine evoked a more extensive cellular infiltrate at the site of inoculation than the monocistronic gp120 DNA vaccine. These results demonstrate that bicistronic DNA vaccines containing GM-CSF elicit remarkably potent CD4+ T cell responses and suggest that optimal Th cell priming requires the precise temporal and spatial codelivery of Ag and GM-CSF.
The Receptor for Heat Shock Protein 60 on Macrophages Is Saturable, Specific, and Distinct from Receptors for Other Heat Shock Proteins
Authors: Habich Christiane , Baumgart Karina , Kolb Hubert , Burkart Volker
Published: April 2014

Previous studies have shown that human heat shock protein (hsp) 60 elicits a strong proinflammatory response in cells of the innate immune system with CD14, Toll-like receptor (TLR) 2, and TLR4 as mediators of signaling, but probably not of binding. In the present study, we directly demonstrate binding of hsp60 to the macrophage surface and find the binding receptor for hsp60 different from the previously described common receptor for several other heat shock proteins, including hsp70, hsp90, and gp96. Fluorescence-labeled human hsp60 bound to cell surfaces of the murine macrophage lines J774 A.1 and RAW264.7 and to mouse bone marrow-derived macrophages. By flow cytometry, we could demonstrate for the first time that hsp60 binding to macrophages occurred at submicromolar concentrations, is saturable, and can be competed by unlabeled hsp60, but not by unrelated proteins, thus confirming the classic characteristics of specific ligand-receptor interactions. Binding of hsp60 at 4°C was followed by endocytosis at 37°C. Hsp60 binding to macrophages could not be competed by excess hsp70, hsp90, or gp96, all of which share the α2-macroglobulin receptor as binding site. Hsp60 binding occurred in the absence of surface TLR4. However, no cytokine response was induced by hsp60 in TLR4-deficient macrophages. We conclude that hsp60 binds to a stereo-specific receptor on macrophages, and that different surface molecules are engaged in binding and signal transduction. Furthermore, the binding site for hsp60 is separate from the common receptor for hsp70, hsp90, and gp96, which suggests an independent role of hsp60 as danger Ag and in immunoregulation.