bluuebunny/crossref_metadata_2025 huggingface dataset:
Divergence of Apoptosis-Inducing and Preventing Signals in Bacteria-Faced Macrophages Through Myeloid Differentiation Factor 88 and IL-1 Receptor-Associated Kinase Members
Authors: Ruckdeschel Klaus , Mannel Oliver , Schröttner Percy
Published: April 2014

The induction of apoptosis in host cells is a common strategy by which pathogenic bacteria interfere with the host immune response. The Yersinia enterocolitica outer protein P (YopP) inhibits activation of transcription factor NF-κB in macrophages, which suppresses NF-κB-dependent antiapoptotic activities. The simultaneous initiation of proapoptotic signaling by yersiniae infection or LPS treatment results in macrophage apoptosis. In this study, we used YopP as a tool to dissect survival- and death-inducing pathways in bacteria-faced macrophages. We cotransfected J774A.1 macrophages with expression plasmids for YopP and dominant-negative mutants of signal transmitters of the NF-κB cascade downstream from the LPS receptor complex. Dominant-negative myeloid differentiation factor 88 (MyD88) or IL-1R-associated kinase (IRAK) 2 diminished LPS-induced apoptosis in YopP-transfected macrophages, suggesting implication of MyD88 and IRAK2 in signaling cell death. In contrast, dominant-negative IRAK1 and TNFR-associated factor 6 (TRAF6) did not provide protection, but augmented LPS-mediated apoptosis in the absence of YopP, which indicates roles of IRAK1 and TRAF6 in the antiapoptotic signal relay of the NF-κB cascade. The distinct functions of IRAK members in macrophage survival were reflected by opposing effects of dominant-negative IRAK1 and IRAK2 on Y. enterocolitica-mediated apoptosis. Yersiniae- and LPS-dependent cell death were substantially attenuated by a specific caspase-8 inhibitory peptide or by dominant negative Fas-associated death domain protein (FADD). This suggests, that Yersinia-induced apoptosis involves a proapoptotic signal relay through MyD88 and IRAK2, which potentially targets the Fas-associated death domain protein/caspase-8 apoptotic pathway, whereas IRAK1 and TRAF6 counteract the bacteria-induced cytotoxic response by signaling macrophage survival.
Enlargement of Secretory Vesicles by Protein Tyrosine Phosphatase PTP-MEG2 in Rat Basophilic Leukemia Mast Cells and Jurkat T Cells
Authors: Wang Xiaodong , Huynh Huong , Gjörloff-Wingren Anette , Monosov Edvard , Stridsberg Mats , Fukuda Minoru , Mustelin Tomas
Published: April 2014

Stimulus-induced secretion of bioactive polypeptides is a fundamental aspect of the immune system. Secretory proteins are synthesized in the endoplasmic reticulum and are transported through the Golgi apparatus to the trans-Golgi network, where they are sorted into transport vesicles that bud off and fuse into condensing vacuoles, which subsequently undergo an editing and concentration process to become mature secretory vesicles. In this study, we report that the PTP-MEG2 protein tyrosine phosphatase is located on these vesicles in mast cells. Expression of PTP-MEG2 caused a striking enlargement of these vesicles in both rat basophilic leukemia mast cells and Jurkat T leukemia cells into giant vesicles with diameters of up to several micrometers. The fused vesicles did not acquire markers for other compartments and were adjacent to the trans-Golgi network, contained carboxypeptidase E, chromogranin C, and IL-2, and had an electron-dense core typical of secretory vesicles. Expression of PTP-MEG2 also caused a reduction in the secretion of IL-2 from stimulated Jurkat cells. The effects of PTP-MEG2 on secretory vesicles required the catalytic activity of PTP-MEG2 and was rapidly reversed by pervanadate. We propose that PTP-MEG2 represents a novel connection between tyrosine dephosphorylation and the regulation of secretory vesicles in hematopoietic cells.
TNF Regulates Chemokine Induction Essential for Cell Recruitment, Granuloma Formation, and Clearance of Mycobacterial Infection
Authors: Roach Daniel R. , Bean Andrew G. D. , Demangel Caroline , France Malcolm P. , Briscoe Helen , Britton Warwick J.
Published: April 2014

Host immunity to mycobacterial infection is dependent on the activation of T lymphocytes and their recruitment with monocytes to form granulomas. These discrete foci of activated macrophages and lymphocytes provide a microenvironment for containing the infection. The cytokine, TNF, is essential for the formation and maintenance of granulomas, but the mechanisms by which TNF regulates these processes are unclear. We have compared the responses of TNF-deficient (TNF−/−) and wild-type C57BL/6 mice to infection with Mycobacterium smegmatis, a potent inducer of TNF, and virulent Mycobacterium tuberculosis to delineate the TNF-dependent and -independent components of the process. The initial clearance of M. smegmatis was TNF independent, but TNF was required for the early expression of mRNA encoding C-C and C-X-C chemokines and the initial recruitment of CD11b+ macrophages and CD4+ T cells to the liver during the second week of infection. Late chemokine expression and cell recruitment developed in TNF−/− mice associated with enhanced Th1-like T cell responses and mycobacterial clearance, but recruited leukocytes did not form tight granulomas. Infection of TNF−/− mice with M. tuberculosis also resulted in an initial delay in chemokine induction and cellular recruitment to the liver. Subsequently, increased mRNA expression was evident in TNF−/− mice, but the loosely associated lymphocytes and macrophages failed to form granulomas and prevent progressive infection. Therefore, TNF orchestrates early induction of chemokines and initial leukocyte recruitment, but has an additional role in the aggregation of leukocytes into functional granulomas capable of controlling virulent mycobacterial infection.
Rapid IL-4 Production by Leishmania Homolog of Mammalian RACK1-Reactive CD4+ T Cells in Resistant Mice Treated Once with Anti-IL-12 or -IFN-γ Antibodies at the Onset of Infection with Leishmania major Instructs Th2 Cell Development, Resulting in Nonhealing Lesions
Authors: Launois Pascal , Gumy Alain , Himmelrich Hayo , Locksley Richard M. , Röcken Martin , Louis Jacques A.
Published: April 2014

Rapid production of IL-4 by Leishmania homolog of mammalian RACK1 (LACK)-reactive CD4+ T cells expressing the Vβ4-Vα8 TCR chains has been shown to drive aberrant Th2 cell development and susceptibility to Leishmania major in BALB/c mice. In contrast, mice from resistant strains fail to express this early IL-4 response. However, administration of either anti-IL-12 or -IFN-γ at the initiation of infection allows the expression of this early IL-4 response in resistant mice. In this work we show that Leishmania homolog of mammalian RACK1-reactive CD4+ T cells also expressing the Vβ4-Vα8 TCR chains are the source of the early IL-4 response to L. major in resistant mice given anti-IL-12 or -IFN-γ Abs only at the onset of infection. Strikingly, these cells were found to be required for the reversal of the natural resistance of C57BL/6 mice following a single administration of anti-IL-12 or -IFN-γ Abs. Together these results suggest that a deficiency in mechanisms capable of down-regulating the early IL-4 response to L. major contributes to the exquisite susceptibility of BALB/c mice to L. major.
Mycobacterium tuberculosisInduces Differential Cytokine Production from Dendritic Cells and Macrophages with Divergent Effects on Naive T Cell Polarization
Authors: Hickman Somia Perdow , Chan John , Salgame Padmini
Published: April 2014

Th1-mediated cellular responses are important for protection in tuberculosis. However, the mechanisms and APC types responsible for initiating Th1 responses are not well understood. These studies show that macrophages and dendritic cells, albeit both being APC, respond differently following Mycobacterium tuberculosis infection and thereby have different consequences for the development of naive T cells. We report that M. tuberculosis-infected dendritic cells bias the polarization of OVA peptide-specific naive transgenic T cells to the Th1 phenotype, and, in contrast, in the presence of infected macrophages naive T cells do not develop a Th1 phenotype. Comparison of the cytokine profile expressed by the infected dendritic cells and macrophages revealed several differences, the most striking being that infected macrophages did not express the Th1-promoting cytokine IL-12. These studies also show that IL-10 is responsible for the failure of IL-12 production by M. tuberculosis-infected macrophages, and that the effects of IL-10 can be overcome by IFN-γ priming. We speculate that the observed difference in response of the two APC types to M. tuberculosis infection may be a reflection of their respective roles in immune initiation and granuloma regulation.
Impaired Th2 Development and Increased Mortality DuringSchistosoma mansoniInfection in the Absence of CD40/CD154 Interaction
Authors: MacDonald Andrew S. , Patton Elisabeth A. , La Flamme Anne C. , Araujo Maria I. , Huxtable Clive R. , Bauman Beverley , Pearce Edward J.
Published: April 2014

The role of CD40/CD154 interaction during infection has primarily focused on pathogens that drive inflammatory Th1 responses. In this study, we show that CD40/CD154 interaction is a fundamental requirement for Th2 response development to the parasitic helminth Schistosoma mansoni. Compared with infected wild-type mice, greatly reduced levels of Th2-associated cytokines were measured both in vitro and in vivo, and no IgE or IgG1 was detected in infected CD154−/− mice. In the absence of an overt Th2 response, no exaggerated Th1 response was mounted by CD154−/− mice. Infected CD154−/− mice suffered severe morbidity and mortality, even though parasitemias in wild-type and CD154−/− mice did not differ significantly. These data indicate that CD40/CD154 interaction is required to allow development of a Th2-dominated immune response to S. mansoni and support the view that failure to develop such a response can have fatal consequences.
Profound Differences in Leukocyte-Endothelial Cell Responses to Lipopolysaccharide Versus Lipoteichoic Acid
Authors: Yipp Bryan G. , Andonegui Graciela , Howlett Christopher J. , Robbins Stephen M. , Hartung Thomas , Ho May , Kubes Paul
Published: April 2014

We have investigated the effects of LPS from Escherichia coli, lipoteichoic acid (LTA), and peptidoglycan (PepG) from Staphylococcus aureus, and live S. aureus on leukocyte-endothelial interactions in vivo using intravital microscopy to visualize muscle microvasculature. Systemic vs local administration of LPS induced very different responses. Local administration of LPS into muscle induced significant leukocyte rolling, adhesion, and emigration in postcapillary venules at the site of injection. LPS given systemically dramatically dropped circulating leukocyte counts and increased neutrophils in the lung. However, the drop in circulating leukocytes was not associated with leukocyte sequestration to the site of injection (peritoneum) nor to peripheral microvessels in muscles. Unlike LPS, various preparations of LTA had no systemic and very minor local effect on leukocyte-endothelial interactions, even at high doses and for prolonged duration. LPS, but not LTA, potently activated human endothelium to recruit leukocytes under flow conditions in vitro. Endothelial adhesion molecule expression was also increased extensively with LPS, but not LTA. Interestingly, systemic administration of live S. aureus induced leukocyte-endothelial cell responses similar to LPS. PepG was able to induce leukocyte-endothelial interactions in muscle and peritoneum, but had no effect systemically (no increase in neutrophils in lungs and no decrease in circulating neutrophil counts). These results demonstrate that: 1) LPS has potent, but divergent local and systemic effects on leukocyte-endothelial interactions; 2) S. aureus can induce a systemic response similar to LPS, but this response is unlikely to be due to LTA, but more likely to be mediated in part by PepG.
Differential Roles of CC Chemokine Ligand 2/Monocyte Chemotactic Protein-1 and CCR2 in the Development of T1 Immunity
Authors: Traynor Tim R. , Herring Amy C. , Dorf Martin E. , Kuziel William A. , Toews Galen B. , Huffnagle Gary B.
Published: April 2014

CCR2 and its major ligand, chemokine ligand 2 (CCL2)/monocyte chemotactic protein-1, have been found to influence T1/T2 immune response polarization. Our objective was to directly compare the roles of CCR2 and CCL2 in T1/T2 immune response polarization using a model of pulmonary Cryptococcus neoformans infection. Either deletion of CCR2 or treatment of wild-type mice with CCL2 neutralizing Ab produced significant and comparable reductions in macrophage and T cell recruitment into the lungs following infection. Both CCL2 neutralization and CCR2 deficiency resulted in significantly diminished IFN-γ production, and increased IL-4 and IL-5 production by lung leukocytes (T1 to T2 switch), but only CCR2 deficiency promoted pulmonary eotaxin production and eosinophilia. In the lung-associated lymph nodes (LALN), CCL2-neutralized mice developed Ag-specific IFN-γ-producing cells, while CCR2 knockout mice did not. LALN from CCR2 knockout mice also had fewer MHCII+CD11c+ and MHCII+CD11b+ cells, and produced significantly less IL-12p70 and TNF-α when stimulated with heat-killed yeast than LALN from wild-type or CCL2-neutralized mice, consistent with a defect in APC trafficking in CCR2 knockout mice. Neutralization of CCL2 in CCR2 knockout mice did not alter immune response development, demonstrating that the high levels of CCL2 in these mice did not play a role in T2 polarization. Therefore, CCR2 (but not CCL2) is required for afferent T1 development in the lymph nodes. In the absence of CCL2, T1 cells polarize in the LALN, but do not traffic from the lymph nodes to the lungs, resulting in a pulmonary T2 response.
Mitogen-Activated Protein Kinase-Activated Protein Kinase 2-Deficient Mice Show Increased Susceptibility to Listeria monocytogenes Infection
Authors: Lehner Martin D. , Schwoebel Frank , Kotlyarov Alexey , Leist Marcel , Gaestel Matthias , Hartung Thomas
Published: April 2014

Mitogen-activated protein kinase-activated protein kinase 2 (MK2) is one of several kinases activated through direct phosphorylation by p38 mitogen-activated protein kinase. MK2 regulates LPS-induced TNF mRNA translation, and targeted mutation of the MK2 gene renders mice more resistant to d-galactosamine plus LPS-induced liver damage. In the present study, we investigated the role of MK2 in immune defense against Listeria monocytogenes infection. MK2-deficient mice displayed diminished resistance to L. monocytogenes due to impaired control of bacterial growth. The increase in bacterial load in MK2−/− mice was associated with normal levels of IL-1β, IL-6, and IFN-γ, whereas TNF production was strongly attenuated. In line, MK2-deficient bone marrow-derived macrophages showed impaired release of TNF, but not of IL-1β, in response to various bacterial stimuli in addition to decreased phagocytosis of fluorescence-labeled bacteria. Furthermore, spleen cells from MK2−/− mice displayed diminished IFN-γ synthesis after stimulation with L. monocytogenes. In contrast, MK2 deficiency had no effect on macrophage generation of NO or on oxidative burst activity in response to L. moocytogenes. These results indicate an essential role of MK2 in host defense against intracellular bacteria probably via regulation of TNF and IFN-γ production required for activation of antibacterial effector mechanisms.
The Role of IL-18 in Blood-Stage Immunity Against Murine MalariaPlasmodium yoelii 265andPlasmodium berghei ANKA
Authors: Singh Ram Pyare , Kashiwamura Shin-ichiro , Rao Prakash , Okamura Haruki , Mukherjee Askok , Chauhan Virander Singh
Published: April 2014

A possible protective role of IL-18 in host defense against blood-stage murine malarial infection was studied in BALB/c mice using a nonlethal strain, Plasmodium yoelii 265, and a lethal strain, Plasmodium berghei ANKA. Infection induced an increase in mRNA expression of IL-18, IL-12p40, IFN-γ, and TNF-α in the case of P. yoelii 265 and an increase of IL-18, IL-12p40, and IFN-γ in the case of P. berghei ANKA. The timing of mRNA expression of IL-18 in both cases was consistent with a role in the induction of IFN-γ protein expression. Histological examination of spleen and liver tissues from infected controls treated with PBS showed poor cellular inflammatory reaction, massive necrosis, a large number of infected parasitized RBCs, and severe deposition of hemozoin pigment. In contrast, IL-18-treated infected mice showed massive infiltration of inflammatory cells consisting of mononuclear cells and Kupffer cells, decreased necrosis, and decreased deposition of the pigment hemozoin. Treatment with rIL-18 increased serum IFN-γ levels in mice infected with both parasites, delayed onset of parasitemia, conferred a protective effect, and thus increased survival rate of infected mice. Administration of neutralizing anti-IL-18 Ab exacerbated infection, impaired host resistance and shortened the mean survival of mice infected with P. berghei ANKA. Furthermore, IL-18 knockout mice were more susceptible to P. berghei ANKA than were wild-type C57BL/6 mice. These data suggest that IL-18 plays a protective role in host defense by enhancing IFN-γ production during blood-stage infection by murine malaria.
The Adapter Molecule Gab2 Regulates FcεRI-Mediated Signal Transduction in Mast Cells
Authors: Xie Zhi-Hui , Ambudkar Indu , Siraganian Reuben P.
Published: April 2014

The recently cloned scaffolding molecule Gab2 can assemble multiple molecules involved in signaling pathways. Bone marrow-derived mast cells isolated from Gab2−/− mice have defective signaling probably due to the lack of the activation of phosphatidylinositol-3 kinase (PI3-kinase). In this study, we investigated the role of Gab2 using the rat basophilic leukemia 2H3 cell line mast cells. FcεRI aggregation induced the tyrosine phosphorylation of Gab2 and translocation of a significant fraction of it from the cytosol to the plasma membrane. As in other cells, Gab2 was found to associate with several signaling molecules including Src homology 2-containing protein tyrosine phosphatase 2, Grb2, Lyn, and phospholipase Cγ (PLCγ). The association of Gab2 with Lyn and PLCγ were enhanced after receptor aggregation. Overexpression of Gab2 in rat basophilic leukemia 2H3 cell line cells inhibited the FcεRI-induced tyrosine phosphorylation of the subunits of the receptor, and the phosphorylation and/or activation of Syk and mitogen-activated protein kinase. Downstream events such as calcium mobilization, degranulation, and induction of TNF-α and IL-6 gene transcripts were decreased in Gab2 overexpressing cells, although Akt phosphorylation as a measure of PI3-kinase activation was unaffected. These results suggest that in addition to the positive effects mediated by PI3-kinase that are apparent in Gab2−/− mast cells, Gab2 by interacting with Lyn and PLCγ may have negative regulatory effects on FcεRI-induced mast cell signaling and functions.
Helicobacter pylori Induces Macrophage Apoptosis by Activation of Arginase II
Authors: Gobert Alain P. , Cheng Yulan , Wang Jian-Ying , Boucher Jean-Luc , Iyer Ramaswamy K. , Cederbaum Stephen D. , Casero Robert A. , Newton Jamie C. , Wilson Keith T.
Published: April 2014

Helicobacter pylori infection induces innate immune responses in macrophages, contributing to mucosal inflammation and damage. Macrophage apoptosis is important in the pathogenesis of mucosal infections but has not been studied with H. pylori. NO derived from inducible NO synthase (iNOS) can activate macrophage apoptosis. Arginase competes with iNOS by converting l-arginine to l-ornithine. Since we reported that H. pylori induces iNOS in macrophages, we now determined whether this bacterium induces arginase and the effect of this activation on apoptosis. NF-κB-dependent induction of arginase II, but not arginase I, was observed in RAW 264.7 macrophages cocultured with H. pylori. The time course of apoptosis matched those of both arginase and iNOS activities. Surprisingly, apoptosis was blocked by the arginase inhibitors Nω-hydroxy-l-arginine or Nω-hydroxy-nor-l-arginine, but not by the iNOS inhibitor N-iminoethyl-l-lysine. These findings were confirmed in peritoneal macrophages from iNOS-deficient mice and were not dependent on bacterial-macrophage contact. Ornithine decarboxylase (ODC), which metabolizes l-ornithine to polyamines, was also induced in H. pylori-stimulated macrophages. Apoptosis was abolished by inhibition of ODC and was restored by the polyamines spermidine and spermine. We also demonstrate that arginase II expression is up-regulated in both murine and human H. pylori gastritis tissues, indicating the likely in vivo relevance of our findings. Therefore, we describe arginase- and ODC-dependent macrophage apoptosis, which implicates polyamines in the pathophysiology of H. pylori infection.
Toll-Like Receptor (TLR)2 and TLR4 in Human Peripheral Blood Granulocytes: A Critical Role for Monocytes in Leukocyte Lipopolysaccharide Responses
Authors: Sabroe Ian , Jones Elizabeth C. , Usher Lynne R. , Whyte Moira K. B. , Dower Steven K.
Published: April 2014

Leukocyte responsiveness to LPS is dependent upon CD14 and receptors of the Toll-like receptor (TLR) family. Neutrophils respond to LPS, but conflicting data exist regarding LPS responses of eosinophils and basophils, and expression of TLRs at the protein level in these granulocyte lineages has not been fully described. We examined the expression of TLR2, TLR4, and CD14 and found that monocytes expressed relatively high levels of cell surface TLR2, TLR4, and CD14, while neutrophils also expressed all three molecules, but at low levels. In contrast, basophils expressed TLR2 and TLR4 but not CD14, while eosinophils expressed none of these proteins. Tested in a range of functional assays including L-selectin shedding, CD11b up-regulation, IL-8 mRNA generation, and cell survival, neutrophils responded to LPS, but eosinophils and basophils did not. In contrast to previous data, we found, using monocyte depletion by negative magnetic selection, that neutrophil responses to LPS were heavily dependent upon the presence of a very low level of monocytes, and neutrophil survival induced by LPS at 22 h was monocyte dependent. We conclude that LPS has little role in the regulation of peripheral blood eosinophil and basophil function, and that, even in neutrophils, monocytes orchestrate many previously observed leukocyte LPS response patterns.
Role of Mitogen-Activated Protein Kinases in CpG DNA-Mediated IL-10 and IL-12 Production: Central Role of Extracellular Signal-Regulated Kinase in the Negative Feedback Loop of the CpG DNA-Mediated Th1 Response
Authors: Yi Ae-Kyung , Yoon Jae-Geun , Yeo Seon-Ju , Hong Soon-Cheol , English B. Keith , Krieg Arthur M.
Published: April 2014

The mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK), and p38, are activated in response to infectious agents and innate immune stimulators such as CpG DNA, and regulate the subsequent initiation and termination of immune responses. CpG DNA activates p38 and ERK with slightly different kinetics in monocytic cells. The present studies investigated the roles of these two key mitogen-activated protein kinases in regulating the CpG DNA-induced production of pro- and anti-inflammatory cytokines in the macrophage-like cell line RAW264.7. p38 activity was essential for the induction of both IL-10 and IL-12 expression by CpG DNA. In contrast, CpG DNA-mediated ERK activation was shown to suppress IL-12 production, but to be essential for the CpG DNA-induced IL-10 production. Studies using rIL-10 and IL-10 gene-deficient mice demonstrated that the inhibitory effect of ERK on CpG DNA-mediated IL-12 production is indirect, due to the role of ERK in mediating IL-10 production. These results demonstrate that ERK and p38 differentially regulate the production of pro- and anti-inflammatory cytokines in APCs that have been activated by CpG DNA. CpG DNA-induced p38 activity is required for the resulting innate immune activation. In contrast, ERK plays a central negative regulatory role in the CpG DNA-mediated Th1 type response by promoting production of the Th2 type cytokine, IL-10.
Endotoxin-Stimulated Nitric Oxide Production Inhibits Expression of Cytochrome c Oxidase in ANA-1 Murine Macrophages
Authors: Wei Junping , Guo Hongtao , Kuo Paul C.
Published: April 2014

In endotoxin (LPS)-mediated states of sepsis, inducible NO synthase expression and NO production are associated with molecular regulatory functions that determine the host inflammatory response. NO inhibits cellular respiration and mitochondrial electron transport by inhibition of cytochrome c oxidase (CcO) activity. CcO is the terminal complex of the mitochondrial respiratory chain, responsible for 90% of cellular oxygen consumption and essential for cellular energy production. Subunit 1 (CcO I) is considered to be the most critical of the 13 CcO component subunits. In this regard little is known of the effect of NO on the transcriptional program for CcO expression. In ANA-1 murine macrophages, LPS-mediated NO synthesis decreases CcO enzyme activity, CcO I protein expression, and CcO I steady mRNA levels. Mitochondrial run-on analysis demonstrates unaltered CcO I mitochondrial gene transcription. Half-life analysis indicates that CcO I mRNA stability is significantly decreased in the presence of LPS-mediated NO synthesis. In this study using LPS-stimulated ANA-1 murine macrophages, we demonstrate that expression of the mitochondrial gene product, CcO I, is significantly decreased as the result of a unique and previously uncharacterized, NO-dependent post-transcriptional regulatory mechanism.
Leukocyte-Endothelial Cell Interactions Are Enhanced in Dermal Postcapillary Venules of MRL/faslpr(Lupus-Prone) Mice: Roles of P- and E-Selectin
Authors: Hickey Michael J. , Bullard Daniel C. , Issekutz Andrew , James Will G.
Published: April 2014

MRL/faslpr mice are affected by a systemic autoimmune disease that results in widespread leukocytic infiltration of the vasculature, including in the skin. The molecular pathways responsible for this leukocyte recruitment are poorly understood. Therefore, the aim of these experiments was to examine the mechanisms of leukocyte trafficking in the dermal microvasculature of MRL/faslpr mice. Intravital microscopy was used to examine leukocyte rolling and adhesion in dermal postcapillary venules of MRL/faslpr mice at 8, 12, and 16 wk of age. When compared with age-matched BALB/c and MRL+/+ (nondiseased) mice, leukocyte rolling and adhesion in MRL/faslpr mice were significantly enhanced at 12 wk of age, and remained elevated at 16 wk of age. At 8 and 12 wk, leukocyte rolling in all three strains was almost entirely inhibited by an anti-P-selectin mAb. In contrast, at 16 wk some (∼10%) leukocyte rolling persisted following P-selectin blockade. This residual rolling was predominantly inhibitable with an anti-E-selectin mAb; however, treatment with anti-E-selectin mAb alone had a minimal effect. P-selectin-deficient MRL/faslpr mice also displayed leukocyte rolling that was significantly lower than in wild-type MRL/faslpr mice. However, in these mice, leukocyte adhesion remained at the elevated levels observed in wild-type MRL/faslpr mice. This adhesion was eliminated by chronic treatment with anti-E-selectin mAb. These findings indicate that leukocyte-endothelial cell interactions are enhanced in the dermal microvasculature of MRL/faslpr mice above the age of 12 wk. Furthermore, the data suggest that the endothelial selectins share overlapping roles in mediating this enhanced leukocyte recruitment.
SHIP Negatively Regulates IgE + Antigen-Induced IL-6 Production in Mast Cells by Inhibiting NF-κB Activity
Authors: Kalesnikoff Janet , Baur Nicole , Leitges Michael , Hughes Michael R. , Damen Jacqueline E. , Huber Michael , Krystal Gerald
Published: April 2014

We demonstrate in this study that IgE + Ag-induced proinflammatory cytokine production is substantially higher in Src homology-2-containing inositol 5′-phosphatase (SHIP)−/− than in SHIP+/+ bone marrow-derived mast cells (BMMCs). Focusing on IL-6, we found that the repression of IL-6 mRNA and protein production in SHIP+/+ BMMCs requires the enzymatic activity of SHIP, because SHIP−/− BMMCs expressing wild-type, but not phosphatase-deficient (D675G), SHIP revert the IgE + Ag-induced increase in IL-6 mRNA and protein down to levels seen in SHIP+/+ BMMCs. Comparing the activation of various signaling pathways to determine which ones might be responsible for the elevated IL-6 production in SHIP−/− BMMCs, we found the phosphatidylinositol 3-kinase/protein kinase B (PKB), extracellular signal-related kinase (Erk), p38, c-Jun N-terminal kinase, and protein kinase C (PKC) pathways are all elevated in IgE + Ag-induced SHIP−/− cells. Moreover, inhibitor studies suggested that all these pathways play an essential role in IL-6 production. Looking downstream, we found that IgE + Ag-induced IL-6 production is dependent on the activity of NF-κB and that IκB phosphorylation/degradation and NF-κB translocation, DNA binding and transactivation are much higher in SHIP−/− BMMCs. Interestingly, using various pathway inhibitors, it appears that the phosphatidylinositol 3-kinase/PKB and PKC pathways elevate IL-6 mRNA synthesis, at least in part, by enhancing the phosphorylation of IκB and NF-κB DNA binding while the Erk and p38 pathways enhance IL-6 mRNA synthesis by increasing the transactivation potential of NF-κB. Taken together, our data are consistent with a model in which SHIP negatively regulates NF-κB activity and IL-6 synthesis by reducing IgE + Ag-induced phosphatidylinositol-3,4,5-trisphosphate levels and thus PKB, PKC, Erk, and p38 activation.
Selective Inhibition of Cyclooxygenase-2 Expression by 15-Deoxy-Δ12,1412,14-prostaglandin J2 in Activated Human Astrocytes, But Not in Human Brain Macrophages
Authors: Janabi Nazila
Published: April 2014

Overexpression of the inducible cyclooxygenase (COX-2) and inducible NO synthase (iNOS) in activated brain macrophages (microglia) and astrocytes appears central to many neuroinflammatory conditions. 15-Deoxy-Δ12,14-PGJ2 (15d-PGJ2) is a ligand for the peroxisome proliferator-activated receptor (PPAR)γ. It has been proposed as an inhibitor of microglial activation, based on the study of iNOS down-regulation in rodent microglia. Because iNOS induction after cytokine activation remains controversial in human microglia, we examined the effect of 15d-PGJ2 and other PPAR agonists on human microglia and astrocytes, using COX-2 induction as an index of activation. We found that PPARα ligands (clofibrate and WY14643) enhanced IL-1β-induced COX-2 expression in human astrocytes and microglia, while inhibiting IL-1β plus IFN-γ induction of iNOS in astrocytes. This is the first description of an inhibition of iNOS uncoupled from that of COX-2. 15d-PGJ2 suppressed COX-2 induction in human astrocytes. It prevented NF-κB binding to the COX-2 promoter through a new pathway that is the repression of NF-κBp50 induction by IL-1β. In contrast, 15d-PGJ2 increased c-Jun and c-Fos DNA-binding activity in astrocytes, which may result in the activation of other inflammatory pathways. In human microglia, no effect of 15d-PGJ2 on COX-2 and NF-κBp65/p50 induction was observed. However, the entry of 15d-PGJ2 occurred in microglia because STAT-1 and c-Jun expression was modulated. Our data suggest the existence of novel pathways mediated by 15d-PGJ2 in human astrocytes. They also demonstrate that, unlike astrocytes and peripheral macrophages or rodent brain macrophages, human microglia are not subject to the anti-inflammatory effect of 15d-PGJ2 in terms of COX-2 inhibition.
IL-16 Promotes Leukotriene C4 and IL-4 Release from Human Eosinophils via CD4- and Autocrine CCR3-Chemokine-Mediated Signaling
Authors: Bandeira-Melo Christianne , Sugiyama Kumiya , Woods Lesley J. , Phoofolo Mojabeng , Center David M. , Cruikshank William W. , Weller Peter F.
Published: April 2014

Human eosinophils are potential sources of inflammatory and immunomodulatory mediators, including cysteinyl leukotrienes, chemokines, and cytokines, which are pertinent to allergic inflammation. We evaluated the means by which IL-16, a recognized eosinophil chemoattractant, might act on eosinophils to affect their capacity to release leukotriene C4 (LTC4) or their preformed stores of chemokines (eotaxin, RANTES) or Th1 (IL-12) or Th2 (IL-4) cytokines. IL-16 dose dependently (0.01–100 nM) elicited new lipid body formation, intracellular LTC4 formation at lipid bodies, and priming for enhanced calcium ionophore-activated LTC4 release. IL-16 also elicited brefeldin A-inhibitable, vesicular transport-mediated release of preformed IL-4, but not IL-12, from eosinophils. CD4 is a recognized IL-16R, and accordingly anti-CD4 Fab, soluble CD4, and a CD4 domain 4-based IL-16 blocking peptide inhibited the actions of IL-16 on eosinophils. Although CD4 is not G-protein coupled, pertussis toxin inhibited IL-16-induced eosinophil activation. IL-16 actions were found to be mediated by the autocrine activity, not of platelet-activating factor, but rather of endogenous CCR3-acting chemokines. IL-16 induced the rapid vesicular transport-mediated release of RANTES. The effects of IL-16 were blocked by CCR3 inhibitors (met-RANTES, anti-CCR3 mAb) and by neutralizing anti-eotaxin and anti-RANTES mAbs, but not by platelet-activating factor receptor antagonists (CV6209, BN52021). RANTES and eotaxin each enhanced LTC4 and IL-4 (but not IL-12) release. Therefore, IL-16 activation of eosinophils is CD4-mediated to elicit the extracellular release of preformed RANTES and eotaxin, which then in an autocrine fashion act on plasma membrane CCR3 receptors to stimulate both enhanced LTC4 production and the preferential release of IL-4, but not IL-12, from within eosinophils.
IFN-γ, But Not Fas, Mediates Reduction of Allergen-Induced Mucous Cell Metaplasia by Inducing Apoptosis
Authors: O-Quan Shi Zha , Fischer Mark J. , De Sanctis George T. , Schuyler Mark R. , Tesfaigzi Yohannes
Published: April 2014

Inflammatory responses induced by allergen exposure cause mucous cell metaplasia (MCM) by differentiation of existing and proliferating epithelial cells into mucus-storing cells. Airway epithelia have various mechanisms that resolve these changes to form normal airway epithelia. In this report, we first investigated the state of mucous cell metaplasia and the mechanisms by which MCM is reduced despite continued exposures to allergen. After 5 days of allergen exposure, extensive MCM had developed but was reduced when allergen challenge was continued for 15 days. During this exposure period, IL-13 levels decreased and IFN-γ levels increased in the bronchoalveolar lavage fluid. In contrast, IL-13 levels decreased but IFN-γ was not detected at any time point during the resolution of MCM following cessation of allergen exposure. Instillation of IFN-γ but not anti-Fas caused accelerated resolution of MCM and MCM was not resolved in Stat1-deficient mice exposed to allergen for 15 days, confirming that IFN-γ is crucial for reducing MCM during prolonged exposures to allergen. IFN-γ but not anti-Fas induced apoptotic cell death in proliferating normal human bronchial epithelial cells and in human bronchial epithelial cells from subjects with asthma. The apoptotic effect of IFN-γ was caspase dependent and was inhibited by IL-13, indicating that the Th2 milieu in asthmatics may maintain MCM by preventing cell death in metaplastic mucous cells. These studies could be useful in the understanding of deficiencies leading to chronicity in airway changes and designing novel therapies to reverse MCM and airway obstruction in asthmatics.
A Functional Role of HLA-G Expression in Human Gliomas: An Alternative Strategy of Immune Escape
Authors: Wiendl Heinz , Mitsdoerffer Meike , Hofmeister Valeska , Wischhusen Jörg , Bornemann Antje , Meyermann Richard , Weiss Elisabeth H. , Melms Arthur , Weller Michael
Published: April 2014

HLA-G is a nonclassical MHC molecule with highly limited tissue distribution that has been attributed chiefly immune regulatory functions. Glioblastoma is paradigmatic for the capability of human cancers to paralyze the immune system. To delineate the potential role of HLA-G in glioblastoma immunobiology, expression patterns and functional relevance of this MHC class Ib molecule were investigated in glioma cells and brain tissues. HLA-G mRNA expression was detected in six of 12 glioma cell lines in the absence of IFN-γ and in 10 of 12 cell lines in the presence of IFN-γ. HLA-G protein was detected in four of 12 cell lines in the absence of IFN-γ and in eight of 12 cell lines in the presence of IFN-γ. Immunohistochemical analysis of human brain tumors revealed expression of HLA-G in four of five tissue samples. Functional studies on the role of HLA-G in glioma cells were conducted with alloreactive PBMCs, NK cells, and T cell subpopulations. Expression of membrane-bound HLA-G1 and soluble HLA-G5 inhibited alloreactive and Ag-specific immune responses. Gene transfer of HLA-G1 or HLA-G5 into HLA-G-negative glioma cells (U87MG) rendered cells highly resistant to direct alloreactive lysis, inhibited the alloproliferative response, and prevented efficient priming of cytotoxic T cells. The inhibitory effects of HLA-G were directed against CD8 and CD4 T cells, but appeared to be NK cell independent. Interestingly, few HLA-G-positive cells within a population of HLA-G-negative tumor cells exerted significant immune inhibitory effects. We conclude that the aberrant expression of HLA-G may contribute to immune escape in human glioblastoma.
Dimethylfumarate Inhibits TNF-Induced Nuclear Entry of NF-κB/p65 in Human Endothelial Cells
Authors: Loewe Robert , Holnthoner Wolfgang , Gröger Marion , Pillinger Manuela , Gruber Florian , Mechtcheriakova Diana , Hofer Erhard , Wolff Klaus , Petzelbauer Peter
Published: April 2014

Fumaric acid esters, mainly dimethylfumarate (DMF), have been successfully used to treat psoriasis. Based on previous observations that DMF inhibited expression of several TNF-induced genes in endothelial cells, we wished to explore the molecular basis of DMF function in greater detail. In first experiments we analyzed DMF effects on tissue factor expression in human endothelial cells in culture, because tissue factor is expressed by two independent sets of transcription factors, by NF-κB via TNF and by early gene response-1 transcription factor via vascular endothelial growth factor (VEGF). We show that DMF inhibits TNF-induced tissue factor mRNA and protein expression as well as TNF-induced DNA binding of NF-κB proteins, but not VEGF-induced tissue factor protein, mRNA expression, or VEGF-induced early gene response-1 transcription factor/DNA binding. To determine where DMF interferes with the TNF/NF-κB signaling cascade, we next analyzed DMF effects on IκB and on the subcellular distribution of NF-κB. DMF does not inhibit TNF-induced IκBα phosphorylation and IκB degradation; thus, NF-κB is properly released from IκB complexes even in the presence of DMF. Importantly, DMF inhibits the TNF-induced nuclear entry of NF-κB proteins, and this effect appears selective for NF-κB after the release from IκB, because the constitutive shuttling of inactive NF-κB/IκB complexes into and out from the nucleus is not blocked by DMF. Moreover, DMF does not block NF-κB/DNA binding. In conclusion, DMF appears to selectively prevent the nuclear entry of activated NF-κB, and this may be the basis of its beneficial effect in psoriasis.
Gene Transfer of Ig-Fusion Proteins Into B Cells Prevents and Treats Autoimmune Diseases
Authors: Melo Marco E. F. , Qian Jiahua , El-Amine Moustapha , Agarwal Rajeev K. , Soukhareva Nadejda , Kang Yubin , Scott David W.
Published: April 2014

Based on the tolerogenic properties of IgG carriers and B cell Ag presentation, we developed a retrovirally mediated gene expression approach for treatment of autoimmune conditions. In this study, we show that the IgG-Ag retroviral constructs, expressing myelin basic protein (MBP) or glutamic acid decarboxylase in B cells, can be used for the treatment of murine models for multiple sclerosis and diabetes. Transduction of syngeneic B cells with MBP-IgG leads to the amelioration of ongoing experimental allergic encephalomyelitis induced by the transfer of primed cells from PL×SJL F1 mice with ongoing disease and could be effective even after symptoms appeared. This effect is specific and does not involve bystander suppression because treatment with MBP-IgG does not affect disease induced after immunization with proteolipid protein immunodominant peptide plus MBP. Interestingly, if donor B cells are derived from gld mice (Fas ligand-negative), then tolerance is not induced with a model Ag although there was no evidence for Fas ligand-mediated deletion of target T cells. In spontaneous diabetes in nonobese diabetic mice, we were able to stop the ongoing autoimmune process by treatment at 7–10 wk with glutamic acid decarboxylase-IgG retrovirally transduced B cells, or attenuate it with B cells transduced with an insulin B chain (B9–23) epitope IgG fusion protein. Furthermore, IgG fusion protein gene therapy can also protect primed recipients from Ag-induced anaphylactic shock, and thus does not cause immune deviation. These results demonstrate proof of principle for future efforts to develop this approach in a clinical setting.
Persistent Decreases in Blood Plasmacytoid Dendritic Cell Number and Function Despite Effective Highly Active Antiretroviral Therapy and Increased Blood Myeloid Dendritic Cells in HIV-Infected Individuals
Authors: Chehimi Jihed , Campbell Donald E. , Azzoni Livio , Bacheller Darlene , Papasavvas Emmanouil , Jerandi Ghassen , Mounzer Karam , Kostman Jay , Trinchieri Giorgio , Montaner Luis J.
Published: April 2014

Dendritic cells (DC) have an instrumental role in the activation and function of both innate and adaptive immune responses. In humans, at least two distinct DC subsets have been characterized based on phenotypic markers: the myeloid DC (MDC) and the plasmacytoid DC (PDC). Both subsets are critical producers of cytokines (IL-12 for MDC and type I/II IFNs for PDC) and are functionally different. We show in this study that HIV+ individuals have a significant decrease in the number of the Lin−HLA-DR+CD123+ and BDCA-2+ PDC compared with uninfected donors (p = 0.0001). HIV+ individuals also have a sustained impairment in viral-induced IFN-α production (p < 0.0001). The decrease of the PDC subsets did not correlate with CD4 count or viral load and was not reversed in subjects under virally suppressive treatment, suggesting an irreversible change after infection. By contrast, the absolute number and median frequency of MDC in HIV-infected individuals were similar to those observed in uninfected controls, while a significant decrease was present in subjects with >5000 HIV-1 copies/ml. The inverse association with viral load of the MDC number, but not of IFN-α secretion or the number of PDC, suggests a role for MDC in viral control. Our data suggest that DC subsets are differentially reconstituted during the immune recovery associated with antiviral therapy. The persistent impairment of certain DC subsets may result in a sustained defect in DC-mediated innate immune functions despite an effective treatment regimen.
A ras-Mutated Peptide Targeted by CTL Infiltrating a Human Melanoma Lesion
Authors: Linard Boris , Bézieau Stéphane , Benlalam Houssem , Labarrière Nathalie , Guilloux Yannick , Diez Elisabeth , Jotereau Francine
Published: April 2014

Ags derived from commonly mutated oncogenic proteins seem ideally suited as targets for tumor immunotherapy. Nonetheless, only a few mutated epitopes efficiently presented by human tumors have thus far been identified. We describe here an approach to identify such epitopes. This approach involves: 1) identifying tumors expressing a ras mutation and isolating the tumor-infiltrating lymphocytes (TIL); 2) transfecting COS cells to induce expression of unknown mutated peptides in the context of a patient’s HLA class I molecules; and 3) screening epitope recognition by using TIL from the tumors expressing a ras mutation. By using this approach, there appeared to be a N-ras mutation (a glutamine-to-arginine exchange at residue 61 (Q61R)), detected in a melanoma lesion, which was recognized specifically by the autologous TIL in the HLA-A*0101 context. The ras peptide 55–64Q61R was the epitope of these TIL and was regularly presented by Q61R-mutated HLA-A*0101+ melanoma cell lines. This peptide and its wild-type homolog (55–64wt) bound to HLA-A*0101 with similar affinities. However, only the mutated peptide could induce specific CTL expansion from PBL. All the CTL clones specific to the mutated peptide, failed to recognize the wild-type sequence on both COS and melanoma cells. These data thus show that oncogenic protein mutations can create shared tumor-specific CTL epitopes, efficiently presented by tumor cells, and that screening for oncogene-transfected COS cell recognition by TIL (from tumors containing mutations) is a powerful approach for the identification of these epitopes.
Cutting Edge: IFN-Producing Cells Respond to CXCR3 Ligands in the Presence of CXCL12 and Secrete Inflammatory Chemokines upon Activation
Authors: Krug Anne , Uppaluri Ravindra , Facchetti Fabio , Dorner Brigitte G. , Sheehan Kathleen C. F. , Schreiber Robert D. , Cella Marina , Colonna Marco
Published: April 2014

Human natural IFN-producing cells (IPC) circulate in the blood and cluster in chronically inflamed lymph nodes around high endothelial venules (HEV). Although L-selectin, CXCR4, and CCR7 are recognized as critical IPC homing mediators, the role of CXCR3 is unclear, since IPC do not respond to CXCR3 ligands in vitro. In this study, we show that migration of murine and human IPC to CXCR3 ligands in vitro requires engagement of CXCR4 by CXCL12. We also demonstrate that CXCL12 is present in human HEV in vivo. Moreover, after interaction with pathogenic stimuli, murine and human IPC secrete high levels of inflammatory chemokines. Thus, IPC migration into inflamed lymph nodes may be initially mediated by L-selectin, CXCL12, and CXCR3 ligands. Upon pathogen encounter, IPC positioning within the lymph node may be further directed by CCR7 and IPC secretion of inflammatory chemokines may attract other IPC, promoting cluster formation in lymph nodes.
The Fate of Effector T Cells In Vivo Is Determined During Activation and Differs for CD4+ and CD8+ Cells
Authors: Bode Ulrike , Sahle Andrea , Sparmann Gisela , Weidner Frauke , Westermann Jürgen
Published: April 2014

Effector T cells generated in the mesenteric lymph nodes (mLN) are known to accumulate in mLN and the tissue drained by them after circulating in the blood. Their accumulation is due less to preferential entry into mLN but more to preferential proliferation within mLN. The factors regulating the proliferation of effector T cells in vivo are unclear, and it is unknown whether they are different for CD4+ and CD8+ effector T cells. Rat T cells from mLN or peripheral lymph nodes (pLN) were stimulated polyclonally via the TCR and CD28 and injected i.v. into congenic recipients. Using three-color flow cytometry and immunohistochemistry, they were identified in mLN, pLN, and blood over time, and proliferation was determined by measuring bromodeoxyuridine incorporation. Only effector mLN T cells showed a significantly increased proliferation rate after entry into mLN compared with that in pLN (2.4 ± 1.8% vs 0.8 ± 0.4%). Proliferation among the injected cells was higher when they had contact with dendritic cells within mLN (9.0 ± 4.3%) than when they did not (4.1 ± 2.1%). Furthermore, effector mLN T cells which were observed 56 days after injection maintained the capacity for preferential proliferation within mLN. Interestingly, CD4+ effector mLN T cells proliferated at a higher rate (4.8 ± 0.7%), remaining in mLN, whereas CD8+ effector mLN T cells proliferated at a lower rate (3.3 ± 1.0%) and were able to leave the mLN into the blood. Elucidating the factors regulating the proliferation of effector T cells in vivo will help to modify their distribution for therapeutic purposes.