bluuebunny/crossref_metadata_2025 huggingface dataset:
Cutting Edge: Susceptibility to Psoriatic Arthritis: Influence of Activating Killer Ig-Like Receptor Genes in the Absence of Specific HLA-C Alleles
Authors: Martin Maureen P. , Nelson George , Lee Jeong-Hee , Pellett Fawnda , Gao Xiaojiang , Wade Judith , Wilson Michael J. , Trowsdale John , Gladman Dafna , Carrington Mary
Published: April 2014

NK cell activity is partially controlled through interactions between killer Ig-like receptors (KIR) on NK cells and their respective HLA class I ligands. Independent segregation of HLA and KIR genes, along with KIR specificity for particular HLA allotypes, raises the possibility that any given individual may express KIR molecules for which no ligand is present. Inhibitory receptor genes KIR2DL2/3 and KIR2DL1 were present in nearly all subjects sampled in this study, whereas their respective activating homologs, KIR2DS2 and KIR2DS1, are each present in about half of the subjects. In this work we report that subjects with activating KIR2DS1 and/or KIR2DS2 genes are susceptible to developing psoriatic arthritis, but only when HLA ligands for their homologous inhibitory receptors, KIR2DL1 and KIR2DL2/3, are missing. Absence of ligands for inhibitory KIRs could potentially lower the threshold for NK (and/or T) cell activation mediated through activating receptors, thereby contributing to pathogenesis of psoriatic arthritis.
Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Signaling in Activated T Cells Abrogates TRAIL-Induced Apoptosis Upstream of the Mitochondrial Amplification Loop and Caspase-8
Authors: Söderström Thomas S. , Poukkula Minna , Holmström Tim H. , Heiskanen Kaisa M. , Eriksson John E.
Published: April 2014

Fas ligand and TNF-related apoptosis-inducing ligand (TRAIL) induce apoptosis in many different cell types. Jurkat T cells die rapidly by apoptosis after treatment with either ligand. We have previously shown that mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) can act as a negative regulator of apoptosis mediated by the Fas receptor. In this study we examined whether MAPK/ERK can also act as a negative regulator of apoptosis induced by TRAIL. Activated Jurkat T cells were efficiently protected from TRAIL-induced apoptosis. The protection was shown to be MAPK/ERK dependent and independent of protein synthesis. MAPK/ERK suppressed TRAIL-induced apoptosis upstream of the mitochondrial amplification loop because mitochondrial depolarization and release of cytochrome c were inhibited. Furthermore, caspase-8-mediated relocalization and activation of Bid, a proapoptotic member of the Bcl family, was also inhibited by the MAPK/ERK signaling. The protection occurred at the level of the apoptotic initiator caspase-8, as the cleavage of caspase-8 was inhibited but the assembly of the death-inducing signaling complex was unaffected. Both TRAIL and Fas ligand have been suggested to regulate the clonal size and persistence of different T cell populations. Our previous results indicate that MAPK/ERK protects recently activated T cells from Fas receptor-mediated apoptosis during the initial phase of an immune response before the activation-induced cell death takes place. The results of this study show clearly that MAPK/ERK also participates in the inhibition of TRAIL-induced apoptosis after T cell activation.
Colocalization of the B Cell Receptor and CD20 Followed by Activation-Dependent Dissociation in Distinct Lipid Rafts
Authors: Petrie Ryan J. , Deans Julie P.
Published: April 2014

The B cell Ag receptor (BCR) and CD20, a putative calcium channel, inducibly associate with cholesterol-dependent membrane microdomains known as lipid rafts. A functional association between the BCR and CD20 is suggested by the effects of CD20-specific mAbs, which can modulate cell cycle transitions elicited by BCR signaling. Using immunofluorescence microscopy we show here that the BCR and CD20 colocalize after receptor ligation and then rapidly dissociate at the cell surface before endocytosis of the BCR. After separation, surface BCR and CD20 were detected in distinct lipid rafts isolated as low density, detergent-resistant membrane fragments. Pretreatment with methyl-β-cyclodextrin, which we have previously shown to enhance receptor-mediated calcium mobilization, did not prevent colocalization of the BCR and CD20, but slowed their dissociation. The data demonstrate rapid dynamics of the BCR in relation to CD20 at the cell surface. Activation-dependent dissociation of the BCR from CD20 occurs before receptor endocytosis and appears to require in part the integrity of lipid rafts.
Innate Immune Response to Malaria: Rapid Induction of IFN-γ from Human NK Cells by LivePlasmodium falciparum-Infected Erythrocytes
Authors: Artavanis-Tsakonas Katerina , Riley Eleanor M.
Published: April 2014

To determine the potential contribution of innate immune responses to the early proinflammatory cytokine response to Plasmodium falciparum malaria, we have examined the kinetics and cellular sources of IFN-γ production in response to human PBMC activation by intact, infected RBC (iRBC) or freeze-thaw lysates of P. falciparum schizonts. Infected erythrocytes induce a more rapid and intense IFN-γ response from malaria-naive PBMC than do P. falciparum schizont lysates correlating with rapid iRBC activation of the CD3−CD56+ NK cell population to produce IFN-γ. IFN-γ+ NK cells are detectable within 6 h of coculture with iRBC, their numbers peaking at 24 h in most donors. There is marked heterogeneity between donors in magnitude of the NK-IFN-γ response that does not correlate with mitogen- or cytokine-induced NK activation or prior malaria exposure. The NK cell-mediated IFN-γ response is highly IL-12 dependent and appears to be partially IL-18 dependent. Exogenous rIL-12 or rIL-18 did not augment NK cell IFN-γ responses, indicating that production of IL-12 and IL-18 is not the limiting factor explaining differences in NK cell reactivity between donors or between live and dead parasites. These data indicate that NK cells may represent an important early source of IFN-γ, a cytokine that has been implicated in induction of various antiparasitic effector mechanisms. The heterogeneity of this early IFN-γ response between donors suggests a variation in their ability to mount a rapid proinflammatory cytokine response to malaria infection that may, in turn, influence their innate susceptibility to malaria infection, malaria-related morbidity, or death from malaria.
Peripheral Immature CD2−/low T Cell Development from Type 2 to Type 1 Cytokine Production
Authors: Loza Matthew J. , Perussia Bice
Published: April 2014

Immature myeloid and NK cells exist, and undergo cytokine-induced differentiation, in the periphery. In this study, we show that also immature CD2−/low T cells exist in peripheral blood. These cells produce the type 2 cytokines IL-13, IL-4, and IL-5, but not IFN-γ or IL-10, and, upon culture with IL-12- and TCR-mediated stimuli, differentiate to IL-13+IFN-γ+ cells producing high IL-2 levels, and finally IL-13−IFN-γ+ cells. The monokine combination IL-12, IL-18, and IFN-α substitutes for TCR-mediated stimulation to induce the same differentiation process in both immature CD2−/low and primary mature CD2+ IL-13+ Τ cells. IFN-α is needed to maintain high level IL-2 production, which is confined to type 2 cytokine-producing cells and lost in the IFN-γ+ ones. Upon TCR-mediated stimulation, IFN-γ+ cells are then induced to produce IL-10 as they undergo apoptosis. These data indicate that peripheral type 2 cytokine+ T cells are immature cells that can differentiate to effector IFN-γ+ cells following a linear monokine-regulated pathway identical with that previously described for NK cells. They define the cellular bases to support that cell-mediated immune responses are regulated not only via Ag-induced activation of mature effector cells, but also via bystander monokine-induced maturation of immature T cells.
Regulation of IFN Regulatory Factor 4 Expression in Human T Cell Leukemia Virus-I-Transformed T Cells
Authors: Sharma Sonia , Grandvaux Nathalie , Mamane Yael , Genin Pierre , Azimi Nazli , Waldmann Thomas , Hiscott John
Published: April 2014

IFN regulatory factor (IRF)-4 is a lymphoid/myeloid-restricted member of the IRF transcription factor family that plays an essential role in the homeostasis and function of mature lymphocytes. IRF-4 expression is tightly regulated in resting primary T cells and is transiently induced at the mRNA and protein levels after activation by Ag-mimetic stimuli such as TCR cross-linking or treatment with phorbol ester and calcium ionophore (PMA/ionomycin). However, IRF-4 is constitutively upregulated in human T cell leukemia virus type I (HTLV-I) infected T cells as a direct gene target for the HTLV-I Tax oncoprotein. In this study we demonstrate that chronic IRF-4 expression in HTLV-I-infected T lymphocytes is associated with a leukemic phenotype, and we examine the mechanisms by which continuous production of IRF-4 is achieved in HTLV-I-transformed T cells. IRF-4 expression in HTLV-1-infected cells is driven through activation of the NF-κB and NF-AT pathways, resulting in the binding of p50, p65, and c-Rel to the κB1 element and p50, c-Rel, and NF-ATp to the CD28RE element within the −617 to −209 region of the IRF-4 promoter. Furthermore, mutation of either the κB1 or CD28RE sites blocks Tax-mediated transactivation of the human IRF-4 promoter in T cells. These experiments constitute the first detailed analysis of human IRF-4 transcriptional regulation within the context of HTLV-I infection and transformation of CD4+ T lymphocytes.
Caspase-9/-3 Activation and Apoptosis Are Induced in Mouse Macrophages upon Ingestion and Digestion ofEscherichia coliBacteria
Authors: Häcker Hans , Fürmann Christine , Wagner Hermann , Häcker Georg
Published: April 2014

A number of highly virulent, intracellular bacteria are known to induce cell death by apoptosis in infected host cells. In this work we demonstrate that phagocytosis of bacteria from the Escherichia coli laboratory strain K12 DH5α is a potent cell death stimulus for mouse macrophages. RAW264.7 mouse macrophages took up bacteria and digested them within 2–4 h as investigated with green fluorescent protein-expressing bacteria. No evidence of apoptosis was seen at 8 h postexposure, but at 24 h ∼70% of macrophages displayed an apoptotic phenotype by a series of parameters. Apoptosis was blocked by inhibition of caspases or by forced expression of the apoptosis-inhibiting protein Bcl-2. Processing of caspase-3 and caspase-9 but not caspase-8 was seen suggesting that the mitochondrial branch of the apoptotic pathway was activated. Active effector caspases could be detected in two different assays. Because the adapter molecule myeloid differentiation factor 88 (MyD88) has been implicated in apoptosis, involvement of the Toll-like receptor pathway was investigated. In RAW264.7 cells, heat-treated bacteria were taken up poorly and failed to induce significant apoptosis. However, cell activation was almost identical between live and heat-inactivated bacteria as measured by extracellular signal-regulated kinase activation, generation of free radicals, and TNF secretion. Furthermore, primary bone marrow-derived macrophages from wild-type as well as from MyD88-deficient mice underwent apoptosis upon phagocytosis of bacteria. These results show that uptake and digestion of bacteria leads to MyD88-independent apoptosis in mouse macrophages. This form of cell death might have implications for the generation of the immune response.
CD80+Gr-1+ Myeloid Cells Inhibit Development of Antifungal Th1 Immunity in Mice with Candidiasis
Authors: Mencacci Antonella , Montagnoli Claudia , Bacci Angela , Cenci Elio , Pitzurra Lucia , Spreca Antonio , Kopf Manfred , Sharpe Arlene H. , Romani Luigina
Published: April 2014

To find out whether polymorphonuclear neutrophils (PMN), abundantly recruited in disseminated Candida albicans infection, could directly affect the activation of Th cells we addressed the issues as to whether murine PMN, like their human counterparts, express costimulatory molecules and the functional consequence of this expression in terms of antifungal immune resistance. To this purpose, we assessed 1) the expression of CD80 (B7-1) and CD86 (B7-2) molecules on peripheral, splenic, and inflammatory murine Gr-1+ PMN; 2) its modulation upon interaction with C. albicans in vitro, in vivo, and in human PMN; 3) the effect of Candida exposure on the ability of murine PMN to affect CD4+ Th1 cell proliferation and cytokine production; and 4) the mechanism responsible for this effect. Murine PMN constitutively expressed CD80 molecules on both the surface and intracellularly; however, in both murine and human PMN, CD80 expression was differentially modulated upon interaction with Candida yeasts or hyphae in vitro as well as in infected mice. The expression of the CD86 molecule was neither constitutive nor inducible upon exposure to the fungus. In vitro, Gr-1+ PMN were found to inhibit the activation of IFN-γ-producing CD4+ T cells and to induce apoptosis through a CD80/CD28-dependent mechanism. A population of CD80+Gr-1+ myeloid cells was found to be expanded in conventional as well as in bone marrow-transplanted mice with disseminated candidiasis, but its depletion increased the IFN-γ-mediated antifungal resistance. These data indicate that alternatively activated PMN expressing CD80 may adversely affect Th1-dependent resistance in fungal infections.
Syntaxins 13 and 7 Function at Distinct Steps During Phagocytosis
Authors: Collins Richard F. , Schreiber Alan D. , Grinstein Sergio , Trimble William S.
Published: April 2014

The phagosome is a dynamic organelle that undergoes progressive changes to acquire the machinery required to kill and degrade internalized foreign particles. This maturation process involves sequential interaction of newly formed phagosomes with several components of the endocytic pathway. The proteins that mediate the ordered fusion of endosomes and lysosomes with the phagosome are not known. In this study, we investigated the possible role of syntaxins present in the endo/lysosomal pathway in directing phagosomal maturation. We show that in phagocytic cells syntaxin 13 is localized to the recycling endosome compartment, while syntaxin 7 is found in late endosomes/lysosomes. Both proteins are recruited to the phagosome, but syntaxin 13 is acquired earlier and rapidly recycles off the phagosome, while syntaxin 7 is recruited later and continues to accumulate throughout the maturation process. Overexpression of truncated (cytosolic) forms of syntaxin 13 or 7 had no effect on phagocytosis, but exerted an inhibitory effect on phagosomal maturation. These results indicate that syntaxins 13 and 7 are both required for interaction of endosomes and/or lysosomes with the phagosome, but play distinct roles in the maturation process.
Enhanced Type 1 Immunity After Secondary Viral Challenge in Mice Primed as Neonates
Authors: Fadel Shaza A. , Ozaki Daniel A. , Sarzotti Marcella
Published: April 2014

The goal of infant immunization against viral infection is to develop protective long term memory responses. Priming neonatal mice with a low dose of Cas-Br-E murine leukemia virus (Cas) results in adult-like, type 1 protective responses. However, other studies suggest that Ag priming of neonates leads to an increase in type 2 secondary responses even when primary responses were type 1. We assessed whether type 1 CD8+ T cell-mediated responses developed in murine neonates are maintained after secondary challenge with Cas in adulthood. Despite the induction of significant anti-viral CD8+-mediated cytotoxic T lymphocyte and IFN-γ responses, initial neonatal priming led to a lower frequency of virus-specific T cells compared with adult priming. Adult frequencies were reached in mice primed as neonates only after secondary challenge in adulthood. A nonspecific and transient CD4+-mediated IL-4 response was present in all groups after secondary challenge with Cas or medium, indicating that this rise in type 2 cytokine production was not unique to mice that had been primed as neonates. Rather, type 1 anti-viral memory CD8+ T cell responses developed in neonatal mice are stable, protective, and enhanced after secondary challenge.
Activation of Monocytic Cells Through Fcγ Receptors Induces the Expression of Macrophage-Inflammatory Protein (MIP)-1α, MIP-1β, and RANTES
Authors: Fernández Nieves , Renedo Marta , García-Rodríguez Carmen , Sánchez Crespo Mariano
Published: April 2014

Monocytic cells were stimulated with IgG-OVA equivalence immune complexes, mAb reacting with FcγRI, FcγRIIA, and FcγRIII, LPS, TNF-α, and the combination of ionomycin and phorbol ester, to address their effects on the expression of the mRNAs encoding for chemokines. Stimulation of monocytes with immune complexes induced a rapid expression of macrophage-inflammatory protein (MIP)-1α, MIP-1β, and IL-8 mRNAs. In contrast, RANTES mRNA was already detectable in resting cells and only increased after 16 h of stimulation. A similar pattern was observed following homotypic stimulation of FcγR with mAb reacting with FcγRI and FcγRIIA, but not with a mAb reacting with FcγRIII, a subtype of receptor not expressed in THP-1 cells, thus indicating that both FcγRI and FcγRIIA are involved in the response. The pattern of chemokine induction elicited by LPS and the combination of ionomycin and PMA showed some similarities to those produced by FcγR cross-linking, although expression of IFN-γ-inducible protein 10 mRNA was also observed in response to those agonists. The production of MIP-1α, MIP-1β, and RANTES proteins encompassing the induction of their mRNAs was confirmed by specific ELISA. Experiments to address the transcription factors involved in the regulation of MIP-1α using pharmacological agents and EMSA showed the possible involvement of CCAAT/enhancer-binding protein β sites and ruled out the functional significance of both NF-AT and AP-1 sites.
Regulation of Phosphatidylinositol 3-Kinase Activity and Phosphatidylinositol 3,4,5-Trisphosphate Accumulation by Neutrophil Priming Agents
Authors: Cadwallader Karen A. , Condliffe Alison M. , McGregor Alex , Walker Trevor R. , White Jessica F. , Stephens Len R. , Chilvers Edwin R.
Published: April 2014

Neutrophil priming by agents such as TNF-α and GM-CSF causes a dramatic increase in the response of these cells to secretagogue agonists and affects the capacity of neutrophils to induce tissue injury. In view of the central role of phosphatidylinositol 3-kinase (PI3-kinase) in regulating NADPH oxidase activity we examined the influence of priming agents on agonist-stimulated phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) accumulation in human neutrophils. Pretreatment of neutrophils with TNF-α or GM-CSF, while not influencing fMLP-stimulated PtdIns(3,4,5)P3 accumulation at 5 s, caused a major increase in PtdIns(3,4,5)P3 at later times (10–60 s), which paralleled the augmented superoxide anion (O2−) response. The intimate relationship between PtdIns(3,4,5)P3 accumulation and O2− release was confirmed using platelet-activating factor, which caused full but transient priming of both responses. Likewise, LY294002, a PI3-kinase inhibitor, and genistein, a tyrosine kinase inhibitor, caused parallel inhibition of O2− generation and PtdIns(3,4,5)P3 accumulation; in contrast, radicicol, which inhibits receptor-mediated activation of p85 PI3-kinase, had no effect on either response. Despite major increases in PI3-kinase activity observed in p85 and anti-phosphotyrosine immunoprecipitates in growth factor-stimulated smooth muscle cells, no such increase was observed in primed/stimulated neutrophils. In contrast, both fMLP and TNF-α alone caused a 3-fold increase in PI3-kinase activity in p110γ PI3-kinase immunoprecipitates. p21ras activation (an upstream regulator of PI3-kinase) was unaffected by priming. These data demonstrate that timing and magnitude of PtdIns(3,4,5)P3 accumulation in neutrophils correlate closely with O2− generation, that PI3-kinase-γ is responsible for the enhanced PtdIns(3,4,5)P3 production seen in primed cells, and that factors other than activation of p21ras underlie this response.
Optimal Colocalization of TCR and CD8 as a Novel Mechanism for the Control of Functional Avidity
Authors: Cawthon Andrew G. , Alexander-Miller Martha A.
Published: April 2014

The improved efficacy of high avidity CTL for clearance of virus has been well-documented. Thus, elucidation of the mechanisms that confer the increased sensitivity to peptide ligand demonstrated by high avidity CTL is critical. Using CTL lines of high and low avidity generated from a TCR transgenic mouse, we have found that functional avidity can be controlled by the expression of CD8αα vs CD8αβ and the ability of CTLs to colocalize the TCR and CD8 in the membrane. Colocalization of these molecules was mediated by lipid rafts and importantly, raft disruption resulted in the conversion of high avidity CTL into a lower functional avidity phenotype. These novel findings provide insights into the control of functional avidity in response to viral infection.
Chronic Exposure to Innocuous Antigen in Sensitized Mice Leads to Suppressed Airway Eosinophilia That Is Reversed by Granulocyte Macrophage Colony-Stimulating Factor
Authors: Swirski Filip K. , Sajic Dusan , Robbins Clinton S. , Gajewska Beata U. , Jordana Manel , Stämpfli Martin R.
Published: April 2014

In this study we investigated the impact of chronic allergen exposure on airway inflammation and humoral responses in sensitized mice. We observed marked eosinophilia in the bronchoalveolar lavage, lung tissue, and peripheral blood after 2 wk of exposure. In contrast, eosinophilia was markedly reduced by 3 wk and completely resolved by 4 wk of exposure, despite the continued presence of Ag. Decreases in airway eosinophilia were associated with a robust humoral response. We observed that levels of OVA-specific IgE, IgG1, and IgG2a increased during the course of exposure. To assess whether continuous exposure to Ag impacts the ability of the lung to respond to subsequent Ag challenge, mice were exposed to either 2 or 4 wk of OVA in the context of GM-CSF. All groups were then rested for 28 days and exposed to OVA on three consecutive days. We observed a significant decrease in airway eosinophilia and IL-5 expression in the bronchoalveolar lavage and serum in mice initially exposed to 4 wk of OVA, compared with animals exposed to 2 wk only. However, in both groups expression of B7.2 on dendritic cells as well as CD25, CD69, and T1/ST2 on CD4+ T cells was enhanced, suggesting immune activation. Delivery of rGM-CSF fully restored airway eosinophilia. This study shows that exposure to innocuous Ag alone does not lead to persistent eosinophilic airway inflammation, but rather to abrogated eosinophilia. This suppression can be reversed by GM-CSF.
Microbial Recognition Via Toll-Like Receptor-Dependent and -Independent Pathways Determines the Cytokine Response of Murine Dendritic Cell Subsets to CD40 Triggering
Authors: Edwards Alexander D. , Manickasingham Shivanthi P. , Spörri Roman , Diebold Sandra S. , Schulz Oliver , Sher Alan , Kaisho Tsuneyasu , Akira Shizuo , Reis e Sousa Caetano
Published: April 2014

Dendritic cells (DC) can produce Th-polarizing cytokines and direct the class of the adaptive immune response. Microbial stimuli, cytokines, chemokines, and T cell-derived signals all have been shown to trigger cytokine synthesis by DC, but it remains unclear whether these signals are functionally equivalent and whether they determine the nature of the cytokine produced or simply initiate a preprogrammed pattern of cytokine production, which may be DC subtype specific. Here, we demonstrate that microbial and T cell-derived stimuli can synergize to induce production of high levels of IL-12 p70 or IL-10 by individual murine DC subsets but that the choice of cytokine is dictated by the microbial pattern recognition receptor engaged. We show that bacterial components such as CpG-containing DNA or extracts from Mycobacterium tuberculosis predispose CD8α+ and CD8α−CD4− DC to make IL-12 p70. In contrast, exposure of CD8α+, CD4+ and CD8α−CD4− DC to heat-killed yeasts leads to production of IL-10. In both cases, secretion of high levels of cytokine requires a second signal from T cells, which can be replaced by CD40 ligand. Consistent with their differential effects on cytokine production, extracts from M. tuberculosis promote IL-12 production primarily via Toll-like receptor 2 and an MyD88-dependent pathway, whereas heat-killed yeasts activate DC via a Toll-like receptor 2-, MyD88-, and Toll/IL-1R domain containing protein-independent pathway. These results show that T cell feedback amplifies innate signals for cytokine production by DC and suggest that pattern recognition rather than ontogeny determines the production of cytokines by individual DC subsets.
Endothelial Cells Stimulate T Cell NFAT Nuclear Translocation in the Presence of Cyclosporin A: Involvement of the wnt/Glycogen Synthase Kinase-3β Pathway
Authors: Murphy Lisa L. Salazar , Hughes Christopher C. W.
Published: April 2014

T cells resistant to the immunosuppressive drug cyclosporin A (CsA) may be important mediators of chronic graft rejection. We previously reported that T cells activated in the presence of endothelial cells (EC) develop resistance to CsA, and initiate IL-2 secretion within 8–12 h of triggering. CsA normally blocks the phosphatase, calcineurin, thus preventing nuclear translocation of the transcription factor, NFAT. We find that in the presence but not the absence of EC, NFAT1 can be detected in the nuclei of CsA-treated T cells within 8 h of triggering, reaching a maximal level of 60% of control by 24 h. Glycogen synthase kinase-3β (GSK-3β), which rephosphorylates NFAT and promotes nuclear export, is inhibited by EC costimulation. GSK-3β is a component of the wnt signaling pathway, and EC express wnt-5a and T cells express frizzled-5, a wnt-5a receptor. Wnt-5a promotes T cell NFAT nuclear accumulation in the presence of CsA, an effect mimicked by Li+, a potent inhibitor of GSK-3β. The protein kinase C agonist PMA dramatically synergizes with both EC and wnt-5a in stimulating T cell IL-2 synthesis, and inhibition of either protein kinase C by Ro-31-8425 or G-proteins by pertussis toxin effectively blocks the actions of wnt-5a on T cells. Finally, a secreted, dominant-negative form of frizzled-5 blocks EC-mediated CsA resistance. Thus, EC promote CsA-resistant nuclear localization of NFAT and subsequent IL-2 synthesis through a noncanonical wnt-dependent pathway.
Molecular Mechanism of the Activation-Induced Cell Death Inhibition Mediated by a p70 Inhibitory Killer Cell Ig-Like Receptor in Jurkat T Cells
Authors: Chwae Yong-Joon , Chang Mi Jung , Park Sang Myun , Yoon Ho , Park Hyun-Joo , Kim Se Jong , Kim Jongsun
Published: April 2014

In this study we investigated the molecular mechanism of the activation-induced cell death (AICD) inhibition mediated by a p70 inhibitory killer cell Ig-like receptor (KIR3DL1, also called NKB1) in Jurkat T cells. Using stable Jurkat transfectants that express KIR or CD8-KIR fusion proteins we have shown for the first time that KIR inhibits, in a ligation-independent manner, the AICD induced by PHA, PMA/ionomycin, or anti-CD3 Ab. The AICD inhibition mediated by KIR appears to result from the blockade of Fas ligand induction upon activation of the Jurkat transfectants. Moreover, the membrane-proximal 20 aa of the KIR cytoplasmic tail were determined to play a crucial role in this process. Since the membrane-proximal portion of the KIR cytoplasmic tail contains a putative protein kinase C (PKC) substrate site, we investigated the molecular interaction between KIR and PKC. Immunoprecipitation analysis demonstrated that KIR constitutively bound both to PKCα, a conventional Ca2+-dependent PKC, and to PKCθ, a novel Ca2+-independent PKC. Furthermore, an in vitro kinase assay revealed that PKC activation was blocked after PHA stimulation in Jurkat transfectants expressing KIR. These observations were supported by the finding that a recombinant KIR cytoplasmic tail also appeared to inhibit PKCα activation in vitro. Taken together these data strongly suggest that KIR inhibits the AICD of T cells by blocking Fas ligand induction upon stimulation, in a process that seems to be accomplished by PKC recruitment to the membrane-proximal PKC binding site and subsequent inhibition of PKC activation against the activating stimuli.
Expression of Antigen on Mature Lymphocytes Is Required to Induce T Cell Tolerance by Gene Therapy
Authors: Tian Chaorui , Bagley Jessamyn , Iacomini John
Published: April 2014

Expression of a retrovirally encoded allogeneic MHC class I gene in bone marrow-derived cells can be used to induce tolerance to the product of the retrovirally transduced gene. In this work we examined whether expression of a retrovirally transduced allogeneic MHC class I gene in bone marrow-derived cells from recombinase-activating gene-1 (RAG-1)-deficient mice was sufficient to induce tolerance when transplanted into conditioned hosts together with bone marrow from MHC-matched wild-type mice. Reconstitution of mice with either MHC-matched RAG-1-deficient or wild-type bone marrow transduced with the allogeneic MHC class I gene H-2Kb led to long-term expression of Kb on the surface of bone marrow-derived hematopoietic lineages. T cells from mice reconstituted with H-2Kb-transduced wild-type bone marrow were tolerant to Kb. In contrast, expression of Kb in the periphery of mice reconstituted with a mixture of retrovirally transduced RAG-1-deficient bone marrow and mock-transduced wild-type bone marrow fell below detectable levels by 4 wk after transplantation. T cells that developed in these mice appeared to be hyporesponsive to Kb, demonstrating that expression of Kb on bone marrow-derived APCs was not sufficient to induce tolerance. Our data suggest that induction of tolerance in molecular chimeras requires expression of the retrovirally transduced allogeneic MHC Ag on the surface of mature lymphocytes that populate the host thymus.
Evidence for Involvement of a Hydrophobic Patch in Framework Region 1 of Human V4-34-Encoded Igs in Recognition of the Red Blood Cell I Antigen
Authors: Potter Kathleen N. , Hobby Paul , Klijn Susanne , Stevenson Freda K. , Sutton Brian J.
Published: April 2014

The monoclonal IgM cold agglutinins that bind to the I/i carbohydrate Ags on the surface of RBCs all have Ig H chains encoded by the V4-34 gene segment. This mandatory use indicates that distinctive amino acid sequences may be involved in recognition. Critical amino acids exist in framework region 1 (FR1) of V4-34-encoded Ig, and these generate a specific Id determinant which apparently lies close to the I binding site. However, I binding by Id-expressing Ig can be modulated by sequences in complementarity-determining region (CDR)H3. Examination of the crystal structure of an anti-I cold agglutinin has revealed a hydrophobic patch in FR1 involving residue W7 on β-strand A and the AVY motif (residues 23–25) on β-strand B. In this study we used mutagenesis to show that each of the strand components of the hydrophobic patch is required for binding the I carbohydrate Ag. In addition, the crystal structure reveals that amino acids in the carboxyl-terminal region of CDRH3 form a surface region adjacent to the hydrophobic patch. We propose that the I carbohydrate Ag interacts simultaneously with the entire hydrophobic patch in FR1 and with the outside surface of CDRH3. This interaction could leave most of the conventional binding site available for binding other Ags.
The Role of Polymerase η in Somatic Hypermutation Determined by Analysis of Mutations in a Patient with Xeroderma Pigmentosum Variant
Authors: Yavuz Sule , Yavuz Akif S. , Kraemer Kenneth H. , Lipsky Peter E.
Published: April 2014

To determine the possible role of polymerase η (pol η) in somatic hypermutation of B cells, a mutational analysis of 24 nonproductive rearrangements from a patient with xeroderma pigmentosum variant with a defect in pol η was conducted. Although the mutational frequency of A and T bases decreased in WA (A/T, A) motifs, regardless of their RGYW (purine, G; pyrimidine, A/T) context, the overall mutational frequency of A or T bases was not affected. Moreover, the overall mutational frequency of the sequences examined was not decreased. There was an apparent increase in the number of insertions and deletions. The results are consistent with the conclusion that pol η specifically targets WA motifs. However, its overall contribution to the somatic hypermutational process does not appear to be indispensable and in its absence other mechanisms maintain mutational activity.
Mycobacterium aviumComplex Promotes Recruitment of Monocyte Hosts for HIV-1 and Bacteria
Authors: Hale-Donze Hollie , Greenwell-Wild Teresa , Mizel Diane , Doherty T. Mark , Chatterjee Delphi , Orenstein Jan M. , Wahl Sharon M.
Published: April 2014

In lymphoid tissues coinfected with Mycobacterium avium complex (MAC) and HIV-1, increased viral replication has been observed. This study investigates the role of MAC in perpetuating both infections through the recruitment of monocytes as potential new hosts for bacteria and HIV-1. Increased numbers of macrophages were present in the lymph nodes of patients with dual infection as compared with lymph nodes from HIV+ patients with no known opportunistic pathogens. In a coculture system, monocyte-derived macrophages were treated with HIV-1 or M. avium and its constituents to further define the mechanism whereby MAC infection of macrophages initiates monocyte migration. Monocyte-derived macrophages treated with bacteria or bacterial products, but not HIV-1, induced a rapid 2- to 3-fold increase in recruitment of monocytes. Pretreatment of the monocytes with pertussis toxin inhibited the migration of these cells, indicating a G protein-linked pathway is necessary for induction of chemotaxis and thus suggesting the involvement of chemokines. Analysis of chemokine mRNA and protein levels from M. avium-treated cultures revealed MAC-induced increases in the expression of IL-8, macrophage-inflammatory protein (MIP)-1α, and MIP-1β with donor-dependent changes in monocyte chemotactic protein-1. Pyrrolidine dithiocarbamate, an antioxidant, inhibited the activation of NF-κB and significantly diminished the MAC-induced chemotaxis, concurrently lowering the levels of monocyte chemotactic protein-1 and MIP-1β. These data demonstrate that MAC induces macrophage production of multiple chemotactic factors via NF-κB to promote monocyte migration to sites of MAC infection. In vivo, opportunistic infection may act as a recruitment mechanism in which newly arrived monocytes serve as naive hosts for both MAC and HIV-1, thus perpetuating both infections.
Divergent Role for TNF-α in IFN-γ-Induced Killing ofToxoplasma gondiiandSalmonella typhimuriumContributes to Selective Susceptibility of Patients with Partial IFN-γ Receptor 1 Deficiency
Authors: Janssen Riny , van Wengen Annelies , Verhard Els , de Boer Tjitske , Zomerdijk Timo , Ottenhoff Tom H. M. , van Dissel Jaap T.
Published: April 2014

Patients with defects in IFN-γ- or IL-12-mediated immunity are susceptible to infections with Salmonella and non-tuberculous mycobacteria, but rarely suffer from infections with other intracellular pathogens such as Toxoplasma gondii. Here we describe macrophage and T cell function in eight individuals with partial IFN-γ receptor 1 (IFN-γR1) deficiency due to a mutation that results in elevated cell surface expression of a truncated IFN-γR1 receptor that lacks the intracellular domain. We show that various effector mechanisms dependent on IFN-γR signaling are affected to different extents. Whereas TNF-α production was normally up-regulated in response to IFN-γ, IL-12 production and CD64 up-regulation were strongly reduced, and IFN-γ-mediated killing of the intracellular pathogens Salmonella typhimurium and T. gondii was completely abrogated in patient’s macrophages. Since these patients suffer selectively from infections with non-tuberculous mycobacteria and Salmonella, but not T. gondii, despite sero-immunity in six of eight patients, which indicates previous contact with this pathogen, we next studied the role of TNF-α as a possible immune compensatory mechanism. IFN-γ-induced killing of T. gondii appeared to be partially mediated by TNF-α, and addition of TNF-α could compensate for the abrogated killing of T. gondii in the patient’s macrophages. In contrast, IFN-γ-mediated killing of S. typhimurium appeared to be independent of TNF-α. We propose that the divergent role of TNF-α in IFN-γ-induced killing of T. gondii and S. typhimurium may at least partially explain the highly selective susceptibility of patients.
Silver Activates Calcium Signals in Rat Basophilic Leukemia-2H3 Mast Cells by a Mechanism That Differs from the FcεRI-Activated Response
Authors: Suzuki Yoshihiro , Yoshimaru Tetsuro , Matsui Takashi , Ra Chisei
Published: April 2014

We previously showed that silver stimulates degranulation and leukotriene (LT) C4 production in rat basophilic leukemia mast cells and now show that silver induces these events by a mechanism that differs from the FcεRI-mediated response. In common with FcεRI cross-linking, silver induced tyrosine phosphorylation of extracellular signal-regulated kinases and furthermore, PD98059, a specific inhibitor of extracellular signal-regulated kinase kinase dose-dependently inhibited the silver-induced LTC4 production. In contrast to FcεRI cross-linking, silver had no effect on the production of IL-4 and TNF-α, indicating that different mechanisms are involved in the activation by these two stimuli. In line with this, silver had no or only marginal effect on the tyrosine phosphorylation of FcεRIβ, Lyn, Syk, and linker for activation of T cells, the early and crucial events in FcεRI signaling. Silver induced calcium signals that were involved in the metal-induced degranulation, but not LTC4 production. Unlike Ag, the silver-induced calcium signals were resistant to the depletion of thapsigargin-sensitive calcium stores and the inhibition of tyrosine kinases and phospholipase Cγ. These findings indicate that silver activates mast cells by bypassing the early signaling events required for the induction of calcium influx. Our data strongly suggest the existence of an alternative pathway bypassing the early signaling events in mast cell activation and indicate that silver may be useful for analyses of such alternative mechanisms.
The Immunomodulatory Actions of Prostaglandin E2 on Allergic Airway Responses in the Rat
Authors: Martin James G. , Suzuki Masaru , Maghni Karim , Pantano Rosa , Ramos-Barbón David , Ihaku Daizo , Nantel François , Denis Danielle , Hamid Qutayba , Powell William S.
Published: April 2014

PGE2 has been reported to inhibit allergen-induced airway responses in sensitized human subjects. The aim of this study was to investigate the mechanism of anti-inflammatory actions of PGE2 in an animal model of allergic asthma. BN rats were sensitized to OVA using Bordetella pertussis as an adjuvant. One week later, an aerosol of OVA was administered. After a further week, animals were anesthetized with urethan, intubated, and subjected to measurements of pulmonary resistance (RL) for a period of 8 h after OVA challenge. PGE2 (1 and 3 μg in 100 μl of saline) was administered by insufflation intratracheally 30 min before OVA challenge. The early response was inhibited by PGE2 (3 μg). The late response was inhibited by both PGE2 (1 and 3 μg). Bronchoalveolar lavage fluid from OVA-challenged rats showed eosinophilia and an increase in the number of cells expressing IL-4 and IL-5 mRNA. These responses were inhibited by PGE2. Bronchoalveolar lavage fluid levels of cysteinyl-leukotrienes were elevated after OVA challenge and were reduced after PGE2 to levels comparable with those of sham challenged animals. We conclude that PGE2 is a potent anti-inflammatory agent that may act by reducing allergen-induced Th2 cell activation and cysteinyl-leukotriene synthesis in the rat.
Orderly Pattern of Development of the Autoantibody Response in (New Zealand White × BXSB)F1 Lupus Mice: Characterization of Target Antigens and Antigen Spreading by Two-Dimensional Gel Electrophoresis and Mass Spectrometry
Authors: Thébault Sandrine , Gilbert Danièle , Hubert Marie , Drouot Laurent , Machour Nadine , Lange Catherine , Charlionet Roland , Tron François
Published: April 2014

Immunoblots of a two-dimensional PAGE-separated HL-60 cell proteomic map and mass spectrometry were combined to characterize proteins targeted by autoantibodies produced by male (New Zealand White × BXSB)F1 (WB) mice that develop lupus and anti-phospholipid syndrome. Analysis of sera sequentially obtained from seven individual mice at different ages showed that six proteins, vimentin, heat shock protein 60, UV excision-repair protein RAD23, α-enolase, heterogeneous nuclear ribonucleoprotein L, and nucleophosmin, were the targets of the B cell autoimmune response, and that autoantibodies to them were synthesized sequentially in an orderly pattern that recurred in all the male WB mice analyzed: anti-vimentin first and anti-nucleophosmin last, with anti-RAD23 and anti-heat shock protein 60, then anti-α-enolase and anti-heterogeneous nuclear ribonucleoprotein L Abs occuring concomitantly. Anti-vimentin reactivity always appeared before anti-cardiolipin and anti-DNA Abs, suggesting that vimentin is the immunogen initiating the autoimmune process. The pattern of HL-60 proteins recognized by female WB sera differed from that of male sera, indicating that the Y chromosome-linked autoimmune acceleration gene is not an accelerator but a strong modifier of the autoimmune response. Thus, 1) combining two-dimensional PAGE and mass spectrometry constitutes a powerful tool to identify the set of Ags bound by autoantibodies present in a single serum and the whole autoantibody pattern of an autoimmune disease; 2) the diversification of the autoimmune response in male WB mice occurs in a predetermined pattern consistent with Ag spreading, and thus provides a useful model to further our understanding of the development of the autoantibody response in lupus.
Neonatal Tolerance in the Absence of Stat4- and Stat6- Dependent Th Cell Differentiation
Authors: Chang Hua-Chen , Zhang Shangming , Kaplan Mark H.
Published: April 2014

Neonatal tolerance to specific Ag is achieved by nonimmunogenic exposure within the first day of life. The mechanism that regulates this tolerance may provide the basis for successful organ transplantation and has recently been thought to be immune deviation from the inflammatory Th1 response to a Th2 response. To test the importance of Th2 cells in the establishment of neonatal tolerance, we examined neonatal tolerance in Stat4- and Stat6-deficient mice, which have reduced Th1 and Th2 cell development, respectively. Neonatal tolerance of both the T and B cell compartments in Stat4- and Stat6-deficient mice was similar to that observed in wild-type mice. Cytokine production shifted from a Th1 to a Th2 response in wild-type mice tolerized as neonates. In contrast, tolerance was observed in Stat6-deficient mice despite maintenance of a Th1 cytokine profile. These results suggest that cells distinct from Stat6-dependent Th2 cells are required for the establishment of neonatal tolerance.
Requirement for the p75 TNF-α Receptor 2 in the Regulation of Airway Hyperresponsiveness by γδ T Cells
Authors: Kanehiro Arihiko , Lahn Michael , Mäkelä Mika J. , Dakhama Azzeddine , Joetham Anthony , Rha Yeong-Ho , Born Willi , Gelfand Erwin W.
Published: April 2014

In a recent study, we found that TNF-α negatively regulates airway responsiveness through the activation of γδ T cells. The biological activities of TNF-α are mediated by two structurally related but functionally distinct receptors, p55 (TNFR1) and p75 (TNFR2), which are independently expressed on the cell surface. However, the relative importance of either TNFR in airway hyperresponsiveness (AHR) is unknown. To investigate the importance of these TNFRs in the development of allergen-induced AHR, p55-deficient and p75-deficient mice were sensitized to OVA by i.p. injection and subsequently challenged with OVA via the airways; airway responsiveness to inhaled methacholine was monitored. p75-deficient mice developed AHR to a similar degree as control mice. In contrast, p55-deficient mice, which were sensitized and challenged with OVA, failed to develop AHR. In p55-deficient mice, both the numbers of eosinophils and levels of IL-5 in bronchoalveolar lavage fluid were significantly lower than in sensitized/challenged control mice (p < 0.05). However, depletion of γδ T cells resulted in significant increases in AHR in the p55-deficient mice, whereas no significant effect of γδ T cell depletion was evident in the p75-deficient mice. These data indicate that, in the absence of TNFR1 (p55), where TNF-α uses the p75 pathway exclusively, the development of AHR is regulated by γδ T cells.
T Cell-Independent Regulation of IgE Antibody Production Induced by Surface-Linked Liposomal Antigen
Authors: Taneichi Maiko , Naito Seishiro , Kato Hiroshi , Tanaka Yuriko , Mori Masahito , Nakano Yoshio , Yamamura Hiroyuki , Ishida Hiroshi , Komuro Katsutoshi , Uchida Tetsuya
Published: April 2014

Control of IgE Ab production is important for the prevention of IgE-related diseases. However, in contrast to the existing information on the induction of IgE production, little is known about the regulation of the production of this isotype, with the exception of the well-documented mechanism involving T cell subsets and their cytokine products. In this study, we demonstrate an alternative approach to interfere with the production of IgE, independent of the activity of T cells, which was discovered during the course of an investigation intended to clarify the mechanism of IgE-selective unresponsiveness induced by surface-coupled liposomal Ags. Immunization of mice with OVA-liposome conjugates induced IgE-selective unresponsiveness without apparent Th1 polarization. Neither IL-12, IL-10, nor CD8+ T cells participated in the regulation. Furthermore, CD4+ T cells of mice immunized with OVA-liposome were capable of inducing Ag-specific IgE synthesis in athymic nude mice immunized with alum-adsorbed OVA. In contrast, immunization of the recipient mice with OVA-liposome did not induce anti-OVA IgE production, even when CD4+ T cells of mice immunized with alum-adsorbed OVA were transferred. In the secondary immune response, OVA-liposome enhanced anti-OVA IgG Ab production, but it did not enhance ongoing IgE production, suggesting that the IgE-selective unresponsiveness induced by the liposomal Ag involved direct effects on IgE, but not IgG switching in vivo. These results suggest the existence of an alternative mechanism not involving T cells in the regulation of IgE synthesis.