bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple Cytokines Regulate the NK Gene Complex-Encoded Receptor Repertoire of Mature NK Cells and T Cells
Authors: Gays Frances , Martin Kimberley , Kenefeck Rupert , Aust Jonathan G. , Brooks Colin G.
Published: April 2014

Mature NK cells comprise a highly diverse population of lymphocytes that express different permutations of receptors to facilitate recognition of diseased cells and perhaps pathogens themselves. Many of these receptors, such as those belonging to the NKRP1, NKG2, and Ly49 families are encoded in the NK gene complex (NKC). It is generally thought that these NKC-encoded receptors are acquired by a poorly understood stochastic mechanism, which operates exclusively during NK cell development, and that following maturation the repertoire is fixed. However, we report a series of observations that demonstrates that the mature NK cell repertoire in mice can in fact be radically remodeled by multiple cytokines. Thus, both IL-2 and IL-15 selectively induce the de novo expression of Ly49E on the majority of mature NK cells. By contrast, IL-4 not only blocks this IL-2-induced acquisition of Ly49E, but reduces the proportion of mature NK cells that expresses pre-existing Ly49 receptors and abrogates the expression of NKG2 receptors while leaving the expression of several NKRP1 receptors unaltered. IL-21 also abrogates NKG2 expression on mature NK cells and selectively down-regulates Ly49F. IL-4 and IL-21 additionally cause dramatic and selective alterations in the NKC-encoded receptor repertoire of IL-2-activated T cells but these are quite different to the changes induced on NK cells. Collectively these findings reveal an unexpected aspect of NKC receptor expression that has important implications for our understanding of the function of these receptors and of the genetic mechanisms that control their expression.
Sphingosine-1-Phosphate Mediates Migration of Mature Dendritic Cells
Authors: Czeloth Niklas , Bernhardt Günter , Hofmann Fred , Genth Harald , Förster Reinhold
Published: April 2014

Sphingosine-1-phosphate (S1P) represents a potent modulator of diverse cellular activities, including lymphocyte trafficking and maintenance of lymphocyte homeostasis. The five known receptors for S1P (S1P1–5) belong to the family of G protein-coupled receptors. Upon binding S1P, they act downstream via heterotrimeric G proteins on members of the small GTPase family (Cdc42/Rac/Rho), evoking a S1P receptor-dependent activation pattern of Cdc42, Rac, and Rho, respectively. This, in turn, triggers cytoskeletal rearrangements determining cellular morphology and movement. In this study we investigated the effects of S1P on murine dendritic cells (DC). Mature DC, but not immature in vitro differentiated DC, were found to migrate to S1P, a phenomenon that correlated to the up-regulation of S1P1 and S1P3 in maturing DC. The same pattern of S1P receptor regulation could be observed in vivo on skin DC after their activation and migration into the lymph node. The migration-inducing effect of S1P could be severely hampered by application of the S1P analogon FTY720 in vitro and in vivo. A similar, yet more pronounced, block was observed upon preventing Cdc42/Rac and/or Rho activation by specific inhibitors. These results suggest that S1P-mediated signaling plays a pivotal role in the life cycle of DC.
Galectin-9 Induces Maturation of Human Monocyte-Derived Dendritic Cells
Authors: Dai Shu-Yan , Nakagawa Ryusuke , Itoh Aiko , Murakami Hiromoto , Kashio Yumiko , Abe Hiroko , Katoh Shigeki , Kontani Keiichi , Kihara Minoru , Zhang Shu-Lan , Hata Toshiyuki , Nakamura Takanori , Yamauchi Akira , Hirashima Mitsuomi
Published: April 2014

Maturation of dendritic cells (DCs) is critical for initiation of immune responses and is regulated by various stimulatory signals. We assessed the role of galectin (Gal)-9 in DC maturation. Culture of immature DCs with exogenous Gal-9 markedly increased the surface expression of CD40, CD54, CD80, CD83, CD86, and HLA-DR in a dose-dependent manner, although Gal-9 had no or little effect on differentiation of human monocytes into immature DCs. Gal-9-treated DCs secreted IL-12 but not IL-10, and they elicited the production of Th1 cytokines (IFN-γ and IL-2) but not that of the Th2 cytokines (IL-4 and IL-5) by allogeneic CD4+ T cells. These effects of Gal-9 on immature DCs were not essentially dependent on its lectin properties, given that they were inhibited only slightly by lactose. We further found that a Gal-9 mutant that lacks β-galactoside binding activity reproduced the above activities and that an anti-Gal-9 mAb suppressed them. Gal-9 induced phosphorylation of the MAPK p38 and ERK1/2 in DCs, and an inhibitor of p38 signaling, but not inhibitors of signaling by either ERK1/2 or PI3K, blocked Gal-9-induced up-regulation of costimulatory molecule expression and IL-12 production. These findings suggest that Gal-9 plays a role not only in innate immunity but also in acquired immunity by inducing DC maturation and promoting Th1 immune responses.
Activation of Vascular Endothelial Growth Factor Receptor-2 in Bone Marrow Leads to Accumulation of Myeloid Cells: Role of Granulocyte-Macrophage Colony-Stimulating Factor
Authors: Larrivée Bruno , Pollet Ingrid , Karsan Aly
Published: April 2014

Vascular endothelial growth factor (VEGF) is a secreted cytokine that plays a major role in the formation and maintenance of the hemopoietic and vascular compartments. VEGF and its receptors, VEGFR-1 and VEGFR-2, have been found to be expressed on subsets of normal and malignant hemopoietic cells, but the role of the individual receptors in hemopoiesis requires further study. Using a VEGFR-2 fusion protein that can be dimerized with a synthetic drug, we were able to specifically examine the effects of VEGFR-2 signaling in hemopoietic cells in vivo. Mice transplanted with bone marrow transduced with this inducible VEGFR-2 fusion protein demonstrated expansion of myeloid cells (Gr-1+, CD11b+). Levels of myeloid progenitors were also increased following VEGFR-2 activation, through autocrine and paracrine mechanisms, as measured by clonogenic progenitor assays. VEGFR-2 activation induced expression of GM-CSF and increased serum levels in vivo. Abrogation of GM-CSF activity, either with neutralizing Abs or by using GM-CSF-null hemopoietic cells, inhibited VEGFR-2-mediated myeloid progenitor activity. Our findings indicate that VEGF signaling through VEGFR-2 promotes myelopoiesis through GM-CSF-dependent and -independent mechanisms.
Natural Recovery and Protection from Autoimmune Encephalomyelitis: Contribution of CD4+CD25+ Regulatory Cells within the Central Nervous System
Authors: McGeachy Mandy J. , Stephens Leigh A. , Anderton Stephen M.
Published: April 2014

Immune regulation of autoimmune disease can function at two sites: at the secondary lymphoid organs or in the target organ itself. In this study, we investigated the natural resolution of autoimmune pathology within the CNS using murine experimental autoimmune encephalomyelitis (EAE). Recovery correlates with the accumulation of IL-10-producing CD4+CD25+ T cells within the CNS. These CD4+CD25+ cells represent as many as one in three of CD4+ cells in the CNS during recovery, they are FoxP3+ and express other markers associated with regulatory cells (CTLA-4, GITR, and αEβ7), and they have regulatory function ex vivo. Depletion of CD25+ cells inhibits the natural recovery from EAE. Also, depletion of CD25+ cells after recovery removes the resistance to reinduction of EAE observed in this model. Furthermore, passive transfer of CNS-derived CD4+CD25+ cells in low numbers provides protection from EAE in recipient mice. These are the first data demonstrating the direct involvement of CD4+CD25+ regulatory T cells in the natural resolution of autoimmune disease within the target organ.
Conditional Fas-Associated Death Domain Protein (FADD):GFP Knockout Mice Reveal FADD Is Dispensable in Thymic Development but Essential in Peripheral T Cell Homeostasis
Authors: Zhang Yuhang , Rosenberg Stephen , Wang Hanming , Imtiyaz Hongxia Z. , Hou Ying-Ju , Zhang Jianke
Published: April 2014

Fas-associated death domain protein (FADD)/mediator of receptor-induced toxicity-1 is required for signaling induced by death receptors such as Fas. In earlier studies, FADD-deficient mice died in utero, and a FADD deficiency in embryonic stem cells inhibited T cell production in viable FADD−/−→RAG-1−/− chimeras. To analyze the temporal requirement of FADD in the development and function in the T lineage, it is necessary to establish viable mutant mice producing detectable FADD-deficient T cells. We generated mice that express a functional FADD:GFP fusion gene reconstituting normal embryogenesis and lymphopoiesis in the absence of the endogenous FADD. Efficient T cell-specific deletion of FADD:GFP was achieved, as indicated by the presence of a high percentage of GFP-negative thymocytes and peripheral T cells in mice expressing Lck-Cre or CD4-Cre. Sorted GFP-negative thymocytes and peripheral T cells contained undetectable levels of FADD and were resistant to apoptosis induced by Fas, TNF, and TCR restimulation. These T cell-specific FADD-deficient mice contain normal thymocyte numbers, but fewer peripheral T cells. Purified peripheral FADD-deficient T cells failed to undergo extensive homeostatic expansion after adoptive transfer into lymphocyte-deficient hosts, and responded poorly to proliferation induced by ex vivo TCR stimulation. Furthermore, deletion of FADD in preactivated mature T cells using retrovirus-Cre resulted in no proliferation. These results demonstrate that FADD plays a dispensable role during thymocyte development, but is essential in maintaining peripheral T cell homeostasis and regulating both apoptotic and proliferation signals.
Restoration by IL-15 of MHC Class I Antigen-Processing Machinery in Human Dendritic Cells Inhibited by Tumor-Derived Gangliosides
Authors: Tourkova Irina L. , Shurin Galina V. , Chatta Gurkamal S. , Perez Lori , Finke James , Whiteside Theresa L. , Ferrone Soldano , Shurin Michael R.
Published: April 2014

We have recently reported that MHC class I Ag-processing machinery (APM) component expression in dendritic cells (DC) might be down-regulated by tumor cells. However, the tumor-derived factors responsible for inhibition of the APM component expression in DC generated in the tumor microenvironment as well as potential protective mechanism have not yet been investigated. In this article, we demonstrate that expression of several MHC class I APM components, including MB1 (β5), LMP2, LMP7, LMP10, and ERp57, is significantly down-regulated in human DC generated in the presence of primary oral squamous cell carcinoma cell lines or coincubated with purified gangliosides. Suppression of MHC class I APM component expression in DC generated in the presence of tumor cells was significantly attenuated by the inhibition of glucosyl transferase in tumor cells, suggesting that tumor-induced MHC class I APM component down-regulation in DC was mediated in part by oral squamous cell carcinoma-derived gangliosides. Furthermore, rIL-15 restored both tumor cell-induced and ganglioside-induced MHC class I APM component expression in DC, as well as their ability to present Ags to autologous Ag-specific T cells. These results demonstrate that IL-15 restores MHC class I APM component expression in DC down-regulated by tumor-derived gangliosides.
Krüppel-Like Factor 2, a Novel Immediate-Early Transcriptional Factor, Regulates IL-2 Expression in T Lymphocyte Activation
Authors: Wu Jinghai , Lingrel Jerry B.
Published: April 2014

Ag presentation to T lymphocytes and subsequent activation are characterized by a cascade of signaling events, some of which result in the transcriptional activation of a diverse set of genes. An important example is the induction of the IL-2 gene, which is a critical event in the escalation of T cell activation. Previous studies have found that expression of Krüppel-like factor 2 (KLF2), a zinc finger transcription factor, is extinguished after T cell activation. However, the biological role of KLF2 during T cell activation is still unknown. In this study we found that KLF2 protein degradation is delayed, and KLF2 expression is up-regulated during the early stage of T cell activation in primary T cells. Within a few hours, this process is reversed, and KLF2 expression is turned off. Next, we found that the expression of KLF2 significantly increases IL-2 production 4-fold in activated T cells, resulting from activation of the IL-2 promoter. By narrowing down the 2.0-kb IL-2 promoter region, we found that the KLF2 responsive element in the IL-2 promoter is a CACCC element, the KLF consensus binding motif. Moreover, KLF2 binds to this promoter in vivo under different conditions. Our studies show that KLF2 regulates IL-2 promoter activity in the earliest stages of T cell activation, indicating that KLF2 may act as a novel immediate-early transcriptional factor to maximally prime T cell activation.
High Sensitivity of CD4+CD25+ Regulatory T Cells to Extracellular Metabolites Nicotinamide Adenine Dinucleotide and ATP: A Role for P2X7 Receptors
Authors: Aswad Fred , Kawamura Hiroki , Dennert Gunther
Published: April 2014

Although regulatory lymphocytes play an important role in the immune system, the regulation of their functions is poorly understood and remains to be elucidated. In this study we demonstrate that micromolar concentrations of the common cell metabolite NAD induce death in murine forkhead/winged helix transcription factor gene-expressing CD4+CD25+ regulatory T cells with high efficiency and within minutes. Similar, but less dramatic, effects are demonstrable with ATP and its nonhydrolysable derivative, benzoylbenzoyl-ATP. Other T cell subsets are more resistant, with CD8 cells being the least sensitive and CD4 cells expressing intermediate sensitivity. The higher sensitivity of CD4+CD25+ cells is demonstrable in vivo. Injection of NAD or benzoylbenzoyl-ATP causes preferential induction of a cell death signal in CD4+CD25+ cells. Transmission of the death signal requires functional P2X7 receptors, pointing to a role for these receptors in regulation and homeostasis of CD4+CD25+ regulatory T cells. Consistent with this, P2X7R gene-deleted mice possess increased levels of forkhead/winged helix transcription factor gene-expressing CD4+CD25+ cells.
CD1d-Restricted NKT Cells Contribute to the Age-Associated Decline of T Cell Immunity
Authors: Faunce Douglas E. , Palmer Jessica L. , Paskowicz Kelly K. , Witte Pamela L. , Kovacs Elizabeth J.
Published: April 2014

NKT cells are known to regulate effector T cell immunity during tolerance, autoimmunity, and antitumor immunity. Whether age-related changes in NKT cell number or function occur remains unclear. Here, we investigated whether young vs aged (3 vs 22 mo old) mice had different numbers of CD1d-restricted NKT cells and whether activation of NKT cells by CD1d in vivo contributed to age-related suppression of T cell immunity. Flow cytometric analyses of spleen and LN cells revealed a 2- to 3-fold increase in the number of CD1d tetramer-positive NKT cells in aged mice. To determine whether NKT cells from aged mice differentially regulated T cell immunity, we first examined whether depletion of NK/NKT cells affected the proliferative capacity of splenic T cells. Compared with those from young mice, intact T cell preparations from aged mice had impaired proliferative responses whereas NK/NKT-depleted preparations did not. To examine the specific contribution of NKT cells to age-related T cell dysfunction, Ag-specific delayed-type hypersensitivity and T cell proliferation were examined in young vs aged mice given anti-CD1d mAb systemically. Compared with young mice, aged mice given control IgG exhibited impaired Ag-specific delayed-type hypersensitivity and T cell proliferation, which could be significantly prevented by systemic anti-CD1d mAb treatment. The age-related impairments in T cell immunity correlated with an increase in the production of the immunosuppressive cytokine IL-10 by splenocytes that was likewise prevented by anti-CD1d mAb treatment. Together, our results suggest that CD1d activation of NKT cells contributes to suppression of effector T cell immunity in aged mice.
Turning On/Off Tumor-Specific CTL Response during Progressive Tumor Growth
Authors: Huang Yujun , Obholzer Nikolaus , Fayad Raja , Qiao Liang
Published: April 2014

Therapeutic vaccinations used to induce CTLs and treat firmly established tumors are generally ineffective. To understand the mechanisms underlying the failure of therapeutic vaccinations, we investigated the fate of tumor-specific CD8+ T cells in tumor-bearing mice with or without vaccinations. Our data demonstrate that tumor-specific CD8+ T cells are activated at the early stage of tumor growth, tumor-specific CTL response reaches a maximal level during progressive tumor growth, and tumor-specific CD8+ T cells lose cytolytic function at the late stage of tumor growth. The early stage therapeutic vaccination induces efficient antitumor activity by amplifying the CTL response, whereas the late-stage therapeutic vaccination is invalid due to tumor-induced dysfunction of CD8+ T cells. However, at the late stage, tumor-specific CD8+ T cells are still present in the periphery. These tumor-specific CD8+ T cells lose cytolytic activity, but retain IFN-γ secretion function. In contrast to in vitro cultured tumor cells, in vivo growing tumor cells are more resistant to tumor-specific CTL killing, despite an increase of tumor Ag gene expression. Both tumor-induced CD8+ T cell dysfunction at the late stage and immune evasion developed by in vivo growing tumor cells contribute to an eventual inefficacy of therapeutic vaccinations. Our study suggests that it is important to design a vaccination regimen according to the stages of tumor growth and the functional states of tumor-specific CD8+ T cells.
Selective Defect in Antigen-Induced TCR Internalization at the Immune Synapse of CD8 T Cells Bearing the ZAP-70(Y292F) Mutation
Authors: Davanture Suzel , Leignadier Julie , Milani Pascale , Soubeyran Philippe , Malissen Bernard , Malissen Marie , Schmitt-Verhulst Anne-Marie , Boyer Claude
Published: April 2014

Cbl proteins have been implicated in ligand-induced TCR/CD3 down-modulation, but underlying mechanisms are unclear. We analyzed the effect of mutation of a cbl-binding site on ZAP-70 (ZAP-Y292F) on dynamics, internalization, and degradation of the TCR/CD3 complex in response to distinct stimuli. Naive CD8 T cells expressing the P14 transgenic TCR from ZAP-Y292F mice were selectively affected in TCR/CD3 down-modulation in response to antigenic stimulation, whereas neither anti-CD3 Ab-, and PMA-induced TCR down-modulation, nor constitutive receptor endocytosis/cycling were impaired. We further established that the defect in TCR/CD3 down-modulation in response to Ag was paralleled by an impaired TCR/CD3 internalization and CD3ζ degradation. Analysis of T/APC conjugates revealed that delayed redistribution of TCR at the T/APC contact zone was paralleled by a delay in TCR internalization in the synaptic zone in ZAP-Y292F compared with ZAP-wild-type T cells. Cbl recruitment to the synapse was also retarded in ZAP-Y292F T cells, although F-actin and LFA-1 redistribution was similar for both cell types. This study identifies a step involving ZAP-70/cbl interaction that is critical for rapid internalization of the TCR/CD3 complex at the CD8 T cell/APC synapse.
Human M-Ficolin Is a Secretory Protein That Activates the Lectin Complement Pathway
Authors: Liu Yu , Endo Yuichi , Iwaki Daisuke , Nakata Munehiro , Matsushita Misao , Wada Ikuo , Inoue Keiichi , Munakata Mitsuru , Fujita Teizo
Published: April 2014

Three types of ficolins have been identified in humans: L-ficolin, M-ficolin, and H-ficolin. Similar to mannose-binding lectin, L-ficolin and H-ficolin are the recognition molecules in the lectin complement pathway. Another human ficolin, M-ficolin, is a nonserum ficolin that is expressed in leukocytes and lung; however, little is known about its physiologic roles. In this study, we report the characterization of M-ficolin in terms of its protein localization and lectin activity. M-ficolin was localized in secretory granules in the cytoplasm of neutrophils, monocytes, and type II alveolar epithelial cells in lung. M-ficolin precipitated with mannose-binding lectin-associated serine proteases (MASP)-1 and MASP-2 in a coimmunoprecipitation assay, indicating that M-ficolin forms complexes with MASP-1 and MASP-2. M-ficolin-MASP complexes activated complement on N-acetylglucosamine (GlcNAc)-coated microplates in a C4 deposition assay. M-ficolin bound to several neoglycoproteins bearing GlcNAc, N-acetylgalactosamine, and sialyl-N-acetyllactosamine, suggesting that M-ficolin can recognize the common carbohydrate residues found in microbes. Indeed, M-ficolin bound to Staphylococcus aureus through GlcNAc. These results indicate that M-ficolin, like its family members, functions as a recognition molecule of the lectin complement pathway and plays an important role in innate immunity.
Leishmania donovani Affects Antigen Presentation of Macrophage by Disrupting Lipid Rafts
Authors: Chakraborty Debabani , Banerjee Subha , Sen Abhik , Banerjee Kalyan K. , Das Pradip , Roy Syamal
Published: April 2014

Leishmania donovani-infected splenic macrophages and P388D1 (P388D1(I)) failed to activate T cells in response to low dose of exogenous peptide. The membrane fluidity of P388D1(I) was greater than that of the normal counterpart P388D1(N), but could be reduced either by exposing the cell below phase transition point or by loading cholesterol into membrane (L-P388D1(I)), and this was associated with enhanced Ag-presenting ability of P388D1(I). Presentation of endogenous leishmanial Ag, kinetoplastid membrane protein-11, was also defective, but could be corrected by loading cholesterol into membrane. Because membrane rafts are important for Ag presentation at a low peptide dose, raft architecture of P388D1(I) was studied using raft (CD48 and cholera toxin-B) and non-raft (CD71) markers in terms of their colocalization with I-Ad. Binding of anti-CD48 mAb and cholera toxin B subunit decreased significantly in P388D1(I), and consequently, colocalization with I-Ad was not seen, but this could be restored in L-P388D1(I). Conversely, colocalization between I-Ad and CD71 remained unaffected regardless of the presence or the absence of intracellular parasites. P388D1(N) and L-P388D1(I), but not P388D1(I), formed peptide-dependent synapse with T cells quite efficiently and this was found to be corroborated with both intracellular Ca2+ mobilization in T cells and IL-2 production. This indicated that intracellular parasites disrupt the membrane rafts, possibly by increasing the membrane fluidity, which could be corrected by making the membrane rigid. This may be a strategy that intracellular L. donovani adopts to evade host immune system.
TNF-α-Dependent and -Independent Maturation of Dendritic Cells and Recruited CD11cintCD11b+ Cells during Oral Salmonella Infection
Authors: Sundquist Malin , Wick Mary Jo
Published: April 2014

Maturation of dendritic cells (DC) is crucial for their ability to induce adaptive immunity. Although several mediators of DC maturation have been found, their contributions to DC maturation during infection are poorly understood. In this study we show that murine conventional (CD11chigh) DC up-regulate costimulatory molecules in a subset-specific manner after oral Salmonella infection. Although both CD8α+ and CD8α− subsets increase CD86 expression, CD40 was preferentially up-regulated on CD8α+ DC, and CD80 was preferentially increased on CD8α− DC. In addition, high levels of CD80 and CD86 were found on CD11cintCD11b+ cells that accumulated in infected organs. Costimulatory molecules were simultaneously induced on CD11chigh and CD11cintCD11b+ cells in Peyer’s patches, mesenteric lymph nodes and spleen 5 days after infection despite different kinetics of peak bacterial burden in these organs. Up-regulation of costimulatory molecules occurred on all DC within the respective subset. Moreover, <1% of CD11c-expressing cells associated with Salmonella expressing enhanced GFP in vivo. Thus, DC maturation did not depend on bacterial uptake. Rather, infection-induced up-regulation of CD80, CD86, and CD40 on CD11c-expressing cells of mesenteric lymph nodes was dependent on TNFR type I (TNFRI) signaling. Although indirect up-regulation of costimulatory molecules on DC and CD11cintCD11b+ cells was TNFRI dependent, cells directly associated with Salmonella were able to mature independently of TNFRI signaling. Thus, Salmonella-induced TNF-α is an important mediator of indirect DC maturation during infection, whereas a TNF-α-independent maturation pathway contributes to direct maturation of bacteria-associated DC.
α-Galactosylceramide Can Act As a Nasal Vaccine Adjuvant Inducing Protective Immune Responses against Viral Infection and Tumor
Authors: Ko Sung-Youl , Ko Hyun-Jeong , Chang Woo-Sung , Park Se-Ho , Kweon Mi-Na , Kang Chang-Yuil
Published: April 2014

α-Galactosylceramide (α-GalCer) is a ligand of invariant Vα14+ NKT cells and is presented by CD1d molecule on APC. NKT cells produce a large amount of Th1 and Th2 cytokines in response to α-GalCer-presented APC. In this study, we assessed whether α-GalCer could act as an effective nasal vaccine adjuvant for mucosal vaccine that would be capable of inducing systemic as well as mucosal immune responses. When α-GalCer was administered with OVA via the intranasal route to C57BL/6 and BALB/c mice, significant OVA-specific mucosal secretory IgA, systemic IgG, and CTL responses were induced with mixed Th1 and Th2 cytokine profiles seen in both strains of mice. Interestingly, as BALB/c mice were intranasally immunized with PR8 hemagglutinin Ag isolated from influenza virus A/PR/8/34 together with α-GalCer, significant protection was afforded against influenza viral infection. When α-GalCer was coimmunized with a replication-deficient live adenovirus to BALB/c mice, it significantly induced both humoral and cellular immune responses. In addition, intranasal administration of OVA with α-GalCer showed complete protection against EG7 tumor challenge in C57BL/6. The adjuvant effects induced by intranasal coadministration with α-GalCer were blocked in CD1d−/− mice, indicating that the immune responses were exclusively mediated by CD1d molecule on APC. Most interestingly, intranasally coadministered α-GalCer activated naive T cells and triggered them to differentiate into functional effector T cells when CFSE-labeled OT-1 cells were adoptively transferred into syngeneic mice. Overall, our results are the first to show that α-GalCer can act as a nasal vaccine adjuvant inducing protective immune responses against viral infections and tumors.
Expression of Many Immunologically Important Genes in Mycobacterium tuberculosis-Infected Macrophages Is Independent of Both TLR2 and TLR4 but Dependent on IFN-αβ Receptor and STAT1
Authors: Shi Shuangping , Blumenthal Antje , Hickey Christopher M. , Gandotra Sheetal , Levy David , Ehrt Sabine
Published: April 2014

Macrophages respond to several subcellular products of Mycobacterium tuberculosis (Mtb) through TLR2 or TLR4. However, primary mouse macrophages respond to viable, virulent Mtb by pathways largely independent of MyD88, the common adaptor molecule for TLRs. Using microarrays, quantitative PCR, and ELISA with gene-disrupted macrophages and mice, we now show that viable Mtb elicits the expression of inducible NO synthase, RANTES, IFN-inducible protein 10, immune-responsive gene 1, and many other key genes in macrophages substantially independently of TLR2, TLR4, their combination, or the TLR adaptors Toll-IL-1R domain-containing adapter protein and Toll-IL-1R domain-containing adapter inducing IFN-β. Mice deficient in both TLR2 and TLR4 handle aerosol infection with viable Mtb as well as congenic controls. Viable Mtb also up-regulates inducible NO synthase, RANTES, IFN-inducible protein 10, and IRG1 in macrophages that lack mannose receptor, complement receptors 3 and 4, type A scavenger receptor, or CD40. These MyD88, TLR2/4-independent transcriptional responses require IFN-αβR and STAT1, but not IFN-γ. Conversely, those genes whose expression is MyD88 dependent do not depend on IFN-αβR or STAT1. Transcriptional induction of TNF is TLR2/4, MyD88, STAT1, and IFN-αβR independent, but TNF protein release requires the TLR2/4-MyD88 pathway. Thus, macrophages respond transcriptionally to viable Mtb through at least three pathways. TLR2 mediates the responses of a numerically minor set of genes that collectively do not appear to affect the course of infection in mice; regulation of TNF requires TLR2/4 for post-transcriptional control, but not for transcriptional induction; and many responding genes are regulated through an unknown, TLR2/4-independent pathway that may involve IFN-αβR and STAT1.
Inhibition of Neutrophil Elastase by α1-Protease Inhibitor at the Surface of Human Polymorphonuclear Neutrophils
Authors: Korkmaz Brice , Attucci Sylvie , Jourdan Marie-Lise , Juliano Luiz , Gauthier Francis
Published: April 2014

The uncontrolled proteolytic activity in lung secretions during lung inflammatory diseases might be due to the resistance of membrane-bound proteases to inhibition. We have used a new fluorogenic neutrophil elastase substrate to measure the activity of free and membrane-bound human neutrophil elastase (HNE) in the presence of α1-protease inhibitor (α1-Pi), the main physiological inhibitor of neutrophil serine proteases in lung secretions. Fixed and unfixed neutrophils bore the same amounts of active HNE at their surface. However, the HNE bound to the surface of unfixed neutrophils was fully inhibited by stoichiometric amounts of α1-Pi, unlike that of fixed neutrophils. The rate of inhibition of HNE bound to the surface of unfixed neutrophils was the same as that of free HNE. In the presence of α1-Pi, membrane-bound elastase is almost entirely removed from the unfixed neutrophil membrane to form soluble irreversible complexes. This was confirmed by flow cytometry using an anti-HNE mAb. HNE activity rapidly reappeared at the surface of HNE-depleted cells when they were triggered with the calcium ionophore A23187, and this activity was fully inhibited by stoichiometric amounts of α1-Pi. HNE was not released from the cell surface by oxidized, inactive α1-Pi, showing that active inhibitor is required to interact with active protease from the cell surface. We conclude that HNE activity at the surface of human neutrophils is fully controlled by α1-Pi when the cells are in suspension. Pericellular proteolysis could be limited to zones of contact between neutrophils and subjacent protease substrates where natural inhibitors cannot penetrate.
Specific Inhibition of MyD88-Independent Signaling Pathways of TLR3 and TLR4 by Resveratrol: Molecular Targets Are TBK1 and RIP1 in TRIF Complex
Authors: Youn Hyung S. , Lee Joo Y. , Fitzgerald Katherine A. , Young Howard A. , Akira Shizuo , Hwang Daniel H.
Published: April 2014

TLRs can activate two distinct branches of downstream signaling pathways. MyD88 and Toll/IL-1R domain-containing adaptor inducing IFN-β (TRIF) pathways lead to the expression of proinflammatory cytokines and type I IFN genes, respectively. Numerous reports have demonstrated that resveratrol, a phytoalexin with anti-inflammatory effects, inhibits NF-κB activation and other downstream signaling pathways leading to the suppression of target gene expression. However, the direct targets of resveratrol have not been identified. In this study, we attempted to identify the molecular target for resveratrol in TLR-mediated signaling pathways. Resveratrol suppressed NF-κB activation and cyclooxygenase-2 expression in RAW264.7 cells following TLR3 and TLR4 stimulation, but not TLR2 or TLR9. Further, resveratrol inhibited NF-κB activation induced by TRIF, but not by MyD88. The activation of IFN regulatory factor 3 and the expression of IFN-β induced by LPS, poly(I:C), or TRIF were also suppressed by resveratrol. The suppressive effect of resveratrol on LPS-induced NF-κB activation was abolished in TRIF-deficient mouse embryonic fibroblasts, whereas LPS-induced degradation of IκBα and expression of cyclooxygenase-2 and inducible NO synthase were still inhibited in MyD88-deficient macrophages. Furthermore, resveratrol inhibited the kinase activity of TANK-binding kinase 1 and the NF-κB activation induced by RIP1 in RAW264.7 cells. Together, these results demonstrate that resveratrol specifically inhibits TRIF signaling in the TLR3 and TLR4 pathway by targeting TANK-binding kinase 1 and RIP1 in TRIF complex. The results raise the possibility that certain dietary phytochemicals can modulate TLR-derived signaling and inflammatory target gene expression and can alter susceptibility to microbial infection and chronic inflammatory diseases.
Stat3 in Resident Macrophages as a Repressor Protein of Inflammatory Response
Authors: Matsukawa Akihiro , Kudo Shinji , Maeda Takako , Numata Kousuke , Watanabe Hiroyuki , Takeda Kiyoshi , Akira Shizuo , Ito Takaaki
Published: April 2014

Inflammation is counterbalanced by anti-inflammatory cytokines such as IL-10, in which Stat3 mediates the signaling pathway. In this study, we demonstrate that resident macrophages, but not other cell types, are important targets of IL-10 in a murine model of acute peritonitis. Injection of thioglycollate i.p. induced a considerable number of neutrophils and macrophages in the peritoneum, which was significantly augmented in mice with a cell-type specific disruption of the Stat3 gene in macrophages and neutrophils (LysMcre/Stat3flox/− mice). The augmented leukocyte infiltration was accompanied by increased peritoneal levels of TNF-α, MIP-2, KC chemokine (KC), and MCP-1/CCL2. Stat3 was tyrosine phosphorylated in peritoneal resident macrophages as well as infiltrating leukocytes in the littermate controls, suggesting that Stat3 in either or both of these cells might play a regulatory role in inflammation. The peritoneal levels of TNF-α, MIP-2, KC, and MCP-1 were similarly elevated in LysMcre/Stat3flox/− mice rendered leukopenic by cyclophosphamide treatment as compared with the controls. Adoptive transfer of resident macrophages from LysMcre/Stat3flox/− mice into the control littermates resulted in increases in the peritoneal level of TNF-α, MIP-2, KC, and MCP-1 after i.p. injection of thioglycollate. Under these conditions, control littermates harboring LysMcre/Stat3flox/− macrophages exhibited an augmented leukocyte infiltration relative to those received control macrophages. Taken together, these data provide evidence that resident macrophages, but not other cell types, play a regulatory role in inflammation through a Stat3 signaling pathway. Stat3 in resident macrophages appears to function as a repressor protein in this model of acute inflammation.
Modulation of Lipopolysaccharide-Induced Gene Transcription and Promotion of Lung Injury by Mechanical Ventilation
Authors: Altemeier William A. , Matute-Bello Gustavo , Gharib Sina A. , Glenny Robb W. , Martin Thomas R. , Liles W. Conrad
Published: April 2014

Mechanical ventilation (MV) with tidal volumes of 10–12 ml/kg is considered safe in the absence of acute lung injury (ALI). However, recent studies show that, when lung injury is already present, tidal volumes of this magnitude increase inflammation and injury in the lungs. We hypothesized that MV with tidal volumes of 10-ml/kg can also function as a cofactor in the initiation of ALI by modulating the transcriptional response to bacterial products. To test this hypothesis, we developed a mouse model in which MV did not independently cause inflammation or injury but augmented the inflammatory response to low-dose aspirated LPS and promoted development of ALI. We analyzed gene expression in lungs from 24 mice assigned to four different groups: control, MV only, intratracheal LPS only, and MV + LPS. There were twice as many differentially regulated genes in the MV + LPS group compared with the LPS-only group and 10 times as many differentially regulated genes compared with the MV-only group. For genes up-regulated by LPS treatment alone, the addition of MV further augmented expression. Cytokine concentrations in bronchoalveolar lavage fluid and tissue distribution of an intracellular protein, GADD45-γ, correlated with mRNA levels. We conclude that MV with conventional tidal volumes enhanced the transcriptional response to LPS and promoted development of ALI.
Local Bradykinin Formation Is Controlled by Glycosaminoglycans
Authors: Renné Thomas , Schuh Kai , Müller-Esterl Werner
Published: April 2014

Bradykinin is a potent inflammatory mediator that induces vasodilation, vascular leakage, and pain sensations. This short-lived peptide hormone is liberated from its large precursor protein high molecular weight kininogen (HK) through the contact system cascade involving coagulation factor XII and plasma kallikrein. Although bradykinin release is well established in vitro, the factors and mechanisms controlling bradykinin generation in vivo are still incompletely understood. In this study we demonstrate that binding of HK to glycosaminoglycans (GAGs) of the heparan and chondroitin sulfate type efficiently interferes with bradykinin release in plasma and on endothelial surfaces. Proteolytic bradykinin production on endothelial cells is restored following degradation of cell surface GAG through heparinase. Alternatively, application of HK fragments D3 or light chain, which compete with uncleaved HK for cell binding, promote kininogen proteolysis and bradykinin release. Intravital microscopy revealed that HK fragments increase bradykinin-mediated mesentery microvascular leakage. Topical application of D3 or light chain enhanced bradykinin generation and edema formation in the mouse skin. Our results demonstrate that bradykinin formation is controlled by HK binding to and detachment from GAGs. Separation of the precursor from cell surfaces is a prerequisite for its efficient proteolytic processing. By this means, fragments arising from HK processing propagate bradykinin generation, revealing a novel regulatory level for the kallikrein-kinin system.
Regulation of Basal and Induced Expression of C-Reactive Protein through an Overlapping Element for OCT-1 and NF-κB on the Proximal Promoter
Authors: Voleti Bhavya , Agrawal Alok
Published: April 2014

C-reactive protein (CRP) is an acute phase protein produced by hepatocytes. A minor elevation in the baseline levels of serum CRP is considered an indicator of chronic inflammation. In hepatoma Hep3B cells, IL-6 induces CRP expression by activating transcription factors STAT3 and C/EBPβ. IL-1 synergistically enhances the effects of IL-6. The first 157 bp of the CRP promoter are sufficient for IL-1 synergy. Previously, NF-κB, a transcription factor activated by IL-1β in Hep3B cells, has been shown to increase endogenous CRP expression. The purpose of this study was to investigate the possible action of NF-κB on the 157 bp of the proximal promoter. In this study we show that NF-κB requires and acts synergistically with C/EBPβ on the CRP-proximal promoter to regulate CRP expression. We located the regulatory element that consisted of overlapping binding sites for NF-κB (p50-p50 and p50-p65) and OCT-1. The κB site was responsible for the synergy between NF-κB and C/EBPβ and was also necessary for the CRP transactivation by C/EBPβ through the C/EBP site. Mutation of the κB site decreased the synergistic effect of IL-1β on IL-6-induced CRP expression. Basal CRP expression increased dramatically when binding of both OCT-1 and NF-κB was abolished. Combined data from luciferase transactivation assays and EMSA lead us to conclude that the binding of OCT-1 to the promoter, facilitated by p50-p50 in a novel way, represses, whereas replacement of OCT-1 by p50-p65 induces CRP transcription in cooperation with C/EBPβ. This model for CRP expression favors the variation seen in baseline serum CRP levels in a normal healthy population.
Dichotomal Role of TNF in Experimental Pulmonary Edema Reabsorption
Authors: Braun Clemens , Hamacher Jürg , Morel Denis R. , Wendel Albrecht , Lucas Rudolf
Published: April 2014

Distinct from its receptor binding sites, TNF carries a lectin-like domain, situated at the tip of the molecule, which specifically binds oligosaccharides, such as N,N′-diacetylchitobiose. In view of the apparently conflicting data concerning TNF actions in pulmonary edema, we investigated the contribution of, on the one hand, the receptor binding sites and, in contrast, the lectin-like domain of the cytokine on pulmonary fluid reabsorption in in situ and in vivo flooded rat lungs. Receptor binding sites were blocked with the human soluble TNFR type 1 construct (sTNFR1), whereas the lectin-like domain was blunted with the oligosaccharide N,N′-diacetylchitobiose. We observed that in situ, TNF failed to stimulate alveolar liquid clearance, but did so together with the sTNFR1, and this activity was neutralized by N,N′-diacetylchitobiose. In vivo TNF inhibited liquid clearance, but activated it when complexed with the sTNFR1. A TNF-derived peptide mimic of the lectin-like domain activated fluid reabsorption in flooded lungs, and this activity was blunted by cotreatment with TNF. Our results thus indicate that in these models the receptor binding sites of TNF inhibit, whereas its lectin-like domain activates, edema reabsorption.
Donor Lymphocyte Infusion Induces Long-Term Donor-Specific Cardiac Xenograft Survival through Activation of Recipient Double-Negative Regulatory T Cells
Authors: Chen Wenhao , Zhou Dejun , Torrealba Jose R. , Waddell Thomas K. , Grant David , Zhang Li
Published: April 2014

Previous studies have shown that pretransplant donor lymphocyte infusion (DLI) can enhance xenograft survival. However, the mechanism by which DLI induces xenograft survival remains obscure. Using T cell subset-deficient mice as recipients we show that CD4+, but not CD8+, T cells are necessary to mediate the rejection of concordant cardiac xenografts. Adoptive transfer of naive CD4+ T cells induces rejection of accepted cardiac xenografts in CD4−/− mice. This rejection can be prevented by pretransplant DLI in the absence of any other treatment. Furthermore, we demonstrate that DLI activates αβ-TCR+CD3+CD4−CD8− double-negative (DN) regulatory T (Treg) cells in xenograft recipients, and that DLI-activated DN Treg cells can inhibit the proliferation of donor-specific xenoreactive CD4+ T cells in vitro. More importantly, adoptive transfer of DLI-activated DN Treg cells from xenograft recipients can suppress the proliferation of xenoreactive CD4+ T cells and their ability to produce IL-2 and IFN-γ in vivo. Adoptive transfer of DLI-activated DN Treg cells also prevents CD4+ T cell-mediated cardiac xenograft rejection in an Ag-specific fashion. These data provide direct evidence that DLI can activate recipient DN Treg cells, which can induce donor-specific long-term cardiac xenograft survival by suppressing the proliferation and function of donor-specific CD4+ T cells in vivo.
Increased Caspase-3 Expression and Activity Contribute to Reduced CD3ζ Expression in Systemic Lupus Erythematosus T Cells
Authors: Krishnan Sandeep , Kiang Juliann G. , Fisher Carolyn U. , Nambiar Madhusoodana P. , Nguyen Hang T. , Kyttaris Vasileios C. , Chowdhury Bhabadeb , Rus Violeta , Tsokos George C.
Published: April 2014

T cells isolated from patients with systemic lupus erythematosus (SLE) express low levels of CD3ζ-chain, a critical molecule involved in TCR-mediated signaling, but the involved mechanisms are not fully understood. In this study we examined caspase-3 as a candidate for cleaving CD3ζ in SLE T cells. We demonstrate that SLE T cells display increased expression and activity of caspase-3. Treatment of SLE T cells with the caspase-3 inhibitor Z-Asp-Glu-Val-Asp-FMK reduced proteolysis of CD3ζ and enhanced its expression. In addition, Z-Asp-Glu-Val-Asp-FMK treatment increased the association of CD3ζ with lipid rafts and simultaneously reversed the abnormal lipid raft preclustering, heightened TCR-induced calcium responses, and reduced the expression of FcRγ-chain exclusively in SLE T cells. We conclude that caspase-3 inhibitors can normalize SLE T cell function by limiting the excessive digestion of CD3ζ-chain and suggest that such molecules can be considered in the treatment of this disease.
NK Cells Can Trigger Allograft Vasculopathy: The Role of Hybrid Resistance in Solid Organ Allografts
Authors: Uehara Shuichiro , Chase Catharine M. , Kitchens William H. , Rose Harris S. , Colvin Robert B. , Russell Paul S. , Madsen Joren C.
Published: April 2014

Progressive arterial stenosis (cardiac allograft vasculopathy (CAV)) is a leading cause of long-term failure of organ transplants. CAV remains intractable, in part because its mechanisms are insufficiently understood. A central proposition is that MHC-driven alloimmune processes play a necessary role in CAV, as shown by the absolute requirement for histoincompatibility between donor and recipient for its production. Two immunological pathways have been implicated involving reactivity to donor MHC Ags by either T or B cells. In this study, we use a novel system of semiallogeneic cardiac transplants between parental donors and F1 hybrid recipients to provide evidence that NK cells, members of the innate immune system, also contribute to the generation of CAV in mice. This finding marks the first demonstration that the hybrid resistance phenomenon occurs in solid organ allografts. Extension of these experiments to recipients deficient in T cells demonstrates that this third pathway of CAV, the NK cell-triggered pathway, involves the recruitment of T cells not responsive to donor alloantigens. Finally, transplants performed with donors or recipients deficient in IFN-γ revealed that recipient-derived IFN-γ is necessary for CAV formation in parental to F1 transplants, suggesting a possible effector mechanism by which NK cells can promote CAV. Together, these results define a previously unknown pathway toward CAV and assign a novel role to NK cells in organ allograft rejection.
Identification of Aldolase as a Target Antigen in Alzheimer’s Disease
Authors: Mor Felix , Izak Marina , Cohen Irun R.
Published: April 2014

Alzheimer’s disease (AD) is the most common human neurodegenerative disease, leading to progressive cognitive decline and eventually death. The prevailing paradigm on the pathogenesis of AD is that abnormally folded proteins accumulate in specific brain areas and lead to neuronal loss via apoptosis. In recent years it has become evident that an inflammatory and possibly autoimmune component exists in AD. Moreover, recent data demonstrate that immunization with amyloid-β peptide is therapeutically effective in AD. The nature of CNS Ags that are the target of immune attack in AD is unknown. To identify potential autoantigens in AD, we tested sera IgG Abs of AD patients in immunoblots against brain and other tissue lysates. We identified a 42-kDa band in brain lysates that was detected with >50% of 45 AD sera. The band was identified by mass spectrometry to be aldolase A. Western blotting with aldolase using patient sera demonstrated a band of identical size. The Ab reactivity was verified with ELISAs using aldolase. One of 25 elderly control patients and 3 of 30 multiple sclerosis patients showed similar reactivity (p < 0.002). In enzymatic assays, anti-aldolase positive sera were found to inhibit the enzyme’s activity, and the presence of the substrate (fructose 1,6-diphosphate) enhanced Ab binding. Immunization of rats and mice with aldolase in complete Freund’s adjuvant was not pathogenic. These findings reveal an autoimmune component in AD, point at aldolase as a common autoantigen in this disease, and suggest a new target for potential immune modulation.