bluuebunny/crossref_metadata_2025 huggingface dataset:
IL-1β Attenuates IFN-αβ-Induced Antiviral Activity and STAT1 Activation in the Liver: Involvement of Proteasome-Dependent Pathway
Authors: Tian Zhigang , Shen Xuening , Feng Hong , Gao Bin
Published: April 2014

IFN-αβ is the only established treatment for viral hepatitis; however, more than 60% of patients are poorly responsive. Because viral hepatitis is associated with inflammation, we hypothesized that inflammation may attenuate the efficacy of IFN therapy. To test this hypothesis, the effect of IL-1β, one of the major proinflammatory cytokines, on IFN signaling pathway in the liver was examined. Administration of IL-1β in vivo attenuated IFN-αβ-induced STAT1 tyrosine phosphorylation in the liver but not in the spleen. The inhibitory action of IL-1β in vivo was not affected by depleting hepatic Kupffer cells, suggesting that IL-1β may directly target IFN-αβ signaling in hepatocytes. Indeed, pretreatment of human hepatocellular carcinoma HepG2 cells with IL-1β suppressed IFN-αβ-induced antiviral activity and antiviral protein MxA mRNA expression. Furthermore, IL-1β attenuated IFN-αβ-induced STAT1 binding and tyrosine phosphorylation without affecting the level of STAT1 protein. This inhibitory effect can be reversed by pretreatment with either proteasome inhibitors or transfection of dominant negative NF-κB inducing kinase mutants. Taken together, these findings suggest that IL-1β attenuates IFN-αβ-induced STAT1 activation by a proteasome-dependent mechanism. In view of high levels of IL-1β in the serum or within the liver of patients with chronic liver diseases, attenuation of IFN-αβ signaling in the liver by IL-1β could be one of the mechanisms underlying the resistance to IFN therapy in chronic hepatitis C, and IL-1β could be a potential therapeutic target for improving the efficacy of IFN therapy.
Impaired IFN-γ Production in IFN Regulatory Factor-1 Knockout Mice During Endotoxemia Is Secondary to a Loss of Both IL-12 and IL-12 Receptor Expression
Authors: Salkowski Cindy A. , Thomas Karen E. , Cody M. Joshua , Vogel Stefanie N.
Published: April 2014

Mice with a targeted mutation in the gene that encodes the transcription factor IFN regulatory factor-1 (IRF-1) were used to assess the contribution of IRF-1 to IL-12-dependent and IL-12-independent pathways of IFN-γ production. In response to LPS, IRF-1−/− mice produced less IL-12 p40, IL-12 p35, and IFN-γ mRNA in the liver than IRF-1+/+ mice. While pulmonary IFN-γ mRNA levels were also mitigated in IRF-1−/− mice, pulmonary IL-12 p40 and IL-12 p35 mRNA were not dysregulated. Circulating IL-12 p70 and IFN-γ levels were profoundly attenuated in LPS-challenged IRF-1−/− mice. Further analysis revealed a major deficiency in hepatic IL-12Rβ1 and IL-12Rβ2 mRNA expression as well as pulmonary IL-12Rβ1 mRNA expression in LPS-challenged IRF-1−/− mice. In vitro, IFN-γ up-regulated IL-12Rβ1 mRNA in macrophages from IRF-1+/+, but not IRF-1−/−, mice. IFN-γ-induced IL-12Rβ2 mRNA expression was also diminished in macrophages from IRF-1−/− mice. In contrast to IRF-1+/+ mice, administration of exogenous IL-12 to IRF-1−/− mice resulted in reduced serum IFN-γ and hepatic and pulmonary IFN-γ mRNA, demonstrating that loss of IL-12R results in diminished IL-12 responsiveness. While LPS-challenged IRF-1−/− mice also had reduced IL-15 mRNA levels, serum IL-18 responses were intact. Finally, induction of IRF-1 mRNA by LPS in livers of IFN-γ knockout mice were markedly attenuated, suggesting a feedback amplification loop. These studies indicate that IRF-1 deficiency disrupts both IL-12-dependent and -independent pathways of IFN-γ production and that IRF-1 is a critical transcription factor involved in the regulation of not only IL-12, but also IL-12R.
Cooperation Between Decay-Accelerating Factor and Membrane Cofactor Protein in Protecting Cells from Autologous Complement Attack
Authors: Brodbeck William G. , Mold Carolyn , Atkinson John P. , Medof M. Edward
Published: April 2014

Decay-accelerating factor (DAF or CD55) and membrane cofactor protein (MCP or CD46) function intrinsically in the membranes of self cells to prevent activation of autologous complement on their surfaces. How these two regulatory proteins cooperate on self-cell surfaces to inhibit autologous complement attack is unknown. In this study, a GPI-anchored form of MCP was generated. The ability of this recombinant protein and that of naturally GPI-anchored DAF to incorporate into cell membranes then was exploited to examine the combined functions of DAF and MCP in regulating complement intermediates assembled from purified alternative pathway components on rabbit erythrocytes. Quantitative studies with complement-coated rabbit erythrocyte intermediates constituted with each protein individually or the two proteins together demonstrated that DAF and MCP synergize the actions of each other in preventing C3b deposition on the cell surface. Further analyses showed that MCP’s ability to catalyze the factor I-mediated cleavage of cell-bound C3b is inhibited in the presence of factors B and D and is restored when DAF is incorporated into the cells. Thus, the activities of DAF and MCP, when present together, are greater than the sum of the two proteins individually, and DAF is required for MCP to catalyze the cleavage of cell-bound C3b in the presence of excess factors B and D. These data are relevant to xenotransplantation, pharmacological inhibition of complement in inflammatory diseases, and evasion of tumor cells from humoral immune responses.
Redundant and Segregated Functions of Granule-Associated Heparin-Binding Group II Subfamily of Secretory Phospholipases A2 in the Regulation of Degranulation and Prostaglandin D2 Synthesis in Mast Cells
Authors: Enomoto Ayako , Murakami Makoto , Valentin Emmanuel , Lambeau Gerard , Gelb Michael H. , Kudo Ichiro
Published: April 2014

We herein demonstrate that mast cells express all known members of the group II subfamily of secretory phospholipase A2 (sPLA2) isozymes, and those having heparin affinity markedly enhance the exocytotic response. Rat mastocytoma RBL-2H3 cells transfected with heparin-binding (sPLA2-IIA, -V, and -IID), but not heparin-nonbinding (sPLA2-IIC), enzymes released more granule-associated markers (β-hexosaminidase and histamine) than mock- or cytosolic PLA2α (cPLA2α)-transfected cells after stimulation with IgE and Ag. Site-directed mutagenesis of sPLA2-IIA and -V revealed that both the catalytic and heparin-binding domains are essential for this function. Confocal laser and electron microscopic analyses revealed that sPLA2-IIA, which was stored in secretory granules in unstimulated cells, accumulated on the membranous sites where fusion between the plasma membrane and granule membranes occurred in activated cells. These results suggest that the heparin-binding sPLA2s bind to the perigranular membranes through their heparin-binding domain, and lysophospholipids produced in situ by their enzymatic action may facilitate the ongoing membrane fusion. In contrast to the redundant role of sPLA2-IIA, -IID, and -V in the regulation of degranulation, only sPLA2-V had the ability to markedly augment IgE/Ag-stimulated immediate PGD2 production, which reached a level comparable to that elicited by cPLA2α. The latter observation reveals an unexplored functional segregation among the three related isozymes expressed in the same cell population.
Eosinophilopoiesis in A Murine Model of Allergic Airway Eosinophilia: Involvement of Bone Marrow IL-5 and IL-5 Receptor α
Authors: Tomaki Masafumi , Zhao Lin-Ling , Lundahl Joachim , Sjöstrand Margareta , Jordana Manel , Lindén Anders , O’Byrne Paul , Lötvall Jan
Published: April 2014

The airway inflammation in asthma is dominated by eosinophils. The aim of this study was to elucidate the contribution of newly produced eosinophils in airway allergic inflammation and to determine mechanisms of any enhanced eosinophilopoiesis. OVA-sensitized BALB/c mice were repeatedly exposed to allergen via airway route. Newly produced cells were identified using a thymidine analog, 5-bromo-2′-deoxyuridine, which is incorporated into DNA during mitosis. Identification of IL-5-producing cells in the bone marrow was performed using FACS. Bone marrow CD3+ cells were enriched to evaluate IL-5-protein release in vitro. Anti-IL-5-treatment (TRFK-5) was given either systemically or directly to the airways. IL-5R-bearing cells were localized by immunocytochemistry. Repeated airway allergen exposure caused prominent airway eosinophilia after three to five exposures, and increased the number of immature eosinophils in the bone marrow. Up to 78% of bronchoalveolar lavage (BAL) granulocytes were 5-bromo-2′-deoxyuridine positive. After three allergen exposures, both CD3+ and non-CD3 cells acquired from the bone marrow expressed and released IL-5-protein. Anti-IL-5 given i.p. inhibited both bone marrow and airway eosinophilia. Intranasal administration of anti-IL-5 also reduced BAL eosinophilia, partly via local effects in the airways. Bone marrow cells, but not BAL eosinophils, displayed stainable amounts of the IL-5R α-chain. We conclude that the bone marrow is activated by airway allergen exposure, and that newly produced eosinophils contribute to a substantial degree to the airway eosinophilia induced by allergen. Airway allergen exposure increases the number of cells expressing IL-5-protein in the bone marrow. The bone marrow, as well as the lung, are possible targets for anti-IL-5-treatment.
Experimentally Induced Recruitment of Plasmacytoid (CD123high) Dendritic Cells in Human Nasal Allergy
Authors: Jahnsen Frode L. , Lund-Johansen Fridtjof , Dunne John F. , Farkas Lorant , Haye Rolf , Brandtzaeg Per
Published: April 2014

Recent evidence suggests that the previously enigmatic cell type designated plasmacytoid monocytes can function as dendritic cells and contribute substantially to both innate and adaptive immunity. This cell type has previously been described only in bone marrow, blood, and organized lymphoid tissue, but not at effector sites with direct Ag exposure such as the mucosae. Plasmacytoid dendritic cells (P-DCs) matured in vitro can induce T cells to produce allergy-promoting Th2 cytokines; therefore, their possible occurrence in nasal mucosa during experimentally elicited allergic rhinitis was examined. Patients with silent nasal allergy were challenged topically with relevant allergen daily for 7 days. Biopsy specimens as well as blood samples were obtained before and during such provocation, and P-DCs were identified by their high expression of CD123 (IL-3R α-chain), together with CD45RA. Our results showed that P-DCs were present in low and variable numbers in normal nasal mucosa but increased dramatically during the allergic reaction. This accumulation concurred with the expression of the L-selectin ligand peripheral lymph node addressin on the mucosal vascular endothelium. The latter observation was particularly interesting in view of the high levels of L-selectin on circulating P-DC precursors and of previous reports suggesting that these cells can enter organized lymphoid tissue via high endothelial venules (which express peripheral lymph node addressin constitutively). Together, our findings suggested that P-DCs are involved in the triggering of airway allergy and that they are directed to allergic lesions by adhesion molecules that normally mediate leukocyte extravasation in organized lymphoid tissue.
Eosinophils Maintain Their Capacity to Signal and Release Eosinophil Cationic Protein Upon Repetitive Stimulation with the Same Agonist
Authors: Simon Hans-Uwe , Weber Martina , Becker Eva , Zilberman Yael , Blaser Kurt , Levi-Schaffer Francesca
Published: April 2014

Eosinophils contain in their granules eosinophil cationic protein (ECP) and other basic proteins that have been implicated in immunity to parasites and pathophysiology of chronic allergic responses. In a model of eosinophil degranulation, we show that eosinophils release ECP upon short-term GM-CSF priming and stimulation with either platelet-activating factor (PAF) or the anaphylatoxin C5a, but not eotaxin. Restimulation with the same agonist (PAF or C5a) was unsuccessful as assessed by monitoring intracellular calcium concentration and ECP release. In contrast, upon an intermediate washing step, eosinophils rapidly transduced PAF and C5a signals followed by significant ECP releases. Ligand-binding studies demonstrated that only a proportion of PAF receptors is internalized upon cell stimulation and that washing of the cells removes the agonist from the cell surface. Upon repetitive stimulation, eosinophils with less than 50% of the original ECP content were obtained. Such eosinophils did not increase cellular ECP levels even in the presence of the eosinophil survival factor GM-CSF in overnight cultures. In vivo studies revealed that eosinophils always express detectable amounts of ECP under chronic inflammatory conditions. In conclusion, we have shown that eosinophils maintain their capacity to degranulate upon repetitive stimulation with the same agonist as long as the receptor is not occupied from a previous stimulation. The cellular content of ECP appears to be a no limiting factor in the case of repetitive stimulation, implying that mature eosinophils may not require a significant ECP resynthesis.
Overexpression of CD1d by Keratinocytes in Psoriasis and CD1d-Dependent IFN-γ Production by NK-T Cells
Authors: Bonish Brian , Jullien Denis , Dutronc Yves , Huang Barbara Bei , Modlin Robert , Spada Franca M. , Porcelli Steven A. , Nickoloff Brian J.
Published: April 2014

The MHC class I-like protein CD1d is a nonpolymorphic molecule that plays a central role in development and activation of a subset of T cells that coexpress receptors used by NK cells (NK-T cells). Recently, T cells bearing NK receptors were identified in acute and chronic lesions of psoriasis. To determine whether NK-T cells could interact with epidermal cells, we examined the pattern of expression of CD1d in normal skin, psoriasis, and related skin disorders, using a panel of CD1d-specific mAbs. CD1d was expressed by keratinocytes in normal skin, although expression was at a relatively low level and was generally confined to upper level keratinocytes immediately beneath the lipid-rich stratum corneum. In contrast, there was overexpression of CD1d in chronic, active psoriatic plaques. CD1d could be rapidly induced on keratinocytes in normal skin by physical trauma that disrupted barrier function or by application of a potent contact-sensitizing agent. Keratinocytes displayed enhanced CD1d following exposure to IFN-γ. Combining CD1d-positive keratinocytes with human NK-T cell clones resulted in clustering of NK-T cells, and while no significant proliferation ensued, NK-T cells became activated to produce large amounts of IFN-γ. We conclude that CD1d can be expressed in a functionally active form by keratinocytes and is up-regulated in psoriasis and other inflammatory dermatoses. The ability of IFN-γ to enhance keratinocyte CD1d expression and the subsequent ability of CD1d-positive keratinocytes to activate NK-T cells to produce IFN-γ, could provide a mechanism that contributes to the pathogenesis of psoriasis and other skin disorders.
Cytokine Dysregulation Induced by Apoptotic Cells Is a Shared Characteristic of Murine Lupus
Authors: Koh Jason S. , Wang Zhiyong , Levine Jerrold S.
Published: April 2014

Of the multiple murine models of autoimmunity, the three most closely resembling human systemic lupus erythematosus (SLE) are the MRL/lpr, New Zealand Black/White F1, and male BXSB. Although these strains share many disease characteristics, no common cellular defect has previously been found in prediseased mice from all these strains. We show in this study that macrophages from prediseased mice of all three SLE-prone strains, as well as macrophages from mice whose genomes contribute to the development of SLE (MRL/+, New Zealand White, New Zealand Black, female BXSB, and LG/J), have an identical and profound defect in cytokine expression that is triggered by apoptotic cells. Strikingly, none of 13 nonautoimmune strains tested exhibited this defect. Given that apoptotic Ags have been increasingly recognized as the target of autoantibodies, a defect in cytokine expression that is triggered by apoptotic cells has broad potential to upset the balance between tolerance and immunity.
The Effects of Prolonged Administration of 5-Bromodeoxyuridine on Cells of the Immune System
Authors: Reome Joyce B. , Johnston Darrin S. , Helmich Brian K. , Morgan Tammy M. , Dutton-Swain Nicholas , Dutton Richard W.
Published: April 2014

We have determined the in vivo effect of 5-bromodeoxyuridine (BrdU) administered to mice in the drinking water for various lengths of time on the performance of T and B lymphocytes in a number of experimental protocols. Young mice continuously exposed to BrdU fail to gain weight, and the lymphocytes recovered after a prolonged period of exposure are fewer in number than in control mice. The recovery of normal levels of T and B lymphocytes after irradiation is severely impaired. Ag-specific cells responding to Ag in an adoptive transfer model fail to expand as much in the presence of BrdU as in the absence, and the Ag-specific effectors produced in the presence of BrdU are less able to secrete cytokines upon restimulation in vitro. Polarized populations of Tc1 and Tc2 effectors generated in vitro proliferate less in the presence of BrdU, and the resulting effectors make less cytokines per cell upon restimulation. Thus, the incorporation of BrdU into T or B lymphocytes can, under some circumstances, seriously impair the performance of the labeled cells, and these findings raise a note of caution in the interpretation of studies that make use of long-term exposure to BrdU.
Immortalization of Human CD8+ T Cell Clones by Ectopic Expression of Telomerase Reverse Transcriptase
Authors: Hooijberg Erik , Ruizendaal Janneke J. , Snijders Peter J. F. , Kueter Esther W. M. , Walboomers Jan M. M. , Spits Hergen
Published: April 2014

Replicative senescence of T cells is correlated with erosion of telomere ends. Telomerase plays a key role in maintaining telomere length. Therefore, it is thought that telomerase regulates the life span of T cells. To test this hypothesis, we have over-expressed human telomerase reverse transcriptase in human CD8+ T cells. Ectopic expression of human telomerase reverse transcriptase led to immortalization of these T cells, without altering the phenotype and without loss of specificity or functionality. As the T cells remained dependent on cytokines and Ag stimulation for their in vitro expansion, we conclude that immortalization was achieved without malignant transformation.
Divergent Response to LPS and Bacteria in CD14-Deficient Murine Macrophages
Authors: Moore Kathryn J. , Andersson Lorna P. , Ingalls Robin R. , Monks Brian G. , Li Rui , Arnaout M. Amin , Golenbock Douglas T. , Freeman Mason W.
Published: April 2014

Gram-negative bacteria and the LPS constituent of their outer membranes stimulate the release of inflammatory mediators believed to be responsible for the clinical manifestations of septic shock. The GPI-linked membrane protein, CD14, initiates the signaling cascade responsible for the induction of this inflammatory response by LPS. In this paper, we report the generation and characterization of CD14-null mice in which the entire coding region of CD14 was deleted. As expected, LPS failed to elicit TNF-α and IL-6 production in macrophages taken from these animals, and this loss in responsiveness is associated with impaired activation of both the NF-κB and the c-Jun N-terminal mitogen-activated protein kinase pathways. The binding and uptake of heat-killed Escherichia coli, measured by FACS analysis, did not differ between CD14-null and wild-type macrophages. However, in contrast to the findings with LPS, whole E. coli stimulated similar levels of TNF-α release from CD14-null and wild-type macrophages at a dose of 10 bioparticles per cell. This effect was dose dependent, and at lower bacterial concentrations CD14-deficient macrophages produced significantly less TNF-α than wild type. Approximately half of this CD14-independent response appeared to be mediated by CD11b/CD18, as demonstrated by receptor blockade using neutrophil inhibitory factor. An inhibitor of phagocytosis, cytochalasin B, abrogated the induction of TNF-α in CD14-deficient macrophages by E. coli. These data indicate that CD14 is essential for macrophage responses to free LPS, whereas other receptors, including CD11b/CD18, can compensate for the loss of CD14 in response to whole bacteria.
Selective Bystander Proliferation of Memory CD4+ and CD8+ T Cells Upon NK T or T Cell Activation
Authors: Eberl Gérard , Brawand Pierre , MacDonald H. Robson
Published: April 2014

Ag-experienced or memory T cells have increased reactivity to recall Ag, and can be distinguished from naive T cells by altered expression of surface markers such as CD44. Memory T cells have a high turnover rate, and CD8+ memory T cells proliferate upon viral infection, in the presence of IFN-αβ and/or IL-15. In this study, we extend these findings by showing that activated NKT cells and superantigen-activated T cells induce extensive bystander proliferation of both CD8+ and CD4+ memory T cells. Moreover, proliferation of memory T cells can be induced by an IFN-αβ-independent, but IFN-γ- or IL-12-dependent pathway. In these conditions of bystander activation, proliferating memory (CD44high) T cells do not derive from activation of naive (CD44low) T cells, but rather from bona fide memory CD44high T cells. Together, these data demonstrate that distinct pathways can induce bystander proliferation of memory T cells.
IL-15 and IL-15 Receptor Selectively Regulate Differentiation of Common Mucosal Immune System-Independent B-1 Cells for IgA Responses
Authors: Hiroi Takachika , Yanagita Manabu , Ohta Noriyuki , Sakaue Gaku , Kiyono Hiroshi
Published: April 2014

We show in this report a new regulatory role for IL-15 and IL-15R in the development of B-1 cells and their differentiation into IgA-producing cells. Mucosal IgA levels were found to be inhibited by anti-IL-15 mAb treatment in vivo, but enhanced by administration of rIL-15, while serum IgA levels remained unaffected. Mucosal B-1 cells preferentially proliferated in response to IL-15 in vitro. When mucosal B-1 and B-2 cells were separated into surface (s)IgM+sIgA− and sIgM−sIgA+ fractions, IL-15R-specific mRNA was found to be predominant in both sIgM+sIgA− and sIgM−sIgA+ B-1 cells at a much higher level than B-2 cells. Further, incubation of these different subsets of B-1 and B-2 cells with IL-15 resulted in greater enhancement of the corresponding receptor expression by B-1 subset when compared with B-2 fraction. Interestingly, de novo isolated sIgM+sIgA− B-1, but not sIgM+sIgA− B-2, cells were already class-switched cells because the germline Cα transcript was detected and was then further enhanced by IL-15. IL-15 also supported differentiation of both sIgM+sIgA− and sIgM−sIgA+ B-1 cells into IgA-producing cells. Taken together, these findings suggest that IL-15 is a critically important cytokine for the differentiation of both sIgM+,IgA− and sIgM−sIgA+ B-1 cells expressing IL-15R into IgA-producing cells in mucosal tissues.
Macrophage-Derived Dendritic Cells Have Strong Th1-Polarizing Potential Mediated by β-Chemokines Rather Than IL-12
Authors: Zou Weiping , Borvak Jozef , Marches Florentina , Wei Shuang , Galanaud Pierre , Emilie Dominique , Curiel Tyler J.
Published: April 2014

Monocyte-derived dendritic cells (MDDCs) activate naive T lymphocytes to induce adaptive immunity, effecting Th1 polarization through IL-12. However, little is known about other potential DC Th1 polarizing mechanisms, or how T cell polarization may be affected by DCs differentiating in, or exposed to, a proinflammatory environment. Macrophages (MΦs) are DC precursors abundant in inflamed tissues, lymph nodes, and tumors. Thus we studied the T cell-activating and -polarizing properties of MΦ-derived DCs (ΦDCs). Monocytes were cultured in MΦ-CSF (M-CSF) to produce MΦs, which were then differentiated into DCs following culture with GM-CSF plus IL-4. ΦDCs activated a significant allogeneic MLR and were significantly better than MDDCs in activating T cells with superantigen. Most strikingly, ΦDCs elicited up to 9-fold more IFN-γ from naive or Ag-specific T cells compared with MDDCs (with equivalent IL-4 secretion), despite producing up to 9-fold less IL-12. Neutralization of MDDC, but not ΦDC IL-12 significantly inhibited T cell IFN-γ induction. ΦDCs produced up to 12-fold more β-chemokines (macrophage-inflammatory protein-1α, -1β, and RANTES) than MDDCs. Ab blockade of CCR5, but not CXC chemokine receptor 4, inhibited T cell IFN-γ induction by ΦDCs significantly greater than by MDDCs. Thus DCs differentiating from MΦs induce T cell IFN-γ through β-chemokines with little or no requirement for IL-12. Myeloid DCs arising from distinct precursor cells may have differing properties, including different mechanisms of Th1 polarization. These data are the first reports of IFN-γ induction through chemokines by DCs.
Reciprocal Expression of the TNF Family Receptor Herpes Virus Entry Mediator and Its Ligand LIGHT on Activated T Cells: LIGHT Down-Regulates Its Own Receptor
Authors: Morel Yannis , Schiano de Colella Jean-Marc , Harrop Jeremy , Deen Keith C. , Holmes Stephen D. , Wattam Trevor A. , Khandekar Sanjay S. , Truneh Alemseged , Sweet Raymond W. , Gastaut Jean-Albert , Olive Daniel , Costello Régis T.
Published: April 2014

The TNF receptor (TNFR) family plays a central role in the development of the immune response. Here we describe the reciprocal regulation of the recently identified TNFR superfamily member herpes virus entry mediator (HVEM) (TR2) and its ligand LIGHT (TL4) on T cells following activation and the mechanism of this process. T cell activation resulted in down-regulation of HVEM and up-regulation of LIGHT, which were both more pronounced in CD8+ than CD4+ T lymphocytes. The analysis of HVEM and LIGHT mRNA showed an increase in the steady state level of both mRNAs following stimulation. LIGHT, which was present in cytoplasm of resting T cells, was induced both in cytoplasm and at the cell surface. For HVEM, activation resulted in cellular redistribution, with its disappearance from cell surface. HVEM down-regulation did not rely on de novo protein synthesis, in contrast to the partial dependence of LIGHT induction. Matrix metalloproteinase inhibitors did not modify HVEM expression, but did enhance LIGHT accumulation at the cell surface. However, HVEM down-regulation was partially blocked by a neutralizing mAb to LIGHT or an HVEM-Fc fusion protein during activation. As a model, we propose that following stimulation, membrane or secreted LIGHT binds to HVEM and induces receptor down-regulation. Degradation or release of LIGHT by matrix metalloproteinases then contributes to the return to baseline levels for both LIGHT and HVEM. These results reveal a self-regulating ligand/receptor system that contributes to T cell activation through the interaction of T cells with each other and probably with other cells of the immune system.
Decreased Neutrophil Adhesion to Human Cytomegalovirus-Infected Retinal Pigment Epithelial Cells Is Mediated by Virus-Induced Up-Regulation of Fas Ligand Independent of Neutrophil Apoptosis
Authors: Cinatl Jindrich , Blaheta Roman , Bittoova Martina , Scholz Martin , Margraf Stefan , Vogel Jens-Uwe , Cinatl Jaroslav , Doerr Hans Wilhelm
Published: April 2014

Human CMV (HCMV) retinitis frequently leads to blindness in iatrogenically immunosuppressed patients and in the end stage of AIDS. Despite the general proinflammatory potential of HCMV, virus infection is associated with a rather mild cellular inflammatory response in the retina. To investigate this phenomenon, the influence of HCMV (strains AD169 or Hi91) infection on C-X-C chemokine secretion, ICAM-1 expression, and neutrophil recruitment in cultured human retinal pigment epithelial (RPE) cells was studied. Supernatants from infected cultures contained enhanced levels of IL-8 and melanoma growth-stimulating activity/Gro α and induced neutrophil chemotaxis compared with supernatants from uninfected RPE cells. Despite HCMV-induced ICAM-1 expression on RPE cells, binding of activated neutrophils to HCMV-infected RPE cells and subsequent transepithelial penetration were significantly reduced. Reduced neutrophil adhesion to infected RPE cells correlated with HCMV-induced up-regulation of constitutive Fas ligand (FasL) expression. Functional blocking of FasL on RPE cells with the neutralizing mAbs NOK-1 and NOK-2 or of the Fas receptor on neutrophils with mAbB-D29 prevented the HCMV-induced impairment of neutrophil/RPE interactions. Fas-FasL-dependent impairment of neutrophil binding had occurred by 10 min after neutrophil/RPE coculture without apoptotic signs. Neutrophil apoptosis was first detected after 4 h. Treatment of neutrophils with a specific inhibitor of caspase-8 suppressed apoptosis, whereas it did not prevent impaired neutrophil binding to infected RPE. The current results suggest a novel role for FasL in the RPE regulation of neutrophil binding. This may be an important feature of virus escape mechanisms and for sustaining the immune-privileged character of the retina during HCMV ocular infection.
Cell-Specific Expression of the Murine CD21 Gene Depends on Accessibility of Promoter and Intronic Elements
Authors: Zabel Mark D. , Byrne Burke L. , Weis Janis J. , Weis John H.
Published: April 2014

The murine complement receptor type 2 gene (Cr2/CD21) is transcriptionally active in murine B and follicular dendritic cells, but not in murine T cells. We have previously shown that altering chromatin structure via histone deacetylase inhibitors results in CD21 expression in murine T cells, and that the minimal CD21 promoter provided appropriate cell-specific expression of luciferase reporter constructs only in the presence of the first third of intron 1, fragment A. We extend this work by showing that replacing the CD21 gene promoter with the SV40 promoter resulted in the loss of this cell-specific control. Further delineation of intronic regulatory elements by fragmentation also resulted in the loss of cell-specific gene expression, suggesting that multiple CD21 promoter and intronic elements interact for appropriate CD21 gene expression. To assess this model, we performed EMSAs to define protein binding sites within promoter and intronic regions and DNase I hypersensitivity assays to determine chromatin accessibility. Multiple DNA binding factors were shown to be present in B and T cell extracts; a minority demonstrated B cell specificity. However, the DNase I sensitivity of T cell CD21 regulatory elements was not comparable to that of B cells until the histone acetylation status of the gene was altered. Taken together, these data suggest that chromatin remodeling facilitates cell-specific CD21 gene expression by modulating access of transcription factors to regulatory elements in the promoter and intron.
Mutational Analysis Reveals Multiple Distinct Sites Within Fcγ Receptor IIB That Function in Inhibitory Signaling
Authors: Fong Dana C. , Brauweiler Anne , Minskoff Stacy A. , Bruhns Pierre , Tamir Idan , Mellman Ira , Daeron Marc , Cambier John C.
Published: April 2014

The low-affinity receptor for IgG, FcγRIIB, functions broadly in the immune system, blocking mast cell degranulation, dampening the humoral immune response, and reducing the risk of autoimmunity. Previous studies concluded that inhibitory signal transduction by FcγRIIB is mediated solely by its immunoreceptor tyrosine-based inhibition motif (ITIM) that, when phosphorylated, recruits the SH2-containing inositol 5′- phosphatase SHIP and the SH2-containing tyrosine phosphatases SHP-1 and SHP-2. The mutational analysis reported here reveals that the receptor’s C-terminal 16 residues are also required for detectable FcγRIIB association with SHIP in vivo and for FcγRIIB-mediated phosphatidylinositol 3-kinase hydrolysis by SHIP. Although the ITIM appears to contain all the structural information required for receptor-mediated tyrosine phosphorylation of SHIP, phosphorylation is enhanced when the C-terminal sequence is present. Additionally, FcγRIIB-mediated dephosphorylation of CD19 is independent of the cytoplasmic tail distal from residue 237, including the ITIM. Finally, the findings indicate that tyrosines 290, 309, and 326 are all sites of significant FcγRIIB1 phosphorylation following coaggregation with B cell Ag receptor. Thus, we conclude that multiple sites in FcγRIIB contribute uniquely to transduction of FcγRIIB-mediated inhibitory signals.
Regulated Production and Molecular Diversity of Human Liver and Activation-Regulated Chemokine/Macrophage Inflammatory Protein-3α from Normal and Transformed Cells
Authors: Schutyser Evemie , Struyf Sofie , Menten Patricia , Lenaerts Jean-Pierre , Conings René , Put Willy , Wuyts Anja , Proost Paul , Van Damme Jo
Published: April 2014

Liver and activation-regulated chemokine (LARC), also designated macrophage inflammatory protein-3α (MIP-3α), Exodus, or CCL20, is a C-C chemokine that attracts immature dendritic cells and memory T lymphocytes, both expressing CCR6. Depending on the cell type, this chemokine was found to be inducible by cytokines (IL-1β) and by bacterial, viral, or plant products (including LPS, dsRNA, and PMA) as measured by a specific ELISA. Although coinduced with monocyte chemotactic protein-1 (MCP-1) and IL-8 by dsRNA, measles virus, and IL-1β in diploid fibroblasts, leukocytes produced LARC/MIP-3α only in response to LPS. However, in myelomonocytic THP-1 cells LARC/MIP-3α was better induced by phorbol ester, whereas in HEp-2 epidermal carcinoma cells IL-1β was the superior inducer. The production levels of LARC/MIP-3α (1–10 ng/ml) were, on the average, 10- to 100-fold lower than those of IL-8 and MCP-1, but were comparable to those of other less abundantly secreted chemokines. Natural LARC/MIP-3α protein isolated from stimulated leukocytes or tumor cell lines showed molecular diversity, in that NH2- and COOH-terminally truncated forms were purified and identified by amino acid sequence analysis and mass spectrometry. In contrast to other chemokines, including MCP-1 and IL-8, the natural processing did not affect the calcium-mobilizing capacity of LARC/MIP-3α through its receptor CCR6. Furthermore, truncated natural LARC/MIP-3α isoforms were equally chemotactic for lymphocytes as intact rLARC/MIP-3α. It is concluded that in addition to its role in homeostatic trafficking of leukocytes, LARC/MIP-3α can function as an inflammatory chemokine during host defense.
Lipopolysaccharide-Binding Protein Is Vectorially Secreted and Transported by Cultured Intestinal Epithelial Cells and Is Present in the Intestinal Mucus of Mice
Authors: Vreugdenhil Anita C. E. , Snoek A. M. Patricia , Greve Jan Willem M. , Buurman Wim A.
Published: April 2014

Lipopolysaccharide-binding protein (LBP) is an important modulator of the host’s response to endotoxin. In a previous study, we found evidence for the synthesis of LBP by intestinal epithelial cells. In this study, we explored the polarity of LBP secretion by these cells. Polarized monolayers of Caco-2 cells were used as intestinal mucosa model. Cells were stimulated apically or basally with cytokines, and LBP secretion was analyzed. Furthermore, the presence of LBP in intestinal mucus of healthy and endotoxemic mice was studied using a mucus-sampling technique. The constitutive unipolar LBP secretion from the apical cell surface was markedly enhanced when cells were exposed to cytokines at their apical surface. However, bioactive LBP was secreted from both cell surfaces after basolateral stimulation of cells. Cytokines also influenced the secretion of the acute phase proteins serum amyloid A, apoA-I, and apoB from both surfaces of Caco-2 cells. Furthermore, transport of exogenous LBP from the basolateral to the apical cell surface was demonstrated. In line with these in vitro data, the presence of LBP in intestinal mucus was strongly enhanced in mice after a challenge with endotoxin. The results indicate that LBP is present at the mucosal surface of the intestine, a phenomenon for which secretion and transport of LBP by intestinal epithelial cells may be responsible.
Partially TAP-Independent Protection AgainstListeria monocytogenesby H2-M3-Restricted CD8+ T Cells
Authors: Rolph Michael S. , Kaufmann Stefan H. E.
Published: April 2014

Effective protection against Listeria monocytogenes requires Ag-specific CD8+ T cells. A substantial proportion of CD8+ T cells activated during L. monocytogenes infection of C57BL/6 mice are restricted by the MHC class Ib molecule H2-M3. In this study, an H2-M3-restricted CD8+ T cell clone specific for a known H2-M3 epitope (fMIGWII) was generated from L. monocytogenes-infected mice. The clone was cytotoxic, produced IFN-γ, and could mediate strong protection against L. monocytogenes when transferred to infected mice. Macrophages pulsed with heat-killed Listeriae presented Ag to the clone in a TAP-independent manner. Both TAP-independent and -dependent processing occurred in vivo, as TAP-deficient mice infected with L. monocytogenes were partially protected by adoptive transfer of the clone. This is the first example of CD8+ T cell-mediated, TAP-independent protection against a pathogen in vivo, confirming the importance of alternative MHC class I processing pathways in the antibacterial immunity.
The Synthetic Peptide Trp-Lys-Tyr-Met-Val-d-Met Is a Potent Chemotactic Agonist for Mouse Formyl Peptide Receptor
Authors: He Rong , Tan Lijun , Browning Darren D. , Wang Ji Ming , Ye Richard D.
Published: April 2014

Formyl peptides are potent neutrophil chemoattractants. In humans and rabbits, the formyl peptide receptor (FPR) binds N-formyl-Met-Leu-Phe (fMLF) with high affinity (Kd ≈ 1 nM). The mouse FPR (mFPR) is a low-affinity receptor for fMLF (Kd ≈ 100 nM); therefore, other agonists for this receptor may exist. Using mFPR-transfected rat basophilic leukemia cells, we found that a recently identified synthetic peptide Trp-Lys-Tyr-Met-Val-d-Met (WKYMVm) is a potent agonist for mFPR. WKYMVm induced calcium mobilization with an EC50 of 1.2–1.5 nM. Optimal chemotaxis was achieved with 1 nM of WKYMVm, but it required 100 nM of fMLF. WKYMVm stimulated rapid and potent phosphorylation of the mitogen-activated protein kinases extracellular signal-related kinases 1 and 2 when used at 50 nM. Pertussis toxin only partially blocked calcium mobilization and production of inositol 1,4,5-trisphosphate in the stimulated mFPR cells, suggesting the possibility that this receptor couples to Gα proteins other than Gi and Go. Competitive binding and desensitization data suggest that both peptides interact with the same receptor but may use nonoverlapping binding sites because WKYMVm was unable to effectively displace [3H]fMLF bound to mFPR. These results provide evidence for the presence of an alternative potent agonist for mFPR, and suggest a potential usage of WKYMVm for probing the ligand-receptor interactions with the murine formyl peptide receptor homologs.
Role of Intracellular Chloride in the Reversible Activation of Neutrophil β2 Integrins: A Lesson from TNF Stimulation
Authors: Menegazzi Renzo , Busetto Sara , Cramer Rita , Dri Pietro , Patriarca Pierluigi
Published: April 2014

The process of β2 integrin activation, which enhances the interaction of these heterodimers with ligands, plays a crucial role in the adherence-dependent neutrophilic polymorphonuclear leukocytes’ (PMN) responses to TNF. Our previous observation, showing that a marked decrease of the high basal Cl− content (Cl−i) is an essential step in the TNF-induced activation of PMN, stimulated this study, which investigates the role of alterations of Cl−i in the activation of β2 integrins triggered by TNF. Here we show that TNF enhances the expression of activation-specific neoepitopes of β2 integrins, namely, epitope 24, a unique epitope present on all three leukocyte integrin α subunits, and epitope CBRM1/5, localized to the I domain on the α-chain of Mac-1 (CD11bCD18). Moreover, we demonstrate that the conformational changes underlying the expression of the neoepitopes are dependent on a drop in Cl−i because 1) inhibition of Cl−i decrease is invariably accompanied by inhibition of β2 integrin activation, 2) Cl−i decrease induced by means other than agonist stimulation, i.e., by placing PMN in Cl−-free buffers, activates β2 integrins, and 3) restoration of the original Cl−i levels is accompanied by deactivation of β2 integrins. We also show that Cl−i decrease is required for TNF-induced cytoplasmic alkalinization, but such a rise in pHi does not seem to be relevant for β2 integrin activation. The results of our study emphasize the role of Cl− as a new PMN “second messenger.”
IL-18 Has IL-12-Independent Effects in Delayed-Type Hypersensitivity: Studies in Cell-Mediated Crescentic Glomerulonephritis
Authors: Kitching A. Richard , Tipping Peter G. , Kurimoto Masashi , Holdsworth Stephen R.
Published: April 2014

IL-18 (formerly known as IFN-γ-inducing factor) enhances Th1 responses via effects that are thought to be dependent on and synergistic with IL-12. The potential for IL-18 to exert IL-12-independent effects in delayed-type hypersensitivity (DTH) responses was studied in a model of Th1-directed, DTH-mediated crescentic glomerulonephritis induced by planting an Ag in glomeruli of sensitized mice as well as in cutaneous DTH. Sensitized genetically normal (IL-12+/+) mice developed proteinuria and crescentic glomerulonephritis with a glomerular influx of DTH effectors (CD4+ T cells, macrophages, and fibrin deposition) in response to the planted glomerular Ag. IL-12p40-deficient (IL-12−/−) mice showed significant reductions in crescent formation, proteinuria, and glomerular DTH effectors. Administration of IL-18 to IL-12−/− mice restored the development of histological (including effectors of DTH) and functional glomerular injury in IL-12−/− mice to levels equivalent to those in IL-12+/+ mice. IL-18 administration to IL-12−/− mice increased glomerular ICAM-1 protein expression, but did not restore Ag-stimulated splenocyte IFN-γ, GM-CSF, IL-2, or TNF-α production. Sensitized IL-12+/+ mice also developed cutaneous DTH following intradermal challenge with the nephritogenic Ag. Cutaneous DTH was inhibited in IL-12−/− mice, but was restored by administration of IL-18. IL-12+/+ mice given IL-18 developed augmented injury, with enhanced glomerular and cutaneous DTH, demonstrating the synergistic effects of IL-18 and IL-12 in DTH responses. These studies demonstrate that even in the absence of IL-12, IL-18 can induce in vivo DTH responses and up-regulate ICAM-1 without inducing IFN-γ, GM-CSF, or TNF-α production.
Cutting Edge: TGF-β Inhibits Th Type 2 Development Through Inhibition of GATA-3 Expression
Authors: Gorelik Leonid , Fields Patrick E. , Flavell Richard A.
Published: April 2014

TGF-β is an important immunomodulatory cytokine that can inhibit differentiation of effector T cells. In this report, we address the molecular mechanisms through which TGF-β inhibits differentiation of CD4+ cells into Th type 2 cells. We demonstrate that TGF-β inhibits GATA-3 expression in developing Th cells. We also show that inhibition of GATA-3 expression by TGF-β is a major mechanism of inhibition of Th2 differentiation by TGF-β as ectopic expression of GATA-3 in developing T cells overcomes the ability of TGF-β to inhibit Th2 differentiation. TGF-β likely inhibits GATA-3 expression at the transcriptional level and does so without interfering with IL-4 signaling.
Cutting Edge: Anti-CD154 Therapeutic Antibodies Induce Infectious Transplantation Tolerance
Authors: Graca Luis , Honey Karen , Adams Elizabeth , Cobbold Stephen P. , Waldmann Herman
Published: April 2014

Nondepleting anti-CD154 (CD40 ligand) mAbs have proven effective in inducing transplantation tolerance in rodents and primates. In the induction phase, anti-CD154 Ab therapy is known to enhance apoptosis of Ag reactive T cells. However, this may not be the sole explanation for tolerance, as we show in this study that tolerance is maintained through a dominant regulatory mechanism which, like tolerance induced with CD4 Abs, manifests as infectious tolerance. Therefore, tolerance induced with anti-CD154 Abs involves not only the deletion of potentially aggressive T cells, but also a contagious spread of tolerance to new cohorts of graft-reactive T cells as they arise.
Cutting Edge: CD1d Deficiency Impairs Murine Host Defense Against the Spirochete, Borrelia burgdorferi
Authors: Kumar Hemant , Belperron Alexia , Barthold Stephen W. , Bockenstedt Linda K.
Published: April 2014

CD1 molecules can present microbial lipid Ag to T cells, suggesting that they participate in host defense against pathogens. In this study, we examined the role of CD1d in resistance to infection with the Lyme disease spirochete, Borrelia burgdorferi (Bb), an organism with proinflammatory lipid Ag. Bb infection of CD1d-deficient (CD1d−/−) mouse strains normally resistant to this pathogen resulted in arthritis. Pathology correlated with an increased prevalence of spirochete DNA in tissues and enhanced production of Bb-specific IgG, including IgG to Ag rapidly down-modulated on spirochetes in vivo. CD1d−/− mice exhibited high-titer Bb-specific IgG2a, an isotype commonly induced in disease-susceptible mice but not in the disease-resistant control mice in this study. These results show that CD1d deficiency impairs host resistance to a spirochete pathogen, and are the first example of a mutation that imparts Bb-resistant mice with the Ab and disease profile of a susceptible mouse strain.
Receptor Engagement on Cells Expressing a Ligand for the Tolerance-Inducing Molecule OX2 Induces an Immunoregulatory Population That Inhibits Alloreactivity In Vitro and In Vivo
Authors: Gorczynski Reginald M. , Yu Kai , Clark David
Published: April 2014

Increased survival of C57BL/6 renal allografts following portal vein donor-specific pretransplant immunization of C3H mice is associated with increased expression of the molecule OX2 seen on host dendritic cells, along with a marked polarization in cytokine production from lymphocytes harvested from the transplanted animals, with preferential production of IL-4, IL-10, and TGF-β on donor-specific restimulation in vitro, and decreased production of IL-2, IFN-γ, and TNF-α compared with non-portal vein-immunized control transplanted mice. The increased renal allograft survival and the altered cytokine production are abolished by infusion of anti-mouse OX2 mAb (3B6). Infusion of a soluble OX2:Fc immunoadhesin can itself produce significant prolongation of xeno- and allografts in mice. We have used FITC-conjugated OX2:Fc to characterize cells expressing a ligand (OX2L) for OX2, and provide evidence that subpopulations of LPS-stimulated splenic macrophages, Con A-activated splenic T cells, and the majority (>80%) of γδTCR+ T cells express this ligand. We show below that F4/80+, OX2L+ splenic macrophages, admixed with OX2:Fc, represent a potent immunosuppressive population capable of causing more profound inhibition of alloreactivity in vitro or in vivo than that seen using either OX2:Fc or OX2+ (or OX2L+) cells alone. Immunoregulation by this OX2L+ population occurs in an MHC-restricted fashion.