bluuebunny/crossref_metadata_2025 huggingface dataset:
Bacterial Lipopolysaccharide Signaling Through Toll-Like Receptor 4 Suppresses Asthma-Like Responses Via Nitric Oxide Synthase 2 Activity
Authors: Rodríguez Dunia , Keller Alexandre C. , Faquim-Mauro Eliana L. , de Macedo Mahasti S. , Cunha Fernando Q. , Lefort Jean , Vargaftig B. Boris , Russo Momtchilo
Published: April 2014

Asthma results from an intrapulmonary allergen-driven Th2 response and is characterized by intermittent airway obstruction, airway hyperreactivity, and airway inflammation. An inverse association between allergic asthma and microbial infections has been observed. Microbial infections could prevent allergic responses by inducing the secretion of the type 1 cytokines, IL-12 and IFN-γ. In this study, we examined whether administration of bacterial LPS, a prototypic bacterial product that activates innate immune cells via the Toll-like receptor 4 (TLR4) could suppress early and late allergic responses in a murine model of asthma. We report that LPS administration suppresses the IgE-mediated and mast cell-dependent passive cutaneous anaphylaxis, pulmonary inflammation, airway eosinophilia, mucus production, and airway hyperactivity. The suppression of asthma-like responses was not due to Th1 shift as it persisted in IL-12−/− or IFN-γ−/− mice. However, the suppressive effect of LPS was not observed in TLR4- or NO synthase 2-deficient mice. Our findings demonstrate, for the first time, that LPS suppresses Th2 responses in vivo via the TLR4-dependent pathway that triggers NO synthase 2 activity.
IL-18 Enhances Collagen-Induced Arthritis by Recruiting Neutrophils Via TNF-α and Leukotriene B4
Authors: Cannetti Claudio A. , Leung Bernard P. , Culshaw Shauna , McInnes Iain B. , Cunha Fernando Q. , Liew Foo Y.
Published: April 2014

IL-18 expression and functional activity have been associated with a range of autoimmune diseases. However, the precise mechanism by which IL-18 induces such pathology remains unclear. In this study we provide direct evidence that IL-18 activates neutrophils via TNF-α induction, which drives the production of leukotriene B4 (LTB4), which in turn leads to neutrophil accumulation and subsequent local inflammation. rIL-18 administered i.p. resulted in the local synthesis of LTB4 and a rapid influx of neutrophils into the peritoneal cavity, which could be effectively blocked by the LTB4 synthesis inhibitor MK-886 (MK) or its receptor antagonist CP-105,696. IL-18-induced neutrophils recruitment and LTB4 production could also be blocked by a neutralizing anti-TNF-α Ab. In addition, IL-18 failed to induce neutrophil accumulation in vivo in TNFRp55−/− mice. In an IL-18-dependent murine collagen-induced arthritis model, administration of MK significantly inhibited disease severity and reduced articular inflammation and joint destruction. Furthermore, MK-886-treated mice also displayed suppressed proinflammatory cytokine production in response to type II collagen in vitro. Finally, we showed that IL-18-activated human peripheral blood neutrophils produced significant amounts of LTB4 that were effectively blocked by the MK. Together, these findings provide a novel mechanism whereby IL-18 can promote inflammatory diseases.
Matrix Metalloproteinase-9-Mediated Dendritic Cell Recruitment into the Airways Is a Critical Step in a Mouse Model of Asthma
Authors: Vermaelen Karim Y. , Cataldo Didier , Tournoy Kurt , Maes Tania , Dhulst An , Louis Renaud , Foidart Jean-Michel , Noël Agnès , Pauwels Romain
Published: April 2014

Dendritic cells (DCs) appear to be strategically implicated in allergic diseases, including asthma. Matrix metalloproteinase (MMP)-9 mediates transmigration of inflammatory leukocytes across basement membranes. This study investigated the role of MMP-9 in airway DC trafficking during allergen-induced airway inflammation. MMP-9 gene deletion affected the trafficking of pulmonary DCs in a specific way: only the inflammatory transmigration of DCs into the airway lumen was impaired, whereas DC-mediated transport of airway Ag to the thoracic lymph nodes remained unaffected. In parallel, the local production of the Th2-attracting chemokine CC chemokine ligand 17/thymus and activation-regulated chemokine, which was highly concentrated in purified lung DCs, fell short in the airways of allergen-exposed MMP-9−/− mice. This was accompanied by markedly reduced peribronchial eosinophilic infiltrates and impaired allergen-specific IgE production. We conclude that the specific absence of MMP-9 activity inhibits the development of allergic airway inflammation by impairing the recruitment of DCs into the airways and the local production of DC-derived proallergic chemokines.
The RhoA Effector mDia Is Induced During T Cell Activation and Regulates Actin Polymerization and Cell Migration in T Lymphocytes
Authors: Vicente-Manzanares Miguel , Rey Mercedes , Pérez-Martínez Manuel , Yáñez-Mó María , Sancho David , Cabrero José Román , Barreiro Olga , de la Fuente Hortensia , Itoh Kazuyuki , Sánchez-Madrid Francisco
Published: April 2014

Regulation of actin polymerization is critical for many different functions of T lymphocytes, including cell migration. Here we show that the RhoA effector mDia is induced in vitro in activated PBL and is highly expressed in vivo in diseased tissue-infiltrating activated lymphocytes. mDia localizes at the leading edge of polarized T lymphoblasts in an area immediately posterior to the leading lamella, in which its effector protein profilin is also concentrated. Overexpression of an activated mutant of mDia results in an inhibition of both spontaneous and chemokine-directed T cell motility. mDia does not regulate the shape of the cell, which involves another RhoA effector, p160 Rho-coiled coil kinase, and is not involved in integrin-mediated cell adhesion. However, mDia activation blocked CD3- and PMA-mediated cell spreading. mDia activation increased polymerized actin levels, which resulted in the blockade of chemokine-induced actin polymerization by depletion of monomeric actin. Moreover, mDia was shown to regulate the function of the small GTPase Rac1 through the control of actin availability. Together, our data demonstrate that RhoA is involved in the control of the filamentous actin/monomeric actin balance through mDia, and that this balance is critical for T cell responses.
Inhibition of Neutrophil Apoptosis by Type 1 IFN Depends on Cross-Talk Between Phosphoinositol 3-Kinase, Protein Kinase C-δ, and NF-κB Signaling Pathways
Authors: Wang KeQing , Scheel-Toellner Dagmar , Wong See Heng , Craddock Rachel , Caamano Jorge , Akbar Arne N. , Salmon Mike , Lord Janet M.
Published: April 2014

Neutrophils are abundant, short-lived leukocytes with a key role in the defense against rapidly dividing bacteria. They enter apoptosis spontaneously within 24–48 h of leaving the bone marrow. However, their life span can be extended during inflammatory responses by several proinflammatory cytokines. Inappropriate survival of neutrophils contributes to chronic inflammation and tissue damage associated with diseases such as rheumatoid arthritis. We have previously reported that type I IFNs can inhibit both cytokine deprivation and Fas-induced apoptosis in activated T cells. IFN-β locally produced by hyperplastic fibroblasts within the pannus tissue of patients with rheumatoid arthritis contributes to the inappropriately extended life span of infiltrating T cells. Type I IFNs are equally effective at delaying spontaneous apoptosis in human neutrophils. In the work presented here we investigated the signaling pathways involved in mediating this effect. The antiapoptotic actions of IFN-β were targeted at an early stage of neutrophil apoptosis, occurring upstream of mitochondrial permeability transition, and were phosphatidylinositol 3-kinase (PI3K) dependent, as they were blocked by the PI3K inhibitor LY294002. Analysis of signaling pathways downstream of PI3K revealed that the antiapoptotic effect of type 1 IFN was inhibited by rottlerin, SN50, and cycloheximide, indicating requirements for activation of protein kinase C-δ, NF-κB, and de novo protein synthesis, respectively. Moreover, EMSA was used to show that the activation of NF-κB occurred downstream of PI3K and protein kinase C-δ activation. We conclude that type I IFNs inhibit neutrophil apoptosis in a PI3K-dependent manner, which requires activation of protein kinase C-δ and induction of NF-κB-regulated genes.
Modulation of CD11b/CD18 Adhesive Activity by Its Extracellular, Membrane-Proximal Regions
Authors: Xiong Yu-Mei , Chen Jian , Zhang Li
Published: April 2014

The integrin receptor CD11b/CD18 is normally kept in a low adhesive state and can be activated by many different agents. However, the mechanism underlying receptor activation is not yet fully understood. We hypothesized that the extracellular, membrane-proximal regions of CD11b/CD18 are critically involved in modulation of its adhesive functions. To test our hypothesis, we perturbed the extracellular, membrane-proximal regions of individual CD11b and CD18 subunits and studied their effect on ligand binding, receptor clustering, and lipid raft association. We report here three major findings: 1) perturbation of the extracellular, membrane-proximal region of either subunit leads to enhanced adhesion, caused by changes in receptor conformation, but not the state of receptor clustering or lipid raft association; 2) the CD11b subunit plays a more important role in confining the receptor in an inactive state; and 3) upon modification of the extracellular, membrane-proximal region, the mutant CD11b/CD18 acquires the ability to respond to stimulation by “inside-out” signaling. Our results suggest that the extracellular, membrane-proximal region of the receptor plays an important role in integrin activation and therefore could be targeted by certain cell surface proteins as a conduit to control the integrin “inside-out” signaling process.
Clara Cell Secretory Protein Modulates Lung Inflammatory and Immune Responses to Respiratory Syncytial Virus Infection
Authors: Wang Shan-Ze , Rosenberger Cynthia L. , Bao Yi-Xiao , Stark James M. , Harrod Kevin S.
Published: April 2014

Clara cell secretory protein (CCSP) has been shown to have anti-inflammatory and immunomodulatory functions in the lung. Respiratory syncytial virus (RSV) is the most common cause of respiratory infection in infants and young children. RSV usually infects small airways and likely interacts with the Clara cells of bronchioles. To determine a possible role for CCSP during acute RSV infection, CCSP-deficient (CCSP−/−) and wild-type (WT) mice were intratracheally infected with RSV and the lung inflammatory and immune responses to RSV infection were assessed. RSV-F gene expression was increased in the lungs of CCSP−/− mice as compared with WT mice following RSV infection, consistent with increased viral persistence. Lung inflammation was significantly increased in CCSP−/− mice as compared with WT mice after infection. Moreover, although the levels of Th1 cytokines were similar, the levels of Th2 cytokines and neutrophil chemokines were increased in the lungs of CCSP−/− mice following infection. Physiologic endpoints of exacerbated lung disease, specifically airway reactivity and mucus production, were increased in CCSP−/− mice after RSV infection. Importantly, restoration of CCSP in the airways of CCSP−/− mice abrogated the increased viral persistence, lung inflammation, and airway reactivity. These findings suggest a role for CCSP and Clara cells in regulating lung inflammatory and immune responses to RSV infection.
TRAIL-R2 (DR5) Mediates Apoptosis of Synovial Fibroblasts in Rheumatoid Arthritis
Authors: Ichikawa Kimihisa , Liu Weimin , Fleck Martin , Zhang Huangge , Zhao Limin , Ohtsuka Toshiaki , Wang Zheng , Liu Di , Mountz John D. , Ohtsuki Masahiko , Koopman William J. , Kimberly Robert , Zhou Tong
Published: April 2014

TRAIL has been proposed as an anti-inflammatory cytokine in animal models of rheumatoid arthritis (RA). Using two agonistic mAbs specific for TRAIL-R1 (DR4) and TRAIL-R2 (DR5), we examined the expression and function of these death receptors in RA synovial fibroblast cells. The synovial tissues and primary synovial fibroblast cells isolated from patients with RA, but not those isolated from patients with osteoarthritis, selectively expressed high levels of cell surface DR5 and were highly susceptible to anti-DR5 Ab (TRA-8)-mediated apoptosis. In contrast, RA synoviocytes did not show increased expression of TRAIL-R1 (DR4), nor was there any difference in expression of Fas between RA and osteoarthritis synovial cells. In vitro TRA-8 induced apoptosis of RA synovial cells and inhibited production of matrix metalloproteinases induced by pro-inflammatory cytokines. In vivo TRA-8 effectively inhibited hypercellularity of a SV40-transformed RA synovial cell line and completely prevented bone erosion and cartilage destruction induced by these cells. These results indicate that increased DR5 expression and susceptibility to DR5-mediated apoptosis are characteristic of the proliferating synovial cells in RA. As highly proliferative transformed-appearing RA synovial cells play a crucial role in bone erosion and cartilage destruction in RA, the specific targeting of DR5 on RA synovial cells with an agonistic anti-DR5 Ab may be a potential therapy for RA.
Induction of Human T Lymphocyte Cytotoxicity and Inhibition of Tumor Growth by Tumor-Specific Diabody-Based Molecules Secreted from Gene-Modified Bystander Cells
Authors: Blanco Belén , Holliger Phillip , Vile Richard G. , Álvarez-Vallina Luis
Published: April 2014

Infiltrating T cells are found in many malignancies, but they appear to be mostly anergic and do not attack the tumor, presumably because of the absence of activation and/or costimulatory signals. We describe a strategy for cellular antitumor immunotherapy by the in situ production of soluble bifunctional Ab-based molecules that activate and retarget T cells to the tumor. We genetically modified cells to simultaneously secrete two bifunctional molecules, a bispecific diabody directed against the carcinoembryonic Ag (CEA) and the CD3ε chain of the TCR (αCEA × αCD3), and a fusion protein comprising the extracellular portion of B7-1 fused to a bivalent anti-CEA diabody (B7-αCEA). Together, αCEA × αCD3 and B7-αCEA proved potent at inducing the activation, proliferation, and survival of primary human T cells. When producer cells were cocultured with primary T cells and CEA+ cancer cells, αCEA × αCD3 and B7-αCEA acted in combination to activate and retarget T cell cytotoxicity and completely abrogate tumor growth in the coculture. Furthermore, the introduction of just a few such producer cells at the tumor site efficiently inhibited the growth of established human colon carcinoma xenografts. Despite a cumbersome generation process, the use of autologous gene-modified producer cells opens the way for a new diabody-based gene therapy strategy of cancer.
A Second Step of Chemotaxis After Transendothelial Migration: Keratinocytes Undergoing Apoptosis Release IFN-γ-Inducible Protein 10, Monokine Induced by IFN-γ, and IFN-γ-Inducible α-Chemoattractant for T Cell Chemotaxis Toward Epidermis in Atopic Dermatitis
Authors: Klunker Sven , Trautmann Axel , Akdis Mübeccel , Verhagen Johan , Schmid-Grendelmeier Peter , Blaser Kurt , Akdis Cezmi A.
Published: April 2014

Activation and skin-selective homing of T cells and their effector functions in the skin represent sequential immunological events in the pathogenesis of atopic dermatitis (AD). Apoptosis of keratinocytes, induced mainly by T cells and mediated by IFN-γ and Fas, is the essential pathogenetic event in eczema formation. Keratinocyte apoptosis appears as activation-induced cell death in AD. By IFN-γ stimulation, chemokines such as IFN-γ-inducible protein 10, monokine induced by IFN-γ, and IFN-γ-inducible α-chemoattractant are strongly up-regulated in keratinocytes. These chemokines attract T cells bearing the specific receptor CXCR3, which is highly expressed on T cells isolated from skin biopsies of AD patients. Accordingly, an increased T cell chemotaxis was observed toward IFN-γ-treated keratinocytes. Supporting these findings, enhanced IFN-γ-inducible protein 10, monokine induced by IFN-γ, and IFN-γ-inducible α-chemoattractant expression was observed in lesional AD skin by immunohistochemical staining. These results indicate a second step of chemotaxis inside the skin after transendothelial migration of the inflammatory cells. Keratinocytes undergoing apoptosis in acute eczematous lesions release chemokines that attract more T cells toward the epidermis, which may further augment the inflammation and keratinocyte apoptosis.
Osteopontin (Eta-1) and Fibroblast Growth Factor-2 Cross-Talk in Angiogenesis
Authors: Leali Daria , Dell’Era Patrizia , Stabile Helena , Sennino Barbara , Chambers Ann F. , Naldini Antonella , Sozzani Silvano , Nico Beatrice , Ribatti Domenico , Presta Marco
Published: April 2014

The cytokine/extracellular matrix protein osteopontin (OPN/Eta-1) is an important component of cellular immunity and inflammation. It also acts as a survival, cell-adhesive, and chemotactic factor for endothelial cells. Here, subtractive suppression hybridization showed that serum-deprived murine aortic endothelial (MAE) cells transfected with the angiogenic fibroblast growth factor-2 (FGF2) overexpress OPN compared with parental cells. This was confirmed by Northern blotting and Western blot analysis of the conditioned media in different clones of endothelial cells overexpressing FGF2 and in endothelial cells treated with the recombinant growth factor. In vivo, FGF2 caused OPN expression in newly formed endothelium of the chick embryo chorioallantoic membrane (CAM) and of murine s.c. Matrigel plug implants. Recombinant OPN (rOPN), the fusion protein GST-OPN, and the deletion mutant GST-ΔRGD-OPN were angiogenic in the CAM assay. Angiogenesis was also triggered by OPN-transfected MAE cells grafted onto the CAM. OPN-driven neovascularization was independent from endothelial αvβ3 integrin engagement and was always paralleled by the appearance of a massive mononuclear cell infiltrate. Accordingly, rOPN, GST-OPN, GST-ΔRGD-OPN, and the conditioned medium of OPN-overexpressing MAE cells were chemotactic for isolated human monocytes. Also, rOPN triggered a proangiogenic phenotype in human monocytes by inducing the expression of the angiogenic cytokines TNF-α and IL-8. OPN-mediated recruitment of proangiogenic monocytes may represent a mechanism of amplification of FGF2-induced neovascularization during inflammation, wound healing, and tumor growth.
HIV-1 Vaccination Administered Intramuscularly Can Induce Both Systemic and Mucosal T Cell Immunity in HIV-1-Uninfected Individuals
Authors: Musey Luwy , Ding Yan , Elizaga Marnie , Ha Richard , Celum Connie , McElrath M. Juliana
Published: April 2014

A vaccine regimen that can rapidly control HIV-1 replication at the site of exposure following sexual contact is likely to be the most effective in preventing HIV-1 infection. As part of a larger, phase II clinical trial, we evaluated the ability of a recombinant canarypox HIV-1 vaccine to induce CTL that can be detected in both the systemic and mucosal compartments following i.m. immunization in 12 low- and high-risk HIV-1 seronegative volunteers. In the 7 volunteers receiving four immunizations with live recombinant canarypox ALVAC-HIV vaccine with or without rgp120/SF-2, HIV-1-specific CTL were detected in the blood of 5 (71%) and in the rectum of 4 (57%). CTL responses were observed in both risk strata. In contrast, 5 volunteers receiving placebo had undetectable responses in both compartments. Vaccine-induced, HIV-1-specific effector activities included IFN-γ secretion and class I MHC-restricted CD8+ CTL. Rectal and systemic CD8+ CTL clones established in 1 vaccine recipient revealed similar Env-specific responses and MHC restriction. These findings indicate that parenteral vaccination can induce HIV-1-specific CTL that localize to sites of HIV-1 acquisition, where their presence may be critical in the control of initial viral replication and eventual dissemination. Determination of the optimal strategy to induce mucosal T cells requires future clinical studies.
Differential Susceptibility to Staphylococcal Superantigen (SsAg)-Induced Apoptosis of CD4+ T Cells from Atopic Dermatitis Patients and Healthy Subjects: The Inhibitory Effect of IL-4 on SsAg-Induced Apoptosis
Authors: Lin Yu-Tsan , Wang Chen-Ti , Hsu Chen-Tong , Wang Li-Fang , Shau Wen-Yi , Yang Yao-Hsu , Chiang Bor-Luen
Published: April 2014

This study had two aims: 1) to determine whether there are differences between atopic dermatitis (AD) patients and healthy subjects in staphylococcal superantigen (SsAg)-induced CD4+ T cell activation, cytokine production, chemokine receptor expression, and apoptosis; and 2) to investigate the effect of IL-4 on SsAg-induced apoptosis. By using immunofluorescence and annexin V staining, we analyzed PBMC with or without staphylococcal enterotoxin B (SEB) stimulation in the presence or absence of rIL-4 or anti-IL-4-neutralizing Abs in 15 healthy subjects and 27 AD patients. We found that SEB preferentially induced production of Th1 cytokine in SEB-reactive (TCRVβ3+ or Vβ12+ or Vβ17+) CD4+ T cells from healthy subjects and Th2 cytokine in those from AD patients. SEB induced up-regulation of CXCR3+ cells in SEB-reactive CD4+ T cells from healthy subjects and CCR4+ cells in those from AD patients. SEB-reactive CD4+ T cells from AD patients were more resistant to SEB-induced apoptosis than those from healthy subjects. There was no significant difference between AD and healthy subjects in SEB-induced activation of CD4+ T cells. CXCR3+ CD4+ T cells were more susceptible to SEB-induced apoptosis than CCR4+ CD4+ T cells in healthy subjects. Exogenously added IL-4 inhibited SEB-induced apoptosis of SEB-reactive CD4+ and CXCR3+ CD4+ T cells but not of CCR4+ CD4+ T cells in healthy subjects. Inhibition of endogenous IL-4 increased SEB-induced apoptosis of SEB-reactive CD4+ T cells from AD patients. These results might provide new clues to the mechanism that SsAgs contribute to the persistence and exacerbation of allergic skin inflammation in AD.
Cutting Edge: Granzymes A and B Are Not Essential for Perforin-Mediated Tumor Rejection
Authors: Smyth Mark J. , Street Shayna E. A. , Trapani Joseph A.
Published: April 2014

Controversy still exists regarding the biological function of granzyme serine proteases released with perforin from the cytotoxic granules of NK cells and CTLs. In particular, it is not clear whether the major granzymes, A and B, play an essential role in tumor rejection mediated by the perforin pathway. We have now examined the relative importance of perforin and granzyme A and B clusters in five different tumor models that stringently distinguish their importance. We conclude that granzyme A and B clusters are not essential for CTL- and NK cell-mediated rejection of spontaneous and experimental tumors, raising the likelihood that either perforin alone or in combination with an additional granzyme or granule component(s) mediates cytotoxicity of tumor cells in vivo.
Cutting Edge: A Chemical Genetic System for the Analysis of Kinases Regulating T Cell Development
Authors: Denzel Angela , Hare Katherine J. , Zhang Chao , Shokat Kevan , Jenkinson Eric J. , Anderson Graham , Hayday Adrian
Published: April 2014

To understand the regulatory activities of kinases in vivo requires their study across a biologically relevant window of activity. To this end, ATP analog-sensitive kinase alleles (ASKAs) specifically sensitive to a competitive inhibitor have been developed. This article tests whether ASKA technology can be applied to complex immunological systems, such as lymphoid development. The results show that when applied to reaggregate thymic organ culture, novel p56Lck ASKAs readily expose a dose-dependent correlation of thymocyte development with a range of p56Lck activity. By regulating kinase activity, rather than amounts of RNA or protein, ASKA technology offers a general means for assessing the quantitative contributions to immunology of numerous kinases emerging from genomics analyses. It can obviate the generation of multiple lines of mice expressing different levels of kinase transgenes and should permit specific biological effects to be associated with defined biochemical activities.
Cutting Edge: TCR δ Gene Is Frequently Rearranged in Adult B Lymphocytes
Authors: Krejci Ondrej , Prouzova Zuzana , Horvath Ondrej , Trka Jan , Hrusak Ondrej
Published: April 2014

TCR gene rearrangement generates diversity of T lymphocytes by V(D)J recombination. Ig genes are rearranged in B cells using the same enzyme machinery. Physiologically, TCR gene is postulated to rearrange exclusively in T lineage, but malignant B precursor lymphoblasts contain rearranged TCR genes in most patients. Several mechanisms by which malignant cells break the regulation of V(D)J recombination have been proposed. In this study we show that incomplete TCR δ rearrangements V2-D3 and D2-D3 occur each in up to 16% alleles in B lymphocytes of all healthy donors studied, but complete VDJ rearrangement was negative at the sensitivity limit of 1%. Data are based on real-time quantitative PCR validated by PAGE and sequencing of the cloned products. Therefore, TCR genes rearrange not exclusively in T lineage. This study opens up further questions regarding the exact extent of the “cross-lineage” TCR or Ig rearrangements in normal lymphocytes, specific subsets in which the cross-lineage rearrangements occur, and the physiological importance of these rearrangements.
Cutting Edge: MHC Class II-Restricted Peptides Containing the Inflammation-Associated Marker 3-Nitrotyrosine Evade Central Tolerance and Elicit a Robust Cell-Mediated Immune Response
Authors: Birnboim H. Chaim , Lemay Anne-Marie , Lam Debbie Ka Yee , Goldstein Rose , Webb John R.
Published: April 2014

Nitrotyrosine is widely recognized as a surrogate marker of up-regulated inducible NO synthase expression at sites of inflammation. However, the potential immunogenicity of autologous proteins containing nitrotyrosine has not previously been investigated. Herein, we used the I-EK-restricted T cell epitope of pigeon/moth cytochrome c (PCC/MCC88–103) to assess the ability of T cells to recognize ligands containing nitrotyrosine. Substitution of the single tyrosine (Y97) in PCC/MCC88–103 with nitrotyrosine abrogates recognition by the MCC88–103-specific T cell hybridoma 2B4. CBA (H2K) mice immunized with MCC88–103 or nitrated MCC88–103 peptides produce T cell responses that are mutually exclusive. Transgenic mice that constitutively express PCC under the control of an MHC class I promoter are tolerant toward immunization with MCC88–103, but exhibited a robust immune response against nitrated MCC88–103. Analysis of T cell hybridomas specific for nitrated-MCC88–103 indicated that subtle differences in TCR VDJ gene usage are sufficient to allow nitrotyrosine-specific T cells to escape the processes of central tolerance.
Cutting Edge: Protective Cell-Mediated Immunity to Listeria monocytogenes in the Absence of Myeloid Differentiation Factor 88
Authors: Way Sing Sing , Kollmann Tobias R. , Hajjar Adeline M. , Wilson Christopher B.
Published: April 2014

In addition to their role in triggering innate immune responses, Toll-like receptors are proposed to play a key role in linking the innate and adaptive arms of the immune response. The majority of cellular responses downstream of Toll-like receptors are mediated through the adapter molecule myeloid differentiation factor 88 (MyD88), and mice with a targeted deletion of MyD88 are highly susceptible to bacterial infections, including primary infection with Listeria monocytogenes (LM). In contrast, herein we demonstrate that MyD88-deficient mice have only a modest impairment in their LM-specific CD4 T cell response, and no impairment in their CD8 T cell response following infection with ActA-deficient LM. Furthermore, CD8 T cells from immunized MyD88-deficient mice protected naive recipient mice following adoptive splenocyte transfer, and immunized MyD88-deficient mice were protected from infection with wild-type LM. These results indicate that adaptive immune responses can be generated and provide protective immunity in the absence of MyD88.
Cutting Edge: The Conversion of Arginine to Citrulline Allows for a High-Affinity Peptide Interaction with the Rheumatoid Arthritis-Associated HLA-DRB1*0401 MHC Class II Molecule
Authors: Hill Jonathan A. , Southwood Scott , Sette Alessandro , Jevnikar Anthony M. , Bell David A. , Cairns Ewa
Published: April 2014

Rheumatoid arthritis (RA) is genetically associated with MHC class II molecules that contain the shared epitope. These MHC molecules may participate in disease pathogenesis by selectively binding arthritogenic peptides for presentation to autoreactive CD4+ T cells. The nature of the arthritogenic Ag is not known, but recent work has identified posttranslationally modified proteins containing citrulline (deiminated arginine) as specific targets of the IgG Ab response in RA patients. To understand how citrulline might evoke an autoimmune reaction, we have studied T cell responses to citrulline-containing peptides in HLA-DRB1*0401 transgenic (DR4-IE tg) mice. In this study, we demonstrate that the conversion of arginine to citrulline at the peptide side-chain position interacting with the shared epitope significantly increases peptide-MHC affinity and leads to the activation CD4+ T cells in DR4-IE tg mice. These results reveal how DRB1 alleles with the shared epitope could initiate an autoimmune response to citrullinated self-Ags in RA patients.
Cutting Edge: Sanglifehrin A, a Novel Cyclophilin-Binding Immunosuppressant Blocks Bioactive IL-12 Production by Human Dendritic Cells
Authors: Steinschulte Christoph , Taner Timucin , Thomson Angus W. , Bein Gregor , Hackstein Holger
Published: April 2014

Sanglifehrin A (SFA) is a novel cyclophilin-binding immunosuppressant with an unknown mechanism of action. IL-12p70 plays a critical role in the pathogenesis of inflammation and autoimmune diseases. We discovered that SFA abrogates bioactive IL-12p70 production by human dendritic cells, the major producers of this cytokine. In direct comparison to the related calcineurin inhibitor cyclosporin A and the mammalian target of rapamycin inhibitor rapamycin, SFA acts uniquely within 1 h to inhibit (80–95%) IL-12p70 production by differentiated dendritic cells. Experiments with Toll-like receptor 3 and 4 ligands show a stimulus-independent suppression. Competitive experiments with a molar excess of cyclosporin A indicate a cyclophilin A-independent blockade of IL-12p70 production. We confirm potent inhibition of IL-12p70 production by SFA using purified human blood DC. Real-time RT-PCR reveals 84–94% suppression of IL-12p40, IL-12p35, and IL-23-specific p19 transcription. These novel insights into the immunosuppressive action of SFA are likely to impact on the clinical use of this agent.
Cutting Edge: Germinal Centers Formed in the Absence of B Cell-Activating Factor Belonging to the TNF Family Exhibit Impaired Maturation and Function
Authors: Vora Kalpit A. , Wang Li Chun , Rao Sambasiva P. , Liu Zhong-Ying , Majeau Gerard R. , Cutler Anne H. , Hochman Paula S. , Scott Martin L. , Kalled Susan L.
Published: April 2014

Germinal centers (GCs) form in B cell follicles and require specific signals for development and maintenance. B cell-activating factor belonging to the TNF family (BAFF) is a fundamental B cell survival factor and therefore may influence GC reactions and subsequent Ab responses. To test this possibility, the effect of BAFF neutralization in immunized mice was assessed. Using B cell maturation Ag-Fc, we demonstrate that BAFF blockade does not inhibit GC formation or somatic hypermutation. However, GCs in B cell maturation Ag-Fc-treated mice dissipated more rapidly than those of control mice and did not form a mature follicular dendritic cell reticulum. Examination of immunized BAFF-null mice validated the BAFF-independent nature of GC formation. Furthermore, Ab responses, including high-affinity responses, were attenuated. This is the first evidence that BAFF is required for maintenance, but not initiation, of the GC reaction, and it further hints that somatic hypermutation within the GC and selection of Ag-specific high-affinity Ab could be uncoupled.
A High Endothelial Venule Secretory Protein, Mac25/Angiomodulin, Interacts with Multiple High Endothelial Venule-Associated Molecules Including Chemokines
Authors: Nagakubo Daisuke , Murai Toshiyuki , Tanaka Toshiyuki , Usui Takeo , Matsumoto Masanori , Sekiguchi Kiyotoshi , Miyasaka Masayuki
Published: April 2014

We previously reported that mac25/angiomodulin (AGM), a 30-kDa secretory protein, is abundantly expressed in high endothelial venules (HEVs), which play a crucial role in lymphocyte trafficking to the lymph nodes and Peyer’s patches. We report that mac25/AGM interacts preferentially with certain molecules that are expressed in or around HEVs. In particular, mac25/AGM interacted with not only the extracellular matrix proteins and glycosaminoglycans that are expressed in most blood vessels including HEVs, but also with some chemokines that are implicated in the regulation of lymphocyte trafficking, such as the secondary lymphoid-tissue chemokine (SLC; CCL21), IFN-γ-inducible protein 10 (IP-10; CXCL10), and RANTES (CCL5). The binding of mac25/AGM to SLC and IP-10 was dose-dependent and saturable. The binding to IP-10 could be inhibited by SLC but not by a non-mac25/AGM-binding chemokine, EBI1-ligand chemokine (ELC; CCL19). Interestingly, mac25/AGM failed to interact with 18 other chemokines, suggesting that it binds to certain chemokines preferentially. Immunohistochemical analysis indicated that mac25/AGM colocalizes at least partially with SLC and IP-10 at the basal lamina of HEVs. Upon binding with mac25/AGM, SLC and IP-10 retained all their Ca2+-signaling activity in vitro, suggesting that mac25/AGM can hold and present chemokines in the basal lamina of HEVs. These results imply that mac25/AGM plays a multifunctional role, serving not only as an adhesion protein to interact with glycosaminoglycans and extracellular matrix proteins but also as a molecule to present chemokines so that lymphocytes extravasating through HEVs receive further directional cues subsequent to the luminal encounter with lymphoid chemokines.
FcγRs Modulate Cytotoxicity of Anti-Fas Antibodies: Implications for Agonistic Antibody-Based Therapeutics
Authors: Xu Yuanyuan , Szalai Alexander J. , Zhou Tong , Zinn Kurt R. , Chaudhuri Tandra R. , Li Xiaoli , Koopman William J. , Kimberly Robert P.
Published: April 2014

Development of anti-Fas Abs to treat diseases with insufficient Fas-mediated apoptosis has been limited by concern about hepatotoxicity. We report here that hepatotoxicity elicited by anti-Fas Ab Jo2 is dependent on FcγRIIB. Thus, following Jo2 treatment, all FcγRIIB−/− mice survived while 80% of wild-type and all FcR-γ−/− mice died from acute liver failure. Microscopic examination suggests that FcγRIIB deficiency protects the hepatic sinusoidal endothelium, a cell type that normally coexpresses Fas and FcγRIIB. In vitro studies showed that FcγRIIB, but not FcγRI and FcγRIII, on neighboring macrophages substantially enhanced Jo2 mediated apoptosis of Fas expressing target cells. However, FcγRI and FcγRIII appeared essential for apoptosis-inducing activity of a non-hepatotoxic anti-Fas mAb HFE7A. These findings imply that by interacting with the Fc region of agonistic Abs, FcγRs can modulate both the desired and undesired consequences of Ab-based therapy. Recognizing this fact should facilitate development of safer and more efficacious agonistic Abs.
Tumor Cells Engineered with IL-12 and IL-15 Genes Induce Protective Antibody Responses in Nude Mice
Authors: Orengo Anna Maria , Di Carlo Emma , Comes Alberto , Fabbi Marina , Piazza Tiziana , Cilli Michele , Musiani Piero , Ferrini Silvano
Published: April 2014

IL-12 and IL-15 stimulate T, B, and NK cell functions through independent mechanisms, and cooperative effects of these cytokines have been reported. The human MHC class I-negative small cell lung cancer cell line, N592, genetically engineered to secrete IL-15, N592/IL-15, showed a reduced tumor growth rate, while N592 cells engineered with IL-12, N592/IL-12, grew similarly to the wild-type N592, N592 parental cells (N592pc), in nude mice. However, N592 cells coexpressing both cytokines, N592/IL-12/IL-15 cells, were completely rejected by 100% of nude mice. Here we show that 60% of nude mice rejecting N592/IL-12/IL-15 cells were resistant to N592pc rechallenge. SCID mice rejected N592/IL-12/IL-15 cells, but did not develop resistance to N592pc rechallenge, suggesting a role of Ab responses. Among nude mice rejecting N592/IL-12/IL-15 cells, those developing resistance to N592pc rechallenge had significantly higher titers of anti-N592 IgG2b Abs than nonresistant nude mice. Induction of an Ig class switch in nude mice was related to the expression of IFN-γ and CD40 ligand in the draining lymph nodes. An IgG2b, anti-N592 mAb, derived from N592/IL-12/IL-15-immunized nude mice splenocytes induced significant protection against N592pc, while an IgM mAb was ineffective. The protective IgG2b mAb, but not the IgM mAb, triggered Ab-dependent cell-mediated cytotoxicity by nude mouse splenocytes against N592pc. These data indicate that IL-12 and IL-15 synergistically trigger innate, immunity-mediated, anti-tumor effects, resulting in cytotoxic IgG Ab responses in T cell-deficient mice. Protective Ab responses may relate to both direct actions of IL-12 and IL-15 on B cells and to the activation of an innate immunity-B cell cross-talk.
Blocking 4-1BB/4-1BB Ligand Interactions Prevents Herpetic Stromal Keratitis
Authors: Seo Su K. , Park Hye Y. , Choi Jae H. , Kim Won Y. , Kim Young H. , Jung Hyo W. , Kwon Byungsuk , Lee Hyeon W. , Kwon Byoung S.
Published: April 2014

Herpetic stromal keratitis (HSK) is a chronic inflammatory process in corneal stroma that results from recurrent HSV type 1 infection. We used the murine model of HSK to demonstrate the importance of the interaction between an inducible T cell costimulatory receptor, 4-1BB, and its ligand, 4-1BB ligand (4-1BBL), in the development of this disease. In BALB/c mice, HSK ordinarily induced by infection with the RE strain of herpes was prevented by blocking 4-1BB/4-1BBL interaction, either by deleting 4-1BB (in mutant 4-1BB−/− mice) or by introducing mAbs against 4-1BBL. The majority of T cells infiltrating the infected corneas were 4-1BB+ activated effector cells that expressed cell surface markers CD44, CD25, and/or CD62L, as well as chemokine receptors CCR1, CCR2, and CCR5, and a limited number of TCR Vβ chains (Vβ8.1/8.2, Vβ8.3, Vβ10b, and Vβ5.1/5.2, in order of abundance). Analysis of cell surface phenotypes showed that the failure to develop HSK in the 4-1BB−/− mice was associated with a reduced expression of CD62L at the time of T cell migration into the corneal stroma.
Increased Resistance of LFA-1-Deficient Mice to Lipopolysaccharide-Induced Shock/Liver Injury in the Presence of TNF-α and IL-12 Is Mediated by IL-10: A Novel Role for LFA-1 in the Regulation of the Proinflammatory and Anti-Inflammatory Cytokine Balance
Authors: Emoto Masashi , Emoto Yoshiko , Brinkmann Volker , Miyamoto Mamiko , Yoshizawa Izumi , Stäber Manuela , van Rooijen Nico , Hamann Alf , Kaufmann Stefan H. E.
Published: April 2014

Challenge with low doses of LPS together with d-galactosamine causes severe liver injury, resulting in lethal shock (low dose LPS-induced shock). We examined the role of LFA-1 in low dose LPS-induced shock. LFA-1−/− mice were more resistant to low dose LPS-induced shock/liver injury than their heterozygous littermates, although serum levels of TNF-α and IL-12 were higher in these mice. C57BL/6 mice were not rescued from lethal effects of LPS by depletion of NK1+ cells, granulocytes, or macrophages, and susceptibility of NKT cell-deficient mice was comparable to that of controls. High numbers of platelets were detected in the liver of LFA-1+/− mice after low dose LPS challenge, whereas liver accumulation of platelets was only marginal in LFA-1−/− mice. Following low dose LPS challenge, serum levels of IL-10 were higher in LFA-1−/− mice than in LFA-1+/− mice, and susceptibility to low dose LPS-induced shock as well as platelet accumulation in the liver of LFA-1−/− mice were markedly increased by IL-10 neutralization. Serum levels of IL-10 in LFA-1+/− mice were only marginally affected by macrophage depletion. However, in LFA-1−/− mice macrophage depletion markedly reduced serum levels of IL-10, and as a corollary, susceptibility of LFA-1−/− mice to low dose LPS-induced shock was markedly elevated despite the fact that TNF-α levels were also diminished. We conclude that LFA-1 participates in LPS-induced lethal shock/liver injury by regulating IL-10 secretion from macrophages and that IL-10 plays a decisive role in resistance to shock/liver injury. Our data point to a novel role of LFA-1 in control of the proinflammatory/anti-inflammatory cytokine network.
Pattern Recognition by TREM-2: Binding of Anionic Ligands
Authors: Daws Michael R. , Sullam Paul M. , Niemi Eréne C. , Chen Thomas T. , Tchao Nadia K. , Seaman William E.
Published: April 2014

We recently described the cloning of murine triggering receptor expressed by myeloid cells (TREM) 2, a single Ig domain DNAX adaptor protein 12-associated receptor expressed by cells of the myeloid lineage. In this study, we describe the identification of ligands for TREM-2 on both bacteria and mammalian cells. First, by using a TREM-2A/IgG1-Fc fusion protein, we demonstrate specific binding to a number of Gram-negative and Gram-positive bacteria and to yeast. Furthermore, we show that fluorescently labeled Escherichia coli and Staphylococcus aureus bind specifically to TREM-2-transfected cells. The binding of TREM-2A/Ig fusion protein to E. coli can be inhibited by the bacterial products LPS, lipoteichoic acid, and peptidoglycan. Additionally, binding can be inhibited by a number of other anionic carbohydrate molecules, including dextran sulfate, suggesting that ligand recognition is based partly on charge. Using a sensitive reporter assay, we demonstrate activation of a TREM-2A/CD3ζ chimeric receptor by both bacteria and dextran sulfate. Finally, we demonstrate binding of TREM-2A/Ig fusion to a series of human astrocytoma lines but not to a variety of other cell lines. The binding to astrocytomas, like binding to bacteria, is inhibited by anionic bacterial products, suggesting either a similar charge-based ligand recognition method or overlapping binding sites for recognition of self- and pathogen-expressed ligands.
Antitumor and Antimetastatic Activity of IL-23
Authors: Lo Chia-Hui , Lee Shan-Chih , Wu Pin-Yi , Pan Wen-Yu , Su Jui , Cheng Chao-Wen , Roffler Steve R. , Chiang Bor-Luen , Lee Chun-Nan , Wu Cheng-Wen , Tao Mi-Hua
Published: April 2014

The structure and T cell stimulatory effects of the recently discovered cytokine IL-23 are similar to, but distinct from, those of IL-12. Although the antitumor activities of IL-12 are well characterized, the effect of IL-23 on tumor growth is not known. In this study, murine CT26 colon adenocarcinoma and B16F1 melanoma cells were engineered using retroviral vectors to release single-chain IL-23 (scIL-23) to evaluate its antitumor activity. In BALB/c mice, scIL-23-transduced CT26 cells grew progressively until day 26 to an average size of 521 ± 333 mm3, then the tumors started to regress in most animals, resulting in a final 70% rate of complete tumor rejection. scIL-23 transduction also significantly suppressed lung metastases of CT26 and B16F1 tumor cells. In addition, mice that rejected scIL-23-transduced tumors developed a memory response against subsequent wild-type tumor challenge. Compared with scIL-12-expressing CT26 cells, scIL-23-transduced tumors lacked the early response, but achieved comparable antitumor and antimetastatic activity. These results demonstrated that IL-23, like IL-12, provided effective protection against malignant diseases, but it probably acted by different antitumor mechanisms. As a first step in identifying these antitumor mechanisms, tumor challenge studies were performed in immunocompromised hosts and in animals selectively depleted of various lymphocyte populations. The results showed that CD8+ T cells, but not CD4+ T cells or NK cells, were crucial for the antitumor activity of IL-23.
IL-21 Activates Both Innate and Adaptive Immunity to Generate Potent Antitumor Responses that Require Perforin but Are Independent of IFN-γ
Authors: Ma Hak-Ling , Whitters Matthew J. , Konz Richard F. , Senices Mayra , Young Deborah A. , Grusby Michael J. , Collins Mary , Dunussi-Joannopoulos Kyriaki
Published: April 2014

IL-21 is a key factor in the transition between innate and adaptive immune responses. We have used the cytokine gene therapy approach to study the antitumor responses mediated by IL-21 in the B16F1 melanoma and MethA fibrosarcoma tumor models in mice. Retrovirally transduced tumor cells secreting biologically functional IL-21 have growth patterns in vitro similar to that of control green fluorescent protein-transduced cells, but are completely rejected in vivo. We show that IL-21 activates NK and CD8+ T cells in vivo, thus mediating complete rejection of poorly immunogenic tumors. Rejection of IL-21-secreting tumors requires the presence of cognate IL-21R and does not depend on CD4+ T cell help. Interestingly, perforin, but not IFN-γ or other major Th1 and Th2 cytokines (IL-12, IL-4, or IL-10), is required for the IL-21-mediated antitumor response. Moreover, IL-21 results in 50% protection and 70% cure of nonimmunogenic tumors when given before and after tumor challenge, respectively, in C57BL/6 mice. We conclude that IL-21 immunotherapy warrants clinical evaluation as a potential treatment for cancer.
Thyrotropin-Mediated Repression of Class IITrans-Activator Expression in Thyroid Cells: Involvement of STAT3 and Suppressor of Cytokine Signaling
Authors: Kim Ho , Suh Jae Mi , Hwang Eun Suk , Kim Dong Wook , Chung Hyo Kyun , Song Jung Hun , Hwang Jung Hwan , Park Ki Cheol , Ro Heung Kyu , Jo Eun-Kyeong , Chang Jong-Soo , Lee Tae-Hoon , Lee Myung-Shik , Kohn Leonard D. , Shong Minho
Published: April 2014

It has been suggested that class I and class II MHC are contributing factors for numerous diseases including autoimmune thyroid diseases, type 1 diabetes, rheumatoid arthritis, Alzheimer’s disease, and multiple sclerosis. The class II trans-activator (CIITA), which is a non-DNA-binding regulator of class II MHC transcription, regulates the constitutive and inducible expression of the class I and class II genes. FRTL-5 thyroid cells incubated in the presence of IFN-γ have a significantly higher level of cell surface rat MHC class II RTI.B. However, the IFN-γ-induced RT1.B expression was suppressed significantly in cells incubated in the presence of thyrotropin. Thyrotropin (TSH) represses IFN-γ-induced CIITA expression by inhibiting type IV CIITA promoter activity through the suppression of STAT1 activation and IFN regulatory factor 1 induction. This study found that TSH induces transcriptional activation of the STAT3 gene through the phosphorylation of STAT3 and CREB activation. TSH induces SOCS-1 and SOCS-3, and TSH-mediated SOCS-3 induction was dependent on STAT3. The cell line stably expressing the wild-type STAT3 showed a higher CIITA induction in response to IFN-γ and also exhibited TSH repression of the IFN-γ-mediated induction of CIITA. However, TSH repression of the IFN-γ-induced CIITA expression was not observed in FRTL-5 thyroid cells, which stably expresses the dominant negative forms of STAT3, STAT3-Y705F, and STAT3-S727A. This report suggests that TSH is also engaged in immunomodulation through signal cross-talk with the cytokines in thyroid cells.