bluuebunny/crossref_metadata_2025 huggingface dataset:
Caspase-3 Is a Component of Fas Death-Inducing Signaling Complex in Lipid Rafts and Its Activity Is Required for Complete Caspase-8 Activation during Fas-Mediated Cell Death
Authors: Aouad Salah M. , Cohen Luchino Y. , Sharif-Askari Ehsan , Haddad Elias K. , Alam Antoine , Sekaly Rafick-Pierre
Published: April 2014

Since its discovery, caspase-8 has been placed at the apex of the proteolytic cascade triggered by death receptor (DR) cross-linking. Because of its capacity to interact with the cytoplasmic portion of DR, it has been suggested that caspase-8 acts independently of other caspases in the initiation of Fas and other DR signaling. In this study, we demonstrate that in Jurkat cells, caspase-3 cleavage is an early step during Fas-induced apoptosis. We show that caspase-3 processing into its p20 occurs rapidly after Fas cross-linking, in the absence of mitochondrial depolarization and caspase-9 activation. Moreover, caspase-3 is present in lipid rafts of untreated Jurkat cells and peripheral T lymphocytes. Caspase-3, caspase-8, and Fas-associated death domain are further recruited to lipid rafts of Jurkat cells following anti-Fas treatment. Fas immunoprecipitation reveals that caspase-3 is a component of the death-inducing signaling complex, suggesting that this cysteine protease is in close proximity to caspase-8. Furthermore, transduction of Jurkat cells with a caspase-3 dominant-negative form inhibits caspase-8 processing and results in inhibition of apoptosis, suggesting that caspase-3 activity is required for caspase-8 activation. Overall, these findings support a model whereby caspase-3 is a component of the death-inducing signaling complex located in lipid rafts, and as such, is involved in the amplification of caspase-8 activity by the mitochondrion.
Activation of Discoidin Domain Receptor 1 Isoform b with Collagen Up-Regulates Chemokine Production in Human Macrophages: Role of p38 Mitogen-Activated Protein Kinase and NF-κB
Authors: Matsuyama Wataru , Wang Lihua , Farrar William L. , Faure Michel , Yoshimura Teizo
Published: April 2014

Macrophages produce an array of proinflammatory mediators at sites of inflammation and contribute to the development of inflammatory responses. Important roles for cytokines, such as IL-1 or TNF-α, and bacterial products, such as LPS, in this process have been well documented; however, the role for the extracellular matrix proteins, such as collagen, remains unclear. We previously reported that discoidin domain receptor 1 (DDR1), a nonintegrin collagen receptor, is expressed during differentiation of human monocytes into macrophages, and the interaction of the DDR1b isoform with collagen facilitates their differentiation via the p38 mitogen-activated protein kinase (MAPK) pathway. In this study, we report that the interaction of DDR1b with collagen up-regulates the production of IL-8, macrophage inflammatory protein-1α, and monocyte chemoattractant protein-1 in human macrophages in a p38 MAPK- and NF-κB-dependent manner. p38 MAPK was critical for DDR1b-mediated, increased NF-κB trans-activity, but not for IκB degradation or NF-κB nuclear translocation, suggesting a role for p38 MAPK in the modification of NF-κB. DDR1b-mediated IκB degradation was mediated through the recruitment of the adaptor protein Shc to the LXNPXY motif of the receptor and the downstream TNFR-associated factor 6/NF-κB activator 1 signaling cascade. Taken together, our study has identified NF-κB as a novel target of DDR1b signaling and provided a novel mechanism by which tissue-infiltrating macrophages produce large amounts of chemokines during the development of inflammatory diseases. Intervention of DDR1b signaling may be useful to control inflammatory diseases in which these proteins play an important role.
FcεRIγ-ITAM Is Differentially Required for Mast Cell Function In Vivo
Authors: Sakurai Daiju , Yamasaki Sho , Arase Kanako , Park Seung Yong , Arase Hisashi , Konno Akiyoshi , Saito Takashi
Published: April 2014

The cross-linking of IgE-bound FcεRI by Ags triggers mast cell activation leading to allergic reactions. The in vivo contribution of FcεRIγ signaling to IgE/FcεRI-mediated mast cell responses has not yet been elucidated. In this study FcεRIγ−/− mast cells were reconstituted with either wild-type or mutant FcεRIγ in transgenic mice and transfected mast cells in vitro. We demonstrate that FcεRIγ-immunoreceptor tyrosine-based activation motif is essential for degranulation, cytokine production, and PG synthesis as well as for passive systemic anaphylaxis. Recent reports have suggested that cell surface FcεRI expression and mast cell survival are regulated by IgE in the absence of Ag, although the molecular mechanism is largely unknown. We also found that the promotion of mast cell survival by IgE without Ags is mediated by signals through the FcεRIγ-immunoreceptor tyrosine-based activation motif. In contrast, the IgE-mediated up-regulation of FcεRI is independent of FcεRIγ signaling. These results indicate that FcεRIγ-mediated signals differentially regulate the receptor expression, activation, and survival of mast cells and systemic anaphylaxis.
Activation/Division of Lymphocytes Results in Increased Levels of Cytoplasmic Activation/Proliferation-Associated Protein-1: Prototype of a New Family of Proteins
Authors: Grill Brock , Wilson Gary M. , Zhang Kai-Xin , Wang Bin , Doyonnas Regis , Quadroni Manfredo , Schrader John W.
Published: April 2014

We purified from activated T lymphocytes a novel, highly conserved, 116-kDa, intracellular protein that occurred at high levels in the large, dividing cells of the thymus, was up-regulated when resting T or B lymphocytes or hemopoietic progenitors were activated, and was down-regulated when a monocytic leukemia, M1, was induced to differentiate. Expression of the protein was highest in the thymus and spleen and lowest in tissues with a low proportion of dividing cells such as kidney or muscle, although expression was high in the brain. The protein was localized to the cytosol and was phosphorylated, which is consistent with a previous report that the Xenopus laevis ortholog was phosphorylated by a mitotically activated kinase (1 ). The cDNA was previously mischaracterized as encoding p137, a 137-kDa GPI-linked membrane protein (2 ). We propose that the authentic protein encoded by this cDNA be called cytoplasmic activation/proliferation-associated protein-1 (caprin-1), and show that it is the prototype of a novel family of proteins characterized by two novel protein domains, termed homology regions-1 and -2 (HR-1, HR-2). Although we have found evidence for caprins only in urochordates and vertebrates, two insect proteins exhibit well-conserved HR-1 domains. The HR-1 and HR-2 domains have no known function, although the HR-1 of caprin-1 appeared necessary for formation of multimeric complexes of caprin-1. Overexpression of a fusion protein of enhanced green fluorescent protein and caprin-1 induced a specific, dose-dependent suppression of the proliferation of NIH-3T3 cells, consistent with the notion that caprin-1 plays a role in cellular activation or proliferation.
Antibody-Dependent Cellular Cytotoxicity via Humoral Immune Epitope of Nef Protein Expressed on Cell Surface
Authors: Yamada Takeshi , Watanabe Nobukazu , Nakamura Tetsuya , Iwamoto Aikichi
Published: April 2014

Antibodies against various proteins of HIV type 1 (HIV-1) can be detected in HIV-1-infected individuals. We previously reported that the level of Ab response against one Nef epitope is correlated with HIV-1 disease progression. To elucidate the mechanism for this correlation, we examined Ab-dependent cellular cytotoxicity (ADCC) against target cells expressing Nef. We observed efficient cytotoxicity against Nef-expressing target cells in the presence of patient plasma and PBMCs. This ADCC activity was correlated with the dilution of plasma from HIV-1-infected patients. Addition of a specific synthetic peptide (peptide 31:FLKEKGGLE) corresponding to the Nef epitope reduced cell lysis to ∼50%. These results suggest that PBMCs of HIV-1-infected patients may exert ADCC via anti-Nef Abs in the patients’ own plasma and serve as a mechanism used by the immune system to regulate HIV-1 replication.
Calcium and Its Role in the Nuclear Translocation and Activation of Cytosolic Phospholipase A2 in Cells Rendered Sensitive to TNF-Induced Apoptosis by Cycloheximide
Authors: Draper David W. , Harris Virginia G. , Culver Carolyn A. , Laster Scott M.
Published: April 2014

In these experiments, we investigated the role of calcium as a second messenger in the apoptotic activation of cytosolic phospholipase A2 (cPLA2). As our model, we used a murine fibroblast cell line (C3HA) that was induced to undergo apoptosis by a combination of TNF and cycloheximide. Using fura 2 Ca2+ imaging, we found strong evidence for an intracellular calcium response after 1 h of treatment, which correlated with the onset of phosphatidylserine externalization, but preceded effector procaspase processing by several hours. The response was strongest in the perinuclear region, where mean levels rose 83% (144 ± 14 nM in untreated cells vs 264 ± 39 nM in treated), while cells displaying morphological evidence of apoptosis had the highest levels of calcium (250–1000 nM). Verapamil blocked this response, indicating an extracellular source for the calcium. Fluorescence microscopy revealed a pattern of nuclear translocation of cPLA2 during apoptosis, which was also blocked by verapamil, indicating an important role for calcium in this process. In addition, we found that verapamil prevented the release of [3H]arachidonic acid from C3HA cells induced to undergo apoptosis by the chemotherapeutic agents vinblastine, melphalan, and cis-platinum. Together, these data suggest that calcium is important for cPLA2 activation by diverse apoptotic stimuli.
Peripheral CD4 T Cells Rapidly Accumulate at the Host:Parasite Interface during an Inflammatory Th2 Memory Response
Authors: Morimoto Motoko , Morimoto Masahiro , Whitmire Jeannette , Xiao Shiyun , Anthony Robert M. , Mirakami Hiroshi , Star Robert A. , Urban Joseph F. , Gause William C.
Published: April 2014

Memory peripheral Th2 immune responses to infectious pathogens are not well studied due to the lack of suitable models and the difficulty of assessing Th2 cytokine expression at sites of inflammation. We have examined the localized immune response to a nematode parasite that encysts in the small intestine. An unexpected architecture was observed on day 4 of the memory response, with granulocytes and macrophages infiltrating the cyst and CD4+, TCR-αβ+ T cells surrounding the cyst. Laser capture microdissection analysis showed a pronounced CD4-dependent Th2 cytokine pattern at the cyst region only during the memory response, demonstrating that the Th2 memory response is readily distinguished from the primary response by the rapid accumulation of Th2 effector cells at the host:parasite interface.
Haemophilus influenzae Type b-Outer Membrane Protein Complex Glycoconjugate Vaccine Induces Cytokine Production by Engaging Human Toll-Like Receptor 2 (TLR2) and Requires the Presence of TLR2 for Optimal Immunogenicity
Authors: Latz Eicke , Franko Jennifer , Golenbock Douglas T. , Schreiber John R.
Published: April 2014

Conjugate vaccines consisting of the capsular polysaccharide (PS) of Haemophilus influenzae type b (Hib) covalently linked to carrier proteins, unlike pure PS, are immunogenic in infants and have significantly reduced Hib infections in the United States, but require multiple doses to induce protective anti-PS Ab titers. Hib-meningococcal outer membrane protein complex (OMPC) conjugate vaccine, however, elicits protective anti-PS Ab titers after one dose. We found that OMPC and Hib-OMPC engaged human Toll-like receptor 2 (TLR2) expressed in human embryonic kidney (HEK) cells, inducing IL-8 production, and engaged mouse TLR2 on bone marrow-derived dendritic cells, inducing TNF release. Hib conjugated to the carrier proteins CRM197 and tetanus toxoid did not engage TLR2 on HEK or dendritic cells. Engagement of TLR2 by Hib-OMPC was MyD88 dependent, as Hib-OMPC-induced TNF production was ablated in MyD88 knockout (KO) mice. Hib-OMPC was significantly less immunogenic in TLR2 KO mice, inducing lower Hib PS IgG and IgM titers compared with those in wild-type mice. Splenocytes from OMPC-immunized TLR2 KO mice also produced significantly less IL-6 and TNF-α than those from wild-type mice. Hib-OMPC is unique among glycoconjugate vaccines by engaging TLR2, and the ability of Hib-OMPC to elicit protective levels of Abs after one dose may be related to TLR2-mediated induction and regulation of cytokines produced by T cells and macrophages in addition to the peptide/MHC II-dependent recruitment of T cell help commonly afforded by carrier proteins. TLR2 engagement by an adjuvant or carrier protein may be a useful strategy for augmentation of the anti-PS Ab response induced by glycoconjugate vaccines.
Parasite Antigen-Driven Basophils Are a Major Source of IL-4 in Human Filarial Infections
Authors: Mitre Edward , Taylor Rebekah T. , Kubofcik Joseph , Nutman Thomas B.
Published: April 2014

Basophil contribution to the IL-4 pool in filarial infections was assessed using PBMC from 20 patients with active filarial infections and from 9 uninfected subjects. Patient basophils released histamine in response to Brugia malayi Ag (BmAg). They also released IL-4 within 2 h after exposure to BmAg, as assessed by intracellular cytokine flow cytometry. This IL-4 induction was Ag specific, as IL-4 was not detected in BmAg-exposed basophils obtained from uninfected subjects. Although there were, on average, 64 times more CD4+ T cells than basophils in the peripheral circulation of filaria-infected patients, the absolute numbers of basophils and CD4+ T cells producing IL-4 per 100,000 PBMC were equivalent (geometric mean: 16 IL-4-producing basophils/100,000 PBMC vs 22 IL-4-producing CD4+ T cells/100,000 PBMC). Basophils also released IL-4 in response to both low and high concentrations of BmAg, whereas CD4+ T cells released IL-4 only after incubation with a high concentration of BmAg, raising the possibility that basophils, due to their lower threshold for activation, may actually release IL-4 more frequently than CD4+ T cells in vivo. Furthermore, IL-4 production in vitro by Ag-stimulated purified basophils or CD4+ T cells provided evidence that basophils release greater quantities of IL-4 per cell than CD4+ T cells in response to BmAg. These results suggest that, when Ag-specific IgE is present in a filaria-infected individual, basophils function to amplify the ongoing Th2 response by releasing IL-4 in greater amounts and possibly more frequently than CD4+ T cells in response to filarial Ag.
The Zinc Finger Mutation C417R of I-κB Kinase γ Impairs Lipopolysaccharide- and TNF-Mediated NF-κB Activation through Inhibiting Phosphorylation of the I-κB Kinase β Activation Loop
Authors: Yang Fan , Yamashita Junro , Tang Eric , Wang Hom-lay , Guan Kunliang , Wang Cun-Yu
Published: April 2014

The activation of the I-κB kinase (IKK) complex by TNF or LPS stimulates phosphorylation and degradation of I-κBα, leading to the nuclear translocation of NF-κB. The IKK complex is mainly composed of two catalytic subunits, IKKα and IKKβ, and a chaperon subunit IKKγ. Although IKKγ does not have catalytic activity, it is essential for IKK activation induced by multiple stimuli. Importantly, the key residue cysteine 417 at the zinc finger domain of IKKγ has been found to be mutated to arginine (IKKγC417R) in a human genetic disorder called the anhydrotic ectodermal dysplasia with immunodeficiency. To understand the underlying mechanisms of immunodeficiency, we examined whether the IKKγC417R mutant modified IKK activation and NF-κB transcription stimulated by LPS or TNF in human monocytes. We found that overexpression of IKKγC417R severely impaired LPS- and TNF-induced I-κBα phosphorylation and degradation in a dominant-negative fashion. Also, LPS- and TNF-induced NF-κB transcription was inhibited by IKKγC417R. The reconstitution of IKKγ, but not IKKγC417R, in IKKγ-deficient cells restored NF-κB signaling, indicating the zinc finger structure of IKKγ plays a key role in IKK activation. Moreover, C417R mutation in IKKγ abolished both LPS- and TNF-induced phosphorylation of the activation loop of IKKβ. Collectively, our results indicated that the zinc finger structure of IKKγ plays a key role in LPS- and TNF-induced NF-κB activation. The anhydrotic ectodermal dysplasia with immunodeficiency patients’ immunodeficiency may be associated with NF-κB defect in response to bacterial stimulation.
Protective T Cell Immunity against Malaria Liver Stage after Vaccination with Live Sporozoites under Chloroquine Treatment
Authors: Belnoue Elodie , Costa Fabio T. M. , Frankenberg Tobias , Vigário Ana Margarida , Voza Tatiana , Leroy Nicolas , Rodrigues Mauricio M. , Landau Irène , Snounou Georges , Rénia Laurent
Published: April 2014

In this study we present the first systematic analysis of the immunity induced by normal Plasmodium yoelii sporozoites in mice. Immunization with sporozoites, which was conducted under chloroquine treatment to minimize the influence of blood stage parasites, induced a strong protection against a subsequent sporozoite and, to a lesser extent, against infected RBC challenges. The protection induced by this immunization protocol proved to be very effective. Induction of this protective immunity depended on the presence of liver stage parasites, as primaquine treatment concurrent with sporozoite immunization abrogated protection. Protection was not found to be mediated by the Abs elicited against pre-erythrocytic and blood stage parasites, as demonstrated by inhibition assays of sporozoite penetration or development in vitro and in vivo assays of sporozoite infectivity or blood stage parasite development. CD4+ and CD8+ T cells were, however, responsible for the protection through the induction of IFN-γ and NO.
TNF Receptor Signaling Contributes to Chemokine Secretion, Inflammation, and Respiratory Deficits duringPneumocystisPneumonia
Authors: Wright Terry W. , Pryhuber Gloria S. , Chess Patricia R. , Wang Zhengdong , Notter Robert H. , Gigliotti Francis
Published: April 2014

CD8+ T cells contribute to the pathophysiology of Pneumocystis pneumonia (PcP) in a murine model of AIDS-related disease. The present studies were undertaken to more precisely define the mechanisms by which these immune cells mediate the inflammatory response that leads to lung injury. Experimental mice were depleted of either CD4+ T cells or both CD4+ and CD8+ T cells and then infected with Pneumocystis. The CD4+-depleted mice had significantly greater pulmonary TNF-α levels than mice depleted of both CD4+ and CD8+ T cells. Elevated TNF-α levels were associated with increased lung concentrations of the chemokines RANTES, monocyte chemoattractant protein 1, macrophage-inflammatory protein 2, and cytokine-induced neutrophil chemoattractant. To determine whether TNFR signaling was involved in the CD8+ T cell-dependent chemokine response, TNFRI- and II-deficient mice were CD4+ depleted and infected with Pneumocystis. TNFR-deficient mice had significantly reduced pulmonary RANTES, monocyte chemoattractant protein 1, macrophage-inflammatory protein 2, and cytokine-induced neutrophil chemoattractant responses, reduced inflammatory cell recruitment to the alveoli, and reduced histological evidence of PcP-related alveolitis as compared with infected wild-type mice. Diminished pulmonary inflammation correlated with improved surfactant activity and improved pulmonary function in the TNFR-deficient mice. These data indicate that TNFR signaling is required for maximal CD8+ T cell-dependent pulmonary inflammation and lung injury during PcP and also demonstrate that CD8+ T cells can use TNFR signaling pathways to respond to an extracellular fungal pathogen.
Involvement of Reactive Oxygen Species in Toll-Like Receptor 4-Dependent Activation of NF-κB
Authors: Asehnoune Karim , Strassheim Derek , Mitra Sanchayita , Kim Jae Yeol , Abraham Edward
Published: April 2014

Although oxidative stress has been thought to play a general role in the activation of NF-κB, the involvement of reactive oxygen species (ROS) in facilitating nuclear translocation of NF-κB in neutrophils has not been described. In addition, the mechanisms by which ROS modulate the transcriptional activity of NF-κB in response to Toll-like receptor 4 (TLR4)-dependent signaling are not well characterized. To examine these issues, oxidant-dependent signaling events downstream of TLR4 were investigated in neutrophils stimulated with LPS. Pretreatment of neutrophils with the antioxidants N-acetylcysteine or α-tocopherol prevented LPS-induced nuclear translocation of NF-κB. Antioxidant treatment of LPS-stimulated neutrophils also inhibited the production of proinflammatory cytokines (TNF-α, macrophage inflammatory protein-2, and IL-1β), as well as activation of the kinases IκB kinase α, IκB kinase β, p38, Akt, and extracellular receptor-activated kinases 1 and 2. The decrease in cytoplasmic levels of IκBα produced by exposure of neutrophils to LPS was prevented by N-acetylcysteine or α-tocopherol. Activation of IL-1R-associated kinase-1 (IRAK-1) and IRAK-4 in response to LPS stimulation was inhibited by antioxidants. These results demonstrate that proximal events in TLR4 signaling, at or antecedent to IRAK-1 and IRAK-4 activation, are oxidant dependent and indicate that ROS can modulate NF-κB-dependent transcription through their involvement in early TLR4-mediated cellular responses.
B7-DC Regulates Asthmatic Response by an IFN-γ-Dependent Mechanism
Authors: Matsumoto Koichiro , Inoue Hiromasa , Nakano Takako , Tsuda Miyuki , Yoshiura Yuki , Fukuyama Satoru , Tsushima Fumihiko , Hoshino Tomoaki , Aizawa Hisamichi , Akiba Hisaya , Pardoll Drew , Hara Nobuyuki , Yagita Hideo , Azuma Miyuki , Nakanishi Yoichi
Published: April 2014

B7-H1 (PD-L1) and B7-DC (PD-L2) are the ligands for programmed death-1 (PD-1), which is a member of the CD28/CTLA-4 family and has been implicated in peripheral tolerance. We investigated the roles of B7-H1 and B7-DC in a murine OVA-induced allergic asthma model. B7-H1 was constitutively expressed on dendritic cells, macrophages, B cells, and T cells in the lungs of naive mice, and its expression could be dramatically increased after allergen challenge. In contrast, B7-DC expression was scarcely expressed on dendritic cells in naive mice, but was up-regulated after allergen challenge, although the up-regulation of B7-DC expression on macrophages was minimal. Treatment of mice with anti-B7-DC mAb at the time of allergen challenge, but not at the time of sensitization, significantly increased their airway hyper-reactivity and eosinophilia. Such treatment also resulted in the increased production of IL-5 and IL-13, and decreased IFN-γ production in the lungs and draining lymph node cells. These changes were diminished when mice were depleted of IFN-γ by anti-IFN-γ mAb pretreatment. Interestingly, treatment with anti-B7-H1 or anti-PD-1 mAb did not significantly affect the asthmatic response. These results suggest a unique role for B7-DC in the regulation of asthmatic response through an IFN-γ-dependent, but PD-1-independent, mechanism.
Microtubule-Associated Serine/Threonine Kinase-205 kDa and Fcγ Receptor Control IL-12 p40 Synthesis and NF-κB Activation
Authors: Zhou Hui , Xiong Huabao , Li Hongxing , Plevy Scott E. , Walden Paul D. , Sassaroli Massimo , Prestwich Glenn D. , Unkeless Jay C.
Published: April 2014

Stimulation of murine macrophages with LPS results in the coordinated activation of a set of proinflammatory cytokines and costimulatory molecules, including TNF-α, IL-6, IL-1, IL-8, IL-12, and CD80. Macrophage LPS-induced synthesis of IL-12 is inhibited following FcγR ligation; TNF-α secretion is unchanged. We report that microtubule-associated serine/threonine kinase-205 kDa (MAST205) is required for LPS-induced IL-12 synthesis. RNA interference-mediated suppression of MAST205 results in the inhibition of LPS-stimulated IL-12 promoter activity and IL-12 secretion, from both J774 cells and bone marrow-derived macrophages. Similarly, dominant-negative MAST205 mutants inhibit LPS-stimulated IL-12 synthesis and NF-κB activation, but do not affect IL-1 or TNF-α signaling. Finally, macrophage FcγR ligation regulates MAST205 by inducing the rapid ubiquitination and proteasomal degradation of the protein.
Inhibition of c-Jun NH2-Terminal Kinase Activity Improves Ischemia/Reperfusion Injury in Rat Lungs
Authors: Ishii Makoto , Suzuki Yukio , Takeshita Kei , Miyao Naoki , Kudo Hiroyasu , Hiraoka Rika , Nishio Kazumi , Sato Nagato , Naoki Katsuhiko , Aoki Takuya , Yamaguchi Kazuhiro
Published: April 2014

Although c-Jun NH2-terminal kinase (JNK) has been implicated in the pathogenesis of transplantation-induced ischemia/reperfusion (I/R) injury in various organs, its significance in lung transplantation has not been conclusively elucidated. We therefore attempted to measure the transitional changes in JNK and AP-1 activities in I/R-injured lungs. Subsequently, we assessed the effects of JNK inhibition by the three agents including SP600125 on the degree of lung injury assessed by means of various biological markers in bronchoalveolar lavage fluid and histological examination including detection of apoptosis. In addition, we evaluated the changes in p38, extracellular signal-regulated kinase, and NF-κB-DNA binding activity. I/R injury was established in the isolated rat lung preserved in modified Euro-Collins solution at 4°C for 4 h followed by reperfusion at 37°C for 3 h. We found that AP-1 was transiently activated during ischemia but showed sustained activation during reperfusion, leading to significant lung injury and apoptosis. The change in AP-1 was generally in parallel with that of JNK, which was activated in epithelial cells (bronchial and alveolar), alveolar macrophages, and smooth muscle cells (bronchial and vascular) on immunohistochemical examination. The change in NF-κB qualitatively differed from that of AP-1. Protein leakage, release of lactate dehydrogenase and TNF-α into bronchoalveolar lavage fluid, and lung injury were improved, and apoptosis was suppressed by JNK inhibition. In conclusion, JNK plays a pivotal role in mediating lung injury caused by I/R. Therefore, inhibition of JNK activity has potential as an effective therapeutic strategy for preventing I/R injury during lung transplantation.
Peptide-Mediated Inhibition of Neutrophil Transmigration by Blocking CD47 Interactions with Signal Regulatory Protein α
Authors: Liu Yuan , O’Connor Miriam B. , Mandell Kenneth J. , Zen Ke , Ullrich Axel , Bühring Hans-Jörg , Parkos Charles A.
Published: April 2014

CD47, a cell surface transmembrane Ig superfamily member, is an extracellular ligand for signal regulatory protein (SIRPα). Interactions between CD47 and SIRPα regulate many important immune cell functions including neutrophil (PMN) transmigration. Here we report identification of a novel function-blocking peptide, CERVIGTGWVRC, that structurally mimics an epitope on CD47 and binds to SIRPα. The CERVIGTGWVRC sequence was identified by panning phage display libraries on the inhibitory CD47 mAb, C5D5. In vitro PMN migration assays demonstrated that peptide CERVIGTGWVRC specifically inhibited PMN migration across intestinal epithelial monolayers and matrix in a dose-dependent fashion. Further studies using recombinant proteins indicated that the peptide specifically blocks CD47 and SIRPα binding in a dose-dependent fashion. Protein binding assays using SIRPα domain-specific recombinant proteins demonstrated that this peptide directly bound to the distal-most Ig loop of SIRPα, the same loop where CD47 binds. In summary, these findings support the relevance of CD47-SIRPα interactions in regulation of PMN transmigration and provide structural data predicting the key residues involved on the surface of CD47. Such peptide reagents may be useful for studies on experimental models of inflammation and provide a template for the design of anti-inflammatory agents.
Matrix Metalloproteinase-9 Deficiency Results in Enhanced Allergen-Induced Airway Inflammation
Authors: McMillan Sarah J. , Kearley Jennifer , Campbell J. Darren , Zhu Xing-Wu , Larbi Karen Y. , Shipley J. Michael , Senior Robert M. , Nourshargh Sussan , Lloyd Clare M.
Published: April 2014

Matrix metalloproteinases (MMPs) are a large family of endopeptidases that proteolytically degrade extracellular matrix. Many different cells produce MMP-9, and levels have been shown to be up-regulated in patients with allergic asthma. The aim of this study was to investigate the in vivo role of MMP-9 during allergen-induced airway inflammation. Acute allergic pulmonary eosinophilia was established in MMP-9 knockout (KO) and wild-type (WT) control mice by sensitization and challenge with OVA. Cell recruitment was significantly increased in both bronchoalveolar lavage (BAL) and lung tissue compartments in MMP-9 KO mice compared with WT mice. This heightened cell recruitment was primarily due to increased eosinophils and Th2 cells in the BAL and lung tissue of MMP-9 KO mice in comparison with WT controls. Moreover, levels of the Th2 cytokines, IL-4 and IL-13, and the chemokines eotaxin/CCL11 and macrophage-derived chemokine/CCL22 were substantially increased in MMP-9 KO mice compared with WT after OVA challenge. Resolution of eosinophilia was similar between MMP-9 KO and WT mice, but Th2 cells persisted in BAL and lungs of MMP-9 KO mice for longer than in WT mice. Our results indicate that MMP-9 is critically involved in the recruitment of eosinophils and Th2 cells to the lung following allergen challenge, and suggest that MMP-9 plays a role in the development of Th2 responses to allergen.
Role of Toll-Like Receptor 4 in Endotoxin-Induced Acute Renal Failure
Authors: Cunningham Patrick N. , Wang Ying , Guo Rongqing , He Gang , Quigg Richard J.
Published: April 2014

Toll-like receptor 4 (TLR4) is present on monocytes and other cell types, and mediates inflammatory events such as the release of TNF after exposure to LPS. C3H/HeJ mice are resistant to LPS-induced mortality, due to a naturally occurring mutation in TLR4. We therefore hypothesized that LPS-induced acute renal failure (ARF) requires systemic TNF release triggered by LPS acting on extrarenal TLR4. We injected C3H/HeJ mice and C3H/HeOuJ controls with 0.25 mg of LPS, and sacrificed them 6 h later for analysis of blood urea nitrogen (BUN) and kidney tissue (n = 8 per group). In contrast to C3H/HeOuJ controls, C3H/HeJ mice were completely resistant to LPS-induced ARF (6-h BUN of 32.3 ± 1.1 vs 61.7 ± 5.6 mg/dl). C3H/HeJ mice released no TNF into the circulation at 2 h (0.00 vs 1.24 ± 0.16 ng/ml), had less renal neutrophil infiltration (6.4 ± 1.0 vs 11.4 ± 1.3 neutrophils per high power field), and less renal apoptosis, as assessed by DNA laddering. Transplant studies showed that C3H/HeJ recipients of wild-type kidneys (n = 9) were protected from LPS-induced ARF, while wild-type recipients of C3H/HeJ kidneys (n = 11) developed severe LPS-induced ARF (24-h BUN 44.0 ± 4.1 vs 112.1 ± 20.0 mg/dl). These experiments support our hypothesis that LPS acts on extrarenal TLR4, thereby leading to systemic TNF release and subsequent ARF. Renal neutrophil infiltration and renal cell apoptosis are potential mechanisms by which endotoxemia leads to functional ARF.
TNF-α Coupled to Membrane of Apoptotic Cells Favors the Cross-Priming to Melanoma Antigens
Authors: Zimmermann Valérie S. , Bondanza Attilio , Monno Antonella , Rovere-Querini Patrizia , Corti Angelo , Manfredi Angelo A.
Published: April 2014

The cross-presentation of Ags derived from apoptotic cell processing contributes to peripheral tolerance. Environmental signals possibly modify this default outcome, favoring cross-priming. In this study, we anchored via a biotin-avidin-biotin bridge soluble TNF-α to the membrane of apoptotic melanoma cells and studied in vivo and in vitro the interaction with Ag-presenting phagocytes. TNF-α-coated apoptotic melanoma cells injected s.c. had a faster and more efficient access to draining lymph nodes, with cross-priming of melanoma-specific CTLs and delayed outgrowth of melanomas in all treated animals. Twenty percent of the animals, in the absence of further adjuvant, did not develop the tumor. Immature dendritic cells challenged with TNF-α-coated apoptotic melanoma cells secreted proinflammatory cytokines in an autocrine/paracrine fashion, efficiently matured, as assessed functionally and by flow cytometry and cross-presented with enhanced efficiency melanoma Ags to MHC class I- and II-restricted T cells. The results indicate that TNF-α targeted to apoptotic membranes, at concentrations that can be safely reached in growing tumors without undue systemic toxicity, influences the outcome of the disposal of dying cells and enhances tumor immunogenicity.
In Vivo Evidence That Peptide Vaccination Can Induce HLA-DR-Restricted CD4+ T Cells Reactive to a Class I Tumor Peptide
Authors: Harada Mamoru , Gohara Rumi , Matsueda Satoko , Muto Akira , Oda Tatsuya , Iwamoto Yoshiko , Itoh Kyogo
Published: April 2014

Vaccination with class I tumor peptides has been performed to induce tumor-reactive CD8+ T cells in vivo. However, the kinds of immune responses that vaccination might elicit in patients are not fully understood. In this study we tried to elucidate the mechanisms by which vaccination of class I binding tumor peptides into an HLA-A2+ lung cancer patient elicited dramatic amounts of IgG1 and IgG2 specific to a nonamer peptide, ubiquitin-conjugated enzyme variant Kua (UBE2V)43–51. The UBE2V43–51 peptide contains cysteine at the sixth position. HLA-DR-restricted and UBE2V43–51 peptide-recognizing CD4+ T cells were induced from postvaccination, but not from prevaccination, PBMCs of the cancer patient. In addition, a CD4+ T cell line (UB-2) and its clone (UB-2.3), both of which recognize the UBE2V43–51 peptide in the context of HLA-DRB1*0403 molecules, were successfully established from postvaccination PBMCs. The peptide vaccination increased the frequency of peptide-specific T cells, especially CD4+ T cells. In contrast, mass spectrometric analysis revealed that the vaccinated UBE2V43–51 peptide contained both monomeric and dimeric forms. Both forms, fractionated by reverse phase HPLC, were recognized by UB-2 and UB-2.3 cells. Recognition by these CD4+ T cells was observed despite the addition of a reduction reagent or the fixation of APC. Overall, these results indicate that vaccination with class I tumor peptides can induce HLA-DR-restricted CD4+ T cells in vivo and elicit humoral immune responses, and that a cysteine-containing peptide can be recognized by CD4+ T cells not only as a monomer, but also as a dimer.
Cutting Edge: Membrane Nanotubes Connect Immune Cells
Authors: Önfelt Björn , Nedvetzki Shlomo , Yanagi Kumiko , Davis Daniel M.
Published: April 2014

We present evidence that nanotubular highways, or membrane nanotubes, facilitate a novel mechanism for intercellular communication in the immune system. Nanotubes were seen to connect multiple cells together and were readily formed between a variety of cell types, including human peripheral blood NK cells, macrophages, and EBV-transformed B cells. Nanotubes could be created upon disassembly of the immunological synapse, as cells move apart. Thus, nanotubular networks could be assembled from transient immunological synapses. Nanotubes were seen to contain GFP-tagged cell surface class I MHC protein expressed in one of the connected cells. Moreover, GPI-conjugated to GFP originating from one cell was transferred onto the surface of another at the connection with a nanotube. Thus, nanotubes can traffic cell surface proteins between immune cells over many tens of microns. Determining whether there are physiological functions for nanotubes is an intriguing new goal for cellular immunology.
Cutting Edge: Innate Production of IFN-γ by NK Cells Is Independent of Epigenetic Modification of the IFN-γ Promoter
Authors: Tato Cristina M. , Martins Gislâine A. , High Frances A. , DiCioccio Catherine B. , Reiner Steven L. , Hunter Christopher A.
Published: April 2014

The ability of NK and T cells to produce IFN-γ is critical for resistance to numerous intracellular pathogens but the kinetics of these responses differ. Consistent with this is a requirement for naive T cells to become activated and undergo proliferation-dependent epigenetic changes to the IFN-γ locus that allow them to produce IFN-γ. The data presented here reveal that unlike T cells, murine NK cells produce IFN-γ under conditions of short-term cytokine stimulation, and these events are independent of proliferation and cell cycle progression. Furthermore, analysis of the IFN-γ locus in NK cells reveals that this locus is constitutively demethylated. The finding that NK cells do not need to remodel the IFN-γ locus to produce IFN-γ, either because they do not exhibit epigenetic repression or they have undergone prior remodeling during development, provides a molecular basis for the innate and adaptive regulation of the production of this cytokine.
Termination of Antigen-Specific Immunity by CD95 Ligand (Fas Ligand) and IL-10
Authors: Barreiro Ramon , Luker Gary , Herndon John , Ferguson Thomas A.
Published: April 2014

Following elimination of a foreign invader, the immune system must return to its normal quiescent levels. This process requires removal of reactive immune cells when they are no longer needed. We have explored the role of Fas/Fas ligand (FasL) in terminating immunity and demonstrate that mice defective in these proteins have prolonged immune responses. Studies demonstrate that termination of immunity occurs via the interaction of Fas+ lymphoid cells with FasL+ nonlymphoid cells at the site of Ag challenge. Our results also show that FasL is absent in quiescent tissue but is rapidly up-regulated during the local immune reaction. This occurs through the production of IL-10. Thus, FasL and IL-10 work in concert to eliminate inflammatory cells and control the duration of an immune response.
Antigen-Processing Machinery in Human Dendritic Cells: Up-Regulation by Maturation and Down-Regulation by Tumor Cells
Authors: Whiteside Theresa L. , Stanson Joanna , Shurin Michael R. , Ferrone Soldano
Published: April 2014

It has been known for some time that functional properties of dendritic cells (DC), and in particular their ability to process and present Ags to T cells, can be modulated by cytokine-induced maturation and by interactions with tumor cells. However, the molecular basis for these functional changes is unknown. We have investigated whether changes in expression of Ag-processing machinery (APM) components in DC are associated with alterations in their ability to present tumor-derived Ags to T cells. Using a panel of mAbs specific for individual APM components and a quantitative flow cytometry-based method, the level of APM components was measured in DC generated from peripheral blood monocytes of 12 normal donors and of 8 patients with cancer. Immature DC had significantly lower (p < 0.01) expression of MB1, LMP-7, LMP-10, TAP-1, and tapasin than mature DC. However, maturation in the presence of a cytokine mixture up-regulated expression of these components in DC obtained from normal donors and patients with cancer. Immature DC incubated with tumor cells had significantly lower (p < 0.001) expression of MB1, LMP-2, LMP-7, LMP-10, and endoplasmic reticulum p75 than controls. These changes were associated with a decreased ability of DC to present tumor-derived Ags to T cells, as measured in ELISPOT assays and with apoptosis of T cells in DC-T cell cultures. Thus, tumor cells have a significant suppressive effect on DC; however, ex vivo maturation of DC derived from patients with cancer in a polarizing cytokine mix restores normal expression of APM components and Ag-processing capabilities.
In Vivo Pattern of Lipopolysaccharide and Anti-CD3-Induced NF-κB Activation Using a Novel Gene-Targeted Enhanced GFP Reporter Gene Mouse
Authors: Magness Scott T. , Jijon Humberto , Van Houten Fisher Nancy , Sharpless Ned E. , Brenner David A. , Jobin Christian
Published: April 2014

NF-κB is a family of transcription factors involved in regulating cell death/survival, differentiation, and inflammation. Although the transactivation ability of NF-κB has been extensively studied in vitro, limited information is available on the spatial and temporal transactivation pattern in vivo. To investigate the kinetics and cellular localization of NF-κB-induced transcription, we created a transgenic mouse expressing the enhanced GFP (EGFP) under the transcriptional control of NF-κB cis elements (cis-NF-κBEGFP). A gene-targeting approach was used to insert a single copy of a NF-κB-dependent EGFP reporter gene 5′ of the X-linked hypoxanthine phosphoribosyltransferase locus in mouse embryonic stem cells. Embryonic fibroblasts, hepatic stellate cells, splenocytes, and dendritic cells isolated from cis-NF-κBEGFP mice demonstrated a strong induction of EGFP in response to LPS, anti-CD3, or TNF-α that was blocked by the NF-κB inhibitors BAY 11-0782 and NEMO-binding peptide. Chromatin immunoprecipitation analysis demonstrated RelA binding to the cis-NF-κBEGFP promoter. Adenoviral delivery of NF-κB-inducing kinase strongly induced EGFP expression in the liver of cis-NF-κBEGFP mice. Similarly, mice injected with anti-CD3 or LPS showed increased EGFP expression in mononuclear cells, lymph node, spleen, and liver as measured by flow cytometry and/or fluorescence microscopy. Using whole organ imaging, LPS selectively induced EGFP expression in the duodenum and proximal jejunum, but not in the ileum and colon. Confocal analysis indicated EGFP expression was primarily found in lamina propria mononuclear cells. In summary, the cis-NF-κBEGFP mouse will serve as a valuable tool to address multiple questions regarding the cell-specific and real-time activation of NF-κB during normal and diseased states.
Differences in the Kinetics, Amplitude, and Localization of ERK Activation in Anergy and Priming Revealed at the Level of Individual Primary T Cells by Laser Scanning Cytometry
Authors: Adams Claire L. , Grierson Angela M. , Mowat Allan M. , Harnett Margaret M. , Garside Paul
Published: April 2014

One of the potential mechanisms of peripheral tolerance is the unresponsiveness of T cells to secondary antigenic stimulation as a result of the induction of anergy. It has been widely reported that antigenic unresponsiveness may be due to uncoupling of MAPK signal transduction pathways. However, such signaling defects in anergic T cell populations have been mainly identified using immortalized T cell lines or T cell clones, which do not truly represent primary Ag-specific T cells. We have therefore attempted to quantify signaling events in murine primary Ag-specific T cells on an individual cell basis, using laser-scanning cytometry. We show that there are marked differences in the amplitude and cellular localization of phosphorylated ERK p42/p44 (ERK1/2) signals when naive, primed and anergic T cells are challenged with peptide-pulsed dendritic cells. Primed T cells display more rapid kinetics of phosphorylation and activation of ERK than naive T cells, whereas anergic T cells display a reduced ability to activate ERK1/2 upon challenge. In addition, the low levels of pERK found in anergic T cells are distributed diffusely throughout the cell, whereas in primed T cells, pERK appears to be targeted to the same regions of the cell as the TCR. These data suggest that the different consequences of Ag recognition by T cells are associated with distinctive kinetics, amplitude, and localization of MAPK signaling.
γδ T Cell Regulation of IFN-γ Production by Central Nervous System-Infiltrating Encephalitogenic T Cells: Correlation with Recovery from Experimental Autoimmune Encephalomyelitis
Authors: Ponomarev Eugene D. , Novikova Marina , Yassai Maryam , Szczepanik Marian , Gorski Jack , Dittel Bonnie N.
Published: April 2014

Interferon-γ has been shown to be important for the resolution of inflammation associated with CNS autoimmunity. Because one of the roles of γδ T cells is the regulation of inflammation, we asked whether γδ T cells were able to regulate CNS inflammation using the autoimmune disease mouse model experimental autoimmune encephalomyelitis (EAE). We show that the presence of γδ T cells was needed to promote the production of IFN-γ by both CD4 and CD8 T cells in the CNS before the onset of EAE. This regulation was shown to be independent of the ability of γδ T cells to produce IFN-γ, and was specific to T cells in the CNS, as no alterations in IFN-γ production were detectable in γδ T cell-deficient mice in the spleen and lymph nodes of mice with EAE or following immunization. Analysis of TCRγδ gene usage in the CNS showed that the only TCRδ V gene families present in the CNS before EAE onset are from the DV7s6 and DV105s1 gene families. We also show that the primary IFN-γ-producing cells in the CNS are the encephalitogenic T cells, and that γδ T cell-deficient mice are unable to resolve EAE disease symptoms like control mice, thus exhibiting a long-term chronic disease course similar to that observed in IFN-γ-deficient mice. These data suggest that CNS resident γδ T cells promote the production of IFN-γ by encephalitogenic T cells in the CNS, which is ultimately required for the recovery from EAE.
Antisense Knockdown of Sphingosine Kinase 1 in Human Macrophages Inhibits C5a Receptor-Dependent Signal Transduction, Ca2+ Signals, Enzyme Release, Cytokine Production, and Chemotaxis
Authors: Melendez Alirio J. , Ibrahim Farazeela Bte Mohd
Published: April 2014

The anaphylatoxin C5a is produced following the activation of the complement system and is associated with a variety of pathologies, including septic shock and adult respiratory distress syndrome, and with immune complex-dependent diseases such as rheumatoid arthritis. C5a has been shown to regulate inflammatory functions by interacting with its receptor, C5aR, which belong to the rhodopsin family of seven-transmembrane GPCRs. However, the intracellular signaling pathways triggered by C5aR on immune-effector cells are not well understood. In this report we present data showing that, in human monocyte-derived macrophages, C5aR uses the intracellular signaling molecule sphingosine kinase (SPHK)1 to trigger various physiological responses. Our data show that C5a rapidly stimulates the generation of sphingosine-1-phosphate, SPHK activity, and membrane translocation of SPHK1. Using an antisense oligonucleotide against SPHK1, we show that knockdown of SPHK1 abolishes the C5a-triggered intracellular Ca2+ signals, degranulation, cytokine generation, and chemotaxis. Our study shows for the first time that SPHK1 not only plays a key role in the generation and release of proinflammatory mediators triggered by anaphylatoxins from human macrophages but is also involved in the process of immune cell motility, thus pointing out SPHK1 as a potential therapeutic target for the treatment of inflammatory and autoimmune diseases.
Regulation of Thymus Size by Competition for Stromal Niches among Early T Cell Progenitors
Authors: Prockop Susan E. , Petrie Howard T.
Published: April 2014

Thymic T cell production is characterized by differentiating waves of non-self-renewing, bone marrow-derived progenitors. The factors constraining new progenitor recruitment, intrathymic precursor expansion, and thymus size remain enigmatic, but are believed to be controlled by a feedback loop responding to lymphoid cellularity and competition for stromal niches. In this study, we show that competition for stromal niches does occur, but is solely limited to cells at the early CD4−8− precursor stages of differentiation. The overall size of the organ is determined both by this limitation on early precursor expansion, and by a second, cell-intrinsic limit on expansion of progenitor cells transiting to the CD4+8+ stage. Together with asymmetric use of marrow-derived progenitors to reconstitute the intrathymic pool, these processes facilitate continuous generation of new T cells while maintaining a relatively stable organ size.