bluuebunny/crossref_metadata_2025 huggingface dataset:
ATP Stimulates Human Macrophages to Kill Intracellular Virulent Mycobacterium tuberculosis Via Calcium-Dependent Phagosome-Lysosome Fusion
Authors: Kusner David J. , Barton James A.
Published: April 2014

Advances in therapy for tuberculosis will require greater understanding of the molecular mechanisms of pathogenesis and the human immune response in this disease. Exposure of Mycobacterium tuberculosis-infected human macrophages to extracellular ATP (ATPe) results in bacterial killing, but the molecular mechanisms remain incompletely characterized. In this study, we demonstrate that ATPe-induced bactericidal activity toward virulent M. tuberculosis requires an increase in cytosolic Ca2+ in infected macrophages. Based on our previous work with primary infection of human macrophages, we hypothesized that the Ca2+ dependence of ATP-induced killing of intracellular M. tuberculosis was linked to promotion of phagosome-lysosome fusion. Using confocal laser-scanning microscopy, we demonstrate that ATPe induces fusion of the M. tuberculosis-containing phagosome with lysosomes, defined by accumulation of three lysosomal proteins and an acidophilic dye. Stimulation of phagosome-lysosome fusion by ATPe exhibited distinct requirements for both Ca2+ and phospholipase D and was highly correlated with killing of intracellular bacilli. Thus, key signal transduction pathways are conserved between two distinct models of human macrophage antituberculous activity: primary infection of naive macrophages and physiologic stimulation of macrophages stably infected with M. tuberculosis.
Predominant Role of Toll-Like Receptor 2 Versus 4 in Chlamydia pneumoniae-Induced Activation of Dendritic Cells
Authors: Prebeck Sigrid , Kirschning Carsten , Dürr Susanne , da Costa Clarissa , Donath Bernadette , Brand Korbinian , Redecke Vanessa , Wagner Hermann , Miethke Thomas
Published: April 2014

Chlamydia pneumoniae is an obligate intracellular human pathogen causing diseases such as pneumonia, bronchitis, and pharyngitis. Because of its intracellular replication, cell-mediated immune responses are needed to mediate successful defenses of the host. Because dendritic cells play a central role in linking innate immunity and Ag-specific cell-mediated immune responses we asked whether dendritic cells are activated upon contact with C. pneumoniae and whether known Toll like receptors (TLR) are involved in this process. Here we show that C. pneumoniae was taken up by bone marrow-derived murine dendritic cells. Ingested C. pneumoniae appeared to be unable to develop mature inclusion inside dendritic cells. Furthermore, upon contact with C. pneumoniae dendritic cells were potently stimulated because NF-κB was activated and translocated to the nucleus, cytokines like IL-12p40 and TNF-α were secreted, and expression of MHC class II molecules, CD40, CD80, and CD86 was up-regulated. Importantly, secretion of cytokines as well as translocation of NF-κB were dependent on the presence of TLR2 and independent from TLR4 with the exception of IL-12p40 secretion, which was attenuated in the absence of either a functional TLR2 or 4. In conclusion, we show here that recognition of the Gram-negative bacterium C. pneumoniae depends largely on TLR2 and only to a minor extent on TLR4.
Sterically Stabilized Cationic Liposomes Improve the Uptake and Immunostimulatory Activity of CpG Oligonucleotides
Authors: Gursel Ihsan , Gursel Mayda , Ishii Ken J. , Klinman Dennis M.
Published: April 2014

Immunostimulatory CpG oligonucleotides (ODN) show promise as immune adjuvants, anti-allergens, and immunoprotective agents. Increasing the bioavailability and duration of action of CpG ODN should improve their therapeutic utility. Encapsulating ODN in sterically stabilized cationic liposomes provides protection from serum nucleases while facilitating uptake by B cells, dendritic cells, and macrophages. In a pathogen challenge model, sterically stabilized cationic liposomes encapsulation doubled the duration of CpG ODN-induced immune protection. In an immunization model, coencapsulation of CpG ODN with protein Ag (OVA) magnified the resultant Ag-specific IFN-γ and IgG responses by 15- to 40-fold compared with Ag plus CpG ODN alone. These findings support the use of sterically stabilized cationic liposomes to significantly enhance the therapeutic efficacy of CpG ODN.
Cathelicidin Family of Antibacterial Peptides CAP18 and CAP11 Inhibit the Expression of TNF-α by Blocking the Binding of LPS to CD14+ Cells
Authors: Nagaoka Isao , Hirota Satoko , Niyonsaba François , Hirata Michimasa , Adachi Yoshiyuki , Tamura Hiroshi , Heumann Didier
Published: April 2014

Mammalian myeloid and epithelial cells express several kinds of antibacterial peptides (α-/β-defensins and cathelicidins) that contribute to the innate host defense by killing invading micro-organisms. In this study we evaluated the LPS-neutralizing activities of cathelicidin peptides human CAP18 (cationic antibacterial proteins of 18 kDa) and guinea pig CAP11 using the CD14+ murine macrophage cell line RAW264.7 and the murine endotoxin shock model. Flow cytometric analysis revealed that CAP18 and CAP11 inhibited the binding of FITC-conjugated LPS to RAW264.7 cells. Likewise, Northern and Western blot analyses indicated that CAP18 and CAP11 suppressed LPS-induced TNF-α mRNA and protein expression by RAW264.7 cells. Interestingly, CAP18 and CAP11 possessed LPS-binding activities, and they strongly suppressed the interaction of LPS with LPS binding protein that mediates the transport of LPS to CD14 to facilitate the activation of CD14+ cells by LPS. Moreover, when CAP18 and CAP11 were preincubated with RAW264.7 cells, they bound to the cell surface CD14 and inhibited the binding of FITC-LPS to the cells. Furthermore, in the murine endotoxin shock model, CAP18 or CAP11 administration inhibited the binding of LPS to CD14+ cells (peritoneal macrophages) and suppressed LPS-induced TNF-α expression by these cells. Together these observations indicate that cathelicidin peptides CAP18 and CAP11 probably exert protective actions against endotoxin shock by blocking the binding of LPS to CD14+ cells, thereby suppressing the production of cytokines by these cells via their potent binding activities for LPS and CD14.
Mycobacteria-Induced TNF-α and IL-10 Formation by Human Macrophages Is Differentially Regulated at the Level of Mitogen-Activated Protein Kinase Activity
Authors: Reiling Norbert , Blumenthal Antje , Flad Hans-Dieter , Ernst Martin , Ehlers Stefan
Published: April 2014

The clinical course of mycobacterial infections is linked to the capacity of pathogenic strains to modulate the initial antimycobacterial response of the macrophage. To elucidate some of the mechanisms involved, we studied early signal transduction events leading to cytokine formation by human monocyte-derived macrophages (MDM) in response to clinical isolates of Mycobacterium avium. TNF-α production induced by M. avium was inhibited by anti-CD14 mAbs, but not by Abs against the macrophage mannose receptor. Analysis of mitogen-activated protein (MAP) kinase activation (extracellular signal-regulated kinase 1/2, p38, and c-Jun NH2-terminal kinase) showed a rapid phosphorylation of all three subfamilies in response to M. avium, which was inhibited by anti-CD14 Abs. Using highly specific inhibitors of p38 (SB203580) and MAP kinase kinase-1 (PD98059), we found that activation of the extracellular signal-regulated kinase pathway, but not of p38, was essential for the M. avium-induced TNF-α formation. In contrast, IL-10 production was abrogated by the p38 inhibitor, but not by the MAP kinase kinase-1 inhibitor. In conclusion, M. avium-induced secretion of TNF-α and IL-10 by human macrophages is differentially regulated at the level of MAP kinase activity.
IL-12-Independent IFN-γ Production by T Cells in Experimental Chagas’ Disease Is Mediated by IL-18
Authors: Müller Uwe , Köhler Gabriele , Mossmann Horst , Schaub Günter A. , Alber Gottfried , Di Santo James P. , Brombacher Frank , Hölscher Christoph
Published: April 2014

IL-12p35-deficient (IL-12p35−/−) mice were highly susceptible to Trypanosoma cruzi infection and succumbed during acute infection, demonstrating the crucial importance of endogenous IL-12 in resistance to experimental Chagas’ disease. Delayed immune responses were observed in mutant mice, although comparable IFN-γ and TNF-α blood levels as in wild-type mice were detected 2 wk postinfection. In vivo and in vitro analysis demonstrated that T cells, but not NK cells, were recruited to infected organs. Analysis of mice double deficient in the recombinase-activating gene 2 (RAG2) and IL-12p35, as well as studies involving T cell depletion, identified CD4+ T cells as the cellular source for IL-12-independent IFN-γ production. IL-18 was induced in IL-12p35−/− mice and was responsible for IFN-γ production, as demonstrated by in vivo IL-18 neutralization studies. In conclusion, evidence is presented for an IL-12-independent IFN-γ production in experimental Chagas’ disease that is T cell and IL-18 dependent.
N-Linked Glycosylations at Asn26 and Asn114 of Human MD-2 Are Required for Toll-Like Receptor 4-Mediated Activation of NF-κB by Lipopolysaccharide
Authors: Ohnishi Takahiro , Muroi Masashi , Tanamoto Ken-ichi
Published: April 2014

MD-2 is physically associated with Toll-like receptor 4 (TLR4) and is required for TLR4-mediated LPS signaling. Western blotting analysis revealed the presence of three forms of human (h)MD-2 with different electrophoretic mobilities. After N-glycosidase treatment of the cellular extract prepared from cells expressing hMD-2, only a single form with the fastest mobility was detected. Mutation of either one of two potential glycosylation sites (Asn26 and Asn114) of MD-2 resulted in the disappearance of the slowest mobility form, and only the fastest form was detected in hMD-2 carrying mutations at both Asn26 and Asn114. Although these mutants were expressed on the cell surface and maintained its ability to associate with human TLR4, these mutations or tunicamycin treatment substantially impaired the ability of MD-2 to complement TLR4-mediated activation of NF-κB by LPS. LPS binding to cells expressing CD14, TLR4, and MD-2 was unaffected by these mutations. These observations demonstrate that hMD-2 undergoes N-linked glycosylation at Asn26 and Asn114, and that these glycosylations are crucial for TLR4-mediated signal transduction of LPS.
Synergistic Induction of Apoptosis in Primary CD4+ T Cells by Macrophage-Tropic HIV-1 and TGF-β1
Authors: Wang Jinhai , Guan Ennan , Roderiquez Gregory , Norcross Michael A.
Published: April 2014

Depletion of CD4+ T lymphocytes is a central immunological characteristic of HIV-1 infection. Although the mechanism of such CD4+ cell loss following macrophage-tropic (R5) HIV-1 infection remains unclear, interactions between viral and host cell factors are thought to play an important role in the pathogenesis of HIV-1 disease. Based on the observation that TGF-β1 enhanced expression of HIV chemokine coreceptors, the role of this host factor in virus effects was investigated using PBLs cultured in a nonmitogen-added system in the absence or presence of TGF-β1. Most CD4 cells in such cultures had the phenotype CD25−CD69−DR−Ki67− and were CD45RObrightCD45RAdim. Cultured cells had increased expression of CCR5 and CXCR4 and supported both HIV-1 entry and completion of viral reverse transcription. Virus production by cells cultured in the presence of IL-2 was inhibited by TGF-β1, and this inhibition was accompanied by a loss of T cells from the culture and an increase in CD4+ T cell apoptosis. Whereas R5X4 and X4 HIV-1 infection was sufficient to induce T cell apoptosis, R5 HIV-1 failed to induce apoptosis of PBLs in the absence of TGF-β1 despite the fact that R5 HIV-1 depletes CD4+ T cells in vivo. Increased apoptosis with HIV and TGF-β1 was associated with reduced levels of Bcl-2 and increased expression of apoptosis-inducing factor, caspase-3, and cleavage of BID, c-IAP-1, and X-linked inhibitor of apoptosis. These results show that TGF-β1 promotes depletion of CD4+ T cells after R5 HIV-1 infection by inducing apoptosis and suggest that TGF-β1 might contribute to the pathogenesis of HIV-1 infection in vivo.
Complementary Antitumor Immunity Induced by Plasmid DNA Encoding Secreted and Cytoplasmic Human ErbB-2
Authors: Piechocki Marie P. , Pilon Shari A. , Wei Wei-Zen
Published: April 2014

A plasmid DNA was constructed to encode the N-terminal 505 aa of human ErbB-2 (E2, HER-2/neu) and designated as secreted ErbB-2 (secE2). Recombinant secE2 protein was detected in the transfected cells and was secreted as an 80-kDa glycoprotein. Vaccination of BALB/c mice with secE2 DNA induced both IgG1 and IgG2a ErbB-2-specific Abs and protected ∼90% of mice against mouse mammary tumor D2F2, which expressed human ErbB-2 (D2F2/E2). The efficacy of secE2 vaccine was comparable with that of wild-type ErbB-2 DNA, which encodes the entire 1258 aa of ErbB-2 protein, induced only IgG2a E2-specific Abs, and stimulated greater CTL activity. Immune lymphocytes were stimulated in vitro with irradiated 3T3 cells, which expressed ErbB-2, Kd, and B7.1. CTL activity was measured by the lysis of E2-positive target cells and by intracellular IFN-γ production. To enhance CTL activation, mice were immunized with a combination of secE2 and cytoplasmic E2 (cytE2); the latter encodes the 1258-aa ErbB-2 protein that was released into the cytoplasm upon synthesis. Significant increase in CTL activity was demonstrated after mice were immunized with the combined vaccines and all mice were protected from D2F2/E2 tumor growth. Therefore, secE2, which induced Th2 Ab and weak CTL, conferred similar protection as E2, which induced Th1 Ab and strong CTL. Combined vaccination with secE2 and cytE2 resulted in Th2 Ab, strong CTL, and the most effective protection against tumor growth. The strategy of coimmunization with DNA that direct Ags to different subcellular compartments may be adapted as appropriate to optimize immune outcome.
The Effect of an Anti-HLA-B27 Immune Response on CTL Recognition ofChlamydia
Authors: Popov Igor , Dela Cruz Charles S. , Barber Brian H. , Chiu Basil , Inman Robert D.
Published: April 2014

The interplay between triggering bacteria and HLA-B27 in the pathogenesis of the spondyloarthropathies remains one of the most active areas of investigation in the rheumatic diseases. This has proved difficult to study systematically in the clinical setting, and in this study we utilized a rat model to address the influence that B27-related immunity may have on the process of generating anti-Chlamydia immunity. When splenocytes from HLA-B27 DNA-immunized Lewis (LEW) animals received restimulation in vitro with Chlamydia-treated cells from B27-transgenic LEW rats, we observed that in addition to the expected CTL recognition of HLA-B27, there was also anti-Chlamydia CTL killing of Chlamydia-sensitized syngeneic fibroblast targets. This was not seen when responding cells in vitro were naive LEW splenocytes. To confirm the existence of CTLs recognizing both HLA-B27 and Chlamydia, LEW rats were immunized with B27-transgenic LEW cells, instead of the B27 DNA construct. Splenocytes from the immune rats were restimulated in vitro with Chlamydia-treated B27-transgenic LEW cells. In this instance, the CTLs retained the allele-specific recognition of HLA-B27, as well as recognition of Chlamydia-sensitized syngeneic fibroblasts. Thus, if there is prior expansion of an immune response against HLA-B27, then the resulting splenocytes demonstrate a reduced threshold for generating a primary anti-Chlamydia CTL response. These studies implicate a dynamic interrelationship between recognition of HLA-B27 and Chlamydia trachomatis. The results may have implications for deciphering the cellular basis of Chlamydia-induced reactive arthritis.
Borrelia burgdorferi-Induced Inflammation Facilitates Spirochete Adaptation and Variable Major Protein-Like Sequence Locus Recombination
Authors: Anguita Juan , Thomas Venetta , Samanta Swapna , Persinski Rafal , Hernanz Carmen , Barthold Stephen W. , Fikrig Erol
Published: April 2014

Spirochete adaptation in vivo is associated with preferential Borrelia burgdorferi gene expression. In this paper, we show that the administration of B. burgdorferi-immune sera to IFN-γR-deficient mice that have been infected with B. burgdorferi N40 for 4 days causes spirochete clearance. In contrast, immune sera-mediated clearance of B. burgdorferi N40 is not apparent in immunocompetent mice, suggesting a role for IFN-γ-mediated responses in B. burgdorferi N40 host adaptation. B. burgdorferi-immune sera also induces clearance of B. burgdorferi N40 that have been passaged in vitro 75 times (B. burgdorferi N40-75), a derivative of B. burgdorferi N40 that does not rapidly adapt in vivo in immunocompetent mice. B. burgdorferi N40-75 produce lower levels of IFN-γ and IL-12 in mice than does B. burgdorferi N40, and the administration of these cytokines to B. burgdorferi N40-75-infected mice results in an increased spirochetal burden, further indicating that IFN-γ-mediated events promote B. burgdorferi survival. Differential immunoscreening and RT-PCR demonstrate that IFN-γ-mediated signals facilitate spirochete recombination at the variable major protein like sequence locus, a site for early antigenic variation in vivo, and that recombination rates by B. burgdorferi N40 are lower in IFN-γR-deficient mice than in control animals. These results suggest that the murine immune response can promote the in vivo adaptation of B. burgdorferi.
Sodium Stibogluconate Is a Potent Inhibitor of Protein Tyrosine Phosphatases and Augments Cytokine Responses in Hemopoietic Cell Lines
Authors: Pathak Manas K. , Yi Taolin
Published: April 2014

Using in vitro protein tyrosine phosphatase (PTPase) assays, we found that sodium stibogluconate, a drug used in treatment of leishmaniasis, is a potent inhibitor of PTPases Src homology PTPase1 (SHP-1), SHP-2, and PTP1B but not the dual-specificity phosphatase mitogen-activated protein kinase phosphatase 1. Sodium stibogluconate inhibited 99% of SHP-1 activity at 10 μg/ml, a therapeutic concentration of the drug for leishmaniasis. Similar degrees of inhibition of SHP-2 and PTP1B required 100 μg/ml sodium stibogluconate, demonstrating differential sensitivities of PTPases to the inhibitor. The drug appeared to target the SHP-1 domain because it showed similar in vitro inhibition of SHP-1 and a mutant protein containing the SHP-1 PTPase domain alone. Moreover, it forms a stable complex with the PTPase: in vitro inhibition of SHP-1 by the drug was not removed by a washing process effective in relieving the inhibition of SHP-1 by the reversible inhibitor suramin. The inhibition of cellular PTPases by the drug was suggested by its rapid induction of tyrosine phosphorylation of cellular proteins in Baf3 cells and its augmentation of IL-3-induced Janus family kinase 2/Stat5 tyrosine phosphorylation and proliferation of Baf3 cells. The augmentation of the opposite effects of GM-CSF and IFN-α on TF-1 cell growth by the drug indicated its broad activities in the signaling of various cytokines. These data represent the first evidence that sodium stibogluconate inhibits PTPases and augments cytokine responses. Our results provide novel insights into the pharmacological effects of the drug and suggest potential new therapeutic applications.
CTA1-DD-Immune Stimulating Complexes: a Novel, Rationally Designed Combined Mucosal Vaccine Adjuvant Effective with Nanogram Doses of Antigen
Authors: Mowat Allan M. I. , Donachie Anne M. , Jägewall Sara , Schön Karin , Löwenadler Björn , Dalsgaard Kristian , Kaastrup Peter , Lycke Nils
Published: April 2014

Mucosally active vaccine adjuvants that will prime a full range of local and systemic immune responses against defined antigenic epitopes are much needed. Cholera toxin and lipophilic immune stimulating complexes (ISCOMS) containing Quil A can both act as adjuvants for orally administered Ags, possibly by targeting different APCs. Recently, we have been successful in separating the adjuvant and toxic effects of cholera toxin by constructing a gene fusion protein, CTA1-DD, that combines the enzymatically active CTA1-subunit with a B cell-targeting moiety, D, derived from Staphylococcus aureus protein A. Here we have extended this work by combining CTA1-DD with ISCOMS, which normally target dendritic cells and/or macrophages. ISCOMS containing a fusion protein comprising the OVA323–339 peptide epitope linked to CTA1-DD were highly immunogenic when given in nanogram doses by the s.c., oral, or nasal routes, inducing a wide range of T cell-dependent immune responses. In contrast, ISCOMS containing the enzymatically inactive CTA1-R7K-DD mutant protein were much less effective, indicating that at least part of the activity of the combined vector requires the ADP-ribosylating property of CTA1. No toxicity was observed by any route. To our knowledge, this is the first report on the successful combination of two mechanistically different principles of adjuvant action. We conclude that rationally designed vectors consisting of CTA1-DD and ISCOMS may provide a novel strategy for the generation of potent and safe mucosal vaccines.
Urokinase Plasminogen Activator and Plasmin Efficiently Convert Hemofiltrate CC Chemokine 1 into Its Active [9–74] Processed Variant
Authors: Vakili Jalal , Ständker Ludger , Detheux Michel , Vassart Gilbert , Forssmann Wolf-Georg , Parmentier Marc
Published: April 2014

We have previously isolated from human hemofiltrate an N-terminally truncated form of the hemofiltrate CC chemokine 1 (HCC-1), and characterized HCC-1[9–74] as a strong agonist of CCR1, CCR5, and to a lower extent CCR3. In this study, we show that conditioned media from human tumor cell lines PC-3 and 143B contain proteolytic activities that convert HCC-1 into the [9–74] form. This activity was fully inhibited by inhibitors of urokinase-type plasminogen activator (uPA), including PA inhibitor-1, an anti-uPA mAb, and amiloride. Pure preparations of uPA processed HCC-1 with high efficiency, without further degrading HCC-1[9–74]. Plasmin could also generate HCC-1[9–74], but degraded the active product as well. The kinetics of HCC-1 cleavage by uPA and plasmin (Michaelis constant, Km, of 0.76 ± 0.4 μM for uPA, and 0.096 ± 0.05 μM for plasmin; catalytic rate constant, kcat: 3.36 ± 0.96 s−1 for uPA and 6 ± 3.6 s−1 for plasmin) are fully compatible with a role in vivo. The activation of an abundant inactive precursor into a broad-spectrum chemokine by uPA and plasmin directly links the production of uPA by numerous tumors and their ability to recruit mononuclear leukocytes, without the need for the transcriptional activation of chemokine genes.
Calcium-Independent Phospholipase A2 Is Required for Human Monocyte Chemotaxis to Monocyte Chemoattractant Protein 1
Authors: Carnevale Kevin A. , Cathcart Martha K.
Published: April 2014

Monocyte chemoattractant protein 1 (MCP-1) has an important influence on monocyte migration into sites of inflammation. Our understanding of the signal transduction pathways involved in the response of monocytes to MCP-1 is quite limited yet potentially significant for understanding and manipulating the inflammatory response. Prior studies have demonstrated a crucial regulatory role for cytosolic phospholipase A2 (cPLA2) in monocyte chemotaxis to MCP-1. In these studies we investigated the role for another PLA2, calcium-independent PLA2 (iPLA2) in comparison to cPLA2. Pharmacological inhibitors of PLA2 were found to substantially inhibit chemotaxis. Using antisense oligodeoxyribonucleotide treatment we found that iPLA2 expression is required for monocyte migration to MCP-1. Complete blocking of the chemotactic response was observed with inhibition of either iPLA2 or cPLA2 expression by their respective antisense oligodeoxyribonucleotide. In reconstitution experiments, lysophosphatidic acid completely restored MCP-1-stimulated migration in iPLA2-deficient monocytes, whereas lysophosphatidic acid was without effect in restoring migration in cPLA2-deficient monocytes. To the contrary, arachidonic acid fully restored migration of cPLA2-deficient monocytes while having no effect on the iPLA2-deficient monocytes. Additional studies revealed that neither enzyme appears to be upstream of the other indicating that iPLA2 and cPLA2 represent parallel regulatory pathways. These data demonstrate novel and distinct roles for these two phospholipases in this critical step in inflammation.
Trypanocidal Drug Benznidazole Impairs Lipopolysaccharide Induction of Macrophage Nitric Oxide Synthase Gene Transcription Through Inhibition of NF-κB Activation
Authors: Piaggio Eliane , Sancéau Josiane , Revelli Silvia , Bottasso Oscar , Wietzerbin Juana , Serra Esteban
Published: April 2014

In murine macrophages, inducible NO synthase II (NOSII) gene expression is promoted at a transcriptional level by LPS and/or IFN-γ with benznidazole (BZL), a trypanocidal drug, acting to down-regulate NOSII gene induction and hence inhibiting NO production. By performing transient transfection experiments, we now report that BZL also inhibited the expression of NOSII gene promoter or multimerized NF-κB binding site controlled reporter genes. By contrast, no effect was observed on the expression of a reporter gene under the control of the NOSII promoter-derived IFN regulatory factor element. EMSAs demonstrated that BZL inhibited the nuclear availability of NF-κB in stimulated macrophages. NF-κB is activated in macrophages by phosphorylation, ubiquitination, and subsequent proteolysis of IκB. Within this setting, Western blot was also performed to show that BZL blocked IκBα degradation. Collectively, these results demonstrate that BZL is able to specifically inhibit macrophage NF-κB activation after LPS plus IFN-γ stimulation.
Drastic Up-Regulation of FcεRI on Mast Cells Is Induced by IgE Binding Through Stabilization and Accumulation of FcεRI on the Cell Surface
Authors: Kubo Shuichi , Matsuoka Kunie , Taya Choji , Kitamura Fujiko , Takai Toshiyuki , Yonekawa Hiromichi , Karasuyama Hajime
Published: April 2014

It has been shown that IgE binding to FcεRI on mast cells results in increased FcεRI expression, which in turn enhances IgE-dependent chemical mediator release from mast cells. Therefore, prevention of the IgE-mediated FcεRI up-regulation would be a promising strategy for management of allergic disorders. However, the mechanism of IgE-mediated FcεRI up-regulation has not been fully elucidated. In this study, we analyzed kinetics of FcεRI on peritoneal mast cells and bone marrow-derived mast cells. In the presence of brefeldin A, which prevented transport of new FcεRI molecules to the cell surface, levels of IgE-free FcεRI on mast cells decreased drastically during culture, whereas those of IgE-bound FcεRI were stable. In contrast, levels of FcγRIII on the same cells were stable even in the absence of its ligand, indicating that FcεRI α-chain, but not β- and γ-chains, was responsible for the instability of IgE-free FcεRI. As far as we analyzed, there was no evidence to support the idea that IgE binding to FcεRI facilitated synthesis and/or transport of FcεRI to the cell surface. Therefore, the stabilization and accumulation of FcεRI on the cell surface through IgE binding appears to be the major mechanism of IgE-mediated FcεRI up-regulation.
The IL-6-Soluble IL-6Rα Autocrine Loop of Endothelial Activation as an Intermediate Between Acute and Chronic Inflammation: an Experimental Model Involving Thrombin
Authors: Marin Valérie , Montero-Julian Felix A. , Grès Sandra , Boulay Vera , Bongrand Pierre , Farnarier Catherine , Kaplanski Gilles
Published: April 2014

Thrombin is a procoagulant and proinflammatory molecule in vivo. In vitro, thrombin has been shown to induce endothelial activation, notably IL-8 secretion and adhesion molecule expression. In this study, we showed that thrombin may induce a new cascade leading from acute to chronic inflammation. Thrombin was able to induce the production of both IL-6 and monocyte chemotactic protein-1 (MCP-1) by HUVEC independently of IL-1αβ and TNF-α. Addition of physiological concentrations of exogenous soluble IL-6Rα (sIL-6Rα) to thrombin-activated HUVEC was sufficient to increase the amounts of MCP-1 produced, but not those of IL-8. These effects could be blocked by anti-IL-6 or anti-sIL-6Rα blocking mAb, demonstrating the existence of an autocrine loop of MCP-1 secretion, involving the IL-6/IL-6Rα/gp130 complex on HUVEC. In addition, we identified IL-8-activated neutrophils as a potential source of sIL-6Rα because IL-8 induced IL-6Rα shedding from the neutrophil membranes and increased in parallel sIL-6Rα concentrations in neutrophil supernatants. Furthermore, addition of neutrophils to thrombin-activated HUVEC significantly increased MCP-1 secretion, which could be decreased by blocking IL-6. Thus, thrombin-activated endothelium may induce a cascade of events characterized by IL-8 secretion, neutrophil local infiltration, and the release of IL-6Rα from neutrophil membranes. sIL-6Rα may then complex with IL-6 and increase the amount of MCP-1 produced by thrombin-activated endothelium, favoring monocyte infiltration, and the transformation of acute into chronic inflammation.
IL-3 Induces Down-Regulation of CCR3 Protein and mRNA in Human Eosinophils
Authors: Dulkys Yasmin , Kluthe Christof , Buschermöhle Thomas , Barg Ines , Knöß Sabine , Kapp Alexander , Proudfoot Amanda E. I. , Elsner Jörn
Published: April 2014

Cytokines and chemokines are responsible for the attraction and activation of eosinophils in allergic and inflammatory diseases. Whereas cytokines such as IL-3, IL-5, and GM-CSF activate eosinophils via heterodimeric receptors containing a distinct α-chain (binding domain) and a common β-chain (signaling domain), chemokines such as eotaxin activate eosinophils via seven-transmembrane Gi protein-coupled CCRs. Recent studies have demonstrated the importance of CCR3 on human eosinophils that undergo receptor recycling after chemokine activation, but the modulation of this receptor by cytokines has not yet been addressed. In this study, we demonstrate that IL-3 induces a dose- and time-dependent down-regulation of CCR3 from the surface of human eosinophils comparable to the CCR3-specific ligand eotaxin, whereas IL-5, GM-CSF, IL-4, IL-10, IL-13, IFN-γ, and TNF-α had no effect. Maximal down-regulation of CCR3 in response to IL-3 was reached at 24 h. Reduction of CCR3 surface protein in response to IL-3 could be prevented by an anti-IL-3 mAb and was neither due to the release of CC chemokines nor to nonspecific binding of IL-3 to CCR3. Moreover, down-regulation was prevented by phenylarsine oxide, a nonspecific inhibitor of receptor internalization. After 24 h, IL-3-induced decrease of CCR3 surface expression correlated with diminished mRNA expression, suggesting a transcriptional regulation mechanism. Since wortmannin partially inhibited IL-3- but not eotaxin-induced CCR3 down-regulation, receptor down-modulation seems to underlie different signaling events. Therefore, these data suggest a novel role for the cytokine IL-3 in the activation process of eosinophils and its predominant chemokine receptor CCR3.
Curcumin Inhibits Activation of Vγ9Vδ2 T Cells by Phosphoantigens and Induces Apoptosis Involving Apoptosis-Inducing Factor and Large Scale DNA Fragmentation
Authors: Cipriani Barbara , Borsellino Giovanna , Knowles Heather , Tramonti Daniela , Cavaliere Fabio , Bernardi Giorgio , Battistini Luca , Brosnan Celia F.
Published: April 2014

Curcumin, in addition to its role as a spice, has been used for centuries to treat inflammatory disorders. Although the mechanism of action remains unclear, it has been shown to inhibit the activation of NF-κB and AP-1, transcription factors required for induction of many proinflammatory mediators. Due to its low toxicity it is currently under consideration as a broad anti-inflammatory, anti-tumor cell agent. In this study we investigated whether curcumin inhibited the response of γδ T cells to protease-resistant phosphorylated derivatives found in the cell wall of many pathogens. The results showed that curcumin levels ≥30 μM profoundly inhibited isopentenyl pyrophosphate-induced release of the chemokines macrophage inflammatory protein-1α and -1β and RANTES. Curcumin also blocked isopentenyl pyrophosphate-induced activation of NF-κB and AP-1. Commencing around 16 h, treatment with curcumin lead to the induction of cell death that could not be reversed by APC, IL-15, or IL-2. This cytotoxicity was associated with increased annexin V reactivity, nuclear expression of active caspase-3, cleavage of poly(ADP-ribose) polymerase, translocation of apoptosis-inducing factor to the nucleus, and morphological evidence of nuclear disintegration. However, curcumin led to only large scale DNA chromatolysis, as determined by a combination of TUNEL staining and pulse-field and agarose gel electrophoresis, suggesting a predominantly apoptosis-inducing factor-mediated cell death process. We conclude that γδ T cells activated by these ubiquitous Ags are highly sensitive to curcumin, and that this effect may contribute to the anti-inflammatory properties of this compound.
Midkine Is Involved in Neutrophil Infiltration into the Tubulointerstitium in Ischemic Renal Injury
Authors: Sato Waichi , Kadomatsu Kenji , Yuzawa Yukio , Muramatsu Hisako , Hotta Nigishi , Matsuo Seiichi , Muramatsu Takashi
Published: April 2014

Midkine (MK) is a multifunctional heparin-binding protein and promotes migration of neutrophils, macrophages, and neurons. In the normal mouse kidney, MK is expressed in the proximal tubules. After renal ischemic reperfusion injury, its expression in proximal tubules was increased. Immediate increase of MK expression was found when renal proximal tubular epithelial cells in culture were exposed to 5 mM H2O2. Histologically defined tubulointerstitial damage was less severe in MK-deficient (Mdk−/−) than in wild-type (Mdk+/+) mice at 2 and 7 days after ischemic reperfusion injury. Within 2 days after ischemic injury, inflammatory leukocytes, of which neutrophils were the major population, were recruited to the tubulointerstitium. The numbers of infiltrating neutrophils and also macrophages were lower in Mdk−/− than in Mdk+/+ mice. Induction of macrophage inflammatory protein-2 and macrophage chemotactic protein-1, chemokines for neutrophils and macrophages, respectively, were also suppressed in Mdk−/− mice. Furthermore, renal tubular epithelial cells in culture expressed macrophage inflammatory protein-2 in response to exogenous MK administration. These results suggested that MK enhances migration of inflammatory cells upon ischemic injury of the kidney directly and also through induction of chemokines, and contributes to the augmentation of ischemic tissue damage.
The Central Nervous System Inflammatory Response to Neurotropic Virus Infection Is Peroxynitrite Dependent
Authors: Hooper D. Craig , Kean Rhonda B. , Scott Gwen S. , Spitsin Sergei V. , Mikheeva Tatiana , Morimoto Kinjiro , Bette Michael , Röhrenbeck Annette M. , Dietzschold Bernhard , Weihe Eberhard
Published: April 2014

We have recently demonstrated that increased blood-CNS barrier permeability and CNS inflammation in a conventional mouse model of experimental allergic encephalomyelitis are dependent upon the production of peroxynitrite (ONOO−), a product of the free radicals NO· and superoxide (O2·−). To determine whether this is a reflection of the physiological contribution of ONOO− to an immune response against a neurotropic pathogen, we have assessed the effects on adult rats acutely infected with Borna disease virus (BDV) of administration of uric acid (UA), an inhibitor of select chemical reactions associated with ONOO−. The pathogenesis of acute Borna disease in immunocompetent adult rats results from the immune response to the neurotropic BDV, rather than the direct effects of BDV infection of neurons. An important stage in the BDV-specific neuroimmune response is the invasion of inflammatory cells into the CNS. UA treatment inhibited the onset of clinical disease, and prevented the elevated blood-brain barrier permeability as well as CNS inflammation seen in control-treated BDV-infected rats. The replication and spread of BDV in the CNS were unchanged by the administration of UA, and only minimal effects on the immune response to BDV Ags were observed. These results indicate that the CNS inflammatory response to neurotropic virus infection is likely to be dependent upon the activity of ONOO− or its products on the blood-brain barrier.
An Antagonist IL-15/Fc Protein Prevents Costimulation Blockade-Resistant Rejection
Authors: Ferrari-Lacraz Sylvie , Zheng Xin Xiao , Kim Yon Su , Li Yongsheng , Maslinski Wlodzimierz , Li Xian Chang , Strom Terry B.
Published: April 2014

IL-15 is a powerful T cell growth factor (TCGF) with particular importance for the maintenance of CD8+ T cells. Because costimulation blockade does not result in universal tolerance, we hypothesized that “escape” from costimulation blockade might represent a CD8+ and IL-15/IL-15R+-dependent process. For this analysis, we have used an IL-15 mutant/Fcγ2a protein, a potentially cytolytic protein that is also a high-affinity receptor site specific antagonist for the IL-15Rα receptor protein, as a therapeutic agent. The IL-15-related fusion protein was used as monotherapy or in combination with CTLA4/Fc in murine islet allograft models. As monotherapies, CTLA4/Fc and an IL-15 mutant/Fcγ2a were comparably effective in a semiallogeneic model system, and combined treatment with IL-15 mutant/Fcγ2a plus CTLA4/Fc produced universal permanent engraftment. In a fully MHC-mismatched strain combination known to be refractory to costimulation blockade treatment, combined treatment with both fusion proteins proved to be highly effective; >70% of recipients were tolerized. The analysis revealed that the IL-15 mutant/Fc treatment confers partial protection from both CD4+ and CD8+ T cell graft infiltration. In rejections occurring despite CTLA4/Fc treatment, concomitant treatment with the IL-15 mutant/Fcγ2a protein blocked a CD8+ T cell-dominated rejection processes. This protection was linked to a blunted proliferative response of alloreactive T cells as well silencing of CTL-related gene expression events. Hence, we have demonstrated that targeting the IL-15/IL-15R pathway represents a new and potent strategy to prevent costimulation blockade-resistant CD8+ T cell-driven rejection.
Terminal Deoxynucleotidyl Transferase Deficiency Decreases Autoimmune Disease in MRL-Fas
lpr Mice
Authors: Feeney Ann J. , Lawson Brian R. , Kono Dwight H. , Theofilopoulos Argyrios N.
Published: April 2014

The neonatal Ab and TCR repertoires are much less diverse, and also very different from, the adult repertoires due to the delayed onset of terminal deoxynucleotidyl transferase (TdT) expression in ontogeny. TdT adds nontemplated N nucleotides to the junctions of Igs and TCRs, and thus its absence removes one of the major components of junctional diversity in complementarity-determining region 3 (CDR3). We have generated TdT-deficient MRL/lpr, Fas-deficient (MRL-Faslpr) mice, and show that they have an increased lifespan, decreased incidence of skin lesions, and much lower serum levels of anti-dsDNA, anti-chromatin, and IgM rheumatoid factors. The generalized hypergammaglobulinemia characteristic of MRL-Faslpr mice is also greatly reduced, as is the percentage of CD4−CD8−B220+ (double-negative) T cells. IgG deposits in the kidney are significantly reduced, although evidence of renal disease is present in many mice at 6 mo. CDR3 regions of both IgH and TCR from peripheral lymphocytes of MRL-Faslpr mice are shorter in the absence of TdT, and there is a paucity of arginines in the IgH CDR3 regions of the MRL-Faslpr TdT−/− mice. Because the amelioration of symptoms is so widespread, it is likely that the absence of N regions has more of an affect than merely decreasing the precursor frequency of anti-dsDNA B cells. Hence, either the T or B cell repertoires, or more likely both, require N region diversity to produce the full spectrum of autoimmune lupus disease.
Monokine Induced by IFN-γ Is a Dominant Factor Directing T Cells into Murine Cardiac Allografts During Acute Rejection
Authors: Miura Masayoshi , Morita Ken , Kobayashi Hirohito , Hamilton Thomas A. , Burdick Marie D. , Strieter Robert M. , Fairchild Robert L.
Published: April 2014

The use of chemokine antagonism as a strategy to inhibit leukocyte trafficking into inflammatory sites requires identification of the dominant chemokines mediating recruitment. The chemokine(s) directing T cells into cardiac allografts during acute rejection remain(s) unidentified. The role of the CXC chemokines IFN-γ inducible protein 10 (IP-10) and monokine induced by IFN-γ (Mig) in acute rejection of A/J (H-2a) cardiac grafts by C57BL/6 (H-2b) recipients was tested. Intra-allograft expression of Mig was observed at day 2 posttransplant and increased to the time of rejection at day 7 posttransplant. IP-10 mRNA and protein production were 2.5- to 8-fold lower than Mig. Whereas allografts were rejected at day 7–9 in control recipients, treatment with rabbit antiserum to Mig, but not to IP-10, prolonged allograft survival up to day 19 posttransplant. At day 7 posttransplant, allografts from Mig antiserum-treated recipients had marked reduction in T cell infiltration. At the time of rejection in Mig antiserum-treated recipients (i.e., days 17–19), intra-allograft expression of macrophage-inflammatory protein-1α, -1β, and their ligand CCR5 was high, whereas expression of CXCR3, the Mig receptor, was virtually absent. Mig was produced by the allograft endothelium as well as by recipient allograft-infiltrating macrophages and neutrophils, indicating the synergistic interactions between innate and adaptive immune compartments during acute rejection. Collectively, these results indicate that Mig is a dominant recruiting factor for alloantigen-primed T cells into cardiac allografts during acute rejection. Although Mig antagonism delays acute heart allograft rejection, the results also suggest that the alloimmune response circumvents Mig antagonism through alternative mechanisms.
IL-10-Deficient B10.Q Mice Develop More Severe Collagen-Induced Arthritis, but Are Protected from Arthritis Induced with Anti-Type II Collagen Antibodies
Authors: Johansson Åsa C. M. , Hansson Ann-Sofie , Nandakumar Kutty S. , Bäcklund Johan , Holmdahl Rikard
Published: April 2014

IL-10 is a pleiotropic cytokine with stimulatory and inhibitory properties, and is thought to have a protective role in rheumatoid arthritis and collagen-induced arthritis (CIA). In this study, we investigated how IL-10 deficiency affects CIA and anti-collagen type II (CII) Ab-transferred arthritis in C57BL/10.Q (B10.Q) mice. The B10.Q.IL-10−/− mice had an 8-cM 129/Ola fragment around the IL-10 gene. The mice were treated with antibiotics, appeared healthy, and had no colitis. T cells from IL-10−/− mice expressed similar levels of IFN-γ, IL-2, and IL-4 after mitogen stimulation; however, macrophages showed a reduced TNF-α production compared with IL-10+/− littermates. IL-10−/− mice had an increased incidence, and a more severe CIA disease than the IL-10+/− littermates. To study the role of IL-10 in T cell tolerance, IL-10−/− were crossed into mice carrying the immunodominant epitope, CII(256–270), in cartilage (MMC) or in skin (TSC). Both IL-10−/− and IL-10+/− MMC and TSC mice were completely tolerized against CIA, indicating that lack of IL-10 in this context did not break tolerance. To investigate whether IL-10 was important in the effector phase of CIA, arthritis was induced with anti-CII Abs. Surprisingly, IL-10−/− were less susceptible to Ab-transferred arthritis, as only 30% showed signs of disease compared with 90% of the littermates. Therefore, IL-10 seemed to have a protective role in CIA, but seemed to exacerbate the arthritogenicity of anti-CII Abs. These data emphasize the importance of studying IL-10 in a defined genetic context in vivo, to understand its role in a complex disease like arthritis.
T Cell Activation by Coxsackievirus B4 Antigens in Type 1 Diabetes Mellitus: Evidence for Selective TCR Vβ Usage Without Superantigenic Activity
Authors: Varela-Calvino Ruben , Sgarbi Gianluca , Wedderburn Lucy R. , Dayan Colin M. , Tremble Jenny , Peakman Mark
Published: April 2014

Numerous clinical and epidemiological studies link enteroviruses such as the Coxsackie virus group with the autoimmune disease type 1 diabetes mellitus (DM). In addition, there are reports that patients with type 1 DM are characterized by skewing of TCR Vβ chain selection among peripheral blood and intraislet T lymphocytes. To examine these issues, we analyzed TCR Vβ chain-specific up-regulation of the early T cell activation marker, CD69, on CD4 T cells after incubation with Coxsackievirus B4 (CVB4) Ags. CD4 T cells bearing the Vβ chains 2, 7, and 8 were the most frequently activated by CVB4. Up-regulation of CD69 by different TCR families was significantly more frequent in new onset type 1 DM patients (p = 0.04), 100% of whom (n = 8) showed activation of CD4 T cells bearing Vβ8, compared with 50% of control subjects (n = 8; p = 0.04). T cell proliferation after incubation with CVB4 Ags required live, nonfixed APCs, suggesting that the selective expansion of CD4 T cells with particular Vβ chains resulted from conventional antigen processing and presentation rather than superantigen activity. Heteroduplex analysis of TCR Vβ chain usage after CVB4 stimulation indicated a relatively polyclonal, rather than oligo- or monoclonal response to viral Ags. These results provide evidence that new-onset patients with type 1 DM and healthy controls are primed against CVB4, and that CD4 T cell responses to the virus have a selective TCR Vβ chain usage which is driven by viral Ags rather than a superantigen.
Severe Perturbations of the Blood T Cell Repertoire in Polymyositis, But Not Dermatomyositis Patients
Authors: Benveniste Olivier , Chérin Patrick , Maisonobe Thierry , Merat Rastine , Chosidow Olivier , Mouthon Luc , Guillevin Loïc , Flahault Antoine , Burland Marie-Christine , Klatzmann David , Herson Serge , Boyer Olivier
Published: April 2014

Polymyositis and dermatomyositis are diseases characterized by muscle weakness and muscle inflammatory infiltrates. Their pathogenesis remains unclear. A central role for endomysial autoaggressive CD8+ T cells is suspected in polymyositis and for perivascular B cells in dermatomyositis. We compared the T cell repertoire of 10 polymyositis and 10 dermatomyositis patients by immunoscope, a method providing a global assessment of the T cell repertoire and a sensitive detection of clonal T cell expansions. Samples were analyzed qualitatively and quantitatively in the blood (unsorted cells and CD4+ and CD8+ cells) and in muscle infiltrates. Dramatic perturbations of the T cell repertoire were observed in the blood of polymyositis but not dermatomyositis patients (p < 0.0005), the latter being undistinguishable from controls. These perturbations were due to oligoclonal expansions of CD8+ T cells and most blood clonal expansions were also found in muscle. These results indicate that the pathogenesis of polymyositis and dermatomyositis is different and reinforce the view that polymyositis but not dermatomyositis is an autoimmune CD8+ T cell-mediated disease. Moreover, this method may be helpful for the differential diagnosis of polymyositis and dermatomyositis and for noninvasive follow-up of polymyositis patients.