bluuebunny/crossref_metadata_2025 huggingface dataset:
A Mechanism for the Impaired IFN-γ Production in C-C Chemokine Receptor 2 (CCR2) Knockout Mice: Role of CCR2 in Linking the Innate and Adaptive Immune Responses
Authors: Peters Wendy , Dupuis Marc , Charo Israel F.
Published: April 2014

We have recently shown that mice with a targeted disruption of CCR2, the receptor for monocyte chemoattractant protein-1, have markedly impaired recruitment of macrophages to sites of inflammation. An unexpected finding in the CCR2−/− mice was a dramatic decrease in the production of IFN-γ after challenge with purified protein derivative of Mycobacterium bovis. In this study, we have investigated the mechanism of this cytokine production defect. In vitro, direct activation of splenocytes with CD3/CD28 Abs failed to reveal any differences in IFN-γ production between CCR2+/+ and CCR2−/− mice. However, after immunization, the number of Ag-specific, IFN-γ-producing cells in the draining lymph nodes was decreased by 70% in the CCR2−/− mice, suggesting an in vivo trafficking defect. Direct measurement of cell trafficking with fluorescently labeled CFA revealed a marked decrease in the number of monocytes/macrophages migrating to the site of immunization and to the draining lymph nodes in the CCR2−/− mice. The data suggest that impaired trafficking of APCs in the CCR2−/− mice contributes to the defect in IFN-γ production. These data support the idea that CCR2-positive monocytes/macrophages are critical in linking the innate and adaptive immune responses.
The Importance of Exogenous Antigen in Priming the Human CD8+ T Cell Response: Lessons from the EBV Nuclear Antigen EBNA1
Authors: Blake Neil , Haigh Tracey , Shaka’a Ghadeer , Croom-Carter Debbie , Rickinson Alan
Published: April 2014

Mouse models suggest that the processing of exogenous Ag by dendritic cells can be important for priming the CD8+ CTL response. To study the situation in humans, we have exploited the CTL response to EBV infection. In this context EBV expresses eight latent proteins, of which EBV-encoded nuclear Ag (EBNA) 3A, 3B, and 3C appear to be immunodominant for CTL responses, whereas another nuclear Ag, EBNA1, which is completely protected from endogenous presentation via the MHC class I pathway, is thought to induce responses rarely, if ever. Here, using EBNA1 peptides and/or EBNA1 protein-loaded dendritic cells as in vitro stimuli, we have identified memory CTL responses to HLA-B*3501, -B7, and -B53-restricted EBNA1 epitopes that can be as strong as those seen in immunodominant epitopes from the “conventionally processed” EBNA3 Ags. Furthermore, we used HLA-peptide tetramers to show that the primary response to one such EBNA1 epitope constituted up to 5% of the CD8+ T cells in infectious mononucleosis blood, the strongest latent Ag-specific response yet detected in this setting. We conclude that exogenous protein represents a significant source of Ag for priming the human CTL response.
Human CD8+ CTL Specific for the Mycobacterial Major Secreted Antigen 85A
Authors: Smith Steven M. , Brookes Roger , Klein Michèl R. , Malin Adam S. , Lukey Pauline T. , King Abigail S. , Ogg Graham S. , Hill Adrian V. S. , Dockrell Hazel M.
Published: April 2014

The role of CD8+ CTL in protection against tuberculosis in human disease is unclear. In this study, we stimulated the peripheral blood mononuclear cells of bacillus Calmette-Guérin (BCG)-vaccinated individuals with live Mycobacterium bovis BCG bacilli to establish short-term cell lines and then purified the CD8+ T cells. A highly sensitive enzyme-linked immunospot (ELISPOT) assay for single cell IFN-γ release was used to screen CD8+ T cells with overlapping peptides spanning the mycobacterial major secreted protein, Ag85A. Three peptides consistently induced a high frequency of IFN-γ responsive CD8+ T cells, and two HLA-A*0201 binding motifs, P48–56 and P242–250, were revealed within the core sequences. CD8+ T cells responding to the 9-mer epitopes were visualized within fresh blood by ELISPOT using free peptide or by binding of HLA-A*0201 tetrameric complexes. The class I-restricted CD8+ T cells were potent CTL effector cells that efficiently lysed an HLA-A2-matched monocyte cell line pulsed with peptide as well as autologous macrophages infected with Mycobacterium tuberculosis or recombinant vaccinia virus expressing the whole Ag85A protein. Tetramer assays revealed a 6-fold higher frequency of peptide-specific T cells than IFN-γ ELISPOT assays, indicating functional heterogeneity within the CD8+ T cell population. These results demonstrate a previously unrecognized, MHC class I-restricted, CD8+ CTL response to a major secreted Ag of mycobacteria and supports the use of Ag85A as a candidate vaccine against tuberculosis.
Synergy and Cross-Tolerance Between Toll-Like Receptor (TLR) 2- and TLR4-Mediated Signaling Pathways
Authors: Sato Shintaro , Nomura Fumiko , Kawai Taro , Takeuchi Osamu , Mühlradt Peter F. , Takeda Kiyoshi , Akira Shizuo
Published: April 2014

A family of Toll-like receptor (TLR) mediates the cellular response to bacterial cell wall components; murine TLR2 and TLR4 recognize mycoplasmal lipopeptides (macrophage-activating lipopeptides, 2 kDa (MALP-2)) and LPS, respectively. Costimulation of mouse peritoneal macrophages with MALP-2 and LPS results in a marked increase in TNF-α production, showing the synergy between TLR2- and TLR4-mediated signaling pathways. Macrophages pretreated with LPS show hyporesponsiveness to the second LPS stimulation, termed LPS tolerance. The LPS tolerance has recently been shown to be primarily due to the down-regulation of surface expression of the TLR4-MD2 complex. When macrophages were treated with MALP-2, the cells showed hyporesponsiveness to the second MALP-2 stimulation, like LPS tolerance. Furthermore, macrophages pretreated with MALP-2 showed reduced production of TNF-α in response to LPS. LPS-induced activation of both NF-κB and c-Jun NH2-terminal kinase was severely impaired in MALP-2-pretreated cells. However, MALP-2-pretreated macrophages did not show any reduction in surface expression of the TLR4-MD2 complex. These findings indicate that LPS-induced LPS tolerance mainly occurs through the down-regulation of surface expression of the TLR4-MD2 complex; in contrast, MALP-2-induced LPS tolerance is due to modulation of the downstream cytoplasmic signaling pathways.
Granulysin, a T Cell Product, Kills Bacteria by Altering Membrane Permeability
Authors: Ernst William A. , Thoma-Uszynski Sybille , Teitelbaum Rachel , Ko Christine , Hanson Dennis A. , Clayberger Carol , Krensky Alan M. , Leippe Matthias , Bloom Barry R. , Ganz Tomas , Modlin Robert L.
Published: April 2014

Granulysin, a protein located in the acidic granules of human NK cells and cytotoxic T cells, has antimicrobial activity against a broad spectrum of microbial pathogens. A predicted model generated from the nuclear magnetic resonance structure of a related protein, NK lysin, suggested that granulysin contains a four α helical bundle motif, with the α helices enriched for positively charged amino acids, including arginine and lysine residues. Denaturation of the polypeptide reduced the α helical content from 49 to 18% resulted in complete inhibition of antimicrobial activity. Chemical modification of the arginine, but not the lysine, residues also blocked the antimicrobial activity and interfered with the ability of granulysin to adhere to Escherichia coli and Mycobacterium tuberculosis. Granulysin increased the permeability of bacterial membranes, as judged by its ability to allow access of cytosolic β-galactosidase to its impermeant substrate. By electron microscopy, granulysin triggered fluid accumulation in the periplasm of M. tuberculosis, consistent with osmotic perturbation. These data suggest that the ability of granulysin to kill microbial pathogens is dependent on direct interaction with the microbial cell wall and/or membrane, leading to increased permeability and lysis.
IL-12-Activated NK Cells Reduce Lung Eosinophilia to the Attachment Protein of Respiratory Syncytial Virus But Do Not Enhance the Severity of Illness in CD8 T Cell-Immunodeficient Conditions
Authors: Hussell Tracy , Openshaw Peter J. M.
Published: April 2014

Bronchiolitis caused by respiratory syncytial virus (RSV) infection is a major cause of hospitalization in children under 1 year of age. RSV causes common colds in older children and adults, but can cause serious disease in immunodeficient patients and the elderly. Development of effective vaccines and treatments for RSV infection is therefore a priority. Because bronchiolitis and vaccine-augmented disease are thought to be caused by exuberant T cell activation, attention has focused on the use of immunomodulators that affect T cell responses. In mice, IL-12 treatment down-regulates type 2 cytokine responses to the attachment protein G of RSV, reducing lung eosinophilia but further enhancing illness. We now show that CD8+ T cells are responsible for enhanced weight loss, whereas IL-12-activated NK cells express high levels of IFN-γ and inhibit lung eosinophilia without causing illness. Moreover, unlike immunocompetent mice, virus is detected in the mediastinal lymph nodes after elimination of both CD8+ T cells and NK cells. These studies show that innate immune responses to viral infections direct the pattern of subsequent specific immunity and are critical to the development of nonpathogenic antiviral effects. We speculate that IL-12 treatment might be beneficial and safe in T cell-deficient patients with RSV pneumonitis.
T Cell-Mediated Tumor Rejection Displays Diverse Dependence Upon Perforin and IFN-γ Mechanisms That Cannot Be Predicted From In Vitro T Cell Characteristics
Authors: Peng Liaomin , Krauss John C. , Plautz Gregory E. , Mukai Shigehiko , Shu Suyu , Cohen Peter A.
Published: April 2014

Experimental pulmonary metastases have been successfully treated by adoptive transfer of tumor-sensitized T cells from perforin knockout (KO) or Fas/APO-1 ligandKO mice, suggesting a prominent role for secretion of cytokines such as IFN-γ. In the present study we confirmed that rejection of established methylcholanthrene-205 (MCA-205) pulmonary metastases displayed a requirement for T cell IFN-γ expression. However, this requirement could be obviated by transferring larger numbers of tumor-sensitized IFN-γ KO T cells or by immunosensitizing sublethal irradiation (500 rad) of the host before adoptive therapy. Extrapulmonary tumors (MCA-205 s.c. and intracranial) that required adjunct sublethal irradiation for treatment efficacy also displayed no requirement for host or T cell expression of IFN-γ. Nonetheless, rejection of MCA-205 s.c. tumors and i.p. EL-4 tumors, but not MCA-205 pulmonary or intracranial tumors, displayed a significant requirement for T cell perforin expression (i.e., CTL participation). The capacity of T cells to lyse tumor targets and secrete IFN-γ in vitro before adoptive transfer was nonpredictive of the roles of these activities in subsequent tumor rejection. Adoptive therapy studies employing KO mice are therefore indispensable for revealing a diversity of tumor rejection mechanisms that may lack in vitro correlation due to delays in their induction. Seemingly contradictory KO data from different studies are reconciled by the capacity of anti-tumor T cells to rely on alternative mechanisms when treated in larger numbers, the variable participation of CTL at different anatomic locations of tumor, and the apparent capacity of sublethal irradiation to provide a therapeutic alternative to host or T cell IFN-γ production.
Evidence for an Accessory Protein Function for Toll-Like Receptor 1 in Anti-Bacterial Responses
Authors: Wyllie D. H. , Kiss-Toth E. , Visintin A. , Smith S. C. , Boussouf S. , Segal D. M. , Duff G. W. , Dower S. K.
Published: April 2014

Members of the Toll-like receptor (TLR) family are components of the mammalian anti-microbial response, signaling with a domain closely related to that of IL-1 receptors. In this report the expression and function of TLR1, a TLR of unknown function, are examined. TLR1 is expressed by monocytes, as demonstrated using a novel mAb. Monocytes also express TLR2. TLR1 transfection of HeLa cells, which express neither TLR1 nor TLR2, was not sufficient to confer responsiveness to several microbial extracts. However, cotransfection of TLR1 and TLR2 resulted in enhanced signaling by HeLa cells to soluble factors released from Neisseria meningitidis relative to the response with either TLR alone. This phenomenon was also seen with high concentrations of some preparations of LPS. The N. meningitidis factors recognized by TLR1/TLR2 were not released by N. meningitidis mutant in the LpxA gene. Although LpxA is required for LPS biosynthesis, because cooperation between TLR1 and TLR2 was not seen with all LPS preparations, the microbial component(s) TLR1/2 recognizes is likely to be a complex of LPS and other molecules or a compound metabolically and chemically related to LPS. The functional IL-1R consists of a heterodimer; this report suggests a similar mechanism for TLR1 and TLR2, for certain agonists. These data further suggest that mammalian responsiveness to some bacterial products may be mediated by combinations of TLRs, suggesting a mechanism for diversifying the repertoire of Toll-mediated responses.
Vγ9Vδ2 T Cells Impair Intracellular Multiplication ofBrucella suisin Autologous Monocytes Through Soluble Factor Release and Contact-Dependent Cytotoxic Effect
Authors: Ottones Florence , Dornand Jacques , Naroeni Aroem , Liautard Jean-Pierre , Favero Jean
Published: April 2014

Human Vγ9Vδ2 T cells are considered to play an important role in brucellosis, as this population is dramatically increased in peripheral blood of patients during the acute phase of the infection. This T lymphocyte population has been largely demonstrated to be activated by small m.w. nonpeptidic molecules from natural or synthetic origin. We recently identified a nonpeptidic fraction of Brucella suis that specifically activates human Vγ9Vδ2 T cells. Using a two-separate-chambers system, we showed that Brucella fraction, as well as isopentenyl pyrophosphate-activated Vγ9Vδ2 T cells, impaired the multiplication of B. suis in differentiated THP-1 cells through TNF-α and IFN-γ release. In the present study, using circulating Vγ9Vδ2 T cells and autologous monocytes infected with B. suis, we provide evidence that 1) intramonocytic multiplication of B. suis is impaired by supernatants of activated Vγ9Vδ2 T cells in part via TNF-α and IFN-γ, this impairment occurring without host cell lysis; 2) unstimulated Vγ9Vδ2 T cells can impair intracellular bacterial multiplication after their activation by soluble factors released by infected monocytes; and 3) activated Vγ9Vδ2 T cells lyse Brucella-infected monocytes in a contact-dependent manner. Taken together, these results provide evidence that Vγ9Vδ2 T cells, in addition to being directly activated by soluble nonpeptidic molecules, can be stimulated to become highly cytotoxic in the specific presence of infected monocytes; moreover, they suggest how Vγ9Vδ2 T cells could be triggered and respond as antibacterial effector cells in the early stages of Brucella infection.
T Cell Expression Cloning of aMycobacterium tuberculosisGene Encoding a Protective Antigen Associated with the Early Control of Infection
Authors: Skeiky Yasir A. W. , Ovendale Pamela J. , Jen Shyian , Alderson Mark R. , Dillon Davin C. , Smith Sherilyn , Wilson Christopher B. , Orme Ian M. , Reed Steven G. , Campos-Neto Antonio
Published: April 2014

Infection of C57BL/6 mice with Mycobacterium tuberculosis results in the development of a progressive disease during the first 2 wk after challenge. Thereafter, the disease is controlled by the emergence of protective T cells. We have used this infection model in conjunction with direct T cell expression cloning to identify Ags involved with the early control of the disease. A protective M. tuberculosis-specific CD4 T cell line derived from mice at 3 wk postchallenge was used to directly screen an M. tuberculosis genomic expression library. This screen resulted in the identification of a genomic clone comprising two putative adjacent genes with predicted open reading frames of 10 and 41 kDa, MTB10 and MTB41, respectively (the products of Rv0916c and Rv0915c, respectively, in the TubercuList H37Rv database). MTB10 and MTB41 belong to the PE and PPE family of proteins recently identified to comprise 10% of the M. tuberculosis genome. Evaluation of the recombinant proteins revealed that MTB41, but not MTB10, is the Ag recognized by the cell line and by M. tuberculosis-sensitized human PBMC. Moreover, C57BL/6 mice immunized with MTB41 DNA developed both CD4- (predominantly Th1) and CD8-specific T cell responses to rMTB41 protein. More importantly, immunization of C57BL/6 mice with MTB41 DNA induced protection against infection with M. tuberculosis comparable to that induced by bacillus Calmette-Guérin. Thus, the use of a proven protective T cell line in conjunction with the T cell expression cloning approach resulted in the identification of a candidate Ag for a subunit vaccine against tuberculosis.
Inhibition of TNF-α Produced by Kupffer Cells Protects Against the Nonspecific Liver Toxicity of Immunotoxin Anti-Tac(Fv)-PE38, LMB-2
Authors: Onda Masanori , Willingham Mark , Wang Qing-cheng , Kreitman Robert J. , Tsutsumi Yasuo , Nagata Satoshi , Pastan Ira
Published: April 2014

LMB-2 (anti-Tac(Fv)-PE38) is a recombinant immunotoxin composed of the Fv fragment of the anti-Tac Ab fused to a 38-kDa form of Pseudomonas exotoxin A. Recent clinical trials showed that LMB-2 is a promising agent for the treatment of patients with Tac-positive leukemia or lymphoma. One major side effect that needs to be overcome is nonspecific liver toxicity. In the current study, we have analyzed the mechanism of this toxicity using a mouse model. Mice that were injected with a lethal dose of LMB-2 showed severe hepatic necrosis. Immunohistochemistry revealed that LMB-2 accumulated in Kupffer cells in the liver, suggesting that the damage to the hepatocytes was indirect. When we examined the effects of LMB-2 on peritoneal macrophages, cells in the same lineage as Kupffer cells, we found that LMB-2 induced the production of TNF-α by these cells. Following LMB-2 administration to mice, the levels of TNF-α in the liver increased to very high levels, whereas the rise in serum levels was modest. In addition, the LMB-2-induced liver toxicity was blocked by a specific TNF binding protein (TNFsRp55). Liver toxicity was also blocked by indomethacin, which also blocked the rise of TNF-α in the liver. Both TNFsRp55 and indomethacin treatment protected mice against a lethal dose of LMB-2. These data indicate that TNF-α produced in the liver by Kupffer cells has an important causal role in the nonspecific liver toxicity of LMB-2. These findings have important clinical implications for the use of immunotoxins in the therapy of patients with cancer.
Molecular Mechanisms of High-Dose Antigen Therapy in Experimental Autoimmune Encephalomyelitis: Rapid Induction of Th1-Type Cytokines and Inducible Nitric Oxide Synthase
Authors: Weishaupt Andreas , Jander Sebastian , Brück Wolfgang , Kuhlmann Tanja , Stienekemeier Martina , Hartung Thomas , Toyka Klaus V. , Stoll Guido , Gold Ralf
Published: April 2014

High-dose Ag administration induces apoptotic death of autoreactive T cells and is an effective therapy of experimental autoimmune diseases of the nervous system. To explore the role of cytokines in Ag-specific immunotherapy, we analyzed mRNA induction and protein expression for the proinflammatory cytokines TNF-α and IFN-γ, the anti-inflammatory cytokine IL-10, and the cytokine-inducible NO synthase (iNOS) during high-dose Ag therapy of adoptive transfer experimental autoimmune encephalomyelitis (AT-EAE) in the Lewis rat. Using semiquantitative and competitive RT-PCR, we found 5- to 6-fold induction of TNF-α mRNA and 3-fold induction of IFN-γ mRNA in the spinal cord that occurred within 1 h after i.v. injection of Ag and was accompanied by a 2-fold increase of iNOS mRNA. Both IFN-γ and iNOS mRNA remained elevated for at least 6 h, whereas TNF-α mRNA was already down-regulated 6 h after Ag injection. A comparable time course was found for circulating serum levels of TNF-α and IFN-γ. IL-10 mRNA levels did not change significantly following Ag injection. Neutralization of TNF-α by anti-TNF-α antiserum in vivo led to a significant decrease in the rate of T cell and oligodendrocyte apoptosis induced by high-dose Ag administration, but did not change the beneficial clinical effect of Ag therapy. Our data suggest profound activation of proinflammatory but not of anti-inflammatory cytokine gene expression by high-dose Ag injection. Functionally, TNF-α contributes to increased apoptosis of both autoaggressive T cells and oligodendrocytes in the target organ and may thereby play a dual role in this model of Ag-specific therapy of CNS autoimmune diseases.
L-Selectin Facilitates Emigration and Extravascular Locomotion of Leukocytes During Acute Inflammatory Responses In Vivo
Authors: Hickey Michael J. , Forster Melanie , Mitchell Debra , Kaur Jaswinder , De Caigny Catherine , Kubes Paul
Published: April 2014

L-selectin has been shown to be important in mediating leukocyte recruitment during inflammatory responses. Although there are numerous in vitro studies demonstrating that engagement of L-selectin leads to the activation of several signaling pathways potentially contributing to subsequent adhesion, emigration, or even migration through the interstitium, whether this actually induces cellular events in vivo is completely unknown. Therefore, we used intravital microscopy to visualize the role of L-selectin in downstream leukocyte adhesion, emigration, and interstitial migration events in wild-type and L-selectin-deficient (L-selectin−/−) mice. The cremaster muscle was superfused with the chemotactic inflammatory mediators platelet-activating factor or KC. Leukocyte rolling, adhesion, and emigration in postcapillary venules were examined, and the migration of emigrated leukocytes was recorded continuously using time-lapse videomicroscopy. Platelet-activating factor increased leukocyte adhesion to a similar level in both wild-type and L-selectin−/− mice. In contrast, both the number of emigrated leukocytes and the distance of extravascular migration were significantly reduced in L-selectin−/− mice. A similar pattern was observed in response to the superfusion of KC. Because superfusion of these mediators induced chemokinesis, we developed a new in vivo chemotaxis assay using slow release of KC from an agarose gel positioned 350 μm from a postcapillary venule. These experiments showed that L-selectin−/− leukocytes were also severely impaired in their ability to respond to a directional cue. These findings indicate that L-selectin is important in enabling leukocytes to respond effectively to chemotactic stimuli in inflamed tissues.
Tat Protein Is an HIV-1-Encoded β-Chemokine Homolog That Promotes Migration and Up-Regulates CCR3 Expression on Human FcεRI+ Cells
Authors: de Paulis Amato , De Palma Raffaele , Di Gioia Luisa , Carfora Maria , Prevete Nella , Tosi Giovanna , Accolla Roberto S. , Marone Gianni
Published: April 2014

Human basophils and mast cells express the chemokine receptor CCR3, which binds the chemokines eotaxin and RANTES. HIV-1 Tat protein is a potent chemoattractant for basophils and lung mast cells obtained from healthy individuals seronegative for Abs to HIV-1 and HIV-2. Tat protein induced a rapid and transient Ca2+ influx in basophils and mast cells, analogous to β-chemokines. Tat protein neither induced histamine release from human basophils and mast cells nor increased IL-3-stimulated histamine secretion from basophils. The chemotactic activity of Tat protein was blocked by preincubation of FcεRI+ cells with anti-CCR3 Ab. Preincubation of Tat with a mAb anti-Tat (aa 1–86) blocked the migration induced by Tat. In contrast, a mAb specific for the basic region (aa 46–60) did not inhibit the chemotactic effect of Tat protein. Tat protein or eotaxin desensitized basophils to a subsequent challenge with the autologous or the heterologous stimulus. Preincubation of basophils with Tat protein up-regulated the level of CCR3 mRNA and the surface expression of the CCR3 receptor. Tat protein is the first identified HIV-1-encoded β-chemokine homologue that influences the directional migration of human FcεRI+ cells and the expression of surface receptor CCR3 on these cells.
Vitamin C Inhibits NF-κB Activation by TNF Via the Activation of p38 Mitogen-Activated Protein Kinase
Authors: Bowie Andrew G. , O’Neill Luke A. J.
Published: April 2014

The transcription factor NF-κB is a central mediator of altered gene expression during inflammation, and is implicated in a number of pathologies, including cancer, atherosclerosis, and viral infection. We report in this study that vitamin C inhibits the activation of NF-κB by multiple stimuli, including IL-1 and TNF in the endothelial cell line ECV304 and in primary HUVECs. The induction of a NF-κB-dependent gene, IL-8, by TNF was also inhibited. The effect requires millimolar concentrations of vitamin C, which occur intracellularly in vivo, particularly during inflammation. Vitamin C was not toxic to cells, did not inhibit another inducible transcription factor, STAT1, and had no effect on the DNA binding of NF-κB. Inhibition by vitamin C was not simply an antioxidant effect, because redox-insensitive pathways to NF-κB were also blocked. Vitamin C was shown to block IL-1- and TNF-mediated degradation and phosphorylation of I-κBα (inhibitory protein that dissociates from NF-κB), due to inhibition of I-κB kinase (IKK) activation. Inhibition of TNF-driven IKK activation was mediated by p38 mitogen-activated protein kinase, because treatment of cells with vitamin C led to a rapid and sustained activation of p38, and the specific p38 inhibitor SB203580 reversed the inhibitory effect of vitamin C on IKK activity, I-κBα phosphorylation, and NF-κB activation. The results identify p38 as an intracellular target for high dose vitamin C.
Endotoxin Activation of Macrophages Does Not Induce ATP Release and Autocrine Stimulation of P2 Nucleotide Receptors
Authors: Beigi Reza D. , Dubyak George R.
Published: April 2014

Receptors for extracellular nucleotides (P2, or purinergic receptors) have previously been implicated in the transduction of endotoxin signaling in macrophages. The most compelling evidence has been the observation that inhibitors of ionotropic nucleotide (P2X) receptors, including periodate-oxidized ATP (oATP), attenuate a subset of endotoxin-induced effects such as activation of NF-κB and up-regulation of inducible NO synthase. We investigated whether endotoxin induces ATP release from a murine macrophage cell line (BAC1.2F5) using sensitive on-line assays for extracellular ATP. These cells constitutively released ATP, producing steady-state extracellular concentrations of ∼1 nM when assayed as monolayers of 106 adherent cells bathed in 1 ml of medium. However, the macrophages did not release additional ATP during either acute or prolonged endotoxin stimulation. In addition, cellular ecto-ATPase activities were measured following prolonged endotoxin activation and were found not to be significantly altered. Although oATP treatment significantly attenuated the endotoxin-induced production of NO, this inhibitory effect was not reproduced when the cells were coincubated with apyrase, a highly effective ATP scavenger. These results indicate that activation of macrophages by endotoxin does not induce autocrine stimulation of P2 nucleotide receptors by endogenous ATP released to extracellular compartments. Moreover, the data suggest that the ability of oATP to interfere with endotoxin signaling is due to its interaction with molecular species other than ATP-binding P2 receptors.
Regulation of IL-6 and IL-8 Expression in Rheumatoid Arthritis Synovial Fibroblasts: the Dominant Role for NF-κB But Not C/EBPβ or c-Jun
Authors: Georganas Constantinos , Liu Hongtao , Perlman Harris , Hoffmann Alexander , Thimmapaya Bayar , Pope Richard M.
Published: April 2014

Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) produce IL-6 and IL-8, which contribute to inflammation and joint damage. The promoters of both cytokines possess binding sites for NF-κB, C/EBPβ, and c-Jun, but the contribution of each to the regulation of IL-6 and IL-8 in RA FLS is unknown. We employed adenoviral-mediated gene delivery of a nondegradable IκBα, or dominant-negative versions of C/EBPβ or c-Jun, to determine the contribution of each transcription factor to IL-6 and IL-8 expression. Inhibition of NF-κB activation significantly reduced the spontaneous and IL-1β-induced secretion of IL-6 and IL-8 by RA FLS and the IL-1β-induced production of IL-6 and IL-8 by human dermal fibroblasts. Inhibition of C/EBPβ modestly reduced constitutive and IL-1β-induced IL-6 by RA FLS, but not by human dermal fibroblasts, and had no effect on IL-8. Inhibition of c-Jun/AP-1 had no effect on the production of either IL-6 or IL-8. Employing gel shift assays, NF-κB, C/EBPβ, and c-Jun were constitutively activated in RA FLS, but only NF-κB and c-Jun activity increased after IL-1β. The reduction of cytokines by IκBα was mediated through inhibition of NF-κB activation, which resulted in decreased IL-6 and IL-8 mRNA. NF-κB was essential for IL-6 expression, because fibroblasts in which both NF-κB p50/p65 genes were deleted failed to express IL-6 in response to IL-1. These findings document the importance of NF-κB for the regulation of the constitutive and IL-1β-stimulated expression of IL-6 and IL-8 by RA FLS and support the role of inhibition of NF-κB as a therapeutic goal in RA.
The Efficacy of Immunotherapy in an Experimental Murine Model of Allergic Asthma Is Related to the Strength and Site of T Cell Activation During Immunotherapy
Authors: Janssen Edith M. , van Oosterhout Antoon J. M. , Nijkamp Frans P. , van Eden Willem , Wauben Marca H. M.
Published: April 2014

In the present study, the relation between the efficacy of immunotherapy, and the strength and site of T cell activation during immunotherapy was evaluated. We used a model of allergic asthma in which OVA-sensitized and OVA-challenged mice display increased airway hyperresponsiveness, airway inflammation, and Th2 cytokine production by OVA-specific T cells. In this model, different immunotherapy strategies, including different routes of administration, or treatment with entire OVA or the immunodominant T cell epitope OVA323–339, or treatment with a peptide analogue of OVA323–339 with altered T cell activation capacity were studied. To gain more insight in how immunotherapy affects allergen-specific T cells, the site of Ag-specific T cell activation and the magnitude of the T cell response induced during different immunotherapy strategies were determined using an adoptive transfer model. Our data suggest that amelioration of airway hyperresponsiveness and inflammation is associated with the induction of a strong, synchronized, and systemic T cell response, resulting in a decreased OVA-specific Th2 response. In contrast, deterioration of the disease after immunotherapy is associated with the induction of a weak nonsynchronized T cell response, resulting in the enhancement of the OVA-specific Th2 response after challenge.
Chemokine Production by G Protein-Coupled Receptor Activation in a Human Mast Cell Line: Roles of Extracellular Signal-Regulated Kinase and NFAT
Authors: Ali Hydar , Ahamed Jasimuddin , Hernandez-Munain Cristina , Baron Jonathan L. , Krangel Michael S. , Patel Dhavalkumar D.
Published: April 2014

Chemoattractants are thought to be the first mediators generated at sites of bacterial infection. We hypothesized that signaling through G protein-coupled chemoattractant receptors may stimulate cytokine production. To test this hypothesis, a human mast cell line (HMC-1) that normally expresses receptors for complement components C3a and C5a at low levels was stably transfected to express physiologic levels of fMLP receptors. We found that fMLP, but not C3a or C5a, induced macrophage inflammatory protein (MIP)-1β (CCL4) and monocyte chemoattractant protein-1 (CCL2) mRNA and protein. Although fMLP stimulated both sustained Ca2+ mobilization and phosphorylation of extracellular signal-regulated kinase (ERK), these responses to C3a or C5a were transient. However, transient expression of C3a receptors in HMC-1 cells rendered the cells responsive to C3a for sustained Ca2+ mobilization and MIP-1β production. The fMLP-induced chemokine production was blocked by pertussis toxin, PD98059, and cyclosporin A, which respectively inhibit Giα activation, mitgen-activated protein kinase kinase-mediated ERK phosphorylation, and calcineurin-mediated activation of NFAT. Furthermore, fMLP, but not C5a, stimulated NFAT activation in HMC-1 cells. These data indicate that chemoattractant receptors induce chemokine production in HMC-1 cells with a selectivity that depends on the level of receptor expression, the length of their signaling time, and the synergistic interaction of multiple signaling pathways, including extracellular signal-regulated kinase phosphorylation, sustained Ca2+ mobilization and NFAT activation.
Basophil Responses to Chemokines Are Regulated by Both Sequential and Cooperative Receptor Signaling
Authors: Heinemann Akos , Hartnell Adele , Stubbs Victoria E. L. , Murakami Kazuki , Soler Dulce , LaRosa Gregory , Askenase Philip W. , Williams Timothy J. , Sabroe Ian
Published: April 2014

To investigate human basophil responses to chemokines, we have developed a sensitive assay that uses flow cytometry to measure leukocyte shape change as a marker of cell responsiveness. PBMC were isolated from the blood of volunteers. Basophils were identified as a single population of cells that stained positive for IL-3Rα (CDw123) and negative for HLA-DR, and their increase in forward scatter (as a result of cell shape change) in response to chemokines was measured. Shape change responses of basophils to chemokines were highly reproducible, with a rank order of potency: monocyte chemoattractant protein (MCP) 4 (peak at <1 nM) ≥ eotaxin-2 = eotaxin-3 ≥ eotaxin > MCP-1 = MCP-3 > macrophage-inflammatory protein-1α > RANTES = MCP-2 = IL-8. The CCR4-selective ligand macrophage-derived chemokine did not elicit a response at concentrations up to 10 nM. Blocking mAbs to CCR2 and CCR3 demonstrated that responses to higher concentrations (>10 nM) of MCP-1 were mediated by CCR3 rather than CCR2, whereas MCP-4 exhibited a biphasic response consistent with sequential activation of CCR3 at lower concentrations and CCR2 at 10 nM MCP-4 and above. In contrast, responses to MCP-3 were blocked only in the presence of both mAbs, but not after pretreatment with either anti-CCR2 or anti-CCR3 mAb alone. These patterns of receptor usage were different from those seen for eosinophils and monocytes. We suggest that cooperation between CCRs might be a mechanism for preferential recruitment of basophils, as occurs in tissue hypersensitivity responses in vivo.
Up-Regulation of the IL-12 Receptor β2 Chain in Crohn’s Disease
Authors: Parrello Tiziana , Monteleone Giovanni , Cucchiara Salvatore , Monteleone Ivan , Sebkova Ladislava , Doldo Patrizia , Luzza Francesco , Pallone Francesco
Published: April 2014

Crohn’ s disease (CD) is a chronic intestinal inflammatory disorder characterized by aberrant mucosal Th1 cell activation and production of IL-12, the major Th1-driving factor. The T cell response to IL-12 is dependent on the expression of a specific receptor composed of two subunits, termed IL-12Rβ1 and IL-12Rβ2. The content of IL-12Rβ2, as measured at the mRNA level, is crucial in regulating Th1 differentiation. In this study we therefore investigated IL-12Rβ2 RNA transcripts in CD. IL-12Rβ2 expression was increased in active CD as well as Helicobacter pylori (HP)-associated gastritis and Salmonella colitis compared with that in inactive CD, ulcerative colitis, noninflammatory controls, and celiac disease. In contrast, IL-12Rβ1 transcripts were expressed at comparable levels in all samples. In CD, IL-12Rβ2 expression strictly correlated with tyrosine phosphorylation of STAT4, a key component of the IL-12-dependent Th1 polarization. This was associated with a pronounced expression of IFN-γ. Transcripts for IL-12/p40 were detected in CD, HP-positive, and Salmonella colitis patients, but not in celiac disease, indicating that IL-12Rβ2 up-regulation occurs only in IL-12-associated Th1 gastrointestinal diseases. Finally, we showed that stimulation of lamina propria mononuclear cells with IL-12 enhanced IL-12Rβ2, suggesting that IL-12 regulates IL-12Rβ2 expression in human gastrointestinal mucosa. The data show that the signaling pathway used by IL-12 to induce Th1 differentiation is increased at the site of disease in CD, further supporting the view that IL-12/IL-12R signals contribute to the inflammatory response in this condition.
Evaluation of TNF-α and IL-1 Blockade in Collagen-Induced Arthritis and Comparison with Combined Anti-TNF-α/Anti-CD4 Therapy
Authors: Williams Richard O. , Marinova-Mutafchieva Lilia , Feldmann Marc , Maini Ravinder N.
Published: April 2014

We have evaluated the effects of anti-TNF-α, anti-IL-1, and combined anti-TNF-α/anti-CD4 therapy in collagen-induced arthritis. Blockade of TNF-α or IL-1 before disease onset delayed, but did not prevent, the induction of arthritis. When treatment was initiated after onset of arthritis, anti-TNF-α, anti-IL-1β, and anti-IL-1R (which blocks IL-1α and IL-1β) were all found to be effective in reducing the severity of arthritis, with anti-IL-1R and anti-IL-1β showing greater efficacy than anti-TNF-α. Anti-IL-1β was equally as effective as anti-IL-1R, indicating that IL-1β plays a more prominent role than IL-1α in collagen-induced arthritis. An additive effect was observed between anti-TNF-α and anti-IL-1R in the prevention of joint erosion and in normalization of the levels of serum amyloid P. Combined anti-TNF-α/anti-CD4 therapy also caused normalization of serum amyloid P levels. The therapeutic effect of anti-TNF-α plus anti-CD4 was comparable to that of anti-TNF-α plus anti-IL-1R, suggesting that combined anti-TNF-α/anti-CD4 therapy prevents both TNF-α- and IL-1-mediated pathology. Anti-TNF-α treatment reduced IL-1β expression in the joint and, conversely, anti-IL-1β treatment reduced TNF-α expression. Combined anti-TNF-α/anti-CD4 treatment almost completely blocked the expression of IL-1β, thereby confirming the ability of this form of combination therapy to prevent IL-1β-mediated pathology.
Adenoviral Transfer of Cyclin-Dependent Kinase Inhibitor Genes Suppresses Collagen-Induced Arthritis in Mice
Authors: Nasu Kimio , Kohsaka Hitoshi , Nonomura Yoshinori , Terada Yoshio , Ito Hiroshi , Hirokawa Katsuiku , Miyasaka Nobuyuki
Published: April 2014

In rheumatoid synovial tissues, synovial fibroblasts are activated by proinflammatory cytokines and proliferate to develop hyperplastic pannus tissues, which irreversibly damage the affected joints. We recently reported that the cyclin-dependent kinase inhibitors p16INK4a and p21Cip1 are not expressed in vivo in rheumatoid synovial fibroblasts, but are readily inducible in vitro. This observation was followed by the successful treatment of rat adjuvant arthritis by local p16INK4a gene transfer, showing that the inhibition of the cell cycle of the synovial cells ameliorates the arthritis. In this study, we show that another animal model of rheumatoid arthritis, murine collagen-induced arthritis, can be effectively treated by local gene transfer of p21Cip1 as well as that of p16INK4a. The anti-arthritic effects were observed even when the treatment was conducted after the arthritis had developed. Furthermore, the effects included suppression of the expression of proinflammatory cytokines such as IL-1β, IL-6, and TNF-α. Our results demonstrate that the ectopic expression of cyclin-dependent kinase inhibitors not only prevents synovial overgrowth but also ameliorates the proinflammatory milieu in the affected joints. The induction of p21Cip1 in rheumatoid synovial tissues by pharmacological agents may also be an effective strategy to treat rheumatoid arthritis.
Efficient Simultaneous Presentation of NY-ESO-1/LAGE-1 Primary and Nonprimary Open Reading Frame-Derived CTL Epitopes in Melanoma
Authors: Rimoldi Donata , Rubio-Godoy Verena , Dutoit Valerie , Lienard Danielle , Salvi Suzanne , Guillaume Philippe , Speiser Daniel , Stockert Elisabeth , Spagnoli Giulio , Servis Catherine , Cerottini Jean-Charles , Lejeune Ferdy , Romero Pedro , Valmori Danila
Published: April 2014

Recent studies have shown that CTL epitopes derived from tumor-associated Ags can be encoded by both primary and nonprimary open reading frames (ORF). In this study we have analyzed the HLA-A2-restricted CD8+ T cell response to a recently identified CTL epitope derived from an alternative ORF product of gene LAGE-1 (named CAMEL), and the highly homologous gene NY-ESO-1 in melanoma patients. Using MHC/peptide tetramers we detected CAMEL1–11-specific CD8+ T cells in peptide-stimulated PBMC as well as among tumor-infiltrated lymph node cells from several patients. Sorting and expansion of tetramer+ CD8+ T cells allowed the isolation of tetramerbright and tetramerdull populations that specifically recognized the peptide Ag with high and low avidity, respectively. Remarkably, only high avidity CAMEL-specific CTL were able to recognize Ag-expressing tumor cells. A large series of HLA-A2-positive melanoma cell lines was characterized for the expression of LAGE-1 and NY-ESO-1 mRNA and protein and tested for recognition by CAMEL-specific CTL as well as CTL that recognize a peptide (NY-ESO-1157–165) encoded by the primary ORF products of the LAGE-1 and NY-ESO-1 genes. This analysis revealed that tumor-associated CD8+ T cell epitopes are simultaneously and efficiently generated from both primary and nonprimary ORF products of LAGE-1 and NY-ESO-1 genes and, importantly, that this occurs in the majority of melanoma tumors. These findings underscore the in vivo immunological relevance of CTL epitopes derived from nonprimary ORF products and support their use as candidate vaccines for inducing tumor specific cell-mediated immunity against cancer.
Early Requirement for B Cells for Development of Spontaneous Autoimmune Thyroiditis in NOD.H-2h4 Mice
Authors: Braley-Mullen Helen , Yu Shiguang
Published: April 2014

B cells are known to play an important role in the pathogenesis of several autoimmune diseases. NOD.H-2h4 mice develop spontaneous autoimmune thyroiditis (SAT) and anti-mouse thyroglobulin (MTg) autoantibodies, the levels of which correlate closely with the severity of thyroid lesions. NOD.H-2h4 mice genetically deficient in B cells (NOD.Kμnull) or rendered B cell-deficient by treatment from birth with anti-IgM develop minimal SAT. B cells were required some time in the first 4–6 wk after birth, because NOD.Kμnull or NOD.H-2h4 mice did not develop SAT when they were reconstituted with B cells as adults. The requirement for B cells was apparently not solely to produce anti-MTg autoantibodies, because passive transfer of anti-MTg Ab did not enable B cell-deficient mice to develop SAT, and mice given B cells as adults produced autoantibodies but did not develop SAT. B cell-deficient mice developed SAT if their T cells developed from bone marrow precursors in the presence of B cells. Because B cells are required early in life and their function cannot be replaced by anti-MTg autoantibodies, B cells may be required for the activation or selection of autoreactive T cells. These autoreactive T cells are apparently unable to respond to Ag if B cells are absent in the first 4–6 wk after birth.
Highly Autoproliferative T Cells Specific for 60-kDa Heat Shock Protein Produce IL-4/IL-10 and IFN-γ and Are Protective in Adjuvant Arthritis
Authors: Paul Alberta G. A. , van Kooten Peter J. S. , van Eden Willem , van der Zee Ruurd
Published: April 2014

Previously we have shown that T cell responses to the mycobacterial 60-kDa heat shock protein (hsp60) peptide M256–270 mediated protection against adjuvant arthritis in Lewis rats. We have demonstrated now that M256–270-primed T cells become highly reactive to naive syngeneic APC upon repetitive restimulation in vitro with peptide M256–265, comprising the conserved core of peptide M256–270. These autoproliferative responses in the absence of added Ag were MHC class II restricted and resulted in the production of IL-4/IL-10 and IFN-γ. Enhanced autoproliferation and expression of the cell surface molecule B7.2 by these T cells were observed in response to syngeneic heat-shocked APC, which indicated that the autoproliferation and expression of B7.2 resulted from the recognition of endogenously expressed and processed hsp. Despite their strong autoreactivity, upon transfer such T cells were found to induce a significant disease reduction in adjuvant arthritis. In contrast, T cells both primed and restimulated with peptide M256–270 became unresponsive toward syngeneic APC as well as toward the conserved core peptide M256–265, and they were devoid of protective capacity. This study demonstrates that the loss of self-tolerance toward hsp60 does not necessarily lead to autoimmune disease, but that hsp60-specific self-reactive and autoproliferative T cells may mediate T cell regulation in arthritis.
The Influence of HLA Class I Alleles and Heterozygosity on the Outcome of Human T Cell Lymphotropic Virus Type I Infection
Authors: Jeffery Katie J. M. , Siddiqui Asna A. , Bunce Mike , Lloyd Alun L. , Vine Alison M. , Witkover Aviva D. , Izumo Shuji , Usuku Koichiro , Welsh Kenneth I. , Osame Mitsuhiro , Bangham Charles R. M.
Published: April 2014

The inflammatory disease human T cell lymphotropic virus type I (HTLV-I)-associated myelopathy (HAM/TSP) occurs in only 1–2% of HTLV-I-infected individuals and is associated with a high provirus load of HTLV-I. We hypothesize that a person’s risk of developing HAM/TSP depends upon the efficiency of their immune response to the virus, which differs between individuals because of polymorphism in genes that influence this response. Previously we showed that the possession of HLA-A*02 was associated with a lower risk of HAM/TSP, and with a lower provirus load in healthy carriers of HTLV-I. However, HLA-A*02 did not account for all the observed difference in the risk of HAM/TSP. Here we present evidence, in the same study population in Japan, that HLA-Cw*08 was also associated with disease protection (probability value, two-tailed test = 0.002) and with a lower proviral load in healthy carriers. Possession of the A*02 and/or Cw*08 genes prevented 36% of potential HAM/TSP cases. In contrast, HLA-B*5401 was associated with a higher susceptibility to HAM/TSP (probability value, two-tailed test = 0.0003) and with a higher provirus load in HAM/TSP patients. At a given provirus load, B*5401 appeared to increase the risk of disease. The fraction of HAM/TSP cases attributable to B*5401 was 17%. Furthermore, individuals who were heterozygous at all three HLA class I loci have a lower HTLV-I provirus load than those who were homozygous at one or more loci. These results are consistent with the proposal that a strong class I-restricted CTL response to HTLV-I reduces the proviral load and hence the risk of disease.