bluuebunny/crossref_metadata_2025 huggingface dataset:
Specific Inhibition of Glucocorticoid-Induced Thymocyte Apoptosis by Substance P
Authors: Dimri Rachel , Sharabi Yedida , Shoham Jacob
Published: April 2014

Glucocorticoids (GC) are strong inducers of thymocyte apoptosis. In the present study we looked into the possibility that the neuropeptide substance P (SP) might serve as an antagonist to GC-induced apoptosis. Indeed, SP inhibited hydrocortisone (HC)-induced apoptosis of CD4+CD8+ thymocytes in mice, both in vivo and in vitro. It also inhibited HC-induced apoptosis in the T cell hybridoma line 2B4.11, which is sensitive to GC. The inhibitory effect was complete if SP was given with HC or within 1 h after it; partial inhibitory effect could be seen at 2 h and no effect at 3 h. The presence of the SP antagonist nullified SP effect. The effect was specific to both components of the system (i.e., HC as apoptosis inducer and SP as its inhibitor), as judged from comparison to three other apoptosis-inducing means (irradiation, thymic epithelial cells, or retinoic acid), and to two other neuropeptides (somatostatin and vasoactive intestinal peptide). SP/HC antagonism was further demonstrated in two relevant molecular events: 1) HC augmented GC receptor production in our cell system and this was inhibited by SP; and 2) HC reduced the expression of the transcription factor NF-κB, SP increased it and when both were present, SP effect dominated. On the other hand, the level of IκB (NF-κB inhibitory molecule) was decreased by SP, preserved at a relatively high level with HC, and when both SP and HC were present, SP effect dominated. The intensity of SP effect, both in vivo and in vitro, its specificity, its inhibition by SP antagonist, as well as the previously documented presence of SP and its receptor in the thymus suggest that SP might be a physiological antagonist of the potent thymocyte apoptosis induced by GC.
Oral Administration of Hapten Inhibits In Vivo Induction of Specific Cytotoxic CD8+ T Cells Mediating Tissue Inflammation: A Role for Regulatory CD4+ T Cells
Authors: Desvignes Cyril , Etchart Nathalie , Kehren Jeanne , Akiba Itoshi , Nicolas Jean-François , Kaiserlian Dominique
Published: April 2014

We investigated whether oral tolerance could block the development of an inflammatory response mediated by CD8+ T cells, using a mouse model of oral tolerance of contact sensitivity (CS) to the hapten 2,4-dinitrofluorobenzene (DNFB). In this system, the skin inflammatory response is initiated by hapten-specific class I-restricted cytotoxic CD8+ T (CTL) cells, independently of CD4 help. Oral delivery of DNFB before skin sensitization blocked the CS response by impairing the development of DNFB-specific CD8+ effector T cells in secondary lymphoid organs. This was shown by complete inhibition of DNFB-specific CTL and proliferative responses of CD8+ T cells, lack of specific IFN-γ-producing CD8+ T cells, and inability of CD8+ T cells to transfer CS in RAG20/0 mice. RT-PCR and immunohistochemical analysis confirmed that recruitment of CD8+ effectors of CS in the skin at the site of hapten challenge was impaired in orally tolerized mice. Sequential anti-CD4 Ab treatment showed that only depletion of CD4+ T cells during the afferent phase of CS abrogated oral tolerance induction by restoring high numbers of specific CD8+ effectors in lymphoid organs, whereas CD4 depletion during the efferent phase of CS did not affect oral tolerance. These data demonstrate that a single intragastric administration of hapten can block in vivo induction of DNFB-specific CD8+ CTL responsible for tissue inflammation and that a subset of regulatory CD4+ T cells mediate oral tolerance by inhibiting expansion of specific CD8+ effectors in lymph nodes.
Significant Role for Fas in the Pathogenesis of Autoimmune Diabetes
Authors: Su Xiao , Hu Qile , Kristan Jane M. , Costa Cristina , Shen Yamin , Gero Demokos , Matis Louis A. , Wang Yi
Published: April 2014

Programmed cell death represents an important pathogenic mechanism in various autoimmune diseases. Type I diabetes mellitus (IDDM) is a T cell-dependent autoimmune disease resulting in selective destruction of the β cells of the islets of Langerhans. β cell apoptosis has been associated with IDDM onset in both animal models and newly diagnosed diabetic patients. Several apoptotic pathways have been implicated in β cell destruction, including Fas, perforin, and TNF-α. Evidence for Fas-mediated lysis of β cells in the pathogenesis of IDDM in nonobese diabetic (NOD) mice includes: 1) Fas-deficient NOD mice bearing the lpr mutation (NOD-lpr/lpr) fail to develop IDDM; 2) transgenic expression of Fas ligand (FasL) on β cells in NOD mice may result in accelerated IDDM; and 3) irradiated NOD-lpr/lpr mice are resistant to adoptive transfer of diabetes by cells from NOD mice. However, the interpretation of these results is complicated by the abnormal immune phenotype of NOD-lpr/lpr mice. Here we present novel evidence for the role of Fas/FasL interactions in the progression of NOD diabetes using two newly derived mouse strains. We show that NOD mice heterozygous for the FasL mutation gld, which have reduced functional FasL expression on T cells but no lymphadenopathy, fail to develop IDDM. Further, we show that NOD-lpr/lpr mice bearing the scid mutation (NOD-lpr/lpr-scid/scid), which eliminates the enhanced FasL-mediated lytic activity induced by Fas deficiency, still have delayed onset and reduced incidence of IDDM after adoptive transfer of diabetogenic NOD spleen cells. These results provide evidence that Fas/FasL-mediated programmed cell death plays a significant role in the pathogenesis of autoimmune diabetes.
An Upstream Oct-1- and Oct-2-Binding Silencer Governs B29 (Igβ) Gene Expression
Authors: Malone Cindy Sue , Patrone Lisa , Buchanan Kent L. , Webb Carol F. , Wall Randolph
Published: April 2014

The B cell-specific B29 (Igβ) gene is activated in the earliest B cell precursors and is expressed throughout B cell development. Tissue-specific expression of the murine B29 gene is controlled by a B cell-specific promoter whose activity is governed by a cassette of upstream transcriptional silencers. This study describes a potent new silencer that is located 5′ of the previously identified B29 silencer elements, FROG and TOAD. Like these known elements, the new B29 silencer is not restricted to the B29 promoter. Nuclear proteins from all cell lines tested interacted with this A+T-rich sequence, which closely resembled a noncanonical octamer binding motif and also conformed to the consensus sequence for nuclear matrix attachment regions. Interaction of Oct-1 and Oct-2 with the B29 A+T-rich sequence was confirmed using octamer-specific Abs. Oct-1/Oct-2 binding was required for the inhibitory activity of this sequence because mutations that blocked Oct-1/Oct-2 binding also eliminated inhibition of the B29 promoter. This B29 A+T-rich sequence specifically interacted with isolated nuclear matrix proteins in vitro, suggesting that it may also function as a matrix attachment region element. Maintenance of the level of B29 gene expression through the interaction of the minimal promoter and the upstream silencer elements FROG, TOAD, and the A+T-rich Oct-1/Oct-2 binding motif may be essential for normal B cell development and/or function.
Identification of a Gene Coding for a New Squamous Cell Carcinoma Antigen Recognized by the CTL
Authors: Nakao Masanobu , Shichijo Shigeki , Imaizumi Toshihiro , Inoue Yoshiko , Matsunaga Kazuko , Yamada Akira , Kikuchi Megumi , Tsuda Naotake , Ohta Keisuke , Takamori Shinzo , Yamana Hideaki , Fujita Hiromasa , Itoh Kyogo
Published: April 2014

Peptide-based specific immunotherapy has resulted in tumor regression in some melanoma patients. However, tumor Ags and peptides for specific immunotherapy, except for treatment of melanomas, have not yet been well identified. In this study, we report a gene encoding a new squamous cell carcinoma (SCC) Ag recognized by cells of the HLA-A24-restricted and tumor-specific CTL line. This gene with 3958-bp length was transcribed from the chromosome 6q22 with six exons, and its mRNA was ubiquitously expressed in both SCCs and normal tissues, and partly expressed in adenocarcinomas. The deduced 958-aa sequence encoded by this gene showed no similarity to any known amino acid sequences. This gene product had a characteristic of an endoplasmic reticulum-resident protein. A 100-kDa protein was detected in the vast majority of SCCs from various tissues, in majority of renal cell carcinomas and brain tumors, and in about one-third of melanomas and adenocarcinomas from various organs other than the breast. In contrast, it was not expressed at all in any of the normal cells or tissues tested, including the testis and fetal liver. Three different peptides at positions 93–101, 161–169, and 899–907 of this Ag were recognized by this CTL line, and all of them induced HLA-A24-restricted and tumor-specific CTLs from PBMCs of SCC patients. Therefore, these peptides may be useful for peptide-based specific immunotherapy of HLA-A24+ patients with SCC in various organs, as well as for treatment of other cancer.
Granulocyte-Macrophage Colony-Stimulating Factor in the Innate Immune Response toPneumocystis cariniiPneumonia in Mice
Authors: Paine Robert , Preston Angela M. , Wilcoxen Steven , Jin Hong , Siu Brian B. , Morris Susan B. , Reed Jacquelyn A. , Ross Gary , Whitsett Jeffrey A. , Beck James M.
Published: April 2014

Innate immunity plays an important role in pulmonary host defense against Pneumocystis carinii, an important pathogen in individuals with impaired cell-mediated immunity. We investigated the role of GM-CSF in host defense in a model of P. carinii pneumonia induced by intratracheal inoculation of CD4-depleted mice. Lung GM-CSF levels increased progressively during the infection and were significantly greater than those in uninfected controls 3, 4, and 5 wk after inoculation. When GM-CSF gene-targeted mice (GM−/−) depleted of CD4+ cells were inoculated with P. carinii, the intensities of infection and inflammation were increased significantly compared with those in CD4-depleted wild-type mice. In contrast, transgenic expression of GM-CSF directed solely in the lungs of GM−/− mice (using the surfactant protein C promoter) dramatically decreased the intensity of infection and inflammation 4 wk after inoculation. The concentrations of surfactant proteins A and D were greater in both uninfected and infected GM−/− mice compared with those in wild-type controls, suggesting that this component of the innate response was preserved in the GM−/− mice. However, alveolar macrophages (AM) from GM−/− mice demonstrated impaired phagocytosis of purified murine P. carinii organisms in vitro compared with AM from wild-type mice. Similarly, AM production of TNF-α in response to P. carinii in vitro was totally absent in AM from GM−/− mice, while GM-CSF-replete mice produced abundant TNF in this setting. Thus, GM-CSF plays a critical role in the inflammatory response to P. carinii in the setting of impaired cell-mediated immunity through effects on AM activation.
Mice Bearing Late-Stage Tumors Have Normal Functional Systemic T Cell Responses In Vitro and In Vivo
Authors: Radoja Sasa , Rao T. Dharma , Hillman Deborah , Frey Alan B.
Published: April 2014

Immune suppression in tumor-bearing hosts is considered to be one factor causally associated with the growth of antigenic tumors. Support for this hypothesis has come from reports that spleen T cells in tumor-bearing mice are deficient in either priming or effector phase functions. We have reexamined this hypothesis in detail using multiple murine tumor models, including transplantable adenocarcinoma, melanoma, sarcoma, and thymoma, and also a transgenic model of spontaneous breast carcinoma. In both in vitro and in vivo assays of T cell function (proliferation, cytokine production, induction of CD8+ alloreactive CTL, and development of anti-keyhole limpet hemocyanin CD4+ T cells, rejection of allogeneic or syngeneic regressor tumors, respectively) we show that mice bearing sizable tumor burdens are not systemically suppressed and do not have diminished T cell functions. Therefore, if immune suppression is a causal function in the growth of antigenic tumor, the basis for escape from immune destruction is likely to be dependent upon tumor-induced T cell dysfunction at the site of tumor growth.
Role of CC Chemokines (Macrophage Inflammatory Protein-1β, Monocyte Chemoattractant Protein-1, RANTES) in Acute Lung Injury in Rats
Authors: Bless Nicolas M. , Huber-Lang Markus , Guo Ren-Feng , Warner Roscoe L. , Schmal Hagen , Czermak Boris J. , Shanley Thomas P. , Crouch Larry D. , Lentsch Alex B. , Sarma Vidya , Mulligan Michael S. , Friedl Hans Peter , Ward Peter A.
Published: April 2014

The role of the CC chemokines, macrophage inflammatory protein-1β (MIP-1β), monocyte chemotactic peptide-1 (MCP-1), and RANTES, in acute lung inflammatory injury induced by intrapulmonary deposition of IgG immune complexes injury in rats was determined. Rat MIP-1β, MCP-1, and RANTES were cloned, the proteins were expressed, and neutralizing Abs were developed. mRNA and protein expression for MIP-1β and MCP-1 were up-regulated during the inflammatory response, while mRNA and protein expression for RANTES were constitutive and unchanged during the inflammatory response. Treatment of rats with anti-MIP-1β Ab significantly decreased vascular permeability by 37% (p = 0.012), reduced neutrophil recruitment into lung by 65% (p = 0.047), and suppressed levels of TNF-α in bronchoalveolar lavage fluids by 61% (p = 0.008). Treatment of rats with anti-rat MCP-1 or anti-rat RANTES had no effect on the development of lung injury. In animals pretreated intratracheally with blocking Abs to MCP-1, RANTES, or MIP-1β, significant reductions in the bronchoalveolar lavage content of these chemokines occurred, suggesting that these Abs had reached their targets. Conversely, exogenously MIP-1β, but not RANTES or MCP-1, caused enhancement of the lung vascular leak. These data indicate that MIP-1β, but not MCP-1 or RANTES, plays an important role in intrapulmonary recruitment of neutrophils and development of lung injury in the model employed. The findings suggest that in chemokine-dependent inflammatory responses in lung CC chemokines do not necessarily demonstrate redundant function.
Lipoxin A4 Inhibits IL-1β-Induced IL-6, IL-8, and Matrix Metalloproteinase-3 Production in Human Synovial Fibroblasts and Enhances Synthesis of Tissue Inhibitors of Metalloproteinases
Authors: Sodin-Semrl Snezna , Taddeo Brunella , Tseng Daniel , Varga John , Fiore Stefano
Published: April 2014

Lipoxins are a novel class of endogenous eicosanoid mediators that potently inhibit inflammatory events by signaling via specific receptors expressed on phagocytic cells. Animal models have shown that lipoxin A4 (LXA4) down-regulates inflammation in vivo. Here we demonstrate, for the first time, the expression of LXA4 receptors, and their up-regulation by IL-1β, in normal human synovial fibroblasts (SF). We examined whether exogenous LXA4 abrogated IL-1β stimulation of SF in vitro. IL-1β induced the synthesis of IL-6, IL-8, and matrix metalloproteinases (MMP)-1 and -3. At nanomolar concentrations, LXA4 inhibited these IL-1β responses with reduction of IL-6 and IL-8 synthesis, by 45 ± 7% and 75 ± 11%, respectively, and prevented IL-1β-induced MMP-3 synthesis without significantly affecting MMP-1 levels. Furthermore, LXA4 induced a 2-fold increase of tissue inhibitor of metalloproteinase (TIMP)-1 and a ∼3-fold increase of TIMP-2 protein levels. LXA4 inhibitory responses were dose dependent and were abrogated by pretreatment with LXA4 receptor antiserum. LXA4-induced changes of IL-6 and TIMP were accompanied by parallel changes in mRNA levels. These results indicate that LXA4 in activated SF inhibits the synthesis of inflammatory cytokines and MMP and stimulates TIMP production in vitro. These findings suggest that LXA4 may be involved in a negative feedback loop opposing inflammatory cytokine-induced activation of SF.
T Lymphocytes Activated by Persistent Viral Infection Differentially Modify the Expression of Metalloproteinases and Their Endogenous Inhibitors, TIMPs, in Human Astrocytes: Relevance to HTLV-I-Induced Neurological Disease
Authors: Giraudon Pascale , Szymocha Raphaël , Buart Stéphanie , Bernard Arlette , Cartier Luis , Belin Marie-Françoise , Akaoka Hideo
Published: April 2014

Activation of T lymphocytes by human pathogens is a key step in the development of immune-mediated neurologic diseases. Because of their ability to invade the CNS and their increased secretion of proinflammatory cytokines, activated CD4+ T cells are thought to play a crucial role in pathogenesis. In the present study, we examined the expression of inflammatory mediators the cytokine-induced metalloproteinases (MMP-2, -3, and -9) and their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMP-1, -2, and -3), in human astrocytes in response to activated T cells. We used a model system of CD4+ T lymphocytes activated by persistent viral infection (human T lymphotropic virus, HTLV-I) in transient contact with human astrocytes. Interaction with T cells resulted in increased production of MMP-3 and active MMP-9 in astrocytes despite increased expression of endogenous inhibitors, TIMP-1 and TIMP-3. These data suggest perturbation of the MMP/TIMP balance. These changes in MMP and TIMP expression were mediated, in part, by soluble factors (presumably cytokines) secreted by activated T cells. Integrin-mediated cell adhesion is also involved in the change in MMP level, since blockade of integrin subunits (α1, α3, α5, and β1) on T cells resulted in less astrocytic MMP-9-induced expression. Interestingly, in CNS tissues from neurological HTLV-I-infected patients, MMP-9 was detected in neural cells within the perivascular space, which is infiltrated by mononuclear cells. Altogether, these data emphasize the importance of the MMP-TIMP axis in the complex interaction between the CNS and invading immune cells in the context of virally mediated T cell activation.
Induction of Chemokine Secretion and Enhancement of Contact-Dependent Macrophage Cytotoxicity by Engineered Expression of Granulocyte-Macrophage Colony-Stimulating Factor in Human Colon Cancer Cells
Authors: Shinohara Hisashi , Yano Seiji , Bucana Corazon D. , Fidler Isaiah J.
Published: April 2014

We investigated the role of tumor cell-derived GM-CSF in recruitment and tumoricidal activation of tissue macrophages. Transfection of the murine GM-CSF gene into KM12SM human colon cancer cells decreased the tumorigenicity of transfected cells and nontransfected bystander colon cancer cells in nude mice. Sequential tissue sections from sites injected with high GM-CSF-producing tumor cells (but not from nontransfected or low GM-CSF-producing cells) demonstrated a dense infiltration of polymorphonuclear cells (PMN), followed by infiltration of macrophages, which correlated with expression of the macrophage-inflammatory protein-1α and the monocyte chemoattractant protein-1 (MCP-1) in mouse PMN and macrophages. GM-CSF-producing KM12SM cells were highly sensitive to lysis by mouse macrophages and also increased macrophage-mediated lysis of bystander nontransfected KM12SM cells. The incubation of macrophages with GM-CSF induced expression of the CD11b surface adhesion molecule, which was associated with increased attachment to tumor cells. All KM12SM cells were sensitive to macrophage-mediated lysis in the presence of rGM-CSF and recombinant MCP-1. Collectively, the results demonstrate that tumor cell-derived GM-CSF stimulates PMN and macrophages to secrete macrophage-inflammatory protein-1α and MCP-1, which triggers recruitment of mononuclear cells, induces expression of adhesion molecules on macrophages, and enhances contact-dependent cytolysis of tumor cells.
Expression and Contribution of Endogenous IL-13 in an Experimental Model of Sepsis
Authors: Matsukawa Akihiro , Hogaboam Cory M. , Lukacs Nickolas W. , Lincoln Pamela M. , Evanoff Holly L. , Strieter Robert M. , Kunkel Steven L.
Published: April 2014

IL-13 has been shown to exert potent anti-inflammatory properties. In this study, we elucidated the functional role of endogenous IL-13 in a murine model of septic peritonitis induced by cecal ligation and puncture (CLP). Initial studies demonstrated that the level of IL-13 increased in tissues including liver, lung, and kidney, whereas no considerable increase was found in either peritoneal fluid or serum after CLP. Immunohistochemically, IL-13-positive cells were Kupffer cells in liver, alveolar macrophages in lung, and epithelial cells of urinary tubules in kidney. IL-13 blockade with anti-IL-13 Abs significantly decreased the survival rate of mice after CLP from 53% to 14% on day 7 compared with control. To determine the potential mechanisms whereby IL-13 exerted a protective role in this model, the effects of anti-IL-13 Abs on both local and systemic inflammation were investigated. Administration of anti-IL-13 Abs did not alter the leukocyte infiltration and bacterial load in the peritoneum after CLP but dramatically increased the neutrophil influx in tissues after CLP, an effect that was accompanied by significant increases in the serum levels of aspartate transaminase, alanine transaminase, blood urea nitrogen, and creatinine. Tissue injury caused by IL-13 blockade was associated with increases in mRNA and the protein levels of CXC chemokines macrophage inflammatory protein-2 and KC as well as the CC chemokine macrophage inflammatory protein-1α and the proinflammatory cytokine TNF-α. Collectively, these results suggest that endogenous IL-13 protected mice from CLP-induced lethality by modulating inflammatory responses via suppression of overzealous production of inflammatory cytokines/chemokines in tissues.
IL-8 Reduced Tumorigenicity of Human Ovarian Cancer In Vivo Due to Neutrophil Infiltration
Authors: Lee Li-Fen , Hellendall Ronald P. , Wang Ying , Haskill J. Stephen , Mukaida Naofumi , Matsushima Kouji , Ting Jenny P.-Y.
Published: April 2014

Paclitaxel is a frontline therapy for ovarian cancer. Our laboratory has shown that paclitaxel induces IL-8, a member of the C-X-C family of chemokines, in subsets of human ovarian cancer cells. However, the critical issue concerns the biological significance of this chemokine in human ovarian cancer. To study the influence of IL-8 on tumor growth, human ovarian cancer cell lines were transfected with an expression vector for human IL-8 and tested for their ability to form tumors in nude mice. IL-8 expression by the transfected cells did not alter their growth properties in vitro. In contrast, tumor growth in vivo was significantly attenuated in animals receiving IL-8-expressing cells when compared with mice injected with control cells. As additional evidence that IL-8 is a crucial factor in tumor growth, it was noted that ovarian cell lines in which constitutive IL-8 expression is elevated did not form tumors. Injection of neutralizing Ab to IL-8 reverted the phenotype and caused tumor growth in vivo. Examination of tissue from the inoculation site revealed a dramatically elevated cellularity, containing neutrophils and macrophages, in mice receiving IL-8-expressing tumor cells. These results suggest that IL-8 production by human ovarian tumor cells can play a role in reducing the rate of tumor growth; this effect may be mediated by the increased targeting of neutrophil and other mononuclear cells to the tumor injection site. These studies indicate a role for IL-8 in ovarian cancer control and suggest that chemotherapy-induced IL-8 may have a positive role in controlling tumor growth.
Gene Therapy for Chronic Relapsing Experimental Allergic Encephalomyelitis Using Cells Expressing a Novel Soluble p75 Dimeric TNF Receptor
Authors: Croxford J. Ludovic , Triantaphyllopoulos Kostas A. , Neve Richard M. , Feldmann Marc , Chernajovsky Yuti , Baker David
Published: April 2014

In a murine relapsing experimental allergic encephalomyelitis (EAE) model, gene therapy to block TNF was investigated with the use of a retroviral dimeric p75 TNF receptor (dTNFR) construct. To effectively produce these TNF inhibitors in vivo, a conditionally immortalized syngeneic fibroblast line was established, using a temperature-sensitive SV40 large T Ag-expressing retrovirus. These cells were subsequently infected with a retrovirus expressing soluble dTNFR. CNS-injected cells could be detected 3 mo after transplantation and were shown to produce the transgene product by immunocytochemistry and ELISA of tissue fluids. These levels of dTNFR protein were biologically active and could significantly ameliorate both acute and relapsing EAE. This cell-based gene-vector approach is ideal for delivering proteins to the CNS and has particular relevance to the control of inflammatory CNS disease.
Naturally Developing Memory T Cell Xenoreactivity to Swine Antigens in Human Peripheral Blood Lymphocytes
Authors: Hartig Carsten V. , Haller Gary W. , Sachs David H. , Kuhlenschmidt Shannon , Heeger Peter S.
Published: April 2014

Naturally developing xenospecific Abs are well-documented barriers to xenograft transplantation in humans, but whether analogous xenoreactive T cell immunity develops is not known. We used an enzyme-linked immunospot assay to determine the frequency and cytokine profiles of xenoreactive PBLs from a panel of human volunteers. Because naive T cells produce only IL-2 in short term culture, IFN-γ production by this approach is a measure of a memory immune response. Stimulation of human PBLs or purified T lymphocytes with stimulator cells from inbred swine revealed a high frequency of IFN-γ producers with 5-fold fewer IL-2 producers. In contrast, lymphocytes obtained from neonatal umbilical cord blood contained swine-specific IL-2 producers but few IFN-γ producers, which is what one would expect to find with a naive phenotype. Moreover, PBLs from adults with a history of abstention from pork consumption responded to swine cells with a significantly lower frequency of IFN-γ producers than PBLs from adults with unrestricted diets did, suggesting that pork consumption may result in priming of swine-specific T cell immunity. Our findings provide the first evidence for naturally occurring xenospecific T cell immunity in humans. The detected strength of this memory response suggests that it will present a formidable barrier to transplantation of swine organs.
Analysis and Significance of Anti-Latent Membrane Protein-1 Antibodies in the Sera of Patients with EBV-Associated Diseases
Authors: Xu Jingwu , Ahmad Ali , D’Addario Mario , Knafo Laurent , Jones James F. , Prasad U. , Dolcetti R. , Vaccher E. , Menezes José
Published: April 2014

Anti-latent membrane protein-1 (LMP-1) is an EBV-encoded type III integral membrane protein with oncogenic potential that is expressed most consistently in various EBV-associated malignancies. Unlike many other EBV proteins, LMP-1 Abs have rarely been demonstrated in EBV-associated disease conditions. We established a high level LMP-1-expressing cell clone and used it for the detection, quantitation, and characterization of these Abs in various human sera in immunoblots and ELISA. Our results demonstrate that, in contrast to the commonly held notion, LMP-1 induces significant humoral immune responses in EBV-associated malignant conditions especially in nasopharyngeal carcinoma (NPC) patients in whom >70% sera are positive for these Abs, and their titers correlate with the clinical condition of the tumors. Interestingly, anti-LMP-1 Abs of IgA isotype were found only in NPC patients. These Abs were absent from the sera of infectious mononucleosis and chronic EBV infection patients, whereas a small fraction (∼5%) of the healthy, EBV-seropositive individuals were positive for them; however, their OD values were much lower than those of NPC patients. These studies demonstrate, for the first time, the potential significance of LMP-1-specific Abs for the diagnosis and prognosis of EBV-associated malignancies, especially of NPC.
Accumulation of Common T Cell Clonotypes in the Salivary Glands of Patients with Human T Lymphotropic Virus Type I-Associated and Idiopathic Sjögren’s Syndrome
Authors: Sasaki Masanori , Nakamura Seiji , Ohyama Yukiko , Shinohara Masanori , Ezaki Ichiko , Hara Hideo , Kadena Tsutomu , Kishihara Kenji , Yamamoto Kazuhiko , Nomoto Kikuo , Shirasuna Kanemitsu
Published: April 2014

To clarify the pathogenesis of human T lymphotropic virus type I (HTLV-I)-associated Sjögren’s syndrome (SS), the TCR Vβ gene usage by the infiltrating lymphocytes in the target organ was examined. The Vβ families predominantly used in the labial salivary gland (LSG) from the HTLV-I-seropositive (HTLV-I+) SS patients were more restricted than those from the HTLV-I-seronegative (idiopathic) SS patients, and were commonly Vβ5.2, Vβ6, and Vβ7. The single-strand conformation polymorphism analysis revealed that T cell clonotypes with Vβ5.2, Vβ6, and Vβ7 accumulate in the LSG from the HTLV-I+ and idiopathic SS patients. Among junctional sequences of the most dominant Vβ7 transcripts, the conserved amino acid motif (QDXG: X is any amino acid) was found in six of the five HTLV-I+ SS patients and was also detected in two of the five idiopathic SS patients. Using the probes specific to the motif, the Vβ7 transcripts with the motif were detected in the LSG from all of the seven HTLV-I+ and five of the six idiopathic SS patients, but not from eight healthy subjects. The Vβ7 transcripts with this motif were also detected in the HTLV-I-infected T cell lines obtained from the LSG of an HTLV-I+ SS patient. The accumulation of HTLV-I-infected T cells expressing TCR with the conserved motif was thus indicated. These T cells were commonly present in patients with idiopathic SS and are strongly suggested to most likely be involved in the pathogenesis of both HTLV-I-associated and idiopathic SS.

1 This work was supported in part by grants from the Ministry of Education, Science, and Culture of Japan. Abbreviations used in this paper: HTLV-I, human T lymphotropic virus type-I; SS, Sjögren’s syndrome; LSG, labial salivary gland; PG, parotid gland; SSCP, single-strand conformation polymorphism; HAM/TSP, HTLV-I-associated myelopathy/tropical spastic paraparesis; HAAP, HTLV-I-associated arthropathy; CDR3, complementarity-determining region 3.
Cutting Edge: Administration of Anti-CD40 Ligand and Donor Bone Marrow Leads to Hemopoietic Chimerism and Donor-Specific Tolerance Without Cytoreductive Conditioning
Authors: Durham Megan M. , Bingaman Adam W. , Adams Andrew B. , Ha Jongwon , Waitze Seung-Yeun , Pearson Thomas C. , Larsen Christian P.
Published: April 2014

Transplantation tolerance, defined as allograft acceptance by an immunocompetent recipient in the absence of long-term immunosuppression, has remained an elusive goal in clinical transplantation. Robust experimental tolerance induction strategies have in common methods to induce mixed hemopoietic chimerism. To date, however, chimerism induction across allogeneic barriers has required recipient conditioning with irradiation or cytoablative agents. In this paper we show that B6 recipients of fully allogeneic BALB/c skin grafts treated with repeated doses of donor bone marrow and anti-CD40 ligand (CD40L) develop durable (>300 days), readily detectable (6–12%) multilineage hemopoietic chimerism, indefinite allograft acceptance (>300 days), and donor-specific tolerance to secondary skin grafts. Analysis of the TCR repertoire of treated mice indicates that the underlying mechanisms of tolerance are in part mediated by deletion of donor-reactive T cells. These data demonstrate that durable hemopoietic chimerism and robust transplantation tolerance can be achieved without cytotoxic conditioning using a potentially clinically applicable regimen.
Cutting Edge: Differential Expression of Chemokines in Th1 and Th2 Cells Is Dependent on Stat6 But Not Stat4
Authors: Zhang Shangming , Lukacs Nicholas W. , Lawless Victoria A. , Kunkel Steven L. , Kaplan Mark H.
Published: April 2014

The in vivo function of Th cell subsets is largely dependent on the ability of differentiated CD4+ T cells to be recruited to specific sites and secrete restricted sets of cytokines. In this paper we demonstrate that Th1 and Th2 cells secrete discrete patterns of chemokines, small m.w. cytokines that function as chemoattractants in inflammatory reactions. Th2 cells secrete macrophage-derived chemokine and T cell activation gene 3, and acquisition of this pattern of expression is dependent on Stat6. In contrast, Th1 cells secrete lymphotactin and RANTES, though unlike IFN-γ, expression of these chemokines is independent of Stat4. We further show that supernatants from activated Th2 cells preferentially induce the chemotaxis of Th2 over Th1 cells, corresponding with Stat6-dependent expression of CCR4 and CCR8 in Th2 cells. These data provide the basis for restricted and direct T cell-mediated cellular recruitment to sites of inflammation.
Lipopolysaccharide Stimulates the Proliferation of Human CD56+CD3− NK Cells: A Regulatory Role of Monocytes and IL-10
Authors: Goodier Martin R. , Londei Marco
Published: April 2014

NK cells recognize and kill tumor cells and normal cells, and these play an important role in immune defense in cancer, infectious disease, and autoimmunity. NK killing is regulated by positive or negative signals derived from the interaction of surface receptors with ligands on the target cells. However, the mechanisms controlling the proliferation and maintenance of NK cells in normal human individuals are less clearly defined. In this study, using an entirely autologous system, we demonstrate that human peripheral blood CD3−CD56+, killer cell-inhibitory receptor (KIR)-expressing cells proliferate and expand in response to LPS. These responses are enhanced in the presence of anti-IL-10 receptor-blocking Abs or on the removal of CD14+ cells from the cultures. This enhancement is also reflected in substantial increases in cytolytic activity and IFN-γ production. The negative effect of CD14+ cells may also be IL-10 mediated, IL-10 being lost from the culture supernatants of CD14-depleted PBMC and rIL-10 reversing the effect of this depletion. On the other hand, mRNA for the p35 and p40 subunits of IL-12 is still induced in CD14-depleted cultures. The expansion of CD3−CD56+ cells was also inhibited by CTLA4-Ig, indicating a role for CD80/86. B lymphocytes were not required for the expansion of CD3−CD56+ cells, whereas removal of MHC class II+ cells from CD14-depleted cultures resulted in a complete abrogation of these responses. Expansion of CD3−CD56+ cells was reconstituted in MHC class II-depleted cell cultures by adding back monocyte-derived dendritic cells. These results indicate that the responses of CD3−CD56+ NK cells to LPS may be driven by a MHC class II+ B7+ CD14− peripheral population, most likely blood dendritic cells.
cGMP-Mediated Inhibition of TNF-α Production by the Atrial Natriuretic Peptide in Murine Macrophages
Authors: Kiemer Alexandra K. , Hartung Thomas , Vollmar Angelika M.
Published: April 2014

The atrial natriuretic peptide (ANP) is suggested to regulate inflammatory response by alteration of macrophage functions. The aim of this study was to investigate whether ANP influences production of TNF-α. TNF-α production in murine bone marrow-derived macrophages was induced by LPS, and TNF-α secretion (±ANP) was determined by L929 bioassay. ANP dose dependently (10−8–10−6 M) inhibited TNF-α release by up to 95%. The effect was mediated via the guanylate cyclase-coupled A receptor, as was shown by employing dibutyryl-cGMP, the cGMP-inhibitory compound Ly-83583, and the A receptor antagonist HS-142-1. A specific ligand of the natriuretic peptide “clearance” receptor inhibited TNF-α production only at 10−7 and 10−8 M, but not at 10−6 M. The B receptor ligand C-type natriuretic peptide showed no TNF-α-inhibitory effect. To investigate the underlying mechanism of ANP-mediated TNF-α inhibition, Northern blot was performed. ANP-treated macrophages displayed decreased TNF-α-mRNA levels. Besides the known inhibition of NF-κB activation, in this study we demonstrated that ANP also attenuates the activation of the proinflammatory transcription factor AP-1 (gel shift assay). ANP did not alter subunit composition of AP-1 complexes, as was shown by supershift assays applying anti-c-jun and anti-c-fos Abs. To get information on the ANP effect for human inflammatory processes, we investigated cytokine production in human LPS-activated blood. ANP significantly attenuated production of TNF-α and IL-1β without affecting production of IL-10 and IL-1ra. In summary, ANP was shown to attenuate TNF-α production of LPS-activated macrophages via cGMP. The inhibition is suggested to involve transcriptional processes that are the result of reduced activation of responsible transcription factors.
Epithelial Cell-Specific Laminin 5 Is Required for Survival of Early Thymocytes
Authors: Kim Moon Gyo , Lee Gwanghee , Lee Suk-Keun , Lolkema Martijn , Yim Jeongbin , Hong Seung H. , Schwartz Ronald H.
Published: April 2014

The gene LamC2 encoding the γ2 chain of laminin 5, an epithelial cell-specific extracellular matrix protein, was identified in a PCR-based subtracted cDNA library from mouse thymic stromal cells. The mRNA existed in two alternative forms (5.1 and 2.4 kb). The full-length message was highly expressed in SCID thymus and in a nurse cell line, but not in other thymic epithelial cell lines, while the short form was more widely expressed. In situ hybridization and immunohistochemical staining revealed laminin 5 expression mostly in the subcapsular region of the adult thymus. Addition to fetal thymic organ cultures of a cell adhesion-blocking mAb to the α3 chain of laminin 5 interrupted T cell development. There was a 40% reduction in the total yield of thymocytes, and the most profound decrease (75–90%) was seen in the CD25+CD44+ and CD25+CD44−subsets of the CD4−CD8− double negative fraction. Most of the surviving double negative thymocytes expressed Sca-1, and there were significant increases in the number of cells with CD69 expression and in the fraction of annexin V-stained cells. None of these changes were observed with a nonblocking anti-laminin α3 chain mAb. These results suggest that the interaction between double negative thymoctyes and laminin 5 made by subcapsular epithelial cells is required for the survival and differentiation of mouse thymocytes.
Systemic Administration of Agonist Peptide Blocks the Progression of Spontaneous CD8-Mediated Autoimmune Diabetes in Transgenic Mice Without Bystander Damage
Authors: Bercovici Nadège , Heurtier Agnès , Vizler Csaba , Pardigon Nathalie , Cambouris Christophe , Desreumaux Pierre , Liblau Roland
Published: April 2014

Insulin-dependent diabetes is an autoimmune disease targeting pancreatic β-islet cells. Recent data suggest that autoreactive CD8+ T cells are involved in both the early events leading to insulitis and the late destructive phase resulting in diabetes. Although therapeutic injection of protein and synthetic peptides corresponding to CD4+ T cell epitopes has been shown to prevent or block autoimmune disease in several models, down-regulation of an ongoing CD8+ T cell-mediated autoimmune response using this approach has not yet been reported. Using CL4-TCR single transgenic mice, in which most CD8+ T cells express a TCR specific for the influenza virus hemagglutinin HA512–520 peptide:Kd complex, we first show that i.v. injection of soluble HA512–520 peptide induces transient activation followed by apoptosis of Tc1-like CD8+ T cells. We next tested a similar tolerance induction strategy in (CL4-TCR × Ins-HA)F1 double transgenic mice that also express HA in the β-islet cells and, as a result, spontaneously develop a juvenile onset and lethal diabetes. Soluble HA512–520 peptide treatment, at a time when pathogenic CD8+ T cells have already infiltrated the pancreas, very significantly prolongs survival of the double transgenic pups. In addition, we found that Ag administration eliminates CD8+ T cell infiltrates from the pancreas without histological evidence of bystander damage. Our data indicate that agonist peptide can down-regulate an autoimmune reaction mediated by CD8+ T cells in vivo and block disease progression. Thus, in addition to autoreactive CD4+ T cells, CD8+ T cells may constitute targets for Ag-specific therapy in autoimmune diseases.
Human Mast Cells Transmigrate Through Human Umbilical Vein Endothelial Monolayers and Selectively Produce IL-8 in Response to Stromal Cell-Derived Factor-1α
Authors: Lin Tong-Jun , Issekutz Thomas B. , Marshall Jean S.
Published: April 2014

Mature mast cells are generally considered to be less mobile cells residing within tissue sites. However, mast cell numbers are known to increase in the context of inflammation, and mast cells are recognized to be important in regulating local neutrophil infiltration. CXC chemokines may play a critical role in this process. In this study two human mast cell-like lines, HMC-1 and KU812, and human cord blood-derived primary cultured mast cells were employed to examine role of stromal cell-derived factor-1 (SDF-1) in regulating mast cell migration and mediator production. It was demonstrated that human mast cells constitutively express mRNA and protein for CXCR4. Stimulation of human mast cells with SDF-1, the only known ligand for CXCR4, induced a significant increase in intracellular calcium levels. In vitro, SDF-1α mediated dose-dependent migration of human cord blood-derived mast cells and HMC-1 cells across HUVEC monolayers. Although SDF-1α did not induce mast cell degranulation, it selectively stimulated production of the neutrophil chemoattractant IL-8 without affecting TNF-α, IL-1β, IL-6, GM-CSF, IFN-γ, or RANTES production, providing further evidence of the selective modulation of mast cell function by this chemokine. These findings provide a novel, SDF-1-dependent mechanism for mast cell transendothelial migration and functional regulation, which may have important implications for the local regulation of mast cells in disease.
Macrophage-Derived Chemokine and EBI1-Ligand Chemokine Attract Human Thymocytes in Different Stage of Development and Are Produced by Distinct Subsets of Medullary Epithelial Cells: Possible Implications for Negative Selection
Authors: Annunziato Francesco , Romagnani Paola , Cosmi Lorenzo , Beltrame Chiara , Steiner Bart H. , Lazzeri Elena , Raport Carol J. , Galli Grazia , Manetti Roberto , Mavilia Carmelo , Vanini Vittorio , Chantry David , Maggi Enrico , Romagnani Sergio
Published: April 2014

The chemoattractant activity of macrophage-derived chemokine (MDC), EBI1-ligand chemokine (ELC), and secondary lymphoid tissue chemokine (SLC) on human thymocytes was analyzed. Both ELC and SLC caused the accumulation of CD4+CD8− or CD4−CD8+ CD45RA+ thymocytes showing high CD3 expression. By contrast, a remarkable proportion of MDC-responsive thymocytes were CD4+CD8+ cells exhibiting reduced levels of CD8 or CD4+CD8− cells showing CD3 and CD45R0, but not CD45RA. MDC-responsive thymocyte suspensions were enriched in cells expressing the MDC receptor, CCR4, selectively localized to the medulla, and in CD30+ cells, whereas ELC-responsive thymocytes never expressed CD30. Reactivity to both MDC and ELC was localized to cells of the medullary areas, but never in the cortex. Double immunostaining showed no reactivity for either MDC or ELC by T cells, macrophages, or mature dendritic cells, whereas many medullary epithelial cells were reactive to MDC or ELC. However, MDC reactivity was consistently localized to the outer wall of Hassal’s corpuscles, whereas ELC reactivity was often found in cells surrounding medullary vessels, but not in Hassal’s corpuscles. Moreover, while most MDC-producing cells also stained positive for CD30L, this molecule was never found on ELC-producing cells. We suggest therefore that CD30L-expressing MDC-producing medullary epithelial cells attract CCR4-expressing thymocytes, thus favoring the CD30/CD30L interaction, and therefore the apoptosis, of cells that are induced to express CD30 by autoantigen activation. By contrast, ELC production by CD30L-lacking medullary epithelial cells may induce the migration into periphery of mature thymocytes that have survived the process of negative selection.
An Essential Contribution by IFN-γ to CD8+ T Cell-Mediated Rejection of Pancreatic Islet Allografts
Authors: Diamond Andrew S. , Gill Ronald G.
Published: April 2014

CD8+ T cells have long been considered to be the prototypical cytotoxic lymphocyte subpopulation. However, whether alloreactive CD8+ T cells require traditional cytolytic pathways such as perforin and Fas ligand (FasL) to mediate graft rejection has been a controversial issue. In the present studies, we examined the role of varied effector pathways in CD8+ T cell-mediated rejection of pancreatic islet allografts. Our goal was to systematically determine the relative requirements, if any, of perforin and FasL as well as the proinflammatory cytokine IFN-γ in triggering graft destruction. To study CD8+ T cell effector pathways independently of other lymphocyte populations, purified alloreactive CD8+ T cells were adoptively transferred into severe combined immune-deficient (SCID) recipients bearing established islet allografts. Results indicate that to reject established islet allografts, primed CD8+ T cells do not require the individual action of the conventional cytotoxic effectors perforin and Fas ligand. In contrast, the ability to produce IFN-γ is critical for efficient CD8+ T cell-mediated rejection of established islet allografts. Furthermore, alloreactive CD8+ TCR transgenic T cells (2C) also show IFN-γ dependence for mediating islet allograft rejection in vivo. We speculate from these results that the production of IFN-γ by alloreactive CD8+ T cells is a rate-limiting step in the process of islet allograft rejection.
The HTLV-I Tax Protein Transcriptionally Modulates OX40 Antigen Expression
Authors: Pankow Rüdiger , Dürkop Horst , Latza Ute , Krause Hans , Kunzendorf Ulrich , Pohl Thomas , Bulfone-Paus Silvia
Published: April 2014

OX40 is a member of the TNF receptor family, expressed on activated T cells. It is the only costimulatory T cell molecule known to be specifically up-regulated in human T cell leukemia virus type-I (HTLV-I)-producing cells. In a T cell line, OX40 surface expression was shown to be induced by HTLV-I Tax alone. To understand molecular mechanisms of OX40 gene regulation and modulation by HTLV-I Tax, we have cloned the human OX40 gene and analyzed its 5′-flanking region. By reporter gene analysis with progressive 5′ deletions from nucleotides −1259 to −64, we have defined a 157-bp DNA fragment as a minimal promoter for constitutive expression. In addition, we show that in the OX40+ cell line, Co, Tax is able to further increase OX40 surface expression. Up-regulation of OX40 promoter activity by Tax requires two upstream NF-κB sites, which are not active in the constitutive OX40 expression. Their deletion abrogates Tax responsiveness in reporter gene analysis. The site-directed mutagenesis of each NF-κB site demonstrates that cooperative NF-κB binding is a prerequisite for Tax-directed activity as neither site alone is sufficient for a full Tax responsiveness of the OX40 promoter. Upon Tax expression, both sites bind p65 and c-Rel. These data provide new insight into the direct regulation of OX40 by Tax and add to our understanding of the possible role of the OX40/OX40 ligand system in the proliferation of HTLV-I+ T cells.