bluuebunny/crossref_metadata_2025 huggingface dataset:
Dexamethasone Inhibits IL-12p40 Production in Lipopolysaccharide-Stimulated Human Monocytic Cells by Down-Regulating the Activity of c-Jun N-Terminal Kinase, the Activation Protein-1, and NF-κB Transcription Factors
Authors: Ma Wei , Gee Katrina , Lim Wilfred , Chambers Kelly , Angel Jonathan B. , Kozlowski Maya , Kumar Ashok
Published: April 2014

IL-12 plays a critical role in the development of cell-mediated immune responses and in the pathogenesis of inflammatory and autoimmune disorders. Dexamethasone (DXM), an anti-inflammatory glucocorticoid, has been shown to inhibit IL-12p40 production in LPS-stimulated monocytic cells. In this study, we investigated the molecular mechanism by which DXM inhibits IL-12p40 production by studying the role of the mitogen-activated protein kinases (MAPKs), and the key transcription factors involved in human IL-12p40 production in LPS-stimulated monocytic cells. A role for c-Jun N-terminal kinase (JNK) MAPK in LPS-induced IL-12p40 regulation in a promonocytic THP-1/CD14 cell line was demonstrated by using specific inhibitors of JNK activation, SP600125 and a dominant-negative stress-activated protein/extracellular signal-regulated kinase kinase-1 mutant. To identify transcription factors regulating IL-12p40 gene transcription, extensive deletion analyses of the IL-12p40 promoter was performed. The results revealed the involvement of a sequence encompassing the AP-1-binding site, in addition to that of NF-κB. The role of AP-1 in IL-12p40 transcription was confirmed by using antisense c-fos and c-jun oligonucleotides. Studies conducted to understand the regulation of AP-1 and NF-κB activation by JNK MAPK revealed that both DXM and SP600125 inhibited IL-12p40 gene transcription by inhibiting the activation of AP-1 and NF-κB transcription factors as revealed by luciferase reporter and gel mobility shift assays. Taken together, our results suggest that DXM may inhibit IL-12p40 production in LPS-stimulated human monocytic cells by down-regulating the activation of JNK MAPK, the AP-1, and NF-κB transcription factors.
Domain Shuffling Has Been the Main Mechanism Forming New Hominoid Killer Cell Ig-Like Receptors
Authors: Rajalingam Raja , Parham Peter , Abi-Rached Laurent
Published: April 2014

The killer cell Ig-like receptor (KIR) gene family encodes MHC class I-specific receptors, which regulate NK cell responses and are also expressed on subpopulations of T cells. KIR haplotypes vary in gene content, which, in combination with allelic polymorphism, extensively diversifies the KIR genotype both within and between human populations. Species comparison indicates that formation of new KIR genes and loss of old ones are frequent events, so that few genes are conserved even between closely related species. In this regard, the hominoids define a time frame that is particularly informative for understanding the processes of KIR evolution and its potential impact on killer cell biology. KIR cDNA were characterized from PBMC of three gorillas, and genomic DNA were characterized for six additional individuals. Eleven gorilla KIR genes were defined. With attainment of these data, a set of 75 KIR sequences representing five hominoid species was assembled, which also included rhesus monkey, cattle, and rodent KIR. Searching this data set for recombination events, and phylogenetic analysis using Bayesian methods, demonstrated that new KIR were usually the result of recombination between loci in which complete protein domains were shuffled. Further phylogenetic analysis of the KIR sequences after removal of confounding recombined segments showed that only two KIR genes, KIR2DL4 and KIR2DL5, have been preserved throughout hominoid evolution, and one of them, KIR2DL4, is also common to rhesus monkey and hominoids. Other KIR genes represent recombinant forms present in a minority of species, often only one, as exemplified by 8 of the 11 gorilla KIR genes.
Molecular Characterization of a Putative Antiretroviral Transcriptional Factor, OTK18
Authors: Carlson Kimberly A. , Leisman Gary , Limoges Jenae , Pohlman Garrett D. , Horiba Masahide , Buescher James , Gendelman Howard E. , Ikezu Tsuneya
Published: April 2014

Elucidation of the factors involved in host defense against human immunodeficiency viral infection remains pivotal if viral control may be achieved. Toward these ends, we investigated the function of a putative antiretroviral factor, OTK18, isolated by differential display of mRNA from HIV type 1-infected primary human monocyte-derived macrophages. Molecular and immunohistochemical analyses showed that the OTK18 nucleotide sequence contains 13 adjacent C2H2-type zinc finger motifs, a Krüppel-associated box, and is localized to both cytosol and nucleus. Mutational analyses revealed that both the Krüppel-associated box and zinc finger regions of OTK18 are responsible for the transcriptional suppressive activities of this gene. OTK18 was copiously expressed in macrophages following HIV type I infection and diminished progeny virion production. A mechanism for this antiretroviral activity was by suppression of HIV type 1 Tat-induced viral long terminal repeat promoter activity. Our findings suggest that one possible function of OTK18 is as a HIV type 1-inducible transcriptional suppresser.
Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin (Cdt): Evidence That the Holotoxin Is Composed of Three Subunits: CdtA, CdtB, and CdtC
Authors: Shenker Bruce J. , Besack Dave , McKay Terry , Pankoski Lisa , Zekavat Ali , Demuth Donald R.
Published: April 2014

We have shown the Actinobacillus actinomycetemcomitans produces an immunosuppressive factor encoded by the cytolethal distending toxin (cdt)B gene, which is homologous to a family of Cdts expressed by several Gram-negative bacteria. We now report that the capacity for CdtB to induce G2 arrest in Jurkat cells is greater in the presence of the other Cdt peptides: CdtA and CdtC. Plasmids containing the cdt operon were constructed and expressed in Escherichia coli; each plasmid contained a modified cdt gene that expressed a Cdt peptide containing a C-terminal His tag. All three Cdt peptides copurified with the His-tagged Cdt peptide. Each of the peptides associated with the complex was truncated; N-terminal amino acid analysis of CdtB and CdtC indicated that the truncation corresponds to cleavage of a previously described signal sequence. CdtA was present in two forms in crude extracts, 25 and 18 kDa; only the 18-kDa fragment copurified with the Cdt complexes. Cdt complexes were also immunoprecipitated from A. actinomycetemcomitans extracts using anti-CdtC mAb. Exposure of Jurkat cells to 40 pg resulted in >50% accumulation of G2 cells. CdtB and CdtC were detected by immunofluorescence on the cell surface after 2-h exposure to the holotoxin. CdtA was not detected by immunofluorescence, but all three peptides were associated with Jurkat cells when analyzed by Western blot. These studies suggest that the active Cdt holotoxin is a heterotrimer composed of truncated CdtA, CdtB, and CdtC, and all three peptides appear to associate with lymphocytes.
Localization of Cystic Fibrosis Transmembrane Conductance Regulator to Lipid Rafts of Epithelial Cells Is Required for Pseudomonas aeruginosa-Induced Cellular Activation
Authors: Kowalski Michael P. , Pier Gerald B.
Published: April 2014

The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein is an epithelial cell receptor for the outer core oligosaccharide of the Pseudomonas aeruginosa LPS. Bacterial binding leads to CFTR-dependent bacterial internalization, initiation of NF-κB nuclear translocation, cellular desquamation, and eventual apoptosis of the infected cells, all of which are critical for innate immune resistance to infection with this pathogen. Lack of this reaction in CF patients underlies their hypersusceptibility to chronic P. aeruginosa infection. In this study we tested whether these epithelial cell responses are dependent upon the localization of CFTR to lipid rafts. Confocal microscopy showed that green fluorescent protein-tagged CFTR (GFP-CFTR) and the lipid raft marker ganglioside GM1 colocalized at sites of P. aeruginosa contact and internalization. GFP-CFTR localized to low density Triton X-100-insoluble fractions in lysates of Madin-Darby canine kidney GFP-CFTR cells, and P. aeruginosa infection increased the levels of GFP-CFTR in these fractions as determined by Western blot. Cells expressing GFP-ΔF508-CFTR did not have rafts with detectable CFTR protein. Extraction of cell surface cholesterol via cyclodextrin treatment of the cells inhibited CFTR entry into rafts. In addition, cyclodextrin treatment of both human and canine epithelial cells inhibited cellular ingestion of P. aeruginosa, NF-κB nuclear translocation, and apoptosis. These results indicate that lipid raft localization of CFTR is required for signaling in response to P. aeruginosa infection. Such signaling is needed for the coordination of innate immunity to P. aeruginosa lung infection, a process that is defective in CF.
Critical Role of T Cell-Dependent Serum Antibody, but Not the Gut-Associated Lymphoid Tissue, for Surviving Acute Mucosal Infection with Citrobacter rodentium, an Attaching and Effacing Pathogen
Authors: Bry Lynn , Brenner Michael B.
Published: April 2014

Citrobacter rodentium uses virulence factors similar to the enteropathogenic Escherichia coli to produce attaching and effacing lesions in the distal colon of mice. We used this infection model to determine components of adaptive immunity needed to survive infection. During acute infection, wild-type mice develop breaks across infected epithelial surfaces but resolve infection. Surprisingly, mice markedly deficient in mucosal lymphocyte populations from β7 integrin deficiency resolve infection, as do CD8α−/− or TCR-δ−/− mice. In contrast, CD4−/− or TCR-β−/− mice develop polymicrobial sepsis and end-organ damage, and succumb during acute infection, despite epithelial damage similar to wild-type mice. B cell-deficient (MuMT−/−) or B and T cell-deficient (recombinase-activating gene 2−/−) mice develop severe pathology in colon and internal organs, and deteriorate rapidly during acute infection. Surviving mice develop robust Citrobacter-specific serum IgM responses during acute infection, whereas mice that succumb do not. However, CD4−/− mice receiving serum Igs from infected wild-type mice survive and clear the infection. Our data show that survival of apparently self-limited and luminal mucosal infections requires a systemic T cell-dependent Ab response against bacteria that enter through damaged mucosa. These findings have implications for understanding host defense against mucosal infections, including the pathogenesis of these diseases in immunocompromised populations.
Redox-Sensing Release of Human Thioredoxin from T Lymphocytes with Negative Feedback Loops
Authors: Kondo Norihiko , Ishii Yasuyuki , Kwon Yong-Won , Tanito Masaki , Horita Hiroyuki , Nishinaka Yumiko , Nakamura Hajime , Yodoi Junji
Published: April 2014

Thioredoxin (TRX) is released from various types of mammalian cells despite no typical secretory signal sequence. We show here that a redox-active site in TRX is essential for its release from T lymphocytes in response to H2O2 and extracellular TRX regulates its own H2O2-induced release. Human T cell leukemia virus type I-transformed T lymphocytes constitutively release a large amount of TRX. The level of TRX release is augmented upon the addition of H2O2, but suppressed upon the addition of N-acetylcysteine. In the culture supernatant of a Jurkat transfectant expressing the tagged TRX-wild type (WT), the tagged TRX protein is rapidly released at 1 h and kept at a constant level until 6 h after the addition of H2O2. In contrast, another type of transfectant expressing the tagged TRX mutant (C32S/C35S; CS) fails to release the protein. H2O2-induced release of TRX from the transfectant is inhibited by the presence of rTRX-WT in a dose-dependent manner. Preincubation of the transfectant with rTRX-WT for 1 h at 37°C, but not 0°C, results in a significant suppression of the TRX release, reactive oxygen species, and caspase-3 activity induced by H2O2, respectively. Confocal microscopy and Western blot analysis show that extracellular rTRX-WT added to the culture does not obviously enter T lymphocytes until 24 h. These results collectively suggest that the oxidative stress-induced TRX release from T lymphocytes depends on a redox-sensitive event and may be regulated by negative feedback loops using reactive oxygen species-mediated signal transductions.
Hyperactivation of STAT3 Is Involved in Abnormal Differentiation of Dendritic Cells in Cancer
Authors: Nefedova Yulia , Huang Mei , Kusmartsev Sergei , Bhattacharya Raka , Cheng Pingyan , Salup Raoul , Jove Richard , Gabrilovich Dmitry
Published: April 2014

Abnormal differentiation of myeloid cells is one of the hallmarks of cancer. However, the molecular mechanisms of this process remain elusive. In this study, we investigated the effect of tumor-derived factors on Janus kinase (Jak)/STAT signaling in myeloid cells during their differentiation into dendritic cells. Tumor cell conditioned medium induced activation of Jak2 and STAT3, which was associated with an accumulation of immature myeloid cells. Jak2/STAT3 activity was localized primarily in these myeloid cells, which prevented the differentiation of immature myeloid cells into mature dendritic cells. This differentiation was restored after removal of tumor-derived factors. Inhibition of STAT3 abrogated the negative effects of these factors on myeloid cell differentiation, and overexpression of STAT3 reproduced the effects of tumor-derived factors. Thus, this is a first demonstration that tumor-derived factors may affect myeloid cell differentiation in cancer via constitutive activation of Jak2/STAT3.
Galectin-3 Mediates IL-4-Induced Survival and Differentiation of B Cells: Functional Cross-Talk and Implications during Trypanosoma cruzi Infection
Authors: Acosta-Rodríguez Eva V. , Montes Carolina L. , Motrán Claudia C. , Zuniga Elina I. , Liu Fu-Tong , Rabinovich Gabriel A. , Gruppi Adriana
Published: April 2014

The role of transcription factors in B cell survival and differentiation has been delineated during the last years. However, little is known about the intermediate signals and the intracellular pathways that control these events. In this study, we provide evidence both in vitro and in vivo, showing that galectin-3 (Gal-3), a β-galactoside-binding protein, is a critical mediator of B cell differentiation and survival. Although Gal-3 is not expressed in resting B cells from normal mice, its expression is markedly induced after activation with stimuli such as IL-4 and CD40 cross-linking. These signals promote survival and block the final differentiation of these cells, thus allowing the rising of a memory B cell phenotype. In addition, Gal-3 is expressed in B cells from Trypanosoma cruzi-infected mice, which received signals for activation and differentiation in vivo. By using an antisense strategy, we determined that Gal-3 is a critical signal mediating the effects of IL-4 on B cell fate. Blockade of intracellular Gal-3 in vitro abrogated IL-4-induced survival of activated B cells, favoring the differentiation toward a plasma cell pathway. Moreover, B cells with restrained endogenous Gal-3 expression failed to down-regulate the Blimp-1 transcription factor after IL-4 stimulation. Finally, inhibition of Gal-3 in vivo skewed the balance toward plasma cell differentiation, which resulted in increased Ig production and parasite clearance during T. cruzi infection. Thus, the present study provides evidence of a novel role for Gal-3 as an intracellular mediator of B cell survival and a checkpoint in IL-4-induced B cell commitment toward a memory phenotype.
Improved Effector Activity and Memory CD8 T Cell Development by IL-2 Expression during Experimental Respiratory Syncytial Virus Infection
Authors: Chang Jun , Choi So Young , Jin Hyun Tak , Sung Young Chul , Braciale Thomas J.
Published: April 2014

Respiratory syncytial virus (RSV) is a major cause of lower respiratory infection in young children and the elderly. Studies of mice suggest that RSV suppresses the effector activity of CD8 T cells and the development of pulmonary CD8 T cell memory, in which the impaired effector activity could be recovered by in vitro IL-2 treatment. To investigate the effect of in vivo IL-2 expression on RSV immunity, mice were infected with RSV followed by administration of replication-defective adenovirus expressing IL-2. The effector activity of RSV M2-specific CD8 T cells and the development of CD8 T cell memory in the lung was significantly increased by IL-2 expression. Furthermore, the Ab responses against RSV were augmented by IL-2. Interestingly, weight loss and illness caused by RSV challenge were substantially reduced by IL-2 priming, suggesting that the pathogenesis of RSV-related disease could be prevented by IL-2-mediated enhancement of beneficial immune responses. Thus, our results show that IL-2 has potential to be used as a vaccine adjuvant against RSV infection.
Neutrophil Serine Proteinases Cleave Bacterial Flagellin, Abrogating Its Host Response-Inducing Activity
Authors: López-Boado Yolanda S. , Espinola Marcia , Bahr Scott , Belaaouaj Abderrazzaq
Published: April 2014

After bacterial infection, neutrophils dominate the cellular infiltrate. Their main function is assumed to be killing invading pathogens and resolving the inflammation they cause. Activated neutrophils are also known to release a variety of molecules, including the neutrophil serine proteinases, extracellularly. The release of these proteinases during inflammation creates a proteolytic environment where degradation of different molecules modulates the inflammatory response. Flagellin, the structural component of flagella on many bacterial species, is a virulence factor with a strong proinflammatory activity on epithelial cells and other cell types. In this study we show that both human and mouse neutrophil serine proteinases cleave flagellin from Pseudomonas aeruginosa and other bacterial species. More important, cleavage of P. aeruginosa flagellin by the neutrophil serine proteinases neutrophil elastase and cathepsin G resulted in loss of the biological activity of this virulence factor, as evidenced by the lack of innate host defense gene expression in human epithelial cells. The finding that flagellin is susceptible to cleavage by neutrophil serine proteinases suggests a novel role for these enzymes in the inflammatory response to infection. Not only can these enzymes kill bacteria, but they also degrade their virulence factors to halt the inflammatory response they trigger.
Murine γ-Herpesvirus-68-Induced IL-12 Contributes to the Control of Latent Viral Burden, but Also Contributes to Viral-Mediated Leukocytosis
Authors: Elsawa Sherine F. , Bost Kenneth L.
Published: April 2014

Early IFN-α/β production, followed by the development of a viral-specific CTL response, are critical factors in limiting the level of murine γ-herpesvirus-68 (γHV-68) infection. Development of a long-lived CTL response requires T cell help, and these CTLs most likely function to limit the extent of infection following reactivation. The importance of IL-12 in the development and/or activity of Th1 cells and CTLs is well documented, and we investigated the kinetics and magnitude of γHV-68-induced IL-12 production. Following intranasal infection, IL-12 and IL-23 mRNA expression was up-regulated in lung and spleen and lung, respectively, followed by increased levels of IL-12p40 in lung homogenates and sera. Exposure of cultured macrophages or dendritic cells to γHV-68 induced secretion of IL-12, suggesting that these cells might be responsible for IL-12 production in vivo. γHV-68 infection of mice made genetically deficient in IL-12p40 expression (IL-12p40−/−) resulted in a leukocytosis and splenomegaly that was significantly less than that observed in syngeneic C57BL/6 mice. IL-12p40−/− mice showed increased levels of infectious virus in the lung, but only at day 9 postinfection. Increased levels of latent virus in the spleen at day 15 postinfection were also observed in IL-12p40−/− mice when compared with syngeneic C57BL/6 mice. An overall reduction in γHV-68-induced IFN-γ production was observed in IL-12p40−/− mice, suggesting that most of the viral-induced IFN-γ in C57BL/6 mice was IL-12 dependent. Taken together, these results suggest that γHV-68-induced IL-12 contributes to the pathophysiology of viral infection while also functioning to limit viral burden.
IFN-Inducible Protein 10/CXC Chemokine Ligand 10-Independent Induction of Experimental Autoimmune Encephalomyelitis
Authors: Klein Robyn S. , Izikson Leonid , Means Terry , Gibson Hilary D. , Lin Eugene , Sobel Raymond A. , Weiner Howard L. , Luster Andrew D.
Published: April 2014

In multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), autoaggressive T cells traffic into the CNS and induce disease. Infiltration of these pathogenic T cells into the CNS has been correlated with the expression of the chemokine IFN-inducible protein (IP)10/CXC chemokine ligand (CXCL)10, a chemoattractant for activated T cells, and its receptor CXCR3, in the CNS of both MS patients and mice with EAE. In the present study, we report that targeted deletion of IP-10 did not diminish the expression, severity, or histopathology of EAE induced by active immunization with 100 μg of myelin oligodendrocyte glycoprotein peptide (MOG)p35–55. However, we found that IP-10-deficient mice had a lower threshold for expression of disease compared with wild-type littermates. EAE induced by immunization with 5 μg of MOGp35–55 resulted in more severe disease characterized by a greater number of CNS lesions and infiltrating mononuclear cells in IP-10-deficient mice compared with wild-type controls. IP-10-deficient mice immunized with MOGp35–55 demonstrated increased levels of IFN-inducible T cell α-chemokine/CXCL11 mRNA in the CNS and decreased levels of monokine induced by IFN-γ/CXCL9 mRNA in draining lymph nodes, suggesting differential compensation for loss of IP-10 in lymphoid vs parenchymal tissue compartments. EAE in IP-10-deficient mice induced by low-dose immunization was associated with enhanced Ag-specific Th1 responses in the draining lymph node, which corresponded with diminished lymph node TGF-β1 expression. Our data demonstrated that IP-10 was not required for the trafficking of pathogenic T cells into the CNS in EAE but played an unexpected role in determining the threshold of disease susceptibility in the periphery.
Neutralizing Endogenous IL-6 Renders Mast Cells of the MCT Type from Lung, but Not the MCTC Type from Skin and Lung, Susceptible to Human Recombinant IL-4-Induced Apoptosis
Authors: Oskeritzian Carole A. , Zhao Wei , Pozez Andrea L. , Cohen Neri M. , Grimes Margaret , Schwartz Lawrence B.
Published: April 2014

Human cord blood-derived mast cells undergo apoptosis upon exposure to recombinant human (rh)IL-4 and become resistant to rhIL-4-induced apoptosis when cultured in the presence of rhIL-6. The current study extends these effects of rhIL-4 to different populations of human mast cells, namely fetal liver-derived mast cells, lung-derived mast cells, and skin-derived mast cells. Endogenous production of IL-6 appears to protect fetal liver-derived mast cells and those of the MCT phenotype from rhIL-4-mediated apoptosis, because neutralization of IL-6 renders these mast cells sensitive. In contrast, mast cells of the MCTC phenotype from skin and lung were resistant to IL-4-mediated apoptosis, even after neutralization of endogenous IL-6. MCTC cells were CD124low, whereas those of the MCT cells were CD124high. These observations extend the phenotypic differences between MCT and MCTC types of human mast cells to include different functional responses to IL-4.
Intercellular Adhesion Molecule-1 Mediates Cellular Cross-Talk between Parenchymal and Immune Cells after Lipopolysaccharide Neutralization
Authors: Lee Jin-Hwa , Del Sorbo Lorenzo , Uhlig Stefan , Porro Giuliana A. , Whitehead Thomas , Voglis Stefanos , Liu Mingyao , Slutsky Arthur S. , Zhang Haibo
Published: April 2014

The mechanisms by which parenchymal cells interact with immune cells, particularly after removal of LPS, remain unknown. Lung explants from rats, mice deficient in the TNF gene, or human lung epithelial A549 cells were treated with LPS and washed, before naive alveolar macrophages, bone marrow monocytes, or PBMC, respectively, were added to the cultures. When the immune cells were cocultured with LPS-challenged explants or A549 cells, TNF production was greatly enhanced. This was not affected by neutralization of LPS with polymyxin B. The LPS-induced increase in the expression of ICAM-1 on A549 cells correlated with TNF production by PBMC. The cellular cross talk leading to the TNF response was blunted by an anti-ICAM-1 Ab and an ICAM-1 antisense oligonucleotide. In A549 cells, a persistent decrease in the concentration of intracellular cAMP was associated with colocalization of LPS into Toll-like receptor 4 and the Golgi apparatus, resulting in increased ICAM-1 expression. Inhibition of LPS internalization by cytochalasin D or treatment with dibutyryl cAMP attenuated ICAM-1 expression and TNF production by PBMC. In conclusion, lung epithelial cells are not bystanders, but possess memory of LPS through the expression of ICAM-1 that interacts with and activates leukocytes. This may provide an explanation for the failure of anti-LPS therapies in sepsis trials.
Corticosteroids Regulate Epithelial Cell Differentiation and Hassall Body Formation in the Human Thymus
Authors: Hale Laura P. , Markert M. Louise
Published: April 2014

The presence of characteristic epithelial swirls called Hassall bodies within the human thymic medulla has been used as an indicator of ongoing or recent thymopoiesis. We present a case where Hassall bodies were present in the absence of current or past thymopoiesis. The patient had been treated with corticosteroids for presumed asthma before his diagnosis of X-linked SCID. Two other cases of nonimmunodeficient patients treated with high-dose corticosteroids had markedly increased numbers of thymic Hassall bodies. To determine whether corticosteroid treatment induces thymic epithelial (TE) differentiation to form Hassall bodies, mAbs reactive with specific cytokeratins (CKs), filaggrin, and involucrin were used to define distinct stages of TE cell differentiation. Treatment of primary TE monolayers with hydrocortisone in vitro induced expression of involucrin and high-molecular-mass CKs that are characteristic of TE differentiation. Treatment of thymic organ cultures with hydrocortisone induced both medullary and subcapsular cortical TE cells to express CK6, a differentiation marker that is normally expressed only by Hassall bodies in vivo. These experimental studies combined with the case observations indicate that exogenous corticosteroids can regulate terminal differentiation of TE cells both in vitro and in vivo. Thus, the presence of Hassall bodies in thymus from corticosteroid-treated patients cannot be taken as an absolute indication of previous thymopoiesis. Because corticosteroids are also made within the thymus under normal physiologic conditions, these studies support the hypothesis that endogenous corticosteroids may play a role in normal TE differentiation and Hassall body formation in vivo.
Gene Expression Profiling Provides Insight into the Pathophysiology of Chronic Granulomatous Disease
Authors: Kobayashi Scott D. , Voyich Jovanka M. , Braughton Kevin R. , Whitney Adeline R. , Nauseef William M. , Malech Harry L. , DeLeo Frank R.
Published: April 2014

Human polymorphonuclear leukocytes (PMNs or neutrophils) kill invading microorganisms with reactive oxygen species (ROS) and cytotoxic granule components. PMNs from individuals with X-linked chronic granulomatous disease (XCGD) do not produce ROS, thereby rendering these individuals more susceptible to infection. In addition, XCGD patients develop tissue granulomas that obstruct vital organs, the mechanism(s) for which are unknown. To gain insight into the molecular processes that contribute to the pathophysiology of XCGD, including formation of granulomas, we compared global gene expression in PMNs from XCGD patients and healthy control individuals. Genes encoding mediators of inflammation and host defense, including CD11c, CD14, CD54, FcγR1, FcαR, CD120b, TLR5, IL-4R, CCR1, p47phox, p40phox, IL-8, CXCL1, Nramp1, and calgranulins A and B, were up-regulated constitutively in unstimulated XCGD patient PMNs. By comparing transcript levels in normal and XCGD PMNs after phagocytosis, we discovered 206 genes whose expression changed in the presence and the absence of ROS, respectively. Notably, altered Bcl2-associated X protein synthesis accompanied defective neutrophil apoptosis in XCGD patients. We hypothesize that granuloma formation in XCGD patients reflects both increased proinflammatory activity and defective PMN apoptosis, and we conclude that ROS contribute directly or indirectly to the resolution of the inflammatory response by influencing PMN gene transcription.
A Novel Mechanism of Alternative Promoter and Splicing Regulates the Epitope Generation of Tumor Antigen CML66-L
Authors: Yan Yan , Phan Leuyen , Yang Fan , Talpaz Moshe , Yang Yu , Xiong Zeyu , Ng Bernard , Timchenko Nikolai A. , Wu Catherine J. , Ritz Jerome , Wang Hong , Yang Xiao-Feng
Published: April 2014

This report describes the difference in the epitope generation of two isoforms of self-tumor Ag CML66 and the regulation mechanism. We identified a new CML66 short isoform, termed CML66-S. The previously identified long CML66 is referred to as CML66-L. CML66-S shares the C terminus with CML66-L but has its unique N terminus. CML66-S is predominantly expressed in testis, but is also expressed in very low levels in tumor cells, whereas CML66-L is expressed in tumor cells and testis. Differential expression of CML66-L and CML66-S in tumor cells resulted from regulation at transcription, although alternative splicing also participated in the generation of the isoforms. In addition, Ab titers to a CML66-L peptide were significantly higher than that to CML66-S peptide in the sera from patients with tumors. Finally, the Abs to full-length CML66-L in the sera from patients with tumors were correlated with the Abs in the sera from these patients to CML66-L-38, which is a fusion protein with a CML66-L-specific N terminus. This suggests that the CML66-L isoform is mainly responsible for the epitope generation. Our studies have identified the alternative promoter in combination with alternative splicing as a novel mechanism for regulation of the epitope generation of a self-tumor Ag.
Antibody Cross-Reactivity between Myelin Oligodendrocyte Glycoprotein and the Milk Protein Butyrophilin in Multiple Sclerosis
Authors: Guggenmos Johannes , Schubart Anna S. , Ogg Sherry , Andersson Magnus , Olsson Tomas , Mather Ian H. , Linington Christopher
Published: April 2014

The etiology of multiple sclerosis (MS) is believed to involve environmental factors, but their identity and mode of action are unknown. In this study, we demonstrate that Ab specific for the extracellular Ig-like domain of myelin oligodendrocyte glycoprotein (MOG) cross-reacts with a homologous N-terminal domain of the bovine milk protein butyrophilin (BTN). Analysis of paired samples of MS sera and cerebrospinal fluid (CSF) identified a BTN-specific Ab response in the CNS that differed in its epitope specificity from that in the periphery. This effect was statistically significant for the Ab response to BTN76–100 (p = 0.0026), which cosequestered in the CSF compartment with Ab to the homologous MOG peptide MOG76–100 in 34% of MS patients (n = 35). These observations suggested that intratheccal synthesis of Ab recognizing BTN peptide epitopes in the CNS was sustained by molecular mimicry with MOG. Formal evidence of molecular mimicry between the two proteins was obtained by analyzing MOG-specific autoantibodies immunopurified from MS sera. The MOG-specific Ab repertoire cross-reacts with multiple BTN peptide epitopes including a MOG/BTN76–100-specific component that occurred at a higher frequency in MS patients than in seropositive healthy controls, as well as responses to epitopes within MOG/BTN1–39 that occur at similar frequencies in both groups. The demonstration of molecular mimicry between MOG and BTN, along with sequestration of BTN-reactive Ab in CSF suggests that exposure to this common dietary Ag may influence the composition and function of the MOG-specific autoimmune repertoire during the course of MS.
A Major B Cell Epitope Present on the Apoptotic but Not the Intact Form of the U1-70-kDa Ribonucleoprotein Autoantigen
Authors: Greidinger Eric L. , Foecking Mark F. , Magee Joseph , Wilson Linda , Ranatunga Sriya , Ortmann Robert A. , Hoffman Robert W.
Published: April 2014

Apoptotically modified forms of autoantigens have been hypothesized to participate in lupus immunopathogenesis. This study identifies a major B cell epitope present on the apoptotic but not the intact form of the U1-70-kDa ribonucleoprotein lupus autoantigen (70k). Human autoimmune sera with strong recognition of apoptotic 70k and minimal recognition of intact 70k were identified and tested for reactivity to truncated forms of 70k by immunoblot and ELISA. Patient sera that preferentially recognized apoptotic 70k were specific for an epitope dependent on residues 180–205 of the protein. This epitope was also recognized by 19 of 28 (68%) intact anti-70k-positive autoimmune human sera with Abs also recognizing apoptotic but not the intact form 70k, but only 1 of 9 (11%) intact 70k-positive sera without such Abs (Fisher’s exact, p = 0.0055). Immunization of HLA-DR4-transgenic C57BL/6 mice with a peptide containing this epitope induced anti-70k immunity in 13 of 15 mice, including Abs recognizing apoptotic but not intact forms of autoantigens in 12 of 15 mice. Anti-70k responder mice also developed spreading of immunity to epitopes on the endogenous form of 70k, and proliferative lung lesions consistent with those described in patients with anti-70k autoimmunity. Thus, a major epitope in the B cell response to U1-70 kDa localizes to the RNA binding domain of the molecule, overlaps with the most common T cell epitope in the anti-70k response, and is not present on the intact form of the 70k molecule. Immunization of mice against this epitope induces an immune response with features seen in human anti-70k autoimmune disease.
DNA Acrobats of the Ig Class Switch
Authors: Wang Clifford L. , Wabl Matthias
Published: April 2014

Small resting B lymphocytes all start out producing IgM Abs. Upon encountering Ag, the cells become activated and make a switch from IgM to other Ig classes. This class switch serves to distribute a particular V region to different Ig C regions. Each C region mediates a specialized effector function, and so, through switching, an organism can guide its Abs to various sites. Creating the new H chain requires loop-out and deletion of DNA between switch regions. These DNA acrobatics require transcription of the switch regions, presumably so that necessary factors can gain access to the DNA. These requisite switching factors include activation-induced cytidine deaminase and components of general DNA repair, including base excision repair, mismatch repair, and double-strand break repair. Despite much recent progress, not all important factors have been discovered, especially those that may guide recombination to a particular subclass.
Cutting Edge: The Natural Ligand for Glucocorticoid-Induced TNF Receptor-Related Protein Abrogates Regulatory T Cell Suppression
Authors: Ji Hong-bin , Liao Gongxian , Faubion William A. , Abadía-Molina Ana C. , Cozzo Cristina , Laroux F. Stephen , Caton Andrew , Terhorst Cox
Published: April 2014

CD4+25+ regulatory T (Treg) cells maintain immunological self-tolerance through mechanisms that are only in part understood. Previous studies suggest that the glucocorticoid-induced TNFR-related protein (GITR), which is preferentially expressed on the surface of Treg cells, potentially provides a signal that abrogates Treg suppression. In this study, we show that a soluble form of mouse GITR ligand (sGITR-L) induces GITR-dependent NF-κB activation and blocks in vitro suppression mediated by both resting and preactivated polyclonal and Ag-specific Treg cells. Since sGITR-L along with rIL-2 induces proliferation of CD4+25+ cells, it appears that sGITR-L can break the anergic state of Treg cells. Because sGITR-L also up-regulates IL-2 secretion by activated CD4+25 −T cells, these two sGITR-L induced signals synergize to interfere with suppressor activity by CD4+25+ Treg cells.
Shift from Systemic to Site-Specific Memory by Tumor-Targeted IL-2
Authors: Schrama David , Xiang Rong , Eggert Andreas O. , Andersen Mads Hald , Pedersen Lars Østergaard , Kämpgen Eckhart , Schumacher Ton N. , Reisfeld Ralph R. , Becker Jürgen C.
Published: April 2014

IL-2 has been approved for treatment of patients with cancer. Moreover, it has been used as a component of vaccines against cancer. In this regard, we have recently demonstrated that dendritic cell-based peptide vaccination in mice required IL-2 to mount an effective immune response against established melanoma metastases. In this study, we confirm this observation by use of tumor-targeted IL-2. However, the development of a protective systemic memory was substantially impaired by this measure, i.e., mice, which successfully rejected s.c. tumors of B16 melanoma after vaccination with dendritic cells pulsed with tyrosinase-related protein 2-derived peptides plus a boost with targeted IL-2, failed to reject a rechallenge with experimental pulmonary metastases. Detailed analysis revealed a change in the distribution of the tumor-reactive T cell population: although targeted IL-2 expanded the local effector population, tyrosinase-related protein 2-reactive T cells were almost completely depleted from lymphatic tissues.
Evolutionary Origins of Lymphocytes: Ensembles of T Cell and B Cell Transcriptional Regulators in a Cartilaginous Fish
Authors: Anderson Michele K. , Pant Rashmi , Miracle Ann L. , Sun Xiao , Luer Carl A. , Walsh Catherine J. , Telfer Janice C. , Litman Gary W. , Rothenberg Ellen V.
Published: April 2014

The evolutionary origins of lymphocytes can be traced by phylogenetic comparisons of key features. Homologs of rearranging TCR and Ig (B cell receptor) genes are present in jawed vertebrates, but have not been identified in other animal groups. In contrast, most of the transcription factors that are essential for the development of mammalian T and B lymphocytes belong to multigene families that are represented by members in the majority of the metazoans, providing a potential bridge to prevertebrate ancestral roles. This work investigates the structure and regulation of homologs of specific transcription factors known to regulate mammalian T and B cell development in a representative of the earliest diverging jawed vertebrates, the clearnose skate (Raja eglanteria). Skate orthologs of mammalian GATA-3, GATA-1, EBF-1, Pax-5, Pax-6, Runx2, and Runx3 have been characterized. GATA-3, Pax-5, Runx3, EBF-1, Spi-C, and most members of the Ikaros family are shown throughout ontogeny to be 1) coregulated with TCR or Ig expression, and 2) coexpressed with each other in combinations that for the most part correspond to known mouse T and B cell patterns, supporting conservation of function. These results indicate that multiple components of the gene regulatory networks that operate in mammalian T cell and B cell development were present in the common ancestor of the mammals and the cartilaginous fish. However, certain factors relevant to the B lineage differ in their tissue-specific expression patterns from their mouse counterparts, suggesting expanded or divergent B lineage characteristics or tissue specificity in these animals.
Differential Gene Expression Patterns by Oligonucleotide Microarray of Basal versus Lipopolysaccharide-Activated Monocytes from Cord Blood versus Adult Peripheral Blood
Authors: Jiang Hong , Van de Ven Carmella , Satwani Prakash , Baxi Laxmi V. , Cairo Mitchell S.
Published: April 2014

Monocytes (Mo) are critically important in the generation of inflammatory mediators, cytokines/chemokines, and regulation of innate and adaptive immune responses. We and others have previously demonstrated significant dysregulated cytokine gene expression and protein production and in vitro functional activities of activated cord blood (CB) vs adult peripheral blood (APB) mononuclear cells (MNC). In this study, we compared, by oligonucleotide microarray, the differential gene expression profiles of basal and LPS-activated APB vs CB Mo. We demonstrated a significant increase in the gene expression of several important functional groups of CB genes compared with basal levels including cytokine (IL-12p40, 5-fold), immunoregulatory (signaling lymphocytic activation molecule, 4-fold), signal transduction (Pim-2, 3-fold), and cell structure (Rho7, 4-fold) among others. Furthermore, there was significantly differentially amplified gene expression in LPS-activated APB vs LPS-activated CB Mo, including cytokine (G-CSF, 14-fold), chemokine (macrophage-inflammatory protein 1α, 5-fold), immunoregulatory (MHC DRB1, 5-fold), transcription factor (JunB, 4-fold), signal transduction (STAT4, 5-fold), apoptotic regulation (BAX, 5-fold), and cell structure (ladinin 1, 6-fold) among others. These results provide insight into the molecular basis for normal genetic regulation of Mo development and cellular function and differential inflammatory and innate and adaptive immune responses between activated CB and APB Mo.
Generation of Anergic and Regulatory T Cells following Prolonged Exposure to a Harmless Antigen
Authors: Chen Tse-Ching , Cobbold Stephen P. , Fairchild Paul J. , Waldmann Herman
Published: April 2014

Regulatory CD4+ T cells are known to develop during the induction of donor-specific peripheral tolerance to transplanted tissues; it is proposed that such tolerance is a consequence of persistent, danger-free stimulation by Ag. To test this hypothesis, male RAG-1−/− mice were recolonized with small numbers of monospecific CD4+ T cells specific for the male H-2Ek-restricted Ag Dby. After 6 wk in the male environment, the monospecific CD4+ T cells, having recolonized the host, had become anergic to stimulation in vitro and had acquired a regulatory capacity. CD4+ T cells in these mice expressed higher levels of CTLA-4 and glucocorticoid-induced TNF-related receptor than naive CD4+ T cells, but only 3% of the recolonizing cells were CD25+ and did not express significant foxP3 mRNA. In vivo, these tolerant T cells could censor accumulation of, and IFN-γ production by, naive T cells, with only a slight inhibition of proliferation. This suppressive effect was not reversed by the addition of fresh bone marrow-derived male dendritic cells. These results suggest that persistent exposure to Ag in conditions that fail to evoke proinflammatory stimuli leads to the development of T cells that are both anergic and regulatory.
IL-2 Induces a Competitive Survival Advantage in T Lymphocytes
Authors: Dooms Hans , Kahn Estelle , Knoechel Birgit , Abbas Abul K.
Published: April 2014

The acquisition of long-term survival potential by activated T lymphocytes is essential to ensure the successful development of a memory population in the competitive environment of the lymphoid system. The factors that grant competitiveness for survival to primed T cells are poorly defined. We examined the role of IL-2 signals during priming of CD4+ T cells in the induction of a long-lasting survival program. We show that Ag-induced cycling of CD4+ IL-2−/− T cells is independent of IL-2 in vitro. However, IL-2−/− T cells failed to accumulate in large numbers and develop in effector cells when primed in the absence of IL-2. More importantly, Ag-activated IL-2−/− T cells were unable to survive for prolonged periods of time after adoptive transfer in unmanipulated, syngeneic mice. IL-2−/− T cells exposed to IL-2 signals during priming, however, acquired a robust and long-lasting survival advantage over cells that cycled in the absence of IL-2. Interestingly, this IL-2-induced survival program was required for long-term persistence of primed IL-2−/− T cells in an intact lymphoid compartment, but was unnecessary in a lymphopenic environment. Therefore, IL-2 enhances competitiveness for survival in CD4+ T cells, thereby facilitating the development of a memory population.
Adult Human Liver Contains CD8pos T Cells with Naive Phenotype, but Is Not a Site for Conventional αβ T Cell Development
Authors: Golden-Mason Lucy , Douek Daniel C. , Koup Richard A. , Kelly Jacinta , Hegarty John E. , O’Farrelly Cliona
Published: April 2014

Normal adult human liver (AHL) contains populations of unconventional lymphocytes that have been shown in the mouse to mature locally. The presence of lymphoid progenitors together with IL-7, recombinase-activating gene, and pre-TCR-α expression in AHL suggests similar local T cell development activity in humans. Flow cytometry was used to characterize potentially naive hepatic αβ-T cells. We looked for evidence of TCR-αβ cell development in AHL by quantifying δ deletion TCR excision circles (TRECs) in CD3pos populations isolated from the liver and matched blood of eight individuals. Phenotypic analysis of hepatic T cells suggests the presence of Ag-inexperienced populations. TRECs were detected in all blood samples (mean, 164.10 TRECs/μg DNA), whereas only two hepatic samples were positive at low levels (59.40 and 1.92). The relatively high level of CD8pos T cells in these livers with a naive phenotype suggests that in addition to its role as a graveyard for Ag-specific activated CD8pos T cells, naive CD8pos T cells may enter the liver without prior activation. The almost complete absence of TRECs suggests that normal AHL is not a site for the development of conventional αβ T cells.
Thymic Expression of a Gastritogenic Epitope Results in Positive Selection of Self-Reactive Pathogenic T Cells
Authors: Laurie Karen L. , La Gruta Nicole L. , Koch Norbert , van Driel Ian R. , Gleeson Paul A.
Published: April 2014

Intrathymic expression of tissue-specific self-Ags can mediate tolerance of self-reactive T cells. However, in this study we define circumstances by which thymic expression of a tissue-specific autoepitope enhances positive selection of disease-causing, self-reactive T cells. An immunodominant gastritogenic epitope, namely the gastric H/K ATPase β subunit253–277 (H/Kβ253–277), was attached to the C terminus of the invariant chain (Ii) and the hybrid Ii (Ii-H/Kβ253–277) expressed in mice under control of the Ii promoter. The Ii-H/Kβ253–277 fusion protein was localized to MHC class II-expressing cells in the thymus and periphery of Ii-H/Kβ253–277 transgenic mice. In one transgenic line the level of presentation in the periphery (spleen) was insufficient to activate naive, low affinity H/Kβ253–277-specific transgenic T cells (1E4-TCR), whereas thymic presentation of H/Kβ253–277 enhanced positive selection of 1E4-TCR cells in Ii-H/Kβ253–277/1E4-TCR double-transgenic mice. Furthermore, Ii-H/Kβ253–277/1E4-TCR double-transgenic mice had an increased incidence of autoimmune gastritis compared with 1E4-TCR single-transgenic mice, demonstrating that the 1E4 T cells that seeded the periphery of Ii-H/Kβ253–277 mice were pathogenic. Therefore, low levels of tissue-specific Ags in the thymus can result in positive selection of low avidity, self-reactive T cells. These findings also suggest that the precise level of tissue-specific Ags in the thymus may be an important consideration in protection against autoimmune disease and that perturbation of the levels of self-Ags may be detrimental.