bluuebunny/crossref_metadata_2025 huggingface dataset:
IFN-α Suppresses Activation of Nuclear Transcription Factors NF-κB and Activator Protein 1 and Potentiates TNF-Induced Apoptosis
Authors: Manna Sunil K. , Mukhopadhyay Asok , Aggarwal Bharat B.
Published: April 2014

We and others have reported that IFN-α potentiates the apoptotic effects of TNF through a mechanism that is not understood. Because the nuclear transcription factors NF-κB and AP-1 have recently been reported to mediate anti-apoptosis and cell survival, we hypothesized that IFN-α potentiates the cytotoxic effects of TNF by suppressing TNF-induced activation of NF-κB and AP-1. We tested this hypothesis by pretreating human Jurkat T cells with IFN-α, which blocked TNF-induced activation of NF-κB and AP-1 in a time- and dose-dependent manner as determined by EMSA. IFN-α blocked TNF-induced phosphorylation and degradation of the inhibitor subunit of NF-κB, and suppressed NF-κB and AP-1 activation induced by various other inflammatory stimuli. NF-κB-dependent reporter gene expression activated by TNF, TNFR1, TNF receptor-associated factor 2, and NF-κB-inducing kinase was also abrogated by IFN-α pretreatment. The suppression of NF-κB and AP-1 correlated with the potentiation of TNF-induced cytotoxicity and caspase activation. Overall our results suggest that IFN-α potentiates the apoptotic effects of TNF possibly by suppressing NF-κB and AP-1 activation.
Emergence of CD8+ T Cells Expressing NK Cell Receptors in Influenza A Virus-Infected Mice
Authors: Kambayashi Taku , Assarsson Erika , Michaëlsson Jakob , Berglund Peter , Diehl Alexander D. , Chambers Benedict J. , Ljunggren Hans-Gustaf
Published: April 2014

Both innate and adaptive immune responses play an important role in the recovery of the host from viral infections. In the present report, a subset of cells coexpressing CD8 and NKR-P1C (NK1.1) was found in the lungs of mice infected with influenza A virus. These cells were detected at low numbers in the lungs of uninfected mice, but represented up to 10% of the total CD8+ T cell population at day 10 postinfection. Almost all of the CD8+NK1.1+ cells were CD8αβ+CD3+TCRαβ+ and a proportion of these cells also expressed the NK cell-associated Ly49 receptors. Interestingly, up to 30% of these cells were virus-specific T cells as determined by MHC class I tetramer staining and by intracellular staining of IFN-γ after viral peptide stimulation. Moreover, these cells were distinct from conventional NKT cells as they were also found at increased numbers in influenza-infected CD1−/− mice. These results demonstrate that a significant proportion of CD8+ T cells acquire NK1.1 and other NK cell-associated molecules, and suggests that these receptors may possibly regulate CD8+ T cell effector functions during viral infection.
Ectopic Human Telomerase Catalytic Subunit Expression Maintains Telomere Length But Is Not Sufficient for CD8+ T Lymphocyte Immortalization
Authors: Migliaccio Marco , Amacker Mario , Just Tom , Reichenbach Patrick , Valmori Danila , Cerottini Jean-Charles , Romero Pedro , Nabholz Markus
Published: April 2014

Like most somatic human cells, T lymphocytes have a limited replicative life span. This phenomenon, called senescence, presents a serious barrier to clinical applications that require large numbers of Ag-specific T cells such as adoptive transfer therapy. Ectopic expression of hTERT, the human catalytic subunit of the enzyme telomerase, permits fibroblasts and endothelial cells to avoid senescence and to become immortal. In an attempt to immortalize normal human CD8+ T lymphocytes, we infected bulk cultures or clones of these cells with a retrovirus transducing an hTERT cDNA clone. More than 90% of transduced cells expressed the transgene, and the cell populations contained high levels of telomerase activity. Measuring the content of total telomere repeats in individual cells (by flowFISH) we found that ectopic hTERT expression reversed the gradual loss of telomeric DNA observed in control populations during long term culture. Telomere length in transduced cells reached the levels observed in freshly isolated normal CD8+ lymphocytes. Nevertheless, all hTERT-transduced populations stopped to divide at the same time as nontransduced or vector-transduced control cells. When kept in IL-2 the arrested cells remained alive. Our results indicate that hTERT may be required but is not sufficient to immortalize human T lymphocytes.
Differentiation of Murine NK Cells into Distinct Subsets Based on Variable Expression of the IL-12Rβ2 Subunit
Authors: Chakir Habiba , Camilucci Allison A. , Filion Lionel G. , Webb John R.
Published: April 2014

The cytokine IL-12 manifests its biological activity via interaction with a heterodimeric receptor (IL-12R) present on activated T and NK cells. The cDNAs for two IL-12R subunits have been cloned from human and mouse and designated IL-12Rβ1 and IL-12Rβ2. The expression of IL-12Rβ2 on T cells is influenced by cytokines, particularly IL-4, IL-12, and IFN-γ; however, little is known regarding regulation of IL-12R expression on NK cells. In this study we show that murine NK cells differentiate into IL-12Rβ2low and IL-12Rβ2high subsets after in vitro stimulation with IL-2 in the absence of exogenous polarizing cytokines. Subset development occurs gradually as NK cells expand in vitro and is generally complete by 8–12 days of culture. Once established, IL-12Rβ2low and IL-12Rβ2high subsets are highly stable in vitro and can be maintained for at least 20 days after FACS sorting. Formation of these NK subsets appears to be strain independent. Flow cytometric analyses demonstrate that both subsets express a number of NK-associated markers, including NK1.1, DX-5, Ly-49A, and Ly-49C, but that the Ly-49G2 class I inhibitory receptor is expressed predominantly on the IL-12Rβ2high population. Both IL-12Rβ2low and IL-12Rβ2high NK cells respond to exogenous IL-12 by rapid production of high levels of IFN-γ and increased lytic activity against NK-sensitive YAC-1 target cells. Analyses of cytokine gene expression by RNase protection assay indicated that similar to the recently described human NK1 subset, both IL-12Rβ2high and IL-12Rβ2low murine NK subsets expressed high levels of IFN-γ, whereas neither subset expressed mRNA for the NK2-associated cytokines IL-5 and IL-13.
NF-κB Is Required for the Positive Selection of CD8+ Thymocytes
Authors: Hettmann Thore , Leiden Jeffrey M.
Published: April 2014

To examine the role of NF-κB in T cell development, we analyzed thymocyte ontogeny in transgenic (mutant I-κBα (mI-κBα)) mice that express a superinhibitory form of the NF-κB inhibitory protein, I-κBα (I-κBαA32/36), under the control of the T cell-specific CD2 promoter and enhancer. Thymi from mI-κBα mice contained increased numbers of double-positive (DP) and decreased numbers of both CD4+ and CD8+ single-positive cells, consistent with a block in DP thymocyte maturation. In addition, expression of CD69, a marker of positive selection, was decreased on DP thymocytes from the mI-κBα mice. To test directly whether NF-κB was required for positive or negative selection, we generated mI-κBα mice expressing the H-Y or 2C αβ TCR transgenes. Expression of the I-κBαA32/36 transgene caused a block in the positive selection of CD8+ single-positive cells in both strains of TCR transgenic animals. In contrast, negative selection was unaffected by expression of the I-κBαA32/36 transgene. Taken together, these results identified a NF-κB-dependent transcriptional pathway that is selectively required for the positive selection of CD8+ thymocytes.
Microbial and T Cell-Derived Stimuli Regulate Antigen Presentation by Dendritic Cells In Vivo
Authors: Manickasingham Shivanthi , Reis e Sousa Caetano
Published: April 2014

B cells and dendritic cells (DC) internalize and degrade exogenous Ags and present them as peptides bound to MHC class II molecules for scrutiny by CD4+ T cells. Here we use an Ab specific for a processed form of the model Ag, hen egg lysozyme (HEL), to demonstrate that this protein is not efficiently presented by lymph node DC following s.c. immunization. HEL presentation by the DC can be dramatically enhanced upon coinjection of a microbial adjuvant, which appears to act by enhancing peptide loading onto MHC class II. CD40 cross-linking or the presence of a high frequency of T cells specific for HEL can similarly improve presentation by DC in vivo. For any of these activating stimuli, CD8α+ DC consistently display the highest proportion of HEL-loaded MHC class II molecules. These data indicate that exogenous Ags can be displayed to T cells in lymphoid tissues by a large cohort of resident DC whose presentation is regulated by innate and adaptive stimuli. Our data further reveal the existence of a feedback mechanism that augments Ag presentation during cognate APC-T cell interactions.
Receptor for Activated C-Kinase (RACK-1), a WD Motif-Containing Protein, Specifically Associates with the Human Type I IFN Receptor
Authors: Croze Ed , Usacheva Anna , Asarnow David , Minshall Richard D. , Perez H. D. , Colamonici Oscar
Published: April 2014

The cytoplasmic domain of the human type I IFN receptor chain 2 (IFNAR2c or IFN-αRβL) was used as bait in a yeast two-hybrid system to identify novel proteins interacting with this region of the receptor. We report here a specific interaction between the cytoplasmic domain of IFN-αRβL and a previously identified protein, RACK-1 (receptor for activated C kinase). Using GST fusion proteins encoding different regions of the cytoplasmic domain of IFN-αRβL, the minimum site for RACK-1 binding was mapped to aa 300–346. RACK-1 binding to IFN-αRβL did not require the first 91 aa of RACK-1, which includes two WD domains, WD1 and WD2. The interaction between RACK-1 and IFN-αRβL, but not the human IFN receptor chain 1 (IFNAR1 or IFN-αRα), was also detected in human Daudi cells by coimmunoprecipitation. RACK-1 was shown to be constitutively associated with IFN-αRβL, and this association was not effected by stimulation of Daudi cells with type I IFNs (IFN-β1b). RACK-1 itself did not become tyrosine phosphorylated upon stimulation of Daudi cells with IFN-β1b. However, stimulation of cells with either IFN-β1b or PMA did result in an increase in detectable immunofluorescence and intracellular redistribution of RACK-1.
DNA Vaccination Against Rat Her-2/Neu p185 More Effectively Inhibits Carcinogenesis Than Transplantable Carcinomas in Transgenic BALB/c Mice
Authors: Rovero Stefania , Amici Augusto , Carlo Emma Di , Bei Roberto , Nanni Patrizia , Quaglino Elena , Porcedda Paola , Boggio Katia , Smorlesi Arianna , Lollini Pier-Luigi , Landuzzi Lorena , Colombo Mario P. , Giovarelli Mirella , Musiani Piero , Forni Guido
Published: April 2014

The ability of vaccination with plasmids coding for the extracellular and the transmembrane domain of the product of transforming rat Her-2/neu oncogene (r-p185) to protect against r-p185+ transplantable carcinoma (TUBO) cells and mammary carcinogenesis was evaluated. In normal BALB/c mice, DNA vaccination elicits anti-r-p185 Ab, but only a marginal CTL reactivity, and protects against a TUBO cell challenge. Massive reactive infiltration is associated with TUBO cell rejection. In BALB/c mice transgenic for the rat Her-2/neu gene (BALB-neuT), DNA vaccination elicits a lower anti-r-p185 Ab response, no CTL activity and only incompletely protects against TUBO cells, but markedly hampers the progression of carcinogenesis. At 33 wk of age, when control BALB-neuT mice display palpable tumors in all mammary glands, about 60% of immunized mice are tumor free, and tumor multiplicity is markedly reduced. Tumor-free mammary glands still display the atypical hyperplasia of the early stages of carcinogenesis, and a marked down-modulation of r-p185, along with a massive reactive infiltrate. However, BALB-neuT mice protected against mammary carcinogenesis fail to efficiently reject a TUBO cell challenge. This suggests that the mechanisms required for the rejection of transplantable tumors may not coincide with those that inhibit the slow progression of carcinogenesis.
Archaeosomes Induce Long-Term CD8+ Cytotoxic T Cell Response to Entrapped Soluble Protein by the Exogenous Cytosolic Pathway, in the Absence of CD4+ T Cell Help
Authors: Krishnan Lakshmi , Sad Subash , Patel Girishchandra B. , Sprott G. Dennis
Published: April 2014

The unique ether glycerolipids of Archaea can be formulated into vesicles (archaeosomes) with strong adjuvant activity for MHC class II presentation. Herein, we assess the ability of archaeosomes to facilitate MHC class I presentation of entrapped protein Ag. Immunization of mice with OVA entrapped in archaeosomes resulted in a potent Ag-specific CD8+ T cell response, as measured by IFN-γ production and cytolytic activity toward the immunodominant CTL epitope OVA257–264. In contrast, administration of OVA with aluminum hydroxide or entrapped in conventional ester-phospholipid liposomes failed to evoke significant CTL response. The archaeosome-mediated CD8+ T cell response was primarily perforin dependent because CTL activity was undetectable in perforin-deficient mice. Interestingly, a long-term CTL response was generated with a low Ag dose even in CD4+ T cell deficient mice, indicating that the archaeosomes could mediate a potent T helper cell-independent CD8+ T cell response. Macrophages incubated in vitro with OVA archaeosomes strongly stimulated cytokine production by OVA-specific CD8+ T cells, indicating that archaeosomes efficiently delivered entrapped protein for MHC class I presentation. This processing of Ag was Brefeldin A sensitive, suggesting that the peptides were transported through the endoplasmic reticulum and presented by the cytosolic MHC class I pathway. Finally, archaeosomes induced a potent memory CTL response to OVA even 154 days after immunization. This correlated to strong Ag-specific up-regulation of CD44 on splenic CD8+ T cells. Thus, delivery of proteins in self-adjuvanting archaeosomes represents a novel strategy for targeting exogenous Ags to the MHC class I pathway for induction of CTL response.
Brucella abortus Lipopolysaccharide in Murine Peritoneal Macrophages Acts as a Down-Regulator of T Cell Activation
Authors: Forestier Claire , Deleuil Fabienne , Lapaque Nicolas , Moreno Edgardo , Gorvel Jean-Pierre
Published: April 2014

Macrophages play a central role in host immune responses against pathogens by acting as both professional phagocytic cells and as fully competent APCs. We report here that the LPS from the facultative intracellular Gram-negative bacteria Brucella abortus interferes with the MHC class II Ag presentation pathway. LPS inhibits the capacity of macrophages to present hen egg lysozyme (HEL) antigenic peptides to specific CD4+ T cells but not those of OVA to specific CD8+ T cells. This defect was neither related to a decrease of MHC class II surface expression nor to a deficient uptake or processing of HEL. In addition, B. abortus LPS did not prevent the formation of SDS-resistant MHC class II complexes induced by HEL peptides. At the cell surface of macrophages, we observed the presence of LPS macrodomains highly enriched in MHC class II molecules, which may be responsible for the significant down-regulation of CD4+ T cell activation. This phenomenon may account for the avoidance of the immune system by certain bacterial pathogens and may explain the immunosuppression observed in individuals with chronic brucellosis.
Role of p38 Mitogen-Activated Protein Kinase in Rhinovirus-Induced Cytokine Production by Bronchial Epithelial Cells
Authors: Griego Sandra D. , Weston Cynthia B. , Adams Jerry L. , Tal-Singer Ruth , Dillon Susan B.
Published: April 2014

The stress-activated protein kinase p38 plays a central role in the regulation of cytokine biosynthesis by various cell types in response to a wide range of stimuli. Because the local inflammatory response and the infiltration of neutrophils is thought to contribute to the symptoms and sequelae of rhinovirus infection, we investigated the role of p38 kinase in cytokine and chemokine elaboration in airway epithelial cells infected with human rhinovirus. Rhinovirus-39 infection of BEAS-2B cells resulted in synthesis of cytokines (IL-1, IL-6, G-CSF, and GM-CSF) and CXC chemokines (IL-8, epithelial neutrophil-activating protein-78, and growth-related oncogene-α), evident 24–72 h postinfection. Rhinovirus infection induced a time- and dose-dependent increase in tyrosine phosphorylation of p38 kinase, which peaked 30 min postinfection and remained elevated for 1 h. Treatment of infected cells with SB 239063, a potent pyridinyl imidazole inhibitor of p38 kinase, resulted in up to 100% inhibition of mediator production and partially reduced levels of IL-8 mRNA as determined by quantitative RT-PCR. Treatment with SB 239063 had no effect on virus replication and was not cytotoxic at concentrations ≤ 70 μM. These studies provide the first evidence that early activation of p38 kinase by rhinovirus infection is a key event in regulation of virus-induced cytokine transcription, and may provide a new target for inhibition of symptoms and airway inflammation associated with rhinovirus infection.
Regulation of Endothelial CD73 by Adenosine: Paracrine Pathway for Enhanced Endothelial Barrier Function
Authors: Narravula Sailaja , Lennon Paul F. , Mueller Beatrice U. , Colgan Sean P.
Published: April 2014

During episodes of inflammation, multiple cell types release adenine nucleotides in the form of ATP, ADP, 5′-AMP, and adenosine. In particular, following activation, polymorphonuclear leukocytes release larger quantities of 5′-AMP. Extracellular 5′-AMP is metabolized to adenosine by surface-expressed 5′-ectonucleotidase (CD73). Adenosine liberated by this process activates surface adenosine A2B receptors, results in endothelial junctional reorganization, and promotes barrier function. We hypothesized that adenosine signaling to endothelia provides a paracrine loop for regulated expression of CD73 and enhanced endothelial barrier function. Using an in vitro microvascular endothelial model, we investigated the influence of 5′-AMP; adenosine; and adenosine analogues on CD73 transcription, surface expression, and function. Initial experiments revealed that adenosine and adenosine analogues induce CD73 mRNA (RT-PCR), surface expression (immunoprecipitation of surface biotinylated CD73), and function (HPLC analysis of etheno-AMP conversion to ethenoadenosine) in a time- and concentration-dependent fashion. Subsequent studies revealed that similar exposure conditions increase surface protein through transcriptional induction of CD73. Analysis of DNA-binding activity by EMSA identified a functional role for CD73 cAMP response element and, moreover, indicated that multiple cAMP agonists induce transcriptional activation of functional CD73. Induced CD73 functioned to enhance 5′-AMP-mediated promotion of endothelial barrier (measured as a paracellular flux of 70-kDa FITC-labeled tracer). These results provide an example of transcriptional induction of enzyme (CD73) by enzymatic product (adenosine) and define a paracrine pathway for the regulated expression of vascular endothelial CD73 and barrier function.
A Genome Scan Using a Novel Genetic Cross Identifies New Susceptibility Loci and Traits in a Mouse Model of Rheumatoid Arthritis
Authors: Otto Jeffrey M. , Chandrasekeran Raman , Vermes Csaba , Mikecz Katalin , Finnegan Alison , Rickert Sarah E. , Enders Jill T. , Glant Tibor T.
Published: April 2014

Proteoglycan-induced arthritis (PGIA) is a murine model for rheumatoid arthritis (RA) both in terms of its pathology and its genetics. PGIA can only be induced in susceptible mouse strains and their F2 progeny. Using the F2 hybrids resulting from an F1 intercross of a newly identified susceptible (C3H/HeJCr) and an established resistant (C57BL/6) strain of mouse, our goals were to: 1) identify the strain-specific loci that confer PGIA susceptibility, 2) determine whether any pathophysiological parameters could be used as markers that distinguish between nonarthritic and arthritic mice, and 3) analyze the effect of the MHC haplotype on quantitative trait loci (QTL) detection. To identify QTLs, we performed a genome scan on the F2 hybrids. For pathophysiological analyses, we measured pro- and antiinflammatory cytokines such as IL-1, IL-6, IFN-γ, IL-4, IL-10, IL-12, Ag-specific T cell proliferation and IL-2 production, serum IgG1 and IgG2 levels of both auto- and heteroantibodies, and soluble CD44. We have identified four new PGIA-linked QTLs (Pgia13 through Pgia16) and confirmed two (Pgia5, Pgia10) from our previous study. All new MHC-independent QTLs were associated with either disease onset or severity. Comprehensive statistical analysis demonstrated that while soluble CD44, IL-6, and IgG1 vs IgG2 heteroantibody levels differed significantly between the arthritic and nonarthritic groups, only Ab-related parameters colocalized with the QTLs. Importantly, the mixed haplotype (H-2b and H-2k) of the C3H × C57BL/6 F2 intercross reduced the detection of several previously identified QTLs to suggestive levels, indicating a masking effect of unmatched MHCs.
Monocyte Chemotactic Protein-1 Receptor CCR2B Is a Glycoprotein That Has Tyrosine Sulfation in a Conserved Extracellular N-Terminal Region
Authors: Preobrazhensky Alexander A. , Dragan Sofya , Kawano Tomonori , Gavrilin Mikhail A. , Gulina Irina V. , Chakravarty Leena , Kolattukudy P. E.
Published: April 2014

Monocyte chemotactic protein-1 (MCP-1) binding to its receptor, CCR2B, plays an important role in a variety of diseases involving infection, inflammation, and/or injury. In our effort to understand the molecular basis of this interaction and its biological consequences, we recognized a conserved hexad of amino acids at the N-terminal extracellular domain of several chemokine receptors, including CCR2B. Human embryonic kidney 293 cells expressing Flag-tagged CCR2B containing site-directed mutations in this region, 21–26, including a consensus tyrosine sulfation site were used to determine MCP-1 binding and its biological consequences. The results showed that several of these amino acids are important for MCP-1 binding and consequent lamellipodium formation, chemotaxis, and signal transduction involving adenylate cyclase inhibition and Ca2+ influx into cytoplasm. Mutations that prevented adenylate cyclase inhibition and Ca2+ influx did not significantly inhibit lamellipodium formation and chemotaxis, suggesting that these signaling events are not involved in chemotaxis. CCR2B was found to be sulfated at Tyr26; this sulfation was abolished by the substitution of Tyr with Ala and severely reduced by substitution of Asp25, a part of the consensus sulfation site. The expressed CCR2B was found to be N-glycosylated, as N-glycosidase F treatment of the receptor or growth of the cells in tunicamycin reduced the receptor size to the same level, from 50 to 45 kDa. Thus, CCR2B is the first member of the CC chemokine receptor family shown to be a glycoprotein that is sulfated at the N-terminal Tyr. These post-translational modifications probably have significant biological functions.
Surges of Increased T Cell Reactivity to an Encephalitogenic Region of Myelin Proteolipid Protein Occur More Often in Patients with Multiple Sclerosis Than in Healthy Subjects
Authors: Pender Michael P. , Csurhes Peter A. , Greer Judith M. , Mowat Paul D. , Henderson Robert D. , Cameron Kaye D. , Purdie David M. , McCombe Pamela A. , Good Michael F.
Published: April 2014

We have previously shown that patients with multiple sclerosis (MS) have increased T cell responses to the immunodominant region (residues 184–209) of myelin proteolipid protein (PLP). The present study investigated whether this reactivity fluctuates over time and correlates with disease activity. We performed monthly limiting dilution assays for 12–16 mo in four healthy subjects and five patients with relapsing-remitting MS to quantify the frequencies of circulating T cells proliferating in response to PLP41–58, PLP184–199, PLP190–209, myelin basic protein (MBP), MBP82–100, and tetanus toxoid. Disease activity was monitored by clinical assessment and gadolinium-enhanced magnetic resonance imaging of the brain. There were fluctuations in the frequencies of autoreactive T cells in all subjects. Compared with healthy controls, MS patients had significantly more frequent surges of T cells reactive to the 184–209 region of PLP, but infrequent surges of T cell reactivity to MBP82–100. There was temporal clustering of the surges of T cell reactivity to MBP82–100 and MBP, suggesting T cell activation by environmental stimuli. Some clinical relapses were preceded by surges of T cell reactivity to PLP184–209, and in one patient there was significant correlation between the frequency of T cells reactive to PLP184–199 and the total number of gadolinium-enhancing magnetic resonance imaging lesions. However, other relapses were not associated with surges of T cell reactivity to the Ags tested. T cells reactive to PLP184–209 may contribute to the development of some of the CNS lesions in MS.
Human Double-Negative T Cells in Systemic Lupus Erythematosus Provide Help for IgG and Are Restricted by CD1c
Authors: Sieling Peter A. , Porcelli Steven A. , Duong Baochau T. , Spada Franca , Bloom Barry R. , Diamond Betty , Hahn Bevra H.
Published: April 2014

To understand the mechanism of T cell help for IgG production in systemic lupus erythematosus (SLE) we investigated the response of CD4- and CD8-negative (double-negative (DN)) T cells because 1) DN T cells are present at unusually high frequency in patients with SLE and can induce pathogenic autoantibodies; 2) the DN T cell repertoire includes cells restricted by CD1 Ag-presenting molecules; and 3) CD1c is expressed on a population of circulating B cells. We derived DN T cell lines from SLE patients and healthy individuals. In the presence of CD1+ APCs, DN T cell lines from SLE patients produced both IL-4 and IFN-γ, whereas DN T cells from healthy donors produced IFN-γ, but no IL-4. In general, cells from patients with highly active disease produced high levels of IFN-γ; cells from those with little activity produced high IL-4. Coculture of CD1c-directly reactive T cells from healthy donors with CD1c+ B cells elicited IgM Abs, but little or no IgG. In contrast, CD1c-directly reactive T cells from SLE patients induced isotype switching, with a striking increase in IgG production. Neutralizing Abs to CD1c inhibited the ability of DN T cells to induce IgG production from CD1c+ B cells, further indicating that CD1c mediated the T and B cell interaction. IgG production was also inhibited by neutralizing Abs to IL-4, correlating with the cytokine pattern of DN T cells derived from these patients. The data suggest that CD1c-restricted T cells from SLE patients can provide help to CD1c+ B cells for IgG production and could therefore promote pathogenic autoantibody responses in SLE.
Evidence For Early Aging in the Mucosal Immune System
Authors: Koga Toshiya , McGhee Jerry R. , Kato Hirotomo , Kato Rie , Kiyono Hiroshi , Fujihashi Kohtaro
Published: April 2014

Despite recent advances in the cellular and molecular analysis of induction and regulation of mucosal immune responses, little is yet known about differences which occur in aging. To address this important issue, we have compared the mucosal and systemic immune responses of aged (12- to 14-mo- or 2-year-old) and young adult (6- to 8-wk-old) C57BL/6 mice. Both aged and young mice were immunized weekly with three oral doses of 1 mg of OVA and 10 μg of cholera toxin (CT) as mucosal adjuvant. Both groups of mice over 1 or 2 years of age showed reduced levels of Ag-specific mucosal or systemic immune responses at day 21. An Ag-specific B cell enzyme-linked immunospot assay confirmed these results at the cellular level. When the Ag-induced cytokine responses were examined at both protein and mRNA levels, CD4+ T cells from spleen and Peyer’s patches of young adult mice revealed elevated levels of IL-4 production; however, these cytokine responses were significantly diminished in aged mice. In contrast to mucosal immunization, mice s.c. immunized with OVA plus CT resulted in impaired OVA-specific but intact CT B subunit-specific immune responses in 12- to 14-mo-old mice although the responses to both Ags were depressed in 2-year-old mice. These results provide the first evidence that the development of age-associated alterations possibly occurs earlier in the mucosal immune system than in the systemic immune compartment.
Cutting Edge: Gln22 of Mouse MD-2 Is Essential for Species-Specific Lipopolysaccharide Mimetic Action of Taxol
Authors: Kawasaki Kiyoshi , Gomi Kazunori , Nishijima Masahiro
Published: April 2014

MD-2 associates with the extracellular domain of Toll-like receptor 4 (TLR4) and greatly enhances LPS signaling via TLR4. Taxol, which mimics the action of LPS on murine macrophages, induces signals via mouse TLR4-MD-2, but not via human TLR4-MD-2. Here we investigated the molecular basis for this species-specific action of Taxol. Expression of mouse MD-2 conferred both LPS and Taxol responsiveness on human embryonic kidney 293 cells expressing mouse TLR4, whereas expression of human MD-2 conferred LPS responsiveness alone, suggesting that MD-2 is responsible for the species-specificity as to Taxol responsiveness. Furthermore, mouse MD-2 mutants, in which Gln22 was changed to other amino acids, showed dramatically reduced ability to confer Taxol responsiveness, although their ability to confer LPS responsiveness was not affected. These results indicated that Gln22 of mouse MD-2 is essential for Taxol signaling but not for LPS signaling.
Outside-to-Inside Signal Through the Membrane TNF-α Induces E-Selectin (CD62E) Expression on Activated Human CD4+ T Cells
Authors: Harashima Shin-ichi , Horiuchi Takahiko , Hatta Nobuaki , Morita Chika , Higuchi Masanori , Sawabe Takuya , Tsukamoto Hiroshi , Tahira Tomoko , Hayashi Kenshi , Fujita Shigeru , Niho Yoshiyuki
Published: April 2014

The membrane TNF-α is known to serve as a precursor of the soluble form of TNF-α. Although it has been reported the biological functions of the membrane TNF-α as a ligand, the outside-to-inside (reverse) signal transmitted through membrane TNF-α is poorly understood. Here we report a novel function mediated by outside-to-inside signal via membrane TNF-α into the cells expressing membrane TNF-α. Activation by anti-TNF-α Ab against membrane TNF-α on human T cell leukemia virus (HTLV) I-infected T cell line, MT-2, or PHA-activated normal human CD4+ T cells resulted in the induction of an adhesion molecule, E-selectin (CD62E), on the cells with the peak of 12–24 h, which completely disappeared by 48 h. When wild-type or mutant membrane TNF-α (R78T/S79T) resistant to proteolytic cleavage was introduced into Jurkat or HeLa cells, E-selectin was induced by the treatment with anti-TNF-α Ab with the similar kinetics. Membrane TNF-α-expressing Jurkat cells also up-regulated E-selectin when brought into cell-to-cell contact with TNF receptor-expressing HeLa cells. Northern blot analysis and RT-PCR analysis showed that the membrane TNF-α-mediated E-selectin expression was up-regulated at the level of transcription. These results not only confirmed our previous findings of reverse signaling through membrane TNF-α, but also presented evidence that E-selectin was inducible in cell types different from endothelial cells. It is strongly suggested that membrane TNF-α is a novel proinflammatory cell surface molecule that transmits bipolar signals in local inflammation.
IgA Immunodeficiency Leads to Inadequate Th Cell Priming and Increased Susceptibility to Influenza Virus Infection
Authors: Arulanandam Bernard P. , Raeder Roberta H. , Nedrud John G. , Bucher Doris J. , Le Jianhua , Metzger Dennis W.
Published: April 2014

IgA is considered to be the principal Ab involved in defense against pathogens in the mucosal compartment. Using mice with a targeted disruption in IgA gene expression (IgA−/− mice), we have examined the precise role of IgA in protective anti-influenza responses after intranasal vaccination. IgA−/− mice immunized intranasally with soluble hemagglutinin (hemagglutinin subtype 1) and neuraminidase (neuraminidase subtype 1) vaccine in the absence of adjuvant were found to be more susceptible to influenza virus infection than IgA+/+ mice (13 vs 75% survival after virus challenge). Inclusion of IL-12 during immunization restored the protective efficacy of the vaccine to that seen in IgA+/+ animals. IgA−/− mice had no detectable IgA expression, but displayed enhanced serum and pulmonary IgM and IgG Ab levels after IL-12 treatment. Assessment of T cell function revealed markedly depressed splenic lymphoproliferative responses to PHA in IgA−/− animals compared with IgA+/+ mice. Furthermore, IgA−/− animals displayed impaired T cell priming to the H1N1 subunit vaccine, with concomitant reduction in recall memory responses due to a defect in APC function. Collectively, these results provide evidence that a major role of IgA is to facilitate presentation of Ag to mucosal T cells. IL-12 treatment can overcome IgA deficiency by providing adequate T cell priming during vaccination.
IFN-γ Production by Th1 Cells Generated from Naive CD4+ T Cells Exposed to Norepinephrine
Authors: Swanson Michelle A. , Lee William T. , Sanders Virginia M.
Published: April 2014

During activation in vivo, naive CD4+ T cells are exposed to various endogenous ligands, such as cytokines and the neurotransmitter norepinephrine (NE). To determine whether NE affects naive T cell differentiation, we used naive CD4+ T cells sort-purified from either BALB/c or DO11.10 TCR-transgenic mouse spleens and activated these cells with either anti-CD3/anti-CD28 mAbs or APC and OVA323–329 peptide, respectively, under Th1-promoting conditions. RT-PCR and functional assays using selective adrenergic receptor (AR) subtype antagonists showed that naive CD4+ T cells expressed only the β2AR subtype to bind NE and that stimulation of this receptor generated Th1 cells that produced 2- to 4-fold more IFN-γ. This increase was due to more IFN-γ produced per cell upon restimulation instead of more IFN-γ-secreting cells, as determined by IFN-γ-specific immunofluorescence and enzyme-linked immunospot. In contrast, Th1 cell differentiation was unaffected when naive T cells were exposed to NE and activated either in the presence of a neutralizing anti-IL-12 mAb or by APC from IL-12-deficient mice. Moreover, the addition of IL-12 to the IL-12-deficient APC cultures restored the ability of NE to increase Th1 differentiation. Taken together, these results indicate that a possible link may exist between the signaling pathways used by NE and IL-12 to increase naive CD4+ T cell differentiation to a Th1 cell.
The Identification of a Novel T Cell Activation State Controlled by a Diabetogenic Gene
Authors: Moore Jodene K. , Scheinman Robert I. , Bellgrau Donald
Published: April 2014

The cyclin-dependent kinase inhibitor p27kip regulates the cell cycle at the G1-S phase restriction point. S phase entry and cell cycle commitment in peripheral T cells requires p27kip degradation, normally initiated by the receipt of costimulatory signals such as those provided by B7.1 or IL-2. We have previously reported that T cells from BioBreeding (BB)-diabetes-prone (DP) rats exhibit decreased costimulatory requirements for activation and cell cycle entry. In the present study, we find that peripheral T cell subsets from BB-DP rats demonstrate activation-like characteristics, including significantly reduced levels of p27kip as well as increased levels of proliferating cell nuclear Ag (PCNA). Since our previous studies have established that expression of extracellular activation markers are relatively low in unmanipulated peripheral BB-DP T cells; this p27low PCNAhigh phenotype represents a novel activation state. Analyses of T cell subsets from congenic rats demonstrate that this phenotype segregates with the lyp diabetogenic locus and that the p27low PCNAhigh phenotype is T cell specific. This p27low PCNAhigh phenotype is not seen in medullary thymocytes, but appears abruptly in the recent thymic emigrant population, suggesting that the lyp locus does not act directly on cell cycle regulators but rather alters the interaction between T cells and the peripheral environment. These results provide a biochemical basis for costimulation-independent activation and suggest a mechanism whereby a diabetes susceptibility gene contributes to disease development.
Regulation of Toll-Like Receptors in Human Monocytes and Dendritic Cells
Authors: Visintin Alberto , Mazzoni Alessandra , Spitzer Jessica H. , Wyllie David H. , Dower Steven K. , Segal David M.
Published: April 2014

A number of pathogens induce immature dendritic cells (iDC) to migrate to lymphoid organs where, as mature DC (mDC), they serve as efficient APC. We hypothesized that pathogen recognition by iDC is mediated by Toll-like receptors (TLRs), and asked which TLRs are expressed during the progression of monocytes to mDC. We first measured mRNA levels for TLRs 1–5 and MD2 (a protein required for TLR4 function) by Northern analysis. For most TLRs, message expression decreased severalfold as monocytes differentiated into iDC, but opposing this trend, TLR3 and MD2 showed marked increases during iDC formation. When iDC were induced to mature with LPS or TNF-α, expression of most TLRs transiently increased and then nearly disappeared. Stimulation of iDC, but not mDC, with LPS resulted in the activation of IL-1 receptor-associated kinase, an early component in the TLR signaling pathway, strongly suggesting that LPS signals through a TLR. Surface expression of TLRs 1 and 4, as measured by mAb binding, was very low, corresponding to a few thousand molecules per cell in monocytes, and a few hundred or less in iDC. We conclude that TLRs are expressed in iDC and are involved in responses to at least one pathogen-derived substance, LPS. If TLR4 is solely responsible for LPS signaling in humans, as it is in mice, then its extremely low surface expression implies that it is a very efficient signal transducer in iDC.
Absence of CD43 Fails to Alter T Cell Development and Responsiveness
Authors: Carlow Douglas A. , Corbel Stéphane Y. , Ziltener Hermann J.
Published: April 2014

Genetic elimination of CD43 has been associated with increased T cell adhesiveness and T cell hyperresponsiveness to mitogens and alloantigens. Therefore, we investigated whether T cell development was perturbed in CD43-deficient mice by breeding CD43null mice with male Ag (Hy)-specific TCR-transgenic mice. Neither positive nor negative thymic selection of male Ag-specific T cells were affected by CD43 status. Furthermore, we did not observe a substantial or consistent hyperresponsive pattern in HY-CD43null lymph node cells compared with littermate HY-CD43+/− lymph node cells upon analysis of in vitro T cell stimulation with male Ag or mitogen. These observations challenged original conclusions associating absence of CD43 with T cell hyperresponsiveness and led us to re-examine this association. Reported phenotypes of CD43null mice have been based on mice with a mixed 129×C57BL/6 genetic background. To exclude a possible influence of genetic background differences among individual mice we analyzed CD43null littermates that had been back-bred onto the C57BL/6 background for seven to eight generations. We found that CD43+ and CD43null littermates with the C57BL/6 background exhibited no differences in response to mitogen or alloantigen, thereby establishing that T cell hyperresponsiveness is not a general correlate of CD43 absence.
CD40 Ligation Ablates the Tolerogenic Potential of Lymphoid Dendritic Cells
Authors: Grohmann Ursula , Fallarino Francesca , Silla Silvia , Bianchi Roberta , Belladonna Maria L. , Vacca Carmine , Micheletti Alessandra , Fioretti Maria C. , Puccetti Paolo
Published: April 2014

The outcome of dendritic cell (DC) presentation of P815AB, a tolerogenic tumor/self peptide, depends on a balance between the respective immunogenic and tolerogenic properties of myeloid (CD8α−) and lymphoid (CD8α+) DC. We have previously shown that CD8− DC can be primed by IL-12 to overcome inhibition by the CD8+ subset and initiate immunogenic presentation in vivo when the two types of peptide-pulsed DC are cotransferred into recipient hosts. IFN-γ enhances the inhibitory activity of CD8+ DC on Ag presentation by the other subset, blocking the ability of IL-12-treated CD8− DC to overcome suppression. We report here that CD40 ligation on lymphoid DC ablated their inhibitory function on Ag presentation as well as IFN-γ potentiation of the effect. CD40 modulation of IFN-γ action on lymphoid DC involved a reduction in IFN-γR expression and tryptophan-degrading ability. This effect was accompanied in vitro by an impaired capacity of the CD40-modulated and IFN-γ-treated DC to initiate T cell apoptosis. In vivo, not only did CD40 triggering on lymphoid DC abrogate their tolerogenic activity, but it also induced the potential for immunogenic presentation of P815AB. Importantly, a pattern similar to P815AB as well as CD40 modulation of lymphoid DC function were observed on testing reactivity to NRP, a synthetic peptide mimotope recognized by diabetogenic CD8+ T cells in nonobese diabetic mice.
Complementary Effects of TNF and Lymphotoxin on the Formation of Germinal Center and Follicular Dendritic Cells
Authors: Wang Yang , Wang Jing , Sun Yonglian , Wu Qiang , Fu Yang-Xin
Published: April 2014

The formation of germinal centers (GC) around follicular dendritic cells (FDC) is a critical step in the humoral immune responses that depends on the cooperative effects of B cells and T cells. Mice deficient in either TNF or lymphotoxin (LT) fail to form both GC and FDC network in B cell follicles. To test a potential complementary effect of TNF and LT, a mixture of bone marrow cells from TNF−/− mice and LTα−/− mice was transferred into irradiated LTα−/− mice or TNF−/− mice. Interestingly, the formation of both GC and FDC clusters in B cell follicles was restored in such chimeric mice, suggesting that TNF and LT from different cells could complement one another. To identify the exact contributions of each subset to the complementary effect of TNF and LT, different sources of T and B cells from LTα−/− mice or TNF−/− mice were used for reconstitution. Our study demonstrates that either T or B cell-derived TNF is sufficient to restore FDC/GC in the presence of LT-expressing B cells. However, TNF itself is not required for GC reactions if the FDC network is already intact. Thus, the development and maintenance of these lymphoid structures depend on a delicate interaction between TNF and LT from different subsets of lymphocytes.
A Subset of Human Dendritic Cells Expresses IgA Fc Receptor (CD89), Which Mediates Internalization and Activation Upon Cross-Linking by IgA Complexes
Authors: Geissmann Frédéric , Launay Pierre , Pasquier Benoit , Lepelletier Yves , Leborgne Michelle , Lehuen Agnès , Brousse Nicole , Monteiro Renato C.
Published: April 2014

Immature dendritic cells (DC) sample Ags within nonlymphoid tissues and acquire exogenous proteins/pathogens via scavenger receptors or Ig FcR such as FcγR and FcεR. IgA is present in a significant proportion among serum Ig and is the main isotype in mucosae, where DC are numerous. We found that a functional FcαR (CD89) was expressed in situ and in vitro on interstitial-type DC but not on Langerhans cell-type DC. Interstitial-type DC expressed CD89 as a 50- to 75-kDa glycoprotein with a 32-kDa protein core, which was down-regulated upon addition of TGF-β1. DC, FcαR specifically, bound IgA1 and IgA2. Cross-linking of CD89 on DC triggered endocytosis in time-dependent manner. In addition, internalization of polymeric IgA complexes induced the production of IL-10 and DC activation, as reflected by up-regulation of CD86 costimulatory molecules, class II MHC expression, and increased allostimulatory activity. Therefore, interstitial-type DC may use FcαR-mediated Ag sampling in the subepithelium to check tissue integrity while Langerhans cells inside epithelial layers may neglect IgA immune complexes.
Group 1 CD1 Genes in Rabbit
Authors: Hayes Sandra M. , Knight Katherine L.
Published: April 2014

CD1 is an Ag-presenting molecule that can present lipids and glycolipids to T cells. The CD1 genes were first identified in the human, and since then, homologs have been identified in every mammalian species examined to date. Over a decade ago, CD1B and CD1D homologs were identified in the rabbit. We have extended this earlier study by identifying additional CD1 genes with the goal of developing the rabbit as an animal model to study the function of CD1 proteins. We constructed a thymocyte cDNA library and screened the library with CD1-specific probes. Based on nucleotide sequence analyses of the CD1+ cDNA clones obtained from the library, we have identified two CD1A genes and one CD1E gene as well as determined the complete sequence of the previously identified CD1B gene. The CD1E+ cDNA clones lacked the transmembrane and cytoplasmic domains and, if translated, would encode for a soluble or secreted CD1E protein. In addition, expression studies demonstrated that the CD1 genes were expressed in peripheral lymphoid tissues as well as in skin, gut, and lung. Of interest is the finding that CD1A2, CD1B, and CD1E genes were found to be expressed by rabbit B cell populations. The rabbit, with a complex CD1 locus composed of at least two CD1A genes, one CD1B gene, one CD1D gene, and one CD1E gene, is an excellent candidate as an animal model to study CD1 proteins.
The Effects of Granulocyte Colony-Stimulating Factor and Neutrophil Recruitment on the Pulmonary Chemokine Response to Intratracheal Endotoxin
Authors: Zhang Ping , Bagby Gregory J. , Kolls Jay K. , Welsh David A. , Summer Warren R. , Andresen Jeff , Nelson Steve
Published: April 2014

Although G-CSF has been shown to increase neutrophil (polymorphonuclear leukocyte, PMN) recruitment into the lung during pulmonary infection, relatively little is known about the local chemokine profiles associated with this enhanced PMN delivery. We investigated the effects of G-CSF and PMN recruitment on the pulmonary chemokine response to intratracheal LPS. Rats pretreated twice daily for 2 days with an s.c. injection of G-CSF (50 μg/kg) were sacrificed at either 90 min or 4 h after intratracheal LPS (100 μg) challenge. Pulmonary recruitment of PMNs was not observed at 90 min post LPS challenge. Macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (CINC) concentrations in bronchoalveolar lavage (BAL) fluid were similar in animals pretreated with or without G-CSF at this time. G-CSF pretreatment enhanced pulmonary recruitment of PMNs (5-fold) and greatly reduced MIP-2 and CINC levels in BAL fluid at 4 h after LPS challenge. In vitro, the presence of MIP-2 and CINC after LPS stimulation of alveolar macrophages was decreased by coculturing with circulating PMNs but not G-CSF. G-CSF had no direct effect on LPS-induced MIP-2 and CINC mRNA expression by alveolar macrophages. Pulmonary recruited PMNs showed a significant increase in cell-associated MIP-2 and CINC. Cell-associated MIP-2 and CINC of circulating PMNs were markedly increased after exposure of these cells to the BAL fluid of LPS-challenged lungs. These data suggest that recruited PMNs are important cells in modulating the local chemokine response. G-CSF augments PMN recruitment and, thereby, lowers local chemokine levels, which may be one mechanism resulting in the subsidence of the host proinflammatory response.