bluuebunny/crossref_metadata_2025 huggingface dataset:
A Microbial TLR2 Agonist Imparts Macrophage-Activating Ability to Apolipoprotein A-1
Authors: Hasebe Akira , Pennock Nathan D. , Mu Hong-Hua , Chan Fok V. , Taylor Michael L. , Cole Barry C.
Published: April 2014

There is increasing epidemiologic evidence implying a role for chronic infection in atherosclerosis and that microbial TLR agonists may contribute to this disease. Mycoplasma arthritidis is an agent of acute and chronic inflammatory disease in rodents, and has been used extensively as a model for defining the mechanisms involved in arthritis and other inflammatory diseases. We have purified a 28-kDa, apolipoprotein A-1 (apoA-1)-like TLR2-dependent macrophage-activating moiety from a culture of a virulent strain of M. arthritidis. ApoA-1 similarly isolated from uninoculated mycoplasma medium was without bioactivity. The activity of the mycoplasma-derived molecule was resistant to heat and to digestion with proteinase K, but was susceptible to alkaline hydrolysis and H2O2 oxidation. Infrared profiles of normal apoA-1 and that derived from mycoplasma were distinct. Unlike the activity of other mycoplasmal TLR2 agonists such as macrophage-activating lipopeptide-2, activity of the M. arthritidis-derived 28-kDa component was dependent upon CD14, a coreceptor for LPS. Finally, we showed that bioactive lipopeptides prepared from M. arthritidis grown in serum-free medium and also from a 41-kDa known bioactive lipoprotein of M. arthritidis, avidly bound to purified apoA-1 that separated out by SDS-PAGE, induced TNF-α and IL-12p40 both in vitro and in vivo. ApoA-1 is a key functional component of the high-density lipoprotein cholesterol complex by scavenging and removing unwanted lipids. Our finding that this molecule can acquire macrophage-activating properties from microbial TLR2-dependent agonists suggests a novel mechanism whereby some microbial agents might reverse the protective role of apoA-1, thus contributing to the genesis of atherosclerosis.
Exposure of the Promonocytic Cell Line THP-1 toEscherichia coliInduces IFN-γ-Inducible Lysosomal Thiol Reductase Expression by Inflammatory Cytokines
Authors: Lackman Rebecca L. , Cresswell Peter
Published: April 2014

IFN-γ-inducible lysosomal thiol reductase (GILT), which plays a role in MHC class II-restricted processing and presentation of Ags containing disulfide bonds, can be induced in various cell types by the cytokine IFN-γ. APCs, including circulating macrophages, constitutively express high levels of GILT, although the pathways regulating its expression in these cells have not been characterized. In this study, we used the promonocytic cell line THP-1, an established model for monocyte to macrophage differentiation, to investigate the induction of GILT upon exposure to bacteria. We show that contact with LPS or intact Escherichia coli causes THP-1 cells to undergo programmed differentiation, characterized by adhesion, cytokine secretion, and up-regulation of Ag processing and presentation components, including GILT. Unlike GILT induction in response to IFN-γ treatment, induction by bacteria is dependent on new protein synthesis, NF-κB signaling, and secretion of the inflammatory cytokines TNF and IL-1β. Furthermore, we show that both cytokines are sufficient for GILT induction in the absence of a microbial stimulus. The majority of GILT synthesized by differentiated THP-1 cells is secreted as the precursor form rather than being transported to, and maturing in, lysosomes, suggesting a novel role for GILT in cells of the macrophage lineage.
Caveolin-1 Regulates NF-κB Activation and Lung Inflammatory Response to Sepsis Induced by Lipopolysaccharide
Authors: Garrean Sean , Gao Xiao-Pei , Brovkovych Victor , Shimizu Jun , Zhao You-Yang , Vogel Stephen M. , Malik Asrar B.
Published: April 2014

Caveolin-1, the principal structural and signaling protein of caveolae, is implicated in NO-mediated cell signaling events, but its precise role in inflammation is not well understood. Using caveolin-1-knockout (Cav-1−/−) mice, we addressed the role of caveolin-1 in the lung inflammatory response to sepsis induced by i.p. injection of LPS. LPS-challenged wild-type (WT) lungs exhibited significant increases in neutrophil sequestration (∼16-fold), lung microvascular permeability Kf,c (∼5.7-fold), and edema formation (∼1.6-fold). Compared with WT, Cav-1−/− lungs showed marked attenuation of LPS-induced neutrophil sequestration (∼11-fold increase) and inhibition of microvascular barrier breakdown and edema formation. Prevention of lung injury in Cav-1−/− mice was associated with decreased mortality in response to LPS challenge. To address the basis of the reduced inflammation and injury in Cav-1−/− lungs, we examined the role of NO because its plasma concentration is known to be increased in Cav-1−/− mice. Cav-1−/− mouse lungs demonstrated a significant increase in endothelial NO synthase (eNOS)-derived NO production relative to WT, which is consistent with the role of caveolin-1 as a negative regulator of eNOS activity. Cav-1−/− lungs concurrently showed suppression of NF-κB activity and decreased transcription of inducible NO synthase and ICAM-1. Coadministration of LPS with the NO synthase inhibitor nitro-l-arginine in Cav-1−/− mice prevented the suppression of NF-κB activity and restored lung polymorphonuclear leukocyte sequestration in response to LPS challenge. Thus, caveolin-1, through its ability to regulate eNOS-derived NO production, is a crucial determinant of NF-κB activation and the lung inflammatory response to LPS.
Essential Role for IL-10 in Resistance to Lipopolysaccharide-Induced Preterm Labor in Mice
Authors: Robertson Sarah A. , Skinner Rebecca J. , Care Alison S.
Published: April 2014

IL-10 is highly expressed in the uterus and placenta and is implicated in controlling inflammation-induced pathologies of pregnancy. To investigate the role of IL-10 in regulating preterm labor, the response of IL-10 null mutant mice to low-dose LPS in late gestation was evaluated. When IL-10 null mutant C57BL/6 (IL-10−/−) and control (IL-10+/+) mice were administered LPS on day 17 of pregnancy, the dose of LPS required to elicit 50% preterm fetal loss was 10-fold lower in IL-10−/− mice than in IL-10+/+ mice. Surviving fetuses in IL-10−/− mice exhibited fetal growth restriction at lower doses of LPS than IL-10+/+ mice. Marked elevation of LPS-induced immunoactive TNF-α and IL-6 was evident in the serum, uterus, and placenta of IL-10−/− mice, and TNF-α and IL-6 mRNA expression was elevated in the uterus and placenta, but not the fetus. Serum IL-1α, IFN-γ, and IL-12p40 were increased and soluble TNFRII was diminished in the absence of IL-10, with these changes also reflected in the gestational tissues. Administration of rIL-10 to IL-10−/− mice attenuated proinflammatory cytokine synthesis and alleviated their increased susceptibility to preterm loss. Exogenous IL-10 also protected IL-10+/+ mice from fetal loss. These data show that IL-10 modulates resistance to inflammatory stimuli by down-regulating proinflammatory cytokines in the uterus and placenta. Abundance of endogenous IL-10 in gestational tissues is therefore identified as a critical determinant of resistance to preterm labor, and IL-10 may provide a useful therapeutic agent in this common condition.
Functional Reversion of Antigen-Specific CD8+ T Cells from Patients with Hodgkin Lymphoma following In Vitro Stimulation with Recombinant Polyepitope
Authors: Smith Corey , Cooper Leanne , Burgess Melinda , Rist Michael , Webb Natasha , Lambley Eleanore , Tellam Judy , Marlton Paula , Seymour John F. , Gandhi Maher , Khanna Rajiv
Published: April 2014

Recent studies on Hodgkin’s lymphoma (HL) have indicated that patients with active disease display functional impairment of Ag-specific CD8+ T cells due to expansion of regulatory T cells at sites of disease and in the peripheral blood. Adoptive cellular immunotherapy based on EBV-specific CD8+ T cells has been explored with limited success to date. It has been proposed that improved targeting of these CD8+ T cells toward viral Ags that are expressed in HL may enhance future therapeutic vaccine strategies. In this study, we have developed a novel replication-deficient adenoviral Ag presentation system that is designed to encode glycine alanine repeat-deleted EBV nuclear Ag 1 covalently linked to multiple CD8+ T cell epitopes from latent membrane proteins 1 and 2. A single stimulation of CD8+ T cells from healthy virus carriers, and patients with HL with this adenoviral construct in combination with IL-2, was sufficient to reverse the functional T cell impairment and restored both IFN-γ production and cytolytic function. More importantly, these activated CD8+ T cells responded to tumor cells expressing membrane proteins and recognized novel EBNA1 epitopes. Flow cytometric analysis revealed that a large proportion of T cells expanded from patients with HL were CD62Lhigh and CD27high, and CCR7low, consistent with early to mid effector T cells. These findings provide an important platform for translation of Ag-specific adoptive immunotherapy for the treatment of EBV-associated malignancies such as HL and nasopharyngeal carcinoma.
ICOS Deficiency Is Associated with a Severe Reduction of CXCR5+CD4 Germinal Center Th Cells
Authors: Bossaller Lukas , Burger Jan , Draeger Ruth , Grimbacher Bodo , Knoth Rolf , Plebani Alessandro , Durandy Anne , Baumann Ulrich , Schlesier Michael , Welcher Andrew A. , Peter Hans Hartmut , Warnatz Klaus
Published: April 2014

ICOS is expressed on activated T cells and particularly on CXCR5+ follicular Th cells in germinal centers (GC). Its deletion leads to a profound deficiency in memory B cell formation and switched Ab response in humans. Here, we show that in ICOS-deficient patients the generation of GCs is severely disturbed, and the numbers of circulating CXCR5+CD45RO+ memory CD4 T cells are significantly reduced, indicating an essential role of ICOS in the differentiation of CXCR5+CD4 T cells. The GC-specific CD57+CXCR5+ subpopulation is virtually absent. In ICOS−/− mice, the decrease of circulating CXCR5+CD4 T cells reflects the reduction of CXCR5+ follicular Th cells in lymph nodes and spleen. Therefore, in concurrence with the absence of CXCR5+ T cells in the blood of CD40L-deficient patients, these data support the hypothesis that circulating CD57+CXCR5+ T cells are GC derived and thus may serve as a surrogate marker for the presence of functional GCs in humans.
Autophagy: Eating for Good Health
Authors: Swanson Michele S.
Published: April 2014

A renaissance in the autophagy field has illuminated many areas of biology, and infectious disease is no exception. By identifying key components of this broadly conserved membrane traffic pathway, yeast geneticists generated tools for microbiologists and immunologists to explore whether autophagy contributes to host defenses. As a result, autophagy is now recognized to be another barrier confronted by microbes that invade eukaryotic cells. Mounting evidence also indicates that autophagy equips cells to deliver cytosolic Ags to the MHC class II pathway. By applying knowledge of the autophagy machinery and exploiting microbes as genetic probes, experimentalists can now examine in detail how this ancient membrane traffic pathway contributes to these and other mechanisms critical for infection and immunity.
Cutting Edge: Hypoxia-Inducible Factor 1α and Its Activation-Inducible Short Isoform I.1 Negatively Regulate Functions of CD4+ and CD8+ T Lymphocytes
Authors: Lukashev Dmitriy , Klebanov Boris , Kojima Hidefumi , Grinberg Alex , Ohta Akiko , Berenfeld Ludmilla , Wenger Roland H. , Ohta Akio , Sitkovsky Michail
Published: April 2014

To evaluate the role of hypoxia-inducible factor 1α (HIF-1α) and its TCR activation-inducible short isoform I.1 in T cell functions, we genetically engineered unique mice with: 1) knockout of I.1 isoform of HIF-1α; 2) T cell-targeted HIF-1α knockdown; and 3) chimeric mice with HIF-1α gene deletion in T and B lymphocytes. In all three types of mice, the HIF-1α-deficient T lymphocytes, which were TCR-activated in vitro, produced more proinflammatory cytokines compared with HIF-1α-expressing control T cells. Surprisingly, deletion of the I.1 isoform, which represents <30% of total HIF-1α mRNA in activated T cells, was sufficient to markedly enhance TCR-triggered cytokine secretion. These data suggest that HIF-1α not only plays a critical role in oxygen homeostasis but also may serve as a negative regulator of T cells.
Role of Galectin-3 in Mast Cell Functions: Galectin-3-Deficient Mast Cells Exhibit Impaired Mediator Release and Defective JNK Expression
Authors: Chen Huan-Yuan , Sharma Bhavya B. , Yu Lan , Zuberi Riaz , Weng I-Chun , Kawakami Yuko , Kawakami Toshiaki , Hsu Daniel K. , Liu Fu-Tong
Published: April 2014

Galectin-3 is a member of the β-galactoside-binding animal lectin family expressed in various cell types, including mast cells. To determine the role of galectin-3 in the function of mast cells, we studied bone marrow-derived mast cells (BMMC) from wild-type (gal3+/+) and galectin-3-deficient (gal3−/−) mice. Cells from the two genotypes showed comparable expression of IgE receptor and c-Kit. However, upon activation by FcεRI cross-linkage, gal3−/− BMMC secreted a significantly lower amount of histamine as well as the cytokine IL-4, compared with gal3+/+ BMMC. In addition, we found significantly reduced passive cutaneous anaphylaxis reactions in gal3−/− mice compared with gal3+/+ mice. These results indicate that there is a defect in the response of mast cells in gal3−/− mice. Unexpectedly, we found that gal3−/− BMMC contained a dramatically lower basal level of JNK1 protein compared with gal3+/+ BMMC, which is probably responsible for the lower IL-4 production. The decreased JNK1 level in gal3−/− BMMC is accompanied by a lower JNK1 mRNA level, suggesting that galectin-3 regulates the transcription of the JNK gene or processing of its RNA. All together, these results point to an important role of galectin-3 in mast cell biology.
Peripheral T Cell Lymphopenia and Concomitant Enrichment in Naturally Arising Regulatory T Cells: The Case of the Pre-Tα Gene-Deleted Mouse
Authors: Bosco Nabil , Agenes Fabien , Rolink Antonius G. , Ceredig Rhodri
Published: April 2014

In pre-Tα (pTα) gene-deleted mice, the positively selectable CD4+CD8+ double-positive thymocyte pool is only 1% that in wild-type mice. Consequently, their peripheral T cell compartment is severely lymphopenic with a concomitant increase in proportion of CD25+FoxP3+ regulatory T cells. Using mixed bone marrow chimeras, where thymic output was 1% normal, the pTα−/− peripheral T cell phenotype could be reproduced with normal cells. In the pTα−/− thymus and peripheral lymphoid organs, FoxP3+CD4+ cells were enriched. Parabiosis experiments showed that many pTα−/−CD4+ single-positive thymocytes represented recirculating peripheral T cells. Therefore, the enrichment of FoxP3+CD4+ single-positive thymocytes was not solely due to increased thymic production. Thus, the pTα−/− mouse serves as a model system with which to study the consequences of chronic decreased thymic T cell production on the physiology of the peripheral T cell compartment.
Evidence for a Role for Notch Signaling in the Cytokine-Dependent Survival of Activated T Cells
Authors: Bheeshmachar Geetha , Purushotaman Divya , Sade Hadassah , Gunasekharan Vigneshkumar , Rangarajan Annapoorni , Sarin Apurva
Published: April 2014

Peripheral T cell homeostasis results from a balance between factors promoting survival and those that trigger deletion of Ag-reactive cells. The cytokine IL-2 promotes T cell survival whereas reactive oxygen species (ROS) sensitize T cells to apoptosis. Two pathways of activated T cell apoptosis–one triggered by Fas ligand and the other by cytokine deprivation–depend on ROS, with the latter also regulated by members of the Bcl-2 family. Notch family proteins regulate several cell-fate decisions in metazoans. Ectopic expression of the Notch1 intracellular domain (NICD) in T cells inhibits Fas-induced apoptosis. The underlying mechanism is not known and the role, if any, of Notch in regulating apoptosis triggered by cytokine deprivation or neglect has not been examined. In this study, we use a Notch1/Fc chimera; a blocking Ab to Notch1 and chemical inhibitors of γ-secretase to investigate the role of Notch signaling in activated T cells of murine origin. We show that perturbing Notch signaling in activated CD4+/CD8+ T cells maintained in IL-2 results in the accumulation of ROS, reduced Akt/protein kinase B activity, and expression of the antiapoptotic protein Bcl-xL, culminating in apoptosis. A broad-spectrum redox scavenger inhibits apoptosis but T cells expressing mutant Fas ligand are sensitive to apoptosis. Activated T cells isolated on the basis of Notch expression (Notch+) are enriched for Bcl-xL expression and demonstrate reduced susceptibility to apoptosis triggered by neglect or oxidative stress. Furthermore, enforced expression of NICD protects activated T cells from apoptosis triggered by cytokine deprivation. Taken together, these data implicate Notch1 signaling in the cytokine-dependent survival of activated T cells.
Identification of a Novel Human Granzyme B Inhibitor Secreted by Cultured Sertoli Cells
Authors: Sipione Simonetta , Simmen Katia C. , Lord Sarah J. , Motyka Bruce , Ewen Catherine , Shostak Irene , Rayat Gina R. , Dufour Jannette M. , Korbutt Greg S. , Rajotte Ray V. , Bleackley R. Chris
Published: April 2014

Sertoli cells have long since been recognized for their ability to suppress the immune system and protect themselves as well as other cell types from harmful immune reaction. However, the exact mechanism or product produced by Sertoli cells that affords this immunoprotection has never been fully elucidated. We examined the effect of mouse Sertoli cell-conditioned medium on human granzyme B-mediated killing and found that there was an inhibitory effect. We subsequently found that a factor secreted by Sertoli cells inhibited killing through the inhibition of granzyme B enzymatic activity. SDS-PAGE analysis revealed that this factor formed an SDS-insoluble complex with granzyme B. Immunoprecipitation and mass spectroscopic analysis of the complex identified a proteinase inhibitor, serpina3n, as a novel inhibitor of human granzyme B. We cloned serpina3n cDNA, expressed it in Jurkat cells, and confirmed its inhibitory action on granzyme B activity. Our studies have led to the discovery of a new inhibitor of granzyme B and have uncovered a new mechanism used by Sertoli cells for immunoprotection.
RANTES Modulates TLR4-Induced Cytokine Secretion in Human Peripheral Blood Monocytes
Authors: Shahrara Shiva , Park Christy C. , Temkin Vladislav , Jarvis Jared W. , Volin Michael V. , Pope Richard M.
Published: April 2014

Monocytes are the key regulators of joint inflammation and destruction in rheumatoid arthritis; hence, suppression of their recruitment into the joint may be therapeutically beneficial. Chemokines, including RANTES, are highly expressed in the joints of patient with rheumatoid arthritis, and they promote leukocyte trafficking into the synovial tissue. Because endogenous TLR4 ligands are expressed in the rheumatoid joint, the TLR4 ligand LPS was used to characterize the effects of RANTES on the TLR4-mediated induction of TNF-α and IL-6. Using peripheral blood (PB) monocytes, RANTES decreased LPS-induced IL-6 transcriptionally, whereas TNF-α was suppressed at the posttranscriptional level. RANTES signaled through p38 MAPK, and this signaling was further enhanced by LPS stimulation in PB monocytes, resulting in the earlier and increased secretion of IL-10. Inhibition of p38 by short-interfering RNA or a chemical inhibitor, as well as neutralization of IL-10, reversed the RANTES-mediated suppression of LPS-induced IL-6 and TNF-α. Further, when rheumatoid arthritis synovial fluid was added to PB monocytes, the neutralization of RANTES in fluid reduced the LPS-induced IL-10 and increased TNF-α. In conclusion, the results of this study suggest that RANTES down-regulates TLR4 ligation-induced IL-6 and TNF-α secretion by enhancing IL-10 production in PB monocytes. These observations suggest that the therapeutic neutralization of RANTES, in addition to decreasing the trafficking of leukocytes, may have a proinflammatory effect at the site of established chronic inflammation.
High Cytotoxic and Specific Migratory Potencies of Senescent CD8+CD57+ Cells in HIV-Infected and Uninfected Individuals
Authors: Le Priol Yannick , Puthier Denis , Lécureuil Cédric , Combadière Christophe , Debré Patrice , Nguyen Catherine , Combadière Béhazine
Published: April 2014

CD8+CD57+ T lymphocytes, present at low levels in the peripheral blood of healthy individuals expand during HIV infection and remain elevated during chronic infection. Their role in the immune response remains unclear. We performed a large-scale gene array analysis (3158 genes) to characterize them and, interestingly, found no distinction in the transcriptional profiles of CD8+CD57+ T lymphocytes from HIV-infected and uninfected subjects. In both groups, these cells showed specificity for multiple Ags and produced large amounts of IFN-γ and TNF-α. The transcriptional profiles of CD8+CD57+ and CD8+CD57− cells, however, differed substantially. We propose that CD8+CD57+ cells were Ag-driven effector cells with very high cytotoxic effector potential including perforin, granzymes, and granulysin, regardless of HIV status. At both the messenger and protein levels, they expressed more adhesion molecules and fewer chemokine receptors (CCR7 and CXCR4) than CD8+CD57− cells but expressed preferentially CX3CR1. The lower expression level of genes involved in cell cycle regulation showed limited proliferation capacities of CD8+CD57+ even in response to TCR and IL-2, IL-7, and IL-15 stimulation. CD8+CD57+ T cells from both HIV and uninfected subjects maintain effective cytotoxic potentials but are destined to migrate to nonlymphoid tissues without further cycling.
A Novel Mechanism for Complement Activation at the Surface of B Cells Following Antigen Binding
Authors: Manderson Anthony P. , Quah Ben , Botto Marina , Goodnow Chris C. , Walport Mark J. , Parish Chris R.
Published: April 2014

Coligation of CD21 with BCR on the surface of B cells provides a costimulatory signal essential for efficient Ab responses to T-dependent Ags. To achieve this, Ag must be directly linked to C3 fragments, but how this occurs in vivo is not fully understood. Using BCR transgenic mice, we demonstrated that C3 was deposited on the surface of B cells following both high- and moderate-affinity Ag binding. This was dependent on the specific binding of IgM to the BCR-bound Ag and can occur independently of soluble immune complex formation. Based on these data, we propose a novel model in which immune complexes can form directly on the surface of the B cell following Ag binding. This model has implications for our understanding of B lymphocyte activation.
The Src Homology 2 Domain-Containing Leukocyte Protein of 76-kDa Adaptor Links Integrin Ligation with p44/42 MAPK Phosphorylation and Podosome Distribution in Murine Dendritic Cells
Authors: Luckashenak Nancy A. , Ryszkiewicz Rebecca L. , Ramsey Kimberley D. , Clements James L.
Published: April 2014

The Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) is an important molecular intermediate in multiple signaling pathways governing immune cell function. In this study, we report that SLP-76 is expressed in CD11c+B220− dendritic cells (DCs) isolated from murine thymus or spleen, and that SLP-76 is rapidly phosphorylated on tyrosine residues upon plating of bone marrow-derived DCs (BMDCs) on integrin agonists. SLP-76 is not required for the in vitro or in vivo generation of DCs, but SLP-76-deficient BMDCs adhere poorly to fibronectin, suggesting impaired integrin function. Consistent with impaired adhesion, cutaneous SLP-76-deficient DCs leave ear tissue at an elevated frequency compared with wild-type DCs. In addition, the pattern and distribution of actin-based podosome formation are visibly altered in BMDCs lacking SLP-76 following integrin engagement. SLP-76-deficient BMDCs manifest multiple signaling defects following integrin ligation, including reduced global tyrosine phosphorylation and markedly impaired phosphorylation of p44/42 MAPK (ERK1/2). These data implicate SLP-76 as an important molecular intermediate in the signaling pathways regulating multiple integrin-dependent DC functions, and add to the growing body of evidence that hemopoietic cells may use unique molecular intermediates and mechanisms for regulating integrin signaling.
Aspergillus fumigatus Generates an Enhanced Th2-Biased Immune Response in Mice with Defective Cystic Fibrosis Transmembrane Conductance Regulator
Authors: Allard Jenna B. , Poynter Matthew E. , Marr Kieren A. , Cohn Lauren , Rincon Mercedes , Whittaker Laurie A.
Published: April 2014

Cystic fibrosis (CF) lung disease is characterized by persistent airway inflammation and airway infection that ultimately leads to respiratory failure. Aspergillus sp. are present in the airways of 20–40% of CF patients and are of unclear clinical significance. In this study, we demonstrate that CF transmembrane conductance regulator (CFTR)-deficient (CFTR knockout, Cftrtm1Unc-TgN(fatty acid-binding protein)CFTR) and mutant (ΔF508) mice develop profound lung inflammation in response to Aspergillus fumigatus hyphal Ag exposure. CFTR-deficient mice also develop an enhanced Th2 inflammatory response to A. fumigatus, characterized by elevated IL-4 in the lung and IgE and IgG1 in serum. In contrast, CFTR deficiency does not promote a Th1 immune response. Furthermore, we demonstrate that CD4+ T cells from naive CFTR-deficient mice produce higher levels of IL-4 in response to TCR ligation than wild-type CD4+ T cells. The Th2 bias of CD4+ T cells in the absence of functional CFTR correlates with elevated nuclear levels of NFAT. Thus, CFTR is important to maintain the Th1/Th2 balance in CD4+ T cells.
DOCK2 is Required for Chemokine-Promoted Human T Lymphocyte Adhesion Under Shear Stress Mediated by the Integrin α4β1
Authors: García-Bernal David , Sotillo-Mallo Elena , Nombela-Arrieta César , Samaniego Rafael , Fukui Yoshinori , Stein Jens V. , Teixidó Joaquin
Published: April 2014

The α4β1 integrin is an essential adhesion molecule for recruitment of circulating lymphocytes into lymphoid organs and peripheral sites of inflammation. Chemokines stimulate α4β1 adhesive activity allowing lymphocyte arrest on endothelium and subsequent diapedesis. Activation of the GTPase Rac by the guanine-nucleotide exchange factor Vav1 promoted by CXCL12 controls T lymphocyte adhesion mediated by α4β1. In this study, we investigated the role of DOCK2, a lymphocyte guanine-nucleotide exchange factor also involved in Rac activation, in CXCL12-stimulated human T lymphocyte adhesion mediated by α4β1. Using T cells transfected with DOCK2 mutant forms defective in Rac activation or with DOCK2 small interfering RNA, we demonstrate that DOCK2 is needed for efficient chemokine-stimulated lymphocyte attachment to VCAM-1 under shear stress. Flow chamber, soluble binding, and cell spreading assays identified the strengthening of α4β1-VCAM-1 interaction, involving high affinity α4β1 conformations, as the adhesion step mainly controlled by DOCK2 activity. The comparison of DOCK2 and Vav1 involvement in CXCL12-promoted Rac activation and α4β1-dependent human T cell adhesion indicated a more prominent role of Vav1 than DOCK2. These results suggest that DOCK2-mediated signaling regulates chemokine-stimulated human T lymphocyte α4β1 adhesive activity, and that cooperation with Vav1 might be required to induce sufficient Rac activation for efficient adhesion. In contrast, flow chamber experiments using lymph node and spleen T cells from DOCK2−/− mice revealed no significant alterations in CXCL12-promoted adhesion mediated by α4β1, indicating that DOCK2 activity is dispensable for triggering of this adhesion in mouse T cells, and suggesting that Rac activation plays minor roles in this process.
Induced Expression of Murine γ2a by CD40 Ligation Independently of IFN-γ
Authors: Collins John T. , Shi Jian , Burrell Bryna E. , Bishop D. Keith , Dunnick Wesley A.
Published: April 2014

IgG2a, with γ2a H chains, is important for protection against viruses and other intracellular pathogens. Although a large portion of IgG2a expression is dependent upon IFN-γ, some germline transcription and switch recombination to the murine γ2a H chain gene expression are independent of IFN-γ. We found that agonistic anti-CD40 Abs injected into IFN-γ-deficient mice induce a >200-fold increase in the amount of serum Ig2a, while other Ig isotypes are increased by 16-fold or less. In vitro, ligation of CD40 on B cells, without the addition of other B cell activators or cytokines, results in germline transcription and switch recombination that are largely restricted to the γ2a gene. These results suggest that some immune responses to infectious agents can result in large amounts of IgG2a expression through ligation of CD40, without the expression of IFN-γ by Th1 or other cells.
Increased Human IgE Induced by Killing Schistosoma mansoni In Vivo Is Associated with Pretreatment Th2 Cytokine Responsiveness to Worm Antigens
Authors: Walter Klaudia , Fulford Anthony J. C. , McBeath Rowena , Joseph Sarah , Jones Frances M. , Kariuki H. Curtis , Mwatha Joseph K. , Kimani Gachuhi , Kabatereine Narcis B. , Vennervald Birgitte J. , Ouma John H. , Dunne David W.
Published: April 2014

In schistosomiasis endemic areas, children are very susceptible to postchemotherapy reinfection, whereas adults are relatively resistant. Different studies have reported that schistosome-specific IL-4 and IL-5 responses, or posttreatment worm-IgE levels, correlate with subsequent low reinfection. Chemotherapy kills i.v. worms providing an in vivo Ag challenge. We measured anti-worm (soluble worm Ag (SWA) and recombinant tegumental Ag (rSm22.6)) and anti-egg (soluble egg Ag) Ab levels in 177 Ugandans (aged 7–50) in a high Schistosoma mansoni transmission area, both before and 7 wk posttreatment, and analyzed these data in relation to whole blood in vitro cytokine responses at the same time points. Soluble egg Ag-Ig levels were unaffected by treatment but worm-IgG1 and -IgG4 increased, whereas worm-IgE increased in many but not all individuals. An increase in worm-IgE was mainly seen in >15-year-olds and, unlike in children, was inversely correlated to pretreatment infection intensities, suggesting this response was associated both with resistance to pretreatment infection, as well as posttreatment reinfection. The increases in SWA-IgE and rSm22.6-IgE positively correlated with pretreatment Th2 cytokines, but not IFN-γ, induced by SWA. These relationships remained significant after allowing for the confounding effects of pretreatment infection intensity, age, and pretreatment IgE levels, indicating a link between SWA-specific Th2 cytokine responsiveness and subsequent increases in worm-IgE. An exceptionally strong relationship between IL-5 and posttreatment worm-IgE levels in <15-year-olds suggested that the failure of younger children to respond to in vivo Ag stimulation with increased levels of IgE, is related to their lack of pretreatment SWA Th2 cytokine responsiveness.
In the Absence of Reactive Oxygen Species, T Cells Default to a Th1 Phenotype and Mediate Protection against Pulmonary Cryptococcus neoformans Infection
Authors: Snelgrove Robert J. , Edwards Lorna , Williams Andrew E. , Rae Aaron J. , Hussell Tracy
Published: April 2014

In recent years, the prevalence of invasive fungal infections has increased, attributed mostly to the rising population of immunocompromised individuals. Cryptococcus neoformans has been one of the most devastating, with an estimated 6–8% of AIDS-infected patients succumbing to Cryptococcus-associated meningitis. Reactive oxygen species (ROS) are potent antimicrobial agents but also play a significant role in regulating immune cell phenotype, but cause immunopathology when produced in excess. We now show that mice lacking phagocyte NADPH oxidase have heightened macrophage and Th1 responses and improved pathogen containment within pulmonary granulomatous lesions. Consequently, dissemination of this fungus to the brain is diminished, an effect that is independent of IL-12. Similar results are described using the metalloporphyrin antioxidant manganese(III) tetrakis(N-ethyl pyridinium-2-yl)porphyrin, which also promoted a protective Th1 response and reduced dissemination to the brain. These findings are in sharp contrast to the protective potential of ROS against other fungal pathogens, and highlight the pivotal role that ROS can fulfill in shaping the profile of the host’s immune response.
Mannose-Binding Lectin Augments the Uptake of Lipid A, Staphylococcus aureus, and Escherichia coli by Kupffer Cells through Increased Cell Surface Expression of Scavenger Receptor A
Authors: Ono Kei , Nishitani Chiaki , Mitsuzawa Hiroaki , Shimizu Takeyuki , Sano Hitomi , Suzuki Hiroshi , Kodama Tatsuhiko , Fujii Nobuhiro , Fukase Koichi , Hirata Koichi , Kuroki Yoshio
Published: April 2014

We investigated roles of scavenger receptor A (SR-A) and mannose-binding lectin (MBL) in the uptake of endotoxin and bacteria by Kupffer cells. When [3H]lipid A was injected into retro-orbital plexus of mice, significantly less accumulation of lipid A in the liver was observed in SR-A-deficient mice and wild-type mice coinjected with fucoidan or acetylated low-density lipoprotein, which are known ligands for SR-A. Isolated Kupffer cells were able to take up [3H]lipid A in a time-dependent manner. The amount of lipid A associated with nonadherent Kupffer cells derived from SR-A-deficient mice was reduced by ∼80% when compared with wild-type cells, indicating an important role of SR-A in endotoxin uptake by Kupffer cells. The lipid A uptake by Kupffer cells was significantly enhanced in the presence of rMBL. Coincubation of fucoidan with [3H]lipid A significantly inhibited the basal and the MBL-stimulated uptake of lipid A by Kupffer cells. Preincubation of MBL with Kupffer cells also increased the uptake of lipid A. These results indicate that MBL augments the SR-A-mediated uptake of lipid A by Kupffer cells. Consistently, the exposure of MBL to Kupffer cells increased cell surface SR-A expression. The phagocytosis of Staphylococcus aureus and Escherichia coli by Kupffer cells was also enhanced by preincubation of MBL with the cells. In addition, MBL bound to lipid A, LPS, and S. aureus, and precipitated S. aureus. This study demonstrates important roles of SR-A and MBL in the uptake of endotoxin and bacteria by Kupffer cells.
Retrovirus-Mediated Over-Expression of Decay-Accelerating Factor Rescues Crry-Deficient Erythrocytes from Acute Alternative Pathway Complement Attack
Authors: Kim David D. , Miwa Takashi , Song Wen-Chao
Published: April 2014

Decay-accelerating factor (DAF) and complement receptor 1-related gene/protein y (Crry) are two membrane-bound complement regulators on murine erythrocytes that inhibit C3/C5 convertases. Previously, we found that Crry- but not DAF-deficient erythrocytes were susceptible to alternative pathway complement-mediated elimination in vivo. To determine whether it is a unique activity or a higher level expression of Crry makes it indispensable on murine erythrocytes, we over-expressed DAF on Crry-deficient (Crry−/−) erythrocytes by retroviral vector-mediated DAF gene transduction of bone marrow stem cells. DAF retrovirus-transduced erythrocytes expressed 846 ± 127 DAF molecules/cell (DAFhigh) compared with 249 ± 94 DAF molecules/cell (DAFlow) and 774 ± 135 Crry molecules/cell on control mouse erythrocytes. DAFhigh-Crry−/− erythrocytes were significantly more resistant than either DAFlow-Crry−/−, DAF−/− -Crry+/+ or wild-type erythrocytes to classical pathway complement-mediated C3 deposition in vitro. Furthermore, increased DAF expression rescued Crry−/− erythrocytes from acute alternative pathway complement attack in vivo. Notably, long term monitoring revealed that DAFhigh-Crry−/− erythrocytes were still more susceptible than wild-type erythrocytes to complement-mediated elimination as they had a shorter half-life in complement-sufficient mice but survived equally well in complement-deficient mice. These results suggest that both a high level expression and a more potent anti-alternative pathway complement activity of Crry contributed to its indispensable role on murine erythrocytes. Additionally, they demonstrate the feasibility of using stem cell gene therapy to correct membrane complement regulator deficiency on blood cells in vivo.
Membrane-Bound CC Chemokine Inhibitor 35K Provides Localized Inhibition of CC Chemokine Activity In Vitro and In Vivo
Authors: Bursill Christina A. , Cash Jenna L. , Channon Keith M. , Greaves David R.
Published: April 2014

CC chemokines mediate mononuclear cell recruitment and activation in chronic inflammation. We have shown previously that gene transfer using recombinant adenoviruses, encoding a soluble CC chemokine-binding protein of vaccinia virus 35K, can dramatically reduce atherosclerosis and vein graft remodeling in apolipoprotein E knockout mice. In this study, we report the development of a membrane-bound form of 35K (m35K), tagged with GFP, which allows for localized, broad-spectrum CC chemokine blockade. In vitro experiments indicate that m35K-expressing cells no longer undergo CC chemokine-induced chemotaxis, and m35K-expressing cells can locally deplete the CC chemokines RANTES (CCL5) and MIP-1α (CCL3) from supernatant medium. This sequestration of CC chemokines can prevent chemotaxis of bystander cells to CC, but not CX3C chemokines. Intraperitoneal injection of mice with an adenovirus-encoding m35K leads to a significant (44%) decrease in leukocyte recruitment into the peritoneal cavity in a sterile peritonitis model. Intravenous adenovirus-encoding m35K delivery leads to m35K expression in hepatocytes, which confers significant protection against liver damage (75% reduction in liver enzymes) in a Con A-induced hepatitis model. In summary, we have generated a membrane-bound CC chemokine-binding protein (m35K) that provides localized broad-spectrum CC chemokine inhibition in vitro and in vivo. m35K may be a useful tool to study the role of CC chemokines in leukocyte trafficking and block the recruitment of monocytes in chronic inflammation.
Deguelin, an Akt Inhibitor, Suppresses IκBα Kinase Activation Leading to Suppression of NF-κB-Regulated Gene Expression, Potentiation of Apoptosis, and Inhibition of Cellular Invasion
Authors: Nair Asha S. , Shishodia Shishir , Ahn Kwang Seok , Kunnumakkara Ajaikumar B. , Sethi Gautam , Aggarwal Bharat B.
Published: April 2014

Deguelin, a constituent of the bark of the African plant Mundulea sericea (Leguminosae), exhibits antiproliferative and anticarcinogenic activities through a mechanism that is not well understood. Because various steps in carcinogenesis are regulated by NF-κB, we postulated that the activity of deguelin is mediated through this transcription factor. We found that deguelin suppressed NF-κB activation induced by carcinogens, tumor promoters, growth factors, and inflammatory stimuli. This suppression was not cell-type specific, because NF-κB activation was suppressed in both lymphoid and epithelial cells. Moreover, constitutive NF-κB activation was also blocked by deguelin. The suppression of TNF-induced NF-κB activation by deguelin occurred through the inhibition of the activation of IκBα kinase, leading to sequential suppression of IκBα phosphorylation, IκBα degradation, p65 phosphorylation, p65 nuclear translocation, and NF-κB-dependent reporter gene expression. Deguelin also suppressed the NF-κB reporter activity induced by TNFR1, TNFR-associated death domain, TNFR-associated factor 2, and IκBα kinase, but not that induced by p65. The inhibition of NF-κB activation thereby led to the down-regulation of gene products involved in cell survival, proliferation, and invasion. Suppression of these gene products by deguelin enhanced the apoptosis induced by TNF and chemotherapeutic agents and suppressed TNF-induced cellular invasion. Our results demonstrate that deguelin inhibits the NF-κB activation pathway, which may explain its role in the suppression of carcinogenesis and cellular proliferation.
Development of Autoimmunity in IL-14α-Transgenic Mice
Authors: Shen Long , Zhang Chongjie , Wang Tao , Brooks Stephen , Ford Richard J. , Lin-Lee Yen Chiu , Kasianowicz Amy , Kumar Vijay , Martin Lisa , Liang Ping , Cowell John , Ambrus Julian L.
Published: April 2014

Multiple genetic loci contribute to the development of systemic lupus erythematosus (SLE). In murine models for SLE, various genes on chromosome four have been implicated. IL-14 is a cytokine originally identified as a B cell growth factor. The il14 gene is located on chromosome 4. IL-14α is a cytokine encoded by the plus strand of the IL-14 gene using exons 3–10. The expression of IL-14α is increased in (NZB × NZW)F1 mice. In this study, we produced IL-14α-transgenic mice to study the role of IL-14α in the development of autoimmunity. At age 3–9 mo, IL-14α-transgenic mice demonstrate increased numbers of B1 cells in the peritoneum, increased serum IgM, IgG, and IgG 2a and show enhanced responses to T-dependent and T-independent Ags compared with littermate controls. At age 9–17 mo, IL-14α-transgenic mice develop autoantibodies, sialadenitis, as in Sjögren’s syndrome, and immune complex-mediated nephritis, as in World Health Organization class II SLE nephritis. Between the ages 14–18 mo, 95% of IL-14α-transgenic mice developed CD5+ B cell lymphomas, consistent with the lymphomas seen in elderly patients with Sjögren’s syndrome and SLE. These data support a role for IL-14α in the development of both autoimmunity and lymphomagenesis. These studies may provide a genetic link between these often related disorders.
Cutting Edge: Deficiency of Macrophage Migration Inhibitory Factor Impairs Murine Airway Allergic Responses
Authors: Wang Bing , Huang Xiaozhu , Wolters Paul J. , Sun Jiusong , Kitamoto Shiro , Yang Min , Riese Richard , Leng Lin , Chapman Harold A. , Finn Patricia W. , David John R. , Bucala Richard , Shi Guo-Ping
Published: April 2014

Increased levels of macrophage migration inhibitory factor (MIF) in serum, sputum, and bronchioalveolar lavage fluid (BALF) from asthmatic patients and time/dose-dependent expression of MIF in eosinophils in response to phorbol myristate acetate suggest the participation of MIF in airway inflammation. In this study, we examined inflammation in OVA-sensitized mouse lungs in wild-type and MIF-deficient mice (MIF−/−). We report increased MIF in the lung and BALF of sensitized wild-type mice. MIF−/− mice demonstrated significant reductions in serum IgE and alveolar inflammatory cell recruitment. Reduced Th1/Th2 cytokines and chemokines also were detected in serum or BALF from MIF−/− mice. Importantly, alveolar macrophages and mast cells, but not dendritic cells or splenocytes, from MIF−/− mice demonstrated impaired CD4+ T cell activation, and the reconstitution of wild-type mast cells in MIF−/− mice restored the phenotype of OVA-induced airway inflammation, revealing a novel and essential role of mast cell-derived MIF in experimentally induced airway allergic diseases.
Receptor Cross-Linking on Human Plasmacytoid Dendritic Cells Leads to the Regulation of IFN-α Production
Authors: Fanning Stacey L. , George Thaddeus C. , Feng Di , Feldman Steven B. , Megjugorac Nicholas J. , Izaguirre Alexander G. , Fitzgerald-Bocarsly Patricia
Published: April 2014

Plasmacytoid dendritic cells (PDC) are the natural type I IFN-producing cells that produce large amounts of IFN-α in response to viral stimulation. During attempts to isolate PDC from human PBMC, we observed that cross-linking a variety of cell surface receptors, including blood DC Ag (BDCA)-2, BDCA-4, CD4, or CD123 with Abs and immunobeads on PDC leads to inhibition of IFN-α production in response to HSV. To understand the mechanisms involved, a number of parameters were investigated. Cross-linking did not inhibit endocytosis of soluble Ag by PDC. Flow cytometry for annexin V and activated caspase-3 indicated that PDC are not undergoing apoptosis after receptor cross-linking. Cross-linking of CD123, but not the other receptors, caused the up-regulation of costimulatory molecules CD80 and CD86, as well as the down-regulation of CD62L, indicating PDC maturation. Thus, anti-CD123 Ab may be acting similar to the natural ligand, IL-3. Anti-phosphotyrosine Ab, as well as Ab to the IFN regulatory factor, IRF-7, was used in intracellular flow cytometry to elucidate the signaling pathways involved. Tyrosine phosphorylation occurred after cross-linking BDCA-2 and BDCA-4, but not CD4. Cross-linking did not affect IRF-7 levels in PDC, however, cross-linking BDCA-2, BDCA-4, and CD4, but not CD123, inhibited the ability of IRF-7 to translocate to the nucleus. Taken together, these results suggest that cross-linking BDCA-2, BDCA-4, and CD4 on PDC regulates IFN-α production at the level of IRF-7, while the decrease in IFN-α production after CD123 cross-linking is due to stimulation of the IL-3R and induction of PDC maturation.
Ly49B Is Expressed on Multiple Subpopulations of Myeloid Cells
Authors: Gays Frances , Aust Jonathan G. , Reid Delyth M. , Falconer Jane , Toyama-Sorimachi Noriko , Taylor Philip R. , Brooks Colin G.
Published: April 2014

Using a novel mAb specific for mouse Ly49B, we report here that Ly49B, the last remaining member of the C57 Ly49 family to be characterized, is expressed at low levels on ∼1.5% of spleen cells, none which are NK cells or T cells but which instead belong to several distinct subpopulations of myeloid cells defined by expression of CD11b and different levels of Gr1. Much larger proportions of bone marrow and peritoneal cells expressed Ly49B, all being CD11b+ and comprising multiple subpopulations defined by light scatter, F4/80, and Gr1 expression. Costaining for Ly49Q, also expressed on myeloid cells, revealed that Ly49B and Ly49Q were most strongly expressed on nonoverlapping subpopulations, Ly49Qhigh cells being mostly B220+CD4+ and/or CD8+, Ly49B+ cells lacking these markers. Myeloid populations that developed from bone marrow progenitors in vitro frequently coexpressed both Ly49B and Ly49Q, and Ly49B expression could be up-regulated by LPS, α-IFN, and γ-IFN, often independently of Ly49Q. PCR analysis revealed that cultured NK cells and T cells contained Ly49B transcripts, and Ly49B expression could be detected on NK cells cultured in IL-12 plus IL-18, and on an immature NK cell line. Immunohistochemical studies showed that Ly49B expression in tissues overlapped with but was distinct from that of all other myeloid molecules examined, being particularly prominent in the lamina propria and dome of Peyer’s patches, implicating an important role of Ly49B in gut immunobiology. In transfected cells, Ly49B was found to associate with SHP-1, SHP-2, and SHIP in a manner strongly regulated by intracellular phosphorylation events.