bluuebunny/crossref_metadata_2025 huggingface dataset:
J Chain in the Nurse Shark: Implications for Function in a Lower Vertebrate
Authors: Hohman Valerie S. , Stewart Sue E. , Rumfelt Lynn L. , Greenberg Andrew S. , Avila David W. , Flajnik Martin F. , Steiner Lisa A.
Published: April 2014

J chain is a small polypeptide covalently attached to polymeric IgA and IgM. In humans and mice, it plays a role in binding Ig to the polymeric Ig receptor for transport into secretions. The putative orthologue of mammalian J chain has been identified in the nurse shark by sequence analysis of cDNA and the polypeptide isolated from IgM. Conservation with J chains from other species is relatively poor, especially in the carboxyl-terminal portion, and, unlike other J chains, the shark protein is not acidic. The only highly conserved segment in all known J chains is a block of residues surrounding an N-linked glycosylation site. Of the eight half-cystine residues that are conserved in mammalian J chains, three are lacking in the nurse shark, including two in the carboxyl-terminal segment that have been reported to be required for binding of human J chain-containing IgA to secretory component. Taken together with these data, the relative abundance of J chain transcripts in the spleen and their absence in the spiral valve (intestine) suggest that J chain in nurse sharks may not have a role in Ig secretion. Analysis of J chain sequences in diverse species is in agreement with accepted phylogenetic relationships, with the exception of the earthworm, suggesting that the reported presence of J chain in invertebrates should be reassessed.
Caspase Activity Is Required for Stimulated B Lymphocytes to Enter the Cell Cycle
Authors: Olson N. Eric , Graves Jonathan D. , Shu Geraldine L. , Ryan Elizabeth J. , Clark Edward A.
Published: April 2014

Following activation with proliferative stimuli, including ligation of CD40, dense human tonsillar B cells (>98% cells in G0) have increased cleavage and activation of caspase-8 and -6 accompanied by decreased caspase-3 activation and apoptosis. Proliferation was blocked by either a broad specificity caspase inhibitor or inhibitors selective for caspase-6 or caspase-8. In contrast, an inhibitor selective for caspase-3 was without effect. Furthermore, induction of cyclin D and cyclin-dependent kinase 4 mRNA and protein was blocked upon inhibition of caspase-6, but not caspase-3. Thus, caspase-6-like activity is required for quiescent B cells to increase the expression of genes required for entry into G1. In support of this model, the transcriptional suppressor special AT-rich sequence-binding protein 1, a preferred caspase-6 substrate, was cleaved upon B cell stimulation. Caspase activity was not required for all signaling events, as caspase inhibitors did not affect the phosphorylation of p42/44 mitogen-activated protein kinase, the expression of the survival factor cellular inhibitor of apoptosis 2, or the production of IL-6 by stimulated G0 B cells. These findings suggest a mechanism by which caspase-6 may selectively allow entry of quiescent B cells into the cell cycle.
Different and Divergent Regulation of the KIR2DL4 and KIR3DL1 Promoters
Authors: Stewart C. Andrew , van Bergen Jeroen , Trowsdale John
Published: April 2014

The killer Ig-like receptors (KIR) are a family of highly related MHC class I receptors that show extreme genetic polymorphism both within the human population and between closely related primate species, suggestive of rapid evolutionary diversification. Most KIR are expressed in a variegated fashion by the NK population, giving rise to an NK repertoire of specificities for MHC class I. We compared the promoter for KIR3DL1, which exhibits variegated gene expression, with that for KIR2DL4, which is expressed by all NK cell clones. Maximum transcriptional activity of each was encoded within ∼270 bp upstream of the translation initiation codon. The KIR2DL4 promoter drove reporter gene expression only in NK cells, while the KIR3DL1 promoter was active in a range of cell types, suggesting that the latter requires other regulatory elements for physiological expression. In NK cells, reporter gene expression driven by the KIR2DL4 promoter was greater than that driven by the KIR3DL1 promoter. DNase I footprinting revealed that transcription factor binding sites differ between the two promoters. The data indicate that while the promoters of these two KIR genes share 67% nucleotide identity, they have evolved distinct properties consistent with different roles in regulating the generation of NK repertoire.
Polyvalent Antigens Stabilize B Cell Antigen Receptor Surface Signaling Microdomains
Authors: Thyagarajan Rathna , Arunkumar Nandini , Song Wenxia
Published: April 2014

The B cell Ag receptor (BCR) can distinguish subtle differences in Ag structure and trigger differential responses. In this study, we analyzed the effects of Ag valency on the signaling and Ag-targeting functions of the BCR. Although both paucivalent and polyvalent Ags induced the redistribution of the surface BCR into polarized caps, polyvalent Ag-induced BCR caps persisted. Ganglioside GM1, a lipid raft marker, and tyrosine-phosphorylated proteins, but not CD45 and transferrin receptor, were concentrated in BCR caps, suggesting BCR caps as surface-signaling microdomains. Prolonged BCR caps were concomitant with an increase in the level and duration of protein tyrosine phosphorylation and a reduction in BCR internalization and movement to late endosomes/lysosomes. Thus, Ag valency influences B cell responses by modulating the stability of BCR-signaling microdomains and BCR trafficking.
OX40 Ligation on Activated T Cells Enhances the Control ofCryptococcus neoformansand Reduces Pulmonary Eosinophilia
Authors: Humphreys Ian R. , Edwards Lorna , Walzl Gerhard , Rae Aaron J. , Dougan Gordon , Hill Sue , Hussell Tracy
Published: April 2014

Pulmonary eosinophilia induced in C57BL/6 mice after Cryptococcus neoformans infection is driven by CD4+ Th2 cells. The immunological mechanisms that protect against eosinophilia are not fully understood. Interaction of OX40 (CD134) and its ligand, OX40L, has been implicated in T cell activation and cell migration. Unlike CD28, OX40 is only expressed on T cells 1–2 days after Ag activation. Manipulation of this pathway would therefore target recently activated T cells, leaving the naive repertoire unaffected. In this study, we show that engagement of OX40 by an OX40L:Ig fusion protein drives IFN-γ production by CD4+ T cells and reduces eosinophilia and C. neoformans burden in the lung. Using gene-depleted mice, we show that reduction of eosinophilia and pathogen burden requires IL-12 and/or IFN-γ. C. neoformans infection itself only partially induces OX40L expression by APCs. Provision of exogenous OX40L reveals a critical role of this pathway in the prevention of C. neoformans-induced eosinophilia.
Central Role of Complement in Passive Protection by Human IgG1 and IgG2 Anti-pneumococcal Antibodies in Mice
Authors: Saeland Eirikur , Vidarsson Gestur , Leusen Jeanette H. W. , van Garderen Evert , Nahm Moon H. , Vile-Weekhout Henriette , Walraven Vanessa , Stemerding Annette M. , Verbeek J. Sjef , Rijkers Ger T. , Kuis Wietse , Sanders Elisabeth A. M. , van de Winkel Jan G. J.
Published: April 2014

Streptococcus pneumoniae is an important cause of morbitity and mortality worldwide. Capsule-specific IgG1 and IgG2 Abs are induced upon vaccination with polysaccharide-based vaccines that mediate host protection. We compared the protective capacity of human recombinant serogroup 6-specific IgG1 and IgG2 Abs in mice deficient for either leukocyte FcR or complement factors. Human IgG1 was found to interact with mouse leukocyte FcR in vitro, whereas human IgG2 did not. Both subclasses induced complement activation, resulting in C3c deposition on pneumococcal surfaces. Passive immunization of C57BL/6 mice with either subclass before intranasal challenge with serotype 6A induced similar degrees of protection. FcγRI- and III-deficient mice, as well as the combined FcγRI, II, and III knockout mice, were protected by passive immunization, indicating FcR not to be essential for protection. C1q or C2/factor B knockout mice, however, were not protected by passive immunization. Passively immunized C2/factor B−/− mice displayed higher bacteremic load than C1q−/− mice, supporting an important protective role of the alternative complement pathway. Spleens from wild-type and C1q−/− mice showed hyperemia and thrombotic vessel occlusion, as a result of septicemic shock. Notably, thrombus formation was absent in spleens of C2/factor B−/− mice, suggesting that the alternative complement pathway contributes to shock-induced intravascular coagulation. These studies demonstrate complement to play a central role in Ab-mediated protection against pneumococcal infection in vivo, as well as in bacteremia-associated thrombotic complications.
Proteins and Their Derived Peptides as Carriers in a Conjugate Vaccine forStreptococcus pneumoniae: Self-Heat Shock Protein 60 and Tetanus Toxoid
Authors: Amir-Kroll Hila , Nussbaum Gabriel , Cohen Irun R.
Published: April 2014

We induced T cell help for vaccination against Streptococcus pneumoniae (Pn) using self and foreign peptides and their source proteins conjugated to the capsular polysaccharide (CPS) of type 4 Pn; the carriers were self-heat shock protein 60 (HSP60) and tetanus toxoid (TT). We measured the production of IgG Abs to the CPS and the carriers, and tested resistance to challenge with highly lethal amounts of Pn injected i.p. (LD50 × 103–106). We now report that vaccination protects old and young mice from bacterial challenge; however, there were significant differences in vaccine efficacy based on the carrier. Self-HSP60 peptide p458m was more effective than the whole HSP60 molecule and was equally effective compared with TT. Both p458m and TT were more protective than the TT-derived peptide p30 after a single vaccination. However, peptide p30 was effective in more MHC genotypes than was p458m. Unlike other vaccines, protection conferred by p458m was not related to the amount of anti-CPS Ab: mice that produced very little Ab were still protected from highly lethal doses of bacteria (LD50 × 105–106). Furthermore, unlike the other carriers, there was no Ab response to the p458m carrier. Thus, peptides, self as well as foreign, can provide T cell help that differs functionally from that provided by the whole parent protein.
Genetic Engineering of a Suboptimal Islet Graft with A20 Preserves β Cell Mass and Function
Authors: Grey Shane T. , Longo Christopher , Shukri Tala , Patel Virendra I. , Csizmadia Eva , Daniel Soizic , Arvelo Maria B. , Tchipashvili Vaja , Ferran Christiane
Published: April 2014

Transplantation of an excessive number of islets of Langerhans (two to four pancreata per recipient) into patients with type I diabetes is required to restore euglycemia. Hypoxia, nutrient deprivation, local inflammation, and the β cell inflammatory response (up-regulation of NF-κB-dependent genes such as inos) result in β cell destruction in the early post-transplantation period. Genetic engineering of islets with anti-inflammatory and antiapoptotic genes may prevent β cell loss and primary nonfunction. We have shown in vitro that A20 inhibits NF-κB activation in islets and protects from cytokine- and death receptor-mediated apoptosis. In vivo, protection of newly transplanted islets would reduce the number of islets required for successful transplantation. Transplantation of 500 B6/AF1 mouse islets into syngeneic, diabetic recipients resulted in a cure rate of 100% within 5 days. Transplantation of 250 islets resulted in a cure rate of only 20%. Transplantation of 250 islets overexpressing A20 resulted in a cure rate of 75% with a mean time to cure of 5.2 days, comparable to that achieved with 500 islets. A20-expressing islets preserve functional β cell mass and are protected from cell death. These data demonstrate that A20 is an ideal cytoprotective gene therapy candidate for islet transplantation.
Aspirin-Triggered Lipoxin A4 and B4 Analogs Block Extracellular Signal-Regulated Kinase-Dependent TNF-α Secretion from Human T Cells
Authors: Ariel Amiram , Chiang Nan , Arita Makoto , Petasis Nicos A. , Serhan Charles N.
Published: April 2014

Lipoxins (LX) and their aspirin-triggered 15-epimer endogenous isoforms are endogenous anti-inflammatory and pro-resolution eicosanoids. In this study, we examined the impact of LX and aspirin-triggered LXA4-stable analogs (ATLa) on human T cell functions. 15-epi-16-(p-fluoro)phenoxy-LXA4 (ATLa1) blocked the secretion of TNF-α from human PBMC after stimulation by anti-CD3 Abs, with the IC50 value of ≈0.05 nM. A similar action was also exerted by the native aspirin-triggered 15-epi-LXA4, a new 15-epi-16-(p-trifluoro)phenoxy-LXA4 analog (ATLa2), as well as LXB4, and its analog 5-(R/S)-methyl-LXB4. The LXA4 receptor (ALX) is expressed in peripheral blood T cells and mediates the inhibition of TNF-α secretion from activated T cells by ATLa1. This action was accomplished by inhibition of the anti-CD3-induced activation of extracellular signal-regulated kinase, which is essential for TNF-α secretion from anti-CD3-activated T cells. These results demonstrate novel roles for LX and aspirin-triggered LX in the regulation of T cell-mediated responses relevant in inflammation and its resolution. Moreover, they provide potential counterregulatory signals in communication(s) between the innate and acquired immune systems.
Transepithelial Migration of Neutrophils in Response to Leukotriene B4 Is Mediated by a Reactive Oxygen Species-Extracellular Signal-Regulated Kinase-Linked Cascade
Authors: Woo Chang-Hoon , Yoo Min-Hyuk , You Hye-Jin , Cho Sung-Hoon , Mun Yeung-Chul , Seong Chu-Myong , Kim Jae-Hong
Published: April 2014

The epithelial cells that form a barrier lining the lung airway are key regulators of neutrophil trafficking into the airway lumen in a variety of lung inflammatory diseases. Although the lipid mediator leukotriene B4 (LTB4) is known to be a principal chemoattractant for recruiting neutrophils to inflamed sites across the airway epithelium, the precise signaling mechanism involved remains largely unknown. In the present study, therefore, we investigated the signaling pathway through which LTB4 induces transepithelial migration of neutrophils. We found that LTB4 induces concentration-dependent transmigration of DMSO-differentiated HL-60 neutrophils and human polymorphonuclear neutrophils across A549 human lung epithelium. This effect was mediated via specific LTB4 receptors and was inhibited by pretreating the cells with N-acetylcysteine (NAC), an oxygen free radical scavenger, with diphenylene iodonium (DPI), an inhibitor of NADPH oxidase-like flavoproteins, or with PD98059, an extracellular signal-regulated kinase (ERK) inhibitor. Consistent with those findings, LTB4-induced ERK phosphorylation was completely blocked by pretreating cells with NAC or DPI. Taken together, our observations suggest LTB4 signaling to transepithelial migration is mediated via generation of reactive oxygen species, which leads to downstream activation of ERK. The physiological relevance of this signaling pathway was demonstrated in BALB/c mice, in which intratracheal instillation of LTB4 led to acute recruitment of neutrophils into the airway across the lung epithelium. Notably, the response to LTB4 was blocked by NAC, DPI, PD98059, or CP105696, a specific LTB4 receptor antagonist.
An Inducible Nitric Oxide Synthase-Luciferase Reporter System for In Vivo Testing of Anti-inflammatory Compounds in Transgenic Mice
Authors: Zhang Ning , Weber Aneil , Li Bonnie , Lyons Richard , Contag Pamela R. , Purchio Anthony F. , West David B.
Published: April 2014

The inducible NO synthase gene (iNOS) plays a role in a number of chronic and acute conditions, including septic shock and contact hypersensitivity autoimmune diseases, such as rheumatoid arthritis, gastrointestinal disorders, and myocardial ischemia. The iNOS gene is primarily under transcriptional control and is induced in a variety of conditions. The ability to monitor and quantify iNOS expression in vivo may facilitate a better understanding of the role of iNOS in different diseases. In this study, we describe a transgenic mouse (iNos-luc) in which the luciferase reporter is under control of the murine iNOS promoter. In an acute sepsis model produced by injection of IFN-γ and LPS, we observed an induction of iNOS-driven luciferase activity in the mouse liver. This transgene induction is dose and time dependent and correlated with an increase of liver iNOS protein and iNOS mRNA levels. With this model, we tested 11 compounds previously shown to inhibit iNOS induction in vitro or in vivo. Administration of dexamethasone, epigallocatechin gallate, α-phenyl-N-tert-butyl nitrone, and ebselen significantly suppressed iNOS transgene induction by IFN-γ and LPS. We further evaluated the use of the iNos-luc transgenic mice in a zymosan-induced arthritis model. Intra-articular injection of zymosan induced iNos-luc expression in the knee joint. The establishment of the iNos-luc transgenic model provides a valuable tool for studying processes in which the iNOS gene is induced and for screening anti-inflammatory compounds in vivo.
Immunoglobulin Activation of T Cell Chemoattractant Expression in Fibroblasts from Patients with Graves’ Disease Is Mediated Through the Insulin-Like Growth Factor I Receptor Pathway
Authors: Pritchard Jane , Han Rui , Horst Noah , Cruikshank William W. , Smith Terry J.
Published: April 2014

Graves’ disease (GD) is associated with T cell infiltration, but the mechanism for lymphocyte trafficking has remained uncertain. We reported previously that fibroblasts from patients with GD express IL-16, a CD4-specific chemoattractant, and RANTES, a C-C chemokine, in response to GD-specific IgG (GD-IgG). We unexpectedly found that these responses result from a functional interaction between GD-IgG and the insulin-like growth factor (IGF)-I receptor (IGF-IR). IGF-I and the IGF-IR-specific IGF-I analog, des(1–3), mimic the effects of GD-IgG. Neither GD-IgG nor IGF-I activates chemoattractant expression in control fibroblasts from donors without GD. Interrupting IGF-IR function with specific receptor-blocking Abs or by transiently transfecting fibroblasts with a dominant negative mutant IGF-IR completely attenuates signaling provoked by GD-IgG. Moreover, GD-IgG displaces specific 125I-labeled IGF-I binding to fibroblasts and attenuates IGF-IR detection by flow cytometry. These findings identify a novel disease mechanism involving a functional GD-IgG/IGF-IR bridge, which potentially explains T cell infiltration in GD. Interrupting this pathway may constitute a specific therapeutic strategy.
Failure of Monocytes of Trauma Patients to Convert to Immature Dendritic Cells is Related to Preferential Macrophage-Colony-Stimulating Factor-Driven Macrophage Differentiation
Authors: De Asit K. , Laudanski Krzysztof , Miller-Graziano Carol L.
Published: April 2014

Following trauma, increased inflammatory monokine activation and depressed APC function can occur simultaneously. These contradictory monocyte (Mφ) dysfunctions could result if postinjury Mφ differentiation preferentially favored inflammatory macrophage (Mac) differentiation over development into the most potent APC, dendritic cells (DC). In this report, Mφ of trauma patients with a depressed MLR induction capacity are, for the first time, shown to be unable to differentiate in vitro to immature CD1a+ DC under the influence of GM-CSF and IL-4. Trauma patient Mφ that retained MLR-inducing capacity had a nonsignificant reduction in DC differentiation capacity. Only patient Mφ populations with depressed differentiation to immature DC (iDC) demonstrated depressed IL-12 and IL-15 production and a continued reduced MLR induction capacity. Neither increased IL-10 production nor decreased CD11c+ DC precursor numbers correlated with depressed Mφ-to-DC differentiation. Instead, these patients’ APC-dysfunctional Mφ populations had increased expression of inflammatory Mac phenotypes (CD64+, CD86low, HLA-DRlow) and up-regulated secretion of M-CSF. M-CSF combined with IL-6 inhibits Mφ-to-iDC differentiation and promotes Mφ-to-Mac differentiation by down-regulating GM-CSFR expression and increasing DC apoptosis. Both depressed GM-CSFR expression and increased Mφ iDC apoptosis, as well as increased expression of CD126 (IL-6R) and CD115 (M-CSFR), were detected in APC-defective patient Mφ. In vitro addition of anti-M-CSF enhanced the IL-4 plus GM-CSF-induced Mφ-to-DC differentiation of these patients. This suggests that, in trauma patients, enhanced Mφ-to-Mac differentiation with concomitant inhibited iDC development is partially due to increased circulating Mφ sensitivity to and production of M-CSF and contributes to postinjury immunoaberrations.
Characterization of the Antigen Specificity and TCR Repertoire, and TCR-Based DNA Vaccine Therapy in Myelin Basic Protein-Induced Autoimmune Encephalomyelitis in DA Rats
Authors: Miyakoshi Akira , Yoon Won Kee , Jee Youngheun , Matsumoto Yoh
Published: April 2014

Like Lewis rats, DA rats are an experimental autoimmune encephalomyelitis (EAE)-susceptible strain and develop severe EAE upon immunization with myelin basic protein (MBP). However, there are several differences between the two strains. In the present study we induced acute EAE in DA rats by immunization with MBP and MBP peptides and examined the Ag specificity and TCR repertoire of encephalitogenic T cells. It was found that although immunization with MBP and a peptide corresponding to its 62–75 sequence (MBP62–75) induced clinical EAE, the responses of lymph node T cells isolated from MBP-immunized rats to MBP62–75 was marginal, indicating that this peptide contains major encephalitogenic, but not immunodominant, epitopes. The TCR analysis by CDR3 spectratyping of spinal cord T cells revealed that Vβ10 and Vβ15 spectratype expansion was always found in MBP62–75-immunized symptomatic rats. On the basis of these findings, we examined the encephalitogenicity of Vβ10- and Vβ15-positive T cells. First, the adoptive transfer experiments revealed that Vβ10-positive T line cells derived from MBP62–75-immunized rats induced clinical EAE in recipients. Second, administration of DNA vaccines encoding Vβ10 and Vβ15, alone or in combination, ameliorated MBP62–75-induced EAE. Collectively, it was strongly suggested that Vβ10- and Vβ15-positive T cells are encephalitogenic. Analyses of the Ag specificity and T cell repertoire of pathogenic T cells performed in this study provide useful information for designing specific immunotherapies against autoimmune diseases.
Role of Vesicle-Associated Membrane Protein-2, Through Q-SolubleN-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor/R-SolubleN-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor Interaction, in the Exocytosis of Specific and Tertiary Granules of Human Neutrophils
Authors: Mollinedo Faustino , Martín-Martín Belén , Calafat Jero , Nabokina Svetlana M. , Lazo Pedro A.
Published: April 2014

We have examined the role of the R-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) synaptobrevin-2/vesicle-associated membrane protein (VAMP)-2 in neutrophil exocytosis. VAMP-2, localized in the membranes of specific and gelatinase-containing tertiary granules in resting human neutrophils, resulted translocated to the cell surface following neutrophil activation under experimental conditions that induced exocytosis of specific and tertiary granules. VAMP-2 was also found on the external membrane region of granules docking to the plasma membrane in activated neutrophils. Specific Abs against VAMP-2 inhibited Ca2+ and GTP-γ-S-induced exocytosis of CD66b-enriched specific and tertiary granules, but did not affect exocytosis of CD63-enriched azurophilic granules, in electropermeabilized neutrophils. Tetanus toxin disrupted VAMP-2 and inhibited exocytosis of tertiary and specific granules. Activation of neutrophils led to the interaction of VAMP-2 with the plasma membrane Q-SNARE syntaxin 4, and anti-syntaxin 4 Abs inhibited exocytosis of specific and tertiary granules in electropermeabilized neutrophils. Immunoelectron microscopy showed syntaxin 4 on the plasma membrane contacting with docked granules in activated neutrophils. These data indicate that VAMP-2 mediates exocytosis of specific and tertiary granules, and that Q-SNARE/R-SNARE complexes containing VAMP-2 and syntaxin 4 are involved in neutrophil exocytosis.
CpG DNA Induces Self and Cross-Hyporesponsiveness of RAW264.7 Cells in Response to CpG DNA and Lipopolysaccharide: Alterations in IL-1 Receptor-Associated Kinase Expression
Authors: Yeo Seon-Ju , Yoon Jae-Geun , Hong Soon-Cheol , Yi Ae-Kyung
Published: April 2014

Exposure of macrophages to LPS induces a state of hyporesponsiveness to subsequent challenge with LPS. It has not been known whether previous exposure to CpG DNA induces a similar suppressive response to subsequent stimulation with CpG DNA. In the present study, we demonstrate that pretreatment with CpG DNA induces suppression of cytokine release in a murine macrophage-like cell RAW264.7 in response to subsequent challenge by CpG DNA. Additionally, CpG DNA-mediated activation of mitogen-activated protein kinases, including c-Jun NH2-terminal kinase, extracellular signal-regulated kinase, and p38, and activation of transcription factors AP-1, CREB, NF-κB, and STAT1 are greatly suppressed in the cells pre-exposed to CpG DNA. Pretreatment with CpG DNA also partially inhibited LPS-mediated production of cytokines and activation of mitogen-activated protein kinases and transcription factors. Neither LPS nor CpG DNA treatment inhibited Toll-like receptor 4, MD2, Toll-like receptor 9, myeloid differentiation factor 88, Toll/IL-1R domain-containing adaptor protein, Tollip, and TNF-α receptor-associated factor 6 expression. Interestingly, CpG DNA or LPS stimulation led to the inhibition of IL-1R-associated kinase expression. These results indicate that CpG DNA-induced refractory of RAW264.7 cells may be, at least in part, due to suppressed IL-1R-associated kinase expression.
Regulation of Cyclooxygenase-2 Expression by Macrophages in Response to Double-Stranded RNA and Viral Infection
Authors: Steer Sarah A. , Moran Jason M. , Maggi Leonard B. , Buller R. Mark L. , Perlman Harris , Corbett John A.
Published: April 2014

In this study the regulation of macrophage expression of cyclooxygenase-2 (COX-2) in response to dsRNA and virus infection was examined. Treatment of RAW 264.7 macrophages with dsRNA results in COX-2 mRNA accumulation and protein expression and the production of PGE2. Similar to dsRNA, encephalomyocarditis virus (EMCV) infection of RAW 264.7 cells stimulates COX-2 expression and PGE2 accumulation. The dsRNA-dependent protein kinase (PKR), which has been shown to participate in the regulation of gene expression in response to dsRNA and virus infection, does not appear to participate in the regulation of COX-2 expression by macrophages. Expression of dominant negative mutants of PKR in RAW 264.7 cells fails to attenuate dsRNA- and EMCV-induced COX-2 expression or PGE2 production. Furthermore, dsRNA and EMCV stimulate COX-2 expression and PGE2 accumulation to similar levels in macrophages isolated from wild-type and PKR-deficient mice. Recently, a novel PKR-independent role for the calcium-independent phospholipase A2 (iPLA2) in the regulation of inducible NO synthase expression by macrophages in response to virus infection has been identified. The selective iPLA2 suicide substrate inhibitor bromoenol lactone prevents dsRNA- and EMCV-stimulated inducible NO synthase expression; however, bromoenol lactone does not attenuate dsRNA- or EMCV-induced COX-2 expression by macrophages. In contrast, inhibition of NF-κB activation prevents dsRNA-stimulated COX-2 expression and PGE2 accumulation by macrophages. These findings indicate that virus infection and treatment with dsRNA stimulate COX-2 expression by a mechanism that requires the activation of NF-κB and that is independent of PKR or iPLA2 activation.
Selective IκB Kinase Expression in Airway Epithelium Generates Neutrophilic Lung Inflammation
Authors: Sadikot Ruxana T. , Han Wei , Everhart M. Brett , Zoia Ornella , Peebles R. Stokes , Jansen E. Duco , Yull Fiona E. , Christman John W. , Blackwell Timothy S.
Published: April 2014

To determine whether NF-κB activation is sufficient to generate lung inflammation in vivo, we selectively expressed a constitutively active form of IκB kinase 1 (cIKK1) or IκB kinase 2 (cIKK2) in airway epithelium. After intratracheal administration of adenoviral vectors expressing cIKK1 or cIKK2 to transgenic reporter mice that express Photinus luciferase under the control of an NF-κB-dependent promoter, we detected significantly increased luciferase activity over time (up to 96 h). Compared with control mice treated with adenoviral vectors expressing β-galactosidase, lung bioluminescence and tissue luciferase activity were increased in NF-κB reporter mice treated with adenovirus (Ad)-cIKK1 or Ad-cIKK2. NF-κB activation in lungs of Ad-cIKK1- and Ad-cIKK2-treated mice was confirmed by immunoblots for RelA and EMSA from lung nuclear protein extracts. Mice treated with Ad-cIKK1 or Ad-cIKK2 showed induction of mRNA expression of several chemokines and cytokines in lung tissue. In lung lavage fluid, mice treated with Ad-cIKK1 or Ad-cIKK2 showed elevated concentrations of NF-κB-dependent chemokines macrophage-inflammatory protein 2 and KC and increased numbers of neutrophils. Coadministration of adenoviral vectors expressing a transdominant inhibitor of NF-κB with Ad-cIKK1 or Ad-cIKK2 resulted in abrogated NF-κB activation and other parameters of lung inflammation, demonstrating that the observed inflammatory effects of Ad-cIKK1 and Ad-cIKK2 were dependent on NF-κB activation by these kinases. These data show that selective expression of IκB kinases in airway epithelium results in NF-κB activation, inflammatory mediator production, and neutrophilic lung inflammation. Therapies targeted to NF-κB in lung epithelium may be beneficial in treating inflammatory lung diseases.
CD8αβ T Cells Are Not Essential to the Pathogenesis of Arthritis or Colitis in HLA-B27 Transgenic Rats
Authors: May Ekkehard , Dorris Martha L. , Satumtira Nimman , Iqbal Imran , Rehman Muhammad I. , Lightfoot Ellis , Taurog Joel D.
Published: April 2014

The class I MHC allele HLA-B27 is highly associated with the human spondyloarthropathies, but the basis for this association remains poorly understood. Transgenic rats with high expression of HLA-B27 develop a multisystem inflammatory disease that includes arthritis and colitis. To investigate whether CD8αβ T cells are needed in this disease, we depleted these cells in B27 transgenic rats before the onset of disease by adult thymectomy plus short-term anti-CD8α mAb treatment. This treatment induced profound, sustained depletion of CD8αβ T cells, but failed to suppress either colitis or arthritis. To address the role of CD8α+β− cells, we studied four additional groups of B27 transgenic rats treated with: 1) continuous anti-CD8α mAb, 2) continuous isotype-matched control mAb, 3) the thymectomy/pulse anti-CD8α regimen, or 4) no treatment. Arthritis occurred in ∼40% of each group, but was most significantly reduced in severity in the anti-CD8α-treated group. In addition to CD8αβ T cells, two sizeable CD8α+β− non-T cell populations were also reduced by the anti-CD8α treatment: 1) NK cells, and 2) a CD4+CD8+CD11b/c+CD161a+CD172a+ monocyte population that became expanded in diseased B27 transgenic rats. These data indicate that HLA-B27-retricted CD8+ T cells are unlikely to serve as effector cells in the transgenic rat model of HLA-B27-associated disease, in opposition to a commonly invoked hypothesis concerning the role of B27 in the spondyloarthropathies. The data also suggest that one or more populations of CD8α+β− non-T cells may play a role in the arthritis that occurs in these rats.
Cutting Edge: Homeostatic Proliferation of Peripheral T Lymphocytes Is Regulated by Clonal Competition
Authors: Troy Amy E. , Shen Hao
Published: April 2014

Homeostatic proliferation functions to maintain peripheral T cell numbers and is regulated by cytokines. In this study, we provide evidence that T cell homeostasis is also regulated by clonal competition. Naive polyclonal T cells divided when transferred to TCR transgenic hosts, as did monoclonal T cells when transferred to TCR transgenic hosts of differing clonotype. However, T cells did not divide in hosts of identical clono-type. Transgenic T cell proliferation was inhibited in irradiated hosts of the same clonotype, while cotransferred nontransgenic T cells proliferated extensively. These results show that clonal competition is a component of homeostasis that may contribute to selection of the peripheral T cell repertoire.
Hypoxia Regulates Expression and Activity of Kv1.3 Channels in T Lymphocytes: A Possible Role in T Cell Proliferation
Authors: Conforti Laura , Petrovic Milan , Mohammad Dina , Lee Susan , Ma Qing , Barone Sharon , Filipovich Alexandra H.
Published: April 2014

T lymphocytes are exposed to hypoxia during their development and also when they migrate to hypoxic pathological sites such as tumors and wounds. Although hypoxia can affect T cell development and function, the mechanisms by which immune cells sense and respond to changes in O2-availability are poorly understood. K+ channels encoded by the Kv1.3 subtype of the voltage-dependent Kv1 gene family are highly expressed in lymphocytes and are involved in the control of membrane potential and cell function. In this study, we investigate the sensitivity of Kv1.3 channels to hypoxia in freshly isolated human T lymphocytes and leukemic Jurkat T cells. Acute exposure to hypoxia (20 mmHg, 2 min) inhibits Kv1.3 currents in both cell types by 20%. Prolonged exposure to hypoxia (1% O2 for 24 h) selectively decreases Kv1.3 protein levels in Jurkat T cells by 47%, but not Kvβ2 and SK2 Ca-activated K+ channel subunit levels. The decrease in Kv1.3 protein levels occurs with no change in Kv1.3 mRNA expression and is associated with a significant decrease in K+ current density. A decrease in Kv1.3 polypeptide levels similar to that obtained during hypoxia is produced by Kv1.3 channel blockage. Our results indicate that hypoxia produces acute and long-term inhibition of Kv1.3 channels in T lymphocytes. This effect could account for the inhibition of lymphocyte proliferation during hypoxia. Indeed, we herein present evidence showing that hypoxia selectively inhibits TCR-mediated proliferation and that this inhibition is associated with a decrease in Kv1.3 proteins.
Program Death-1 Engagement Upon TCR Activation Has Distinct Effects on Costimulation and Cytokine-Driven Proliferation: Attenuation of ICOS, IL-4, and IL-21, But Not CD28, IL-7, and IL-15 Responses
Authors: Bennett Frann , Luxenberg Deborah , Ling Vincent , Wang I-Ming , Marquette Kim , Lowe David , Khan Nighat , Veldman Geertruida , Jacobs Kenneth A. , Valge-Archer Viia E. , Collins Mary , Carreno Beatriz M.
Published: April 2014

The program death 1 (PD-1) receptor and its ligands, PD-1 ligand (PD-L)1 and PD-L2, define a novel regulatory pathway with potential inhibitory effects on T, B, and monocyte responses. In the present study, we show that human CD4+ T cells express PD-1, PD-L1, and PD-L2 upon activation, and Abs to the receptor can be agonists or antagonists of the pathway. Under optimal conditions of stimulation, ICOS but not CD28 costimulation can be prevented by PD-1 engagement. IL-2 levels induced by costimulation are critical in determining the outcome of the PD-1 engagement. Thus, low to marginal IL-2 levels produced upon ICOS costimulation account for the greater sensitivity of this pathway to PD-1-mediated inhibition. Interestingly, exogenous IL-2, IL-7, and IL-15 but not IL-4 and IL-21 can rescue PD-1 inhibition, suggesting that among these cytokines only those that activate STAT5 can rescue PD-1 inhibition. As STAT5 has been implicated in the maintenance of IL-2Rα expression, these results suggest that IL-7 and IL-15 restore proliferation under conditions of PD-1 engagement by enhancing high-affinity IL-2R expression and hence, IL-2 responsiveness.
Either IL-2 or IL-12 Is Sufficient to Direct Th1 Differentiation by Nonobese Diabetic T Cells
Authors: Zhou Weisong , Zhang Feng , Aune Thomas M.
Published: April 2014

Th cell differentiation from naive precursors is a tightly controlled process; the most critical differentiation factor is the action of the driving cytokine: IL-12 for Th1 development, IL-4 for Th2 development. We found that CD4+ T cells from nonobese diabetic mice spontaneously differentiate into IFN-γ-producing Th1 cells in response to polyclonal TCR stimulation in the absence of IL-12 and IFN-γ. Instead, IL-2 was necessary and sufficient to direct T cell differentiation to the Th1 lineage by nonobese diabetic CD4+ T cells. Its ability to direct Th1 differentiation of both naive and memory CD4+ T cells was clearly uncoupled from its ability to stimulate cell division. Autocrine IL-2-driven Th1 differentiation of nonobese diabetic T cells may represent a genetic liability that favors development of IFN-γ-producing autoreactive T cells.
Down-Modulation of Responses to Type I IFN Upon T Cell Activation
Authors: Dondi Elisabetta , Rogge Lars , Lutfalla Georges , Uzé Gilles , Pellegrini Sandra
Published: April 2014

The immunomodulatory role of type I IFNs (IFN-α/β) in shaping T cell responses has been demonstrated, but the direct effects of IFN on T cells are still poorly characterized. Particularly, because IFN exert an antiproliferative activity, it remains elusive how the clonal expansion of effector T cells can paradoxically occur in the event of an infection when large amounts of IFN are produced. To address this issue, we have studied the effects of type I IFN in an in vitro differentiation model of human primary CD4+ T cells. We found that IFN-α treatment of resting naive T cells delayed their entry into the cell cycle after TCR triggering. Conversely, the ongoing expansion of effector T cells was not inhibited by the presence of IFN. Moreover, activated T cells showed a significantly reduced induction of IFN-sensitive genes, as compared with naive precursors, and this decline occurred independently of subset-specific polarization. The residual type I IFN response measured in activated T cells was found sufficient to inhibit replication of the vesicular stomatitis virus. Our data suggest that the activation of T lymphocytes includes regulatory processes that restrain the transcriptional response to IFN and allow the proliferation of effector cells in the presence of this cytokine.
Protection Against Experimental Autoimmune Encephalomyelitis Generated by a Recombinant Adenovirus Vector Expressing the Vβ8.2 TCR Is Disrupted by Coadministration with Vectors Expressing Either IL-4 or -10
Authors: Braciak Todd A. , Pedersen Brian , Chin Judy , Hsiao Clay , Ward E. Sally , Maricic Igor , Jahng Alex , Graham Frank L. , Gauldie Jack , Sercarz Eli E. , Kumar Vipin
Published: April 2014

Adenovirus vectors are increasingly being used for genetic vaccination and may prove highly suitable for intervention in different pathological conditions due to their capacity to generate high level, transient gene expression. In this study, we report the use of a recombinant adenovirus vector to induce regulatory responses for the prevention of autoimmune diseases through transient expression of a TCR β-chain. Immunization of B10.PL mice with a recombinant adenovirus expressing the TCR Vβ8.2 chain (Ad5E1 mVβ8.2), resulted in induction of regulatory type 1 CD4 T cells, directed against the framework region 3 determinant within the B5 peptide (aa 76–101) of the Vβ8.2 chain. This determinant is readily processed and displayed in an I-Au context, on ambient APC. Transient genetic delivery of the TCR Vβ8.2 chain protected mice from Ag-induced experimental autoimmune encephalomyelitis. However, when the Ad5E1 mVβ8.2 vector was coadministered with either an IL-4- or IL-10-expressing vector, regulation was disrupted and disease was exacerbated. These results highlight the importance of the Th1-like cytokine requirement necessary for the generation and activity of effective regulatory T cells in this model of experimental autoimmune encephalomyelitis.
Long Term Maintenance of IgE-Mediated Memory in Mast Cells in the Absence of Detectable Serum IgE
Authors: Kubo Shuichi , Nakayama Toshinori , Matsuoka Kunie , Yonekawa Hiromichi , Karasuyama Hajime
Published: April 2014

Mast cells and basophils involved in allergic responses do not have clonotypic Ag receptors. However, they can acquire Ag specificity through binding of Ag-specific IgE to FcεRI expressed on their surface. Previous studies demonstrated that IgE binding induced the stabilization and accumulation of FcεRI on the cell surface and resulted in up-regulation of FcεRI. In this study we have further analyzed the maintenance of IgE-mediated memory in mast cells and basophils in vivo by comparing kinetics of serum IgE levels, FcεRI expression, and ability to induce systemic anaphylaxis. A single i.v. injection of trinitrophenyl-specific IgE induced 8-fold up-regulation of FcεRI expression on peritoneal mast cells in B cell-deficient (μm−/−) mice. Serum IgE levels became undetectable by day 6, but the treatment of mice with anti-IgE mAb induced a significant drop in body temperature on days 14, 28, and 42. The administration of trinitrophenyl -BSA, but not BSA, in place of anti-IgE mAb gave similar results, indicating the Ag specificity of the allergic response. This long term maintenance of Ag-specific reactivity in the allergic response was also observed in normal mice passively sensitized with IgE even though the duration was shorter than that in B cell-deficient mice. The appearance of IgE with a different specificity did not interfere with the maintenance of IgE-mediated memory of mast cells and basophils. These results suggest that IgE-mediated stabilization and up-regulation of FcεRI enables mast cells and basophils not only to acquire Ag specificity, but also to maintain memory in vivo for lengthy periods of time.
Differential Requirements for Expression of CD80/86 and CD40 on B Cells for T-Dependent Antibody Responses In Vivo
Authors: Lumsden Joanne M. , Williams Joy A. , Hodes Richard J.
Published: April 2014

The CD80/86-CD28 and CD40-CD40 ligand costimulatory pathways are essential for Th cell-dependent B cell responses that generate high-affinity, class-switched Ab in vivo. Disruption of either costimulatory pathway results in defective in vivo humoral immune responses, but it remains unclear to what extent this is due to deficient activation of Th cells and/or of B cells. To address this issue, we generated mixed chimeras in which CD80/86- or CD40-deficient bone marrow-derived cells coexist with wild-type (WT) cells, thereby providing the functional T cell help and accessory cell functions required for fully competent B cell responses. We were then able to assess the requirement for CD80/86 or CD40 expression on B cells producing class-switched Ig in response to a T-dependent Ag. In CD80/86 WT plus CD80/86 double-knockout mixed chimeras, both WT- and CD80/86-deficient B cells produced IgG1 and IgE responses, indicating that direct signaling by CD80/86 is not essential for efficient B cell activation. In marked contrast, only WT IgG1 and IgE responses were detected in the chimeras containing CD40-deficient cells, demonstrating that CD40 expression on B cells is essential for class switching by those B cells. Thus, while disrupting either the CD80/86-CD28 or the CD40-CD40 ligand costimulatory pathway abrogates T-dependent B cell immune responses, the two pathways are nonredundant and mediated by distinct mechanisms.
Complement Component 3 Is Required for Optimal Expansion of CD8 T Cells During a Systemic Viral Infection
Authors: Suresh M. , Molina Hector , Salvato Maria S. , Mastellos Dimitrios , Lambris John D. , Sandor Matyas
Published: April 2014

In addition to its established role in innate immune mechanisms, complement component C3 is also of critical importance in B cell activation and T cell-dependent Ab responses. In this study, we have examined the requirement for C3 in the generation of primary CD8 T cell responses to an acute systemic viral infection. We compared Ag-specific CD8 T cell responses to lymphocytic choriomeningitis virus (LCMV) between wild-type (+/+) and C3-deficient (C3−/−) mice on both 129/B6 and B6 backgrounds. These studies revealed that C3 activity is required for optimal expansion of LCMV-specific effector CD8 T cells in an epitope-dependent fashion, which is influenced by the genetic background of the mice. Studies in complement receptor 1/2 (CR1/CR2)-deficient mice showed that regulation of LCMV-specific CD8 T cell responses by C3 is not dependent upon CR1/CR2. These findings may have implications in vaccine development, therapy of autoimmune diseases, and prevention of graft rejection.
Endotoxin/Lipopolysaccharide Activates NF-κB and Enhances Tumor Cell Adhesion and Invasion Through a β1 Integrin-Dependent Mechanism
Authors: Wang Jiang Huai , Manning Brian J. , Wu Qiong Di , Blankson Siobhan , Bouchier-Hayes D. , Redmond H. Paul
Published: April 2014

β1 integrins play a crucial role in supporting tumor cell attachment to and invasion into the extracellular matrix. Endotoxin/LPS introduced by surgery has been shown to enhance tumor metastasis in a murine model. Here we show the direct effect of LPS on tumor cell adhesion and invasion in extracellular matrix proteins through a β1 integrin-dependent pathway. The human colorectal tumor cell lines SW480 and SW620 constitutively expressed high levels of the β1 subunit, whereas various low levels of α1, α2, α4, and α6 expression were detected. SW480 and SW620 did not express membrane-bound CD14; however, LPS in the presence of soluble CD14 (sCD14) significantly up-regulated β1 integrin expression; enhanced tumor cell attachment to fibronectin, collagen I, and laminin; and strongly promoted tumor cell invasion through the Matrigel. Anti-β1 blocking mAbs (4B4 and 6S6) abrogated LPS- plus sCD14-induced tumor cell adhesion and invasion. Furthermore, LPS, when combined with sCD14, resulted in NF-κB activation in both SW480 and SW620 cells. Inhibition of the NF-κB pathway significantly attenuated LPS-induced up-regulation of β1 integrin expression and prevented tumor cell adhesion and invasion. These results provide direct evidence that although SW480 and SW620 cells do not express membrane-bound CD14, LPS in the presence of sCD14 can activate NF-κB, up-regulate β1 integrin expression, and subsequently promote tumor cell adhesion and invasion. Moreover, LPS-induced tumor cell attachment to and invasion through extracellular matrix proteins is β1 subunit-dependent.