bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional Heterogeneity of Cytokines and Cytolytic Effector Molecules in Human CD8+ T Lymphocytes
Authors: Sandberg Johan K. , Fast Noam M. , Nixon Douglas F.
Published: April 2014

CD8+ T cells use a number of effector mechanisms to protect the host against infection. We have studied human CD8+ T cells specific for CMV pp65495–503 epitope, or for staphylococcal enterotoxin B, for the expression patterns of five cytokines and cytolytic effector molecules before and after antigenic stimulation. Ex vivo, the cytolytic molecule granzyme B was detected in a majority of circulating CMV-specific CD8+ T cells, whereas perforin was rarely expressed. Both were highly expressed after Ag-specific activation accompanied by CD45RO up-regulation. TNF-α, IFN γ, and IL-2 were sequentially acquired on recognition of Ag, but surprisingly, only around half of the CMV-specific CD8+ T cells responded to antigenic stimuli with production of any cytokine measured. A dominant population coexpressed TNF-α and IFN-γ, and cells expressing TNF-α only, IFN-γ only, or all three cytokines together also occurred at lower but clearly detectable frequencies. Interestingly, perforin expression and production of IFN-γ and TNF-α in CD8+ T cells responding to staphylococcal enterotoxin B appeared to be largely segregated, and no IL-2 was detected in perforin-positive cells. Together, these data indicate that human CD8+ T cells can be functionally segregated in vivo and have implications for the understanding of human CD8+ T cell differentiation and specialization and regulation of effector mechanisms.
Partially Distinct Molecular Mechanisms Mediate Inhibitory FcγRIIB Signaling in Resting and Activated B Cells
Authors: Brauweiler Anne , Tamir Idan , Marschner Susanne , Helgason Cheryl D. , Cambier John C.
Published: April 2014

FcγRIIB functions as an inhibitory receptor to dampen B cell Ag receptor signals and immune responses. Accumulating evidence indicates that ex vivo B cells require the inositol 5-phosphatase, Src homology domain 2-containing inositol 5-phosphatase (SHIP), for FcγRIIB-mediated inhibitory signaling. However, we report here that LPS-activated primary B cells do not require SHIP and thus differ from resting B cells. SHIP-deficient B cell blasts display efficient FcγRIIB-dependent inhibition of calcium mobilization as well as Akt and extracellular signal-related protein kinase phosphorylation. Surprisingly, FcγRIIB-dependent degradation of phosphatidylinositol 3,4,5-trisphosphate and conversion into phosphatidylinositol 3,4-bisphosphate occur in SHIP-deficient B cell blasts, demonstrating the function of an additional inositol 5-phosphatase. Further analysis reveals that while resting cells express only SHIP, B cell blasts also express the recently described inositol 5-phosphatase, SHIP-2. Finally, data suggest that both SHIP-2 and SHIP can mediate downstream biologic consequences of FcγRIIB signaling, including inhibition of the proliferative response.
Memory T Cells Constitute a Subset of the Human CD8+CD45RA+ Pool with Distinct Phenotypic and Migratory Characteristics
Authors: Faint Jeffery M. , Annels Nicola E. , Curnow S. John , Shields Philip , Pilling Darrell , Hislop Andrew D. , Wu Lijun , Akbar Arne N. , Buckley Christopher D. , Moss Paul A. H. , Adams David H. , Rickinson Alan B. , Salmon Mike
Published: April 2014

Using HLA class I-viral epitope tetramers to monitor herpes virus-specific CD8+ T cell responses in humans, we have shown that a significant fraction of responding cells revert from a CD45RO+ to a CD45RA+ state after priming. All tetramer-binding CD45RA+ cells, regardless of epitope specificity, expressed a phenotype LFA-1highCCR7low that was stable for at least 10 years in infectious mononucleosis patients and indefinitely in asymptomatic carriers. CD8+CD45RA+LFA-1high cells were not present in cord blood but in adults account for up to 50% of CD8+CD45RA+ cells. These CD45RA+LFA-1high cells have significantly shorter telomeres than CD45RA+LFA-1low cells, suggesting that the latter represent a naive population, while the former are memory cells. CD45RA+ memory cells are a stable population of noncycling cells, but on stimulation they are potent producers of IFN-γ, while naive CD8+ cells produce only IL-2. The chemokine receptor profile and migratory potential of CD45RA+ memory cells is very similar to CD45RO+ cells but different to naive CD8 cells. In accord with this, CD45RA+ memory cells were significantly underrepresented in lymph nodes, but account for virtually all CD8+CD45RA+ T cells in peripheral tissues of the same individuals.
Down-Regulation of IL-12 p40 Gene in Plasmodium berghei-Infected Mice
Authors: Xu Xin , Sumita Kohsuke , Feng Chiguang , Xiong Xiaozhong , Shen Hua , Maruyama Saho , Kanoh Makoto , Asano Yoshihiro
Published: April 2014

We analyzed the mechanism that causes suppression of IL-12 p40 gene induction during Plasmodium berghei infection. Although IL-12 together with IFN-γ plays an important role in protection against pathogenic infection, the IL-12 p70 protein production of infected macrophages is lower than that by the uninfected macrophages. We showed in the present study that the induction of IL-12 p40 gene but not IL-12 p35 gene in macrophages of P. berghei-infected mice was profoundly inhibited. The inhibition was induced by interaction with macrophages that had contacted with P. berghei-infected erythrocytes and was mediated by a soluble factor, IL-10. There was comparable activation of NF-κB in uninfected and infected cells. The induction of IFN-regulatory factor-1 gene was comparable in transcription level in uninfected and infected cells, while the unidentified complex formation of IFN-regulatory factor-1 was observed in infected cells. Therefore, the inhibition of the IL-12 p40 gene induction appeared to be regulated at transcriptional regulation level of the gene.
IL-1 Induced Chemokine Production Through the Association of Syk with TNF Receptor-Associated Factor-6 in Nasal Fibroblast Lines
Authors: Yamada Takechiyo , Fujieda Shigeharu , Yanagi Shigeru , Yamamura Hirohei , Inatome Ryoko , Yamamoto Hideyuki , Igawa Hideki , Saito Hitoshi
Published: April 2014

The fibroblasts stimulated by cytokines released the chemokine and recruited the infiltrating cells, including eosinophils, that play a key role in the pathogenesis of airway disease. We established the human fibroblast lines showing high Syk expression and the lines showing low Syk expression from pieces of nasal polyp. IL-1 induces the interaction of TNFR-associated factor (TRAF) 6 with IL-1R-associated kinase, which is rapidly recruited to the IL-1R after IL-1 induction, whereas TRAF2 participates in TNF-α-signaling. In the present study, we found that Syk played a different role in IL-1- and TNF-α-induced chemokine production through a signaling complex involving Syk and TRAF6. Overexpression of wild-type Syk by gene transfer enhanced RANTES production from nasal fibroblasts stimulated with IL-1. The decrease of Syk expression by the administration of Syk antisense inhibited RANTES production in response to IL-1. However, the change of Syk expression did not affect RANTES production by TNF-α stimulation. We concluded that Syk is required for the IL-1-induced chemokine production through the association with TRAF-6 in fibroblasts of nasal polyps.
Structural Effects of Framework Mutations on a Humanized Anti-Lysozyme Antibody
Authors: Holmes Margaret A. , Buss Timothy N. , Foote Jefferson
Published: April 2014

A humanized version of the mouse anti-lysozyme Ab D1.3 was previously constructed as an Fv fragment and its structure was crystallographically determined in the free form and in complex with lysozyme. Here we report five new crystal structures of single-amino acid substitution mutants of the humanized Fv fragment, four of which were determined as Fv-lysozyme complexes. The crystals were isomorphous with the parent forms, and were refined to free R values of 28–31% at resolutions of 2.7–2.9 Å. Residue 27 in other Abs has been implicated in stabilizing the conformation of the first complementarity-determining region (CDR) of the H chain, residues 31–35. We find that a Phe-to-Ser mutation at 27 alters the conformation of immediately adjacent residues, but this change is only weakly transmitted to Ag binding residues in the nearby CDR. Residue 71 of the H chain has been proposed to control the relative disposition of H chain CDRs 1 and 2, based on the bulk of its side chain. However, in structures we determined with Val, Ala, or Arg substituted in place of Lys at position 71, no significant change in the conformation of CDRs 1 and 2 was observed.
The Smad3 Protein Is Involved in TGF-β Inhibition of Class II Transactivator and Class II MHC Expression
Authors: Dong Yuanshu , Tang Liping , Letterio John J. , Benveniste Etty N.
Published: April 2014

TGF-β is a immunoregulatory cytokine that inhibits class II MHC expression in a variety of cell types. Previous studies have shown that the class II MHC transactivator (CIITA), a master regulator that controls class II MHC expression, is targeted by TGF-β for repression of IFN-γ-induced class II MHC expression in astrocytes. The mechanism(s) underlying the TGF-β inhibitory effect is not understood. In this study, we demonstrate that TGF-β inhibition of CIITA expression occurs at the transcriptional level, and that both constitutive and IFN-γ-induced human CIITA type IV promoter activity is inhibited by TGF-β. TGF-β does not affect the signaling events that mediate IFN-γ activation of CIITA expression; i.e, TGF-β does not inhibit IFN-γ-induced STAT-1α phosphorylation and/or DNA binding ability, nor is IFN-γ induction of IFN regulatory factor affected. The inhibitory effect of TGF-β on the type IV CIITA promoter is mediated through a promoter region within 80 bp from the transcription start site. Elimination of TGF-β inhibition of class II MHC and CIITA expression in Smad3-deficient astrocytes, as well as restoration of the inhibitory effect by overexpression of the Smad3 protein, demonstrates that Smad3 is essential in mediating TGF-β inhibition of CIITA and class II MHC expression.
Induction of Germline Transcription in the Human TCRγ Locus by STAT5
Authors: Lee Hai-Chon , Ye Sang-Kyu , Honjo Tasuku , Ikuta Koichi
Published: April 2014

TCR and Ig genes are assembled by V(D)J recombination during lymphocyte development. The enhancer and the germline promoter control the accessibility of each locus for the common recombinase activity. In the mouse TCRγ locus, STAT5 proteins activated by the IL-7R interact with consensus motifs in 5′ regions of Jγ segments and induce germline transcription. To evaluate the role of STAT5 in controlling the accessibility of the TCRγ locus, we characterized the germline transcription of human TCRγ genes and compared it with mouse. We first demonstrated that Jγ-Cγ germline transcripts are induced in a cytokine-dependent human erythroleukemia cell line. STAT consensus motifs are present in 5′ regions of Jγ1.1 and Jγ2.1 gene segments, and activated STAT5 binds to these motifs. By using a reporter assay, we showed that the Jγ1.1 germline promoter is transactivated by STAT5 and that mutations in any of the two STAT motifs abrogate this activity. Thus, this study demonstrates that STAT5 induces germline transcription in the TCRγ locus of both mouse and human and suggests the possibility that this mechanism may play an essential role in controlling the TCRγ locus accessibility. In addition, STAT motifs are conserved among 5′ Jγ germline promoters, 3′ enhancers, and a locus control region-like element, HsA, in both mouse and human TCRγ loci, indicating the possibility that IL-7R/STAT5 signaling probably controls the locus-wide accessibility through these elements.
Decreased Frequency of Somatic Hypermutation and Impaired Affinity Maturation but Intact Germinal Center Formation in Mice Expressing Antisense RNA to DNA Polymerase ζ
Authors: Diaz Marilyn , Verkoczy Laurent K. , Flajnik Martin F. , R. Klinman Norman
Published: April 2014

To examine a role of DNA polymerase ζ in somatic hypermutation, we generated transgenic mice that express antisense RNA to a portion of mouse REV3, the gene encoding this polymerase. These mice express high levels of antisense RNA, significantly reducing the levels of endogenous mouse REV3 transcript. Following immunization to a hapten-protein complex, transgenic mice mounted vigorous Ab responses, accomplished the switch to IgG, and formed numerous germinal centers. However, in most transgenic animals, the generation of high affinity Abs was delayed. In addition, accumulation of somatic mutations in the VH genes of memory B cells from transgenic mice was decreased, particularly among those that generate amino acid replacements that enhance affinity of the B cell receptor to the hapten. These data implicate DNA polymerase ζ, a nonreplicative polymerase, in the process of affinity maturation, possibly through a role in somatic hypermutation, clonal selection, or both.
Unstimulated Human CD4 Lymphocytes Express a Cytoplasmic Immature Form of the Common Cytokine Receptor γ-Chain
Authors: Bani Lynda , Pasquier Virginie , Kryworuchko Marko , Salamero Jean , Thèze Jacques
Published: April 2014

As a component of various cytokine receptors, common cytokine receptor γ-chain (γc) is essential in the development of the immune system and plays an important role in different stages of inflammatory and immune responses. Here we establish that resting CD4 T cells and the Jurkat CD4 T cell line do not express the mature form of γc (64 kDa) recognized by mAb Tugh4. However, these cells constitutively transcribe the corresponding γc gene. This apparent paradox was solved by the demonstration that polyclonal anti-γc Abs detected endoglycosidase-H-sensitive immature forms of γc (54–58 kDa) expressed by quiescent CD4 T lymphocytes and Jurkat cells. Immature γc is characterized as an intracellular component localized in the endoplasmic reticulum. Pulse-chase analysis shows that the immature γc is rapidly degraded after synthesis. After activation of CD4 T lymphocytes, and as seen in the CD4 T cell line Kit 225, the endoglycosidase-H-resistant mature form of γc is detectable at the cell surface and in the endosomal compartment. For the first time, our results demonstrate that a cytokine receptor chain may be constitutively produced as an immature form. Furthermore, this supports the notion that expression of the functional form of γc may require intracellular interactions with lineage- or subset-specific molecular partners.
A Distinct Pathway of Cell-Mediated Apoptosis Initiated by Granulysin
Authors: Kaspar Allan A. , Okada Satoshi , Kumar Jayant , Poulain Francis R. , Drouvalakis Katerina A. , Kelekar Ameeta , Hanson Dennis A. , Kluck Ruth M. , Hitoshi Yasumichi , Johnson Daniel E. , Froelich Christopher J. , Thompson Craig B. , Newmeyer Donald D. , Anel Alberto , Clayberger Carol , Krensky Alan M.
Published: April 2014

Granulysin is an antimicrobial and tumoricidal molecule expressed in granules of CTL and NK cells. In this study, we show that granulysin damages cell membranes based upon negative charge, disrupts the transmembrane potential (Δψ) in mitochondria, and causes release of cytochrome c. Granulysin-induced apoptosis is blocked in cells overexpressing Bcl-2. Despite the release of cytochrome c, procaspase 9 is not processed. Nevertheless, activation of caspase 3 is observed in granulysin-treated cells, suggesting that granulysin activates a novel pathway of CTL- and NK cell-mediated death distinct from granzyme- and death receptor-induced apoptosis.
Protection Against Murine Listeriosis by Oral Vaccination with Recombinant Salmonella Expressing Hybrid Yersinia Type III Proteins
Authors: Rüssmann Holger , Igwe Emeka I. , Sauer Jeannette , Hardt Wolf-Dietrich , Bubert Andreas , Geginat Gernot
Published: April 2014

In the present study, we have investigated the possibility to engage the Yersinia outer protein E (YopE) as a carrier molecule for heterologous Ag delivery by the type III secretion system of Salmonella typhimurium. Defined secretion and translocation domains of YopE were fused to the immunodominant T cell Ags listeriolysin O and p60 of Listeria monocytogenes. In vitro experiments showed that S. typhimurium allows secretion and translocation of large hybrid YopE proteins in a type III-dependent fashion. Translocation and cytosolic delivery of these chimeric proteins into host cells, but not secretion into endosomal compartments, led to efficient MHC class I-restricted Ag presentation of listerial nonamer peptides. Mice orally vaccinated with a single dose of attenuated S. typhimurium expressing translocated hybrid YopE proteins revealed high numbers of IFN-γ-producing cells reactive with listeriolysin O 91–99 or p60 217–225, respectively. This CD8 T cell response protected mice against a challenge with L. monocytogenes. In conclusion, these findings suggest that YopE is a versatile carrier molecule for type III-mediated foreign Ag delivery by Salmonella vaccine strains.
Structural Features of Nonpeptide Prenyl Pyrophosphates That Determine Their Antigenicity for Human γδ T Cells
Authors: Morita Craig T. , Lee Hoi K. , Wang Hong , Li Hongmin , Mariuzza Roy A. , Tanaka Yoshimasa
Published: April 2014

Human Vγ2Vδ2+ T cells proliferate in vivo during many microbial infections. We have found that Vγ2Vδ2+ T cells recognize nonpeptide prenyl pyrophosphates and alkylamines. We now have defined structural features that determine the antigenicity of prenyl pyrophosphates by testing synthetic analogs for bioactivity. We find that the carbon chain closest to the pyrophosphate moiety plays the major role in determining bioactivity. Changes in this area, such as the loss of a double bond, abrogated bioactivity. The loss of a phosphate from the pyrophosphate moiety also decreased antigenicity 100- to 200-fold. However, nucleotide monophosphates could be added with minimal changes in bioactivity. Longer prenyl pyrophosphates also retained bioactivity. Despite differences in CDR3 sequence, Vγ2Vδ2+ clones and a transfectant responded similarly. Ag docking into a Vγ2Vδ2 TCR model reveals a potential binding site in germline regions of the Vγ2Jγ1.2 CDR3 and Vδ2 CDR2 loops. Thus, Vγ2Vδ2+ T cells recognize a core carbon chain and pyrophosphate moiety. This recognition is relatively unaffected by additions at distal positions to the core Ag unit.
IL-8 Production in Human Lung Fibroblasts and Epithelial Cells Activated by the Pseudomonas Autoinducer N-3-Oxododecanoyl Homoserine Lactone Is Transcriptionally Regulated by NF-κB and Activator Protein-2
Authors: Smith Roger S. , Fedyk Eric R. , Springer T. A. , Mukaida N. , Iglewski Barbara H. , Phipps Richard P.
Published: April 2014

The destructive pulmonary inflammation associated with Pseudomonas aeruginosa colonization is caused, in part, by the production of the chemokine IL-8, which recruits neutrophils into the lung. The Pseudomonas autoinducer, N-3-oxododecanoyl homoserine lactone (3-O-C12-HSL), is a small lipid-soluble molecule that is essential in the regulation of many P. aeruginosa virulence factors, but little is known about how it affects eukaryotic cells. In this report we demonstrate that 3-O-C12-HSL is a potent stimulator of both IL-8 mRNA and protein from human fibroblasts and epithelial cells in vitro. The IL-8 produced from these 3-O-C12-HSL-stimulated cells was found to be functionally active by inducing the chemotaxis of neutrophils. To determine a mechanism for this IL-8 induction, deletion constructs of the IL-8 promoter were examined. It was found that the DNA region between nucleotides −1481 and −546 and the transcription factor NF-κB were essential for the maximal induction of IL-8 by 3-O-C12-HSL. This was confirmed by EMSAs, where 3-O-C12-HSL induced a shift with both AP-2 and NF-κB consensus DNA. The activation of NF-κB and subsequent production of IL-8 were found to be regulated by a mitogen-activated protein kinase pathway. These findings support the concept that the severe lung damage that accompanies P. aeruginosa infections is caused by an exuberant neutrophil response stimulated by 3-O-C12-HSL-induced IL-8. Understanding the mechanisms of 3-O-C12-HSL activation of lung structural cells may provide a means to help control lung damage during infections with P. aeruginosa.
IFN-γ-Independent Autocrine Cytokine Regulatory Mechanism in Reprogramming of Macrophage Responses to Bacterial Lipopolysaccharide
Authors: Brewington Ryan , Chatterji Manjil , Zoubine Mikhail , Miranda Roberto N. , Norimatsu Mari , Shnyra Alex
Published: April 2014

Macrophages are now well recognized to have a critical role in both innate and acquired immunity. The sentinel macrophage function is highly regulated and serves to allow for intrinsic plasticity of the innate immune responses to potential environmental signals. However, the mechanisms underlying the dynamic properties of the cellular arm of innate immunity are poorly understood. Therefore, we have conducted a series of in vitro studies to evaluate the contribution of immunoregulatory cytokines, such as IFN-γ, IL-10, and IL-12, in modulation of macrophage responses. We found that macrophages from IFN-γ knockout (IFN-γ−/−) mice exhibit only marginal LPS-induced TNF-α, IL-12, and NO responses, all of which can be fully restored in the presence of rIFN-γ. Pretreatment with substimulatory LPS concentrations led to reprogramming of IFN-γ−/− macrophage responses in a dose-dependent manner that manifested by an increased TNF-α and IL-12, but not NO, production upon the subsequent LPS challenge. These reprogramming effects were substantially attenuated and profoundly enhanced in macrophages from IL-12−/− and IL-10−/− mice, respectively, as compared with those modulated in macrophages from the congenic wild-type mice. LPS-dependent reprogramming was also fully reproduced in macrophages isolated from SCID mice after immunodepletion of NK cells. Our data strongly imply that cytokine (TNF-α and IL-12), but not NO, responses in macrophages may, at least in part, be governed by an autocrine IFN-γ-independent regulatory mechanism reciprocally controlled by IL-10 and IL-12. This mechanism may serve as an alternative/coherent pathway to the canonical IFN-γ-dependent induction of antimicrobial and tumoricidal activity in macrophages.
Impaired Mucosal Immunity in L-Selectin-Deficient Mice Orally Immunized with aSalmonellaVaccine Vector
Authors: Pascual David W. , White Michelle D. , Larson Trina , Walters Nancy
Published: April 2014

Lymphocyte trafficking in the gastrointestinal tract is primarily mediated by interactions with the mucosal addressin cell adhesion molecule 1 and its lymphocyte ligand, α4β7, and partly by L-selectin (L-Sel) interactions with peripheral node addressin coexpressed on some mucosal addressin cell adhesion molecule 1. We inquired whether intestinal responses in mice lacking L-Sel would be enhanced. L-Sel-deficient (L-Sel−/−) mice were orally immunized with either Salmonella vaccine vector or Salmonella vector-expressing colonization factor Ag I (CFA/I) from enterotoxigenic Escherichia coli. In L-Sel−/− mice, mucosal IgA anti-CFA/I fimbrial responses were greatly reduced, and systemic IgG2a anti-CFA/I fimbrial responses were 26-fold greater compared with C57BL/6 (L-Sel+/+) mice. L-Sel−/− Peyer’s patch (PP) CD4+ Th cells revealed IFN-γ-dominated responses and an unprecedented absence of IL-4, whereas the expected mixed Th cell phenotype developed in L-Sel+/+ mice. PP CD4+ Th cell anti-Salmonella responses were nearly nonexistent in L-Sel−/− mice immunized with either Salmonella vaccine. Splenic CD4+ Th cell anti-Salmonella responses were reduced but did show cytokine production in Ag restimulation assays. Increased colonization of PP and spleen was noted only with the Salmonella vector in L-Sel−/− mice, resulting in increased splenomegaly, suggesting that the Salmonella-CFA/I vaccine was not as infectious or that the presence of the fimbriae improved clearance, possibly because of reduced neutrophil recruitment. However, sufficient anti-Salmonella immunity was induced, because Salmonella vector-immunized L-Sel−/− mice showed complete protection against wild-type Salmonella challenge, unlike L-Sel+/+ mice. This evidence shows that L-Sel is important for development of mucosal immunity, and absence of L-Sel is protective against salmonellosis.
Activation of Toll-Like Receptor-2 by Glycosylphosphatidylinositol Anchors from a Protozoan Parasite
Authors: Campos Marco A. S. , Almeida Igor C. , Takeuchi Osamu , Akira Shizuo , Valente Eneida P. , Procópio Daniela O. , Travassos Luiz R. , Smith Jason A. , Golenbock Douglas T. , Gazzinelli Ricardo T.
Published: April 2014

Glycosylphosphatidylinositol (GPI) anchors and glycoinositolphospholipids (GIPLs) from parasitic protozoa have been shown to exert a wide variety of effects on cells of the host innate immune system. However, the receptor(s) that are triggered by these protozoan glycolipids has not been identified. Here we present evidence that Trypanosoma cruzi-derived GPI anchors and GIPLs trigger CD25 expression on Chinese hamster ovary-K1 cells transfected with CD14 and Toll-like receptor-2 (TLR-2), but not wild-type (TLR-2-deficient) Chinese hamster ovary cells. The protozoan-derived GPI anchors and GIPLs containing alkylacylglycerol and saturated fatty acid chains or ceramide were found to be active in a concentration range of 100 nM to 1 μM. More importantly, the GPI anchors purified from T. cruzi trypomastigotes, which contain a longer glycan core and unsaturated fatty acids in the sn-2 position of the alkylacylglycerolipid component, triggered TLR-2 at subnanomolar concentrations. We performed experiments with macrophages from TLR-2 knockout and TLR-4 knockout mice, and found that TLR-2 expression appears to be essential for induction of IL-12, TNF-α, and NO by GPI anchors derived from T. cruzi trypomastigotes. Thus, highly purified GPI anchors from T. cruzi parasites are potent activators of TLR-2 from both mouse and human origin. The activation of TLR-2 may initiate host innate defense mechanisms and inflammatory response during protozoan infection, and may provide new strategies for immune intervention during protozoan infections.
Expression of Active Protein Kinase B in T Cells Perturbs Both T and B Cell Homeostasis and Promotes Inflammation
Authors: Parsons Michael J. , Jones Russell G. , Tsao Ming-Sound , Odermatt Bernard , Ohashi Pamela S. , Woodgett James R.
Published: April 2014

The molecular mechanisms that contribute to autoimmunity remain poorly defined. While inflammation is considered to be one of the major checkpoints in autoimmune disease progression, very little is known about the initiating events that trigger inflammation. We have studied transgenic mice expressing the prosurvival molecule protein kinase B/Akt under control of a T cell-specific CD2 promoter. In this study, we demonstrate that aged mice develop lymphadenopathy and splenomegaly that result from an accumulation of CD4, CD8, and unexpectedly B cells. An increased proportion of T cells express activation markers, while T cell proliferative responses remain normal. B cells are hyperproliferative in response to anti-IgM F(ab′)2 and anti-CD40, and increased IgA and IgG2a were found in the sera. In addition, a profound multiorgan lymphocytic infiltration is observed, and T cells from these mice display a defect in Fas-mediated apoptosis, which may be the mechanism underlying this phenotype. Therefore, T cell expression of active protein kinase B can alter T cell homeostasis, indirectly influence B cell homeostasis, and promote inflammation in vivo.
Role of Effector Cell-Derived IL-4, IL-5, and Perforin in Early and Late Stages of Type 2 CD8 Effector Cell-Mediated Tumor Rejection
Authors: Dobrzanski Mark J. , Reome Joyce B. , Dutton Richard W.
Published: April 2014

Type 2 CD8 T cells (Tc2) secrete IL-4 and IL-5 and display perforin-dependent cytolysis in vitro. Using an OVA-transfected B16-melanoma model, we show that tumor-reactive Tc2 effector cells accumulated at the tumor site and induced tumor regression that enhanced survival in mice with pulmonary tumors. Transfer of perforin-deficient Tc2 cells generated from perforin gene knockout mice showed no differences in therapeutic efficiency when compared with wild-type Tc2 cells. In contrast, Tc2 cells derived from select cytokine gene-deficient mice showed that therapeutic effects were dependent on effector cell-derived IL-4 and IL-5 that led to a local elevation in lung-derived chemoattractants and accumulation of activated host-derived CD8/CD44high, CD4/CD44high, and OVA-specific tetramer-positive CD8 cells in vivo. Host-derived T and non-T immune cells increased in the lung over time and correlated with an elevated production of type 1-related chemokines. Conversely, donor Tc2 cell numbers markedly diminished at later times, suggesting that prolonged therapeutic responses were due to host-derived mechanisms. Moreover, type 1 host responses were detectable with increased levels of IFN-γ production by lung-derived CD4 and CD8 T cells from surviving Tc2-treated mice. Transfer of Tc2 cells into IFN-γ-deficient tumor-bearing mice was markedly less effective then into wild-type mice, suggesting that host-derived IFN-γ-dependent mechanisms play a role in Tc2-mediated antitumor responses.
Induction of Apoptosis in Human T Cells by Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin Is a Consequence of G2 Arrest of the Cell Cycle
Authors: Shenker Bruce J. , Hoffmaster Roselle H. , Zekavat Ali , Yamaguchi Noboru , Lally Edward T. , Demuth Donald R.
Published: April 2014

We have previously shown that Actinobacillus actinomycetemcomitans produces an immunosuppressive factor that is encoded by the cdtB gene, which is homologous to a family of cytolethal distending toxins (Cdt) expressed by several Gram-negative bacteria. Moreover, we have shown that CdtB impairs lymphocyte function by inducing G2 arrest of the cell cycle. We now report that both CdtB as well as an extract prepared from an Escherichia coli strain that expresses all three of the A. actinomycetemcomitans cdt genes (rCdtABC) induce apoptosis. Pretreatment of lymphocytes with either CdtB or rCdtABC leads to DNA fragmentation in activated lymphocytes at 72 and 96 h. No DNA fragmentation was induced in nonactivated cells. Flow cytometric analysis of the Cdt-treated lymphocytes demonstrates a reduction in cell size and an increase in nuclear condensation. Mitochondrial function was also perturbed in cells pretreated with either CdtB or rCdtABC. An increase in the expression of the mitochondria Ag, Apo 2.7, was observed along with evidence of the development of a mitochondrial permeability transition state; this includes a decrease in the transmembrane potential and elevated generation of reactive oxygen species. Activation of the caspase cascade, which is an important biochemical feature of the apoptotic process, was also observed in Cdt-treated lymphocytes. Overexpression of the bcl-2 gene in the human B lymphoblastoid cell line, JY, led to a decrease in Cdt-induced apoptosis. Interestingly, Bcl-2 overexpression did not block Cdt-induced G2 arrest. The implications of our results with respect to the immunosuppressive functions of Cdt proteins are discussed.
Role of Mitogen-Activated Protein Kinase-Mediated Cytosolic Phospholipase A2 Activation in Arachidonic Acid Metabolism in Human Eosinophils
Authors: Zhu Xiangdong , Sano Hiroyuki , Kim Kwang Pyo , Sano Akiko , Boetticher Evan , Muñoz Nilda M. , Cho Wonhwa , Leff Alan R.
Published: April 2014

The objective of this investigation was to determine the role of secretory and cytosolic isoforms of phospholipase A2 (PLA2) in the induction of arachidonic acid (AA) and leukotriene synthesis in human eosinophils and the mechanism of PLA2 activation by mitogen-activated protein kinase (MAPK) isoforms in this process. Pharmacological activation of eosinophils with fMLP caused increased AA release in a concentration (EC50 = 8.5 nM)- and time-dependent (t1/2 = 3.5 min) manner. Both fMLP-induced AA release and leukotriene C4 (LTC4) secretion were inhibited concentration dependently by arachidonic trifluoromethyl ketone, a cytosolic PLA2 (cPLA2) inhibitor; however, inhibition of neither the 14-kDa secretory phospholipase A2 by 3-(3-acetamide-1-benzyl-2-ethylindolyl-5-oxy)propanephosphonic acid nor cytosolic Ca2+-independent phospholipase A2 inhibition by bromoenol lactone blocked hydrolysis of AA or subsequent leukotriene synthesis. Pretreatment of eosinophils with a mitogen-activated protein/extracellular signal-regulated protein kinase (ERK) kinase inhibitor, U0126, or a p38 MAPK inhibitor, SB203580, suppressed both AA production and LTC4 release. fMLP induced phosphorylation of MAPK isoforms, ERK1/2 and p38, which were evident after 30 s, maximal at 1–5 min, and declined thereafter. fMLP stimulation also increased cPLA2 activity in eosinophils, which was inhibited completely by 30 μM arachidonic trifluoromethyl ketone. Preincubation of eosinophils with U0126 or SB203580 blocked fMLP-enhanced cPLA2 activity. Furthermore, inhibition of Ras, an upstream GTP-binding protein of ERK, also suppressed fMLP-stimulated AA release. These findings demonstrate that cPLA2 activation causes AA hydrolysis and LTC4 secretion. We also find that cPLA2 activation caused by fMLP occurs subsequent to and is dependent upon ERK1/2 and p38 MAPK activation. Other PLA2 isoforms native to human eosinophils possess no significant activity in the stimulated production of AA or LTC4.
Diphosphoryl Lipid A from Rhodobacter sphaeroides Blocks the Binding and Internalization of Lipopolysaccharide in RAW 264.7 Cells
Authors: Kutuzova Galina D. , Albrecht Ralph M. , Erickson Chris M. , Qureshi Nilofer
Published: April 2014

Diphosphoryl lipid A derived from the nontoxic LPS of Rhodobacter sphaeroides (RsDPLA) has been shown to be a powerful LPS antagonist in both human and murine cell lines. In addition, RsDPLA also can protect mice against the lethal effects of toxic LPS. In this study, we complexed both the deep rough LPS from Escherichia coli D31 m4 (ReLPS) and RsDPLA with 5- and 30-nm colloidal gold and compared their binding to the RAW 264.7 cell line by electron microscopy. Both ReLPS and RsDPLA bound to the cells with the following observations. First, binding studies revealed that pretreatment with RsDPLA completely blocked the binding and thus internalization of ReLPS-gold conjugates to these cells at both 37°C and 4°C. Second, ReLPS was internalized via micropinocytosis (noncoated plasma membrane invaginations) involving formation of caveolae-like structures and leading to the formation of micropinocytotic vesicles, macropinocytosis (or phagocytosis), formation of clathrin-coated pits (receptor mediated), and penetration through plasma membrane into cytoplasm. Third, in contrast, RsDPLA was internalized predominantly via macropinocytosis. These studies show for the first time that RsDPLA blocks the binding and thus internalization of LPS as observed by scanning and transmission electron microscopy.
Gammalinolenic Acid, an Unsaturated Fatty Acid with Anti-Inflammatory Properties, Blocks Amplification of IL-1β Production by Human Monocytes
Authors: Furse Robert K. , Rossetti Ronald G. , Zurier Robert B.
Published: April 2014

Administration of gammalinolenic acid (GLA), an unsaturated fatty acid, reduces joint inflammation in patients with rheumatoid arthritis. Addition of GLA in vitro suppresses release of IL-1β from human monocytes stimulated with LPS. LPS-induced IL-1β release is followed by IL-1-induced IL-1β release, an amplification process termed autoinduction. We show here with peripheral blood monocytes from normal volunteers and from patients with rheumatoid arthritis by using IL-1R antagonist to block autoinduction and IL-1α stimulation to simulate autoinduction that ∼40% of IL-1β released from LPS-stimulated cells is attributable to autoinduction and that GLA reduces autoinduction of IL-1β while leaving the initial IL-1β response to LPS intact. Experiments with cells in which transcription and protein synthesis were blocked suggest that GLA induces a protein that reduces pro-IL-1β mRNA stability. IL-1β is important to host defense, but the amplification mechanism may be excessive in genetically predisposed patients. Thus, reduction of IL-1β autoinduction may be protective in some patients with endotoxic shock and with diseases characterized by chronic inflammation.
Both E6 and E7 Oncoproteins of Human Papillomavirus 16 Inhibit IL-18-Induced IFN-γ Production in Human Peripheral Blood Mononuclear and NK Cells
Authors: Lee Shin-Je , Cho Young-Sik , Cho Min-Chul , Shim Jung-Hyun , Lee Kyung-Ae , Ko Kwang-Kjune , Choe Yong Kyung , Park Sue-Nie , Hoshino Tomoaki , Kim SooHyun , Dinarello Charles A. , Yoon Do-Young
Published: April 2014

Cervical carcinoma is the predominant cancer among malignancies in women throughout the world, and human papillomavirus (HPV) 16 is the most common agent linked to human cervical carcinoma. The present study was performed to investigate the mechanisms of immune escape in HPV-induced cervical cancer cells. The presence of HPV oncoproteins E6 and E7 in the extracellular fluids of HPV-containing cervical cancer cell lines SiHa and CaSki was demonstrated by ELISA. The effect of HPV 16 oncoproteins E6 and E7 on the production of IFN-γ by IL-18 was assessed. E6 and E7 proteins reduced IL-18-induced IFN-γ production in both primary PBMCs and the NK0 cell line. FACS analysis revealed that the viral oncoproteins reduced the binding of IL-18 to its cellular surface receptors on NK0 cells, whereas there was no effect of oncoproteins on IL-1 binding to its surface IL-1 receptors on D10S, a subclone of the murine Th cell D10.G4.1. In vitro pull-down assays also revealed that the viral oncoproteins and IL-18 bound to IL-18R α-chain competitively. These results suggest that the extracellular HPV 16 E6 and E7 proteins may inhibit IL-18-induced IFN-γ production locally in HPV lesions through inhibition of IL-18 binding to its α-chain receptor. Down-modulation of IL-18-induced immune responses by HPV oncoproteins may contribute to viral pathogenesis or carcinogenesis.
Estrogen Treatment Down-Regulates TNF-α Production and Reduces the Severity of Experimental Autoimmune Encephalomyelitis in Cytokine Knockout Mice
Authors: Ito Atsushi , Bebo Bruce F. , Matejuk Agata , Zamora Alex , Silverman Marc , Fyfe-Johnson Amber , Offner Halina
Published: April 2014

A shift toward Th2 cytokine production has been demonstrated during pregnancy and high dose estrogen therapy and is thought to be the primary mechanism by which estrogen suppresses the development of experimental autoimmune encephalomyelitis. However, low dose estrogen treatment is equally protective in the absence of a significant shift in cytokine production. In this study cytokine-deficient mice were treated with estrogen to determine whether a shift in Th2 cytokine production was required for the protective effects of hormone therapy. Estrogen effectively suppressed the development of experimental autoimmune encephalomyelitis in IL-4 and IL-10 knockout mice and in wild type littermate mice with a similar potency of protection. Significant disease suppression was also seen in IFN-γ-deficient mice. The decrease in disease severity was accompanied by a concomitant reduction in the number of proinflammatory cytokine- and chemokine-producing cells in the CNS. Although there was no apparent increase in compensatory Th2 cytokine production in cytokine-deficient mice, there was a profound decrease in the frequency of TNF-α-producing cells in the CNS and the periphery. Therefore, we propose that one mechanism by which estrogen protects females from the development of cell-mediated autoimmunity is through a hormone-dependent regulation of TNF-α production.
Suppression of IL-2-Induced T Cell Proliferation and Phosphorylation of STAT3 and STAT5 by Tumor-Derived TGFβ Is Reversed by IL-15
Authors: Campbell John D. M. , Cook Gordon , Robertson Susan E. , Fraser Alasdair , Boyd Kelly S. , Gracie J. Alastair , Franklin Ian M.
Published: April 2014

IL-2 responses are susceptible to suppression by TGFβ, a cytokine widely implicated in suppression of inflammatory responses and secreted by many different tumor cell types. There have been conflicting reports regarding inhibition of IL-2-induced STAT3 and STAT5 phosphorylation by TGFβ and subsequent suppression of immune responses. Using TGFβ-producing multiple myeloma tumor cells we demonstrate that tumor-derived TGFβ can block IL-2-induced proliferation and STAT3 and STAT5 phosphorylation in T cells. High affinity IL-2R expression was required for the suppression of IL-2 responses as a novel CD25− T cell line proliferated and phosphorylated STAT3 when cultured with tumor cells or rTGFβ1. Activating T cells with IL-15, which does not use the high affinity IL-2R, completely restored the ability of T cells to phosphorylate STAT3 and STAT5 when cultured with tumor cells. IL-15-treated T cells proliferated normally when cocultured with tumor cells or rTGFβ1, whereas IL-2 responses were consistently inhibited. Preincubation with IL-15 also restored the ability of T cells to respond to IL-2 by phosphorylating STAT3 and STAT5, and proliferating normally in the presence of tumor cells. IL-2 pretreatment did not restore T cell function. IL-15 also restored T cell responses by T cells from multiple myeloma patients, and against freshly isolated bone marrow tumor samples. Thus, activation of T cells by IL-15 renders T cells resistant to suppression by TGFβ1-producing tumor cells and rTGFβ1. This finding may be exploited in the design of new immunotherapy approaches that will rely on T cells avoiding tumor-induced suppression.
Plasmid DNAs Encoding Insulin and Glutamic Acid Decarboxylase 65 Have Distinct Effects on the Progression of Autoimmune Diabetes in Nonobese Diabetic Mice
Authors: Weaver Donald J. , Liu Bo , Tisch Roland
Published: April 2014

We previously demonstrated that administration of plasmid DNAs (pDNAs) encoding IL-4 and a fragment of glutamic acid decarboxylase 65 (GAD65) fused to IgGFc induces GAD65-specific Th2 cells and prevents insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice. To assess the general applicability of pDNA vaccination to mediate Ag-specific immune deviation, we examined the immunotherapeutic efficacy of recombinants encoding murine insulin A and B chains fused to IgGFc. Insulin was chosen based on studies demonstrating that administration of insulin or insulin B chain by a variety of strategies prevents IDDM in NOD mice. Surprisingly, young NOD mice receiving i.m. injections of pDNA encoding insulin B chain-IgGFc with or without IL-4 exhibited an accelerated progression of insulitis and developed early diabetes. Exacerbation of IDDM correlated with an increased frequency of IFN-γ-secreting CD4+ and CD8+ T cells in response to insulin B chain-specific peptides compared with untreated mice. In contrast, treatment with pDNAs encoding insulin A chain-IgGFc and IL-4 elicited a low frequency of IL-4-secreting Th cells and had no effect on the progression of IDDM. Vaccination with pDNAs encoding GAD65-IgGFc and IL-4, however, prevented IDDM. These results demonstrate that insulin- and GAD65-specific T cell reactivity induced by pDNA vaccination has distinct effects on the progression of IDDM.
Fas Aggregation Does Not Correlate with Fas-Mediated Apoptosis
Authors: Lee Yang-ja , Shacter Emily
Published: April 2014

Cross-linking of cell surface Fas molecules by Fas ligand or by agonistic anti-Fas Abs induces cell death by apoptosis. We found that a serine protease inhibitor, N-tosyl-l-lysine chloromethyl ketone (TLCK), dramatically enhances Fas-mediated apoptosis in the human T cell line Jurkat and in various B cell lines resistant to Fas-mediated apoptosis. The enhancing effect of TLCK is specific to Fas-induced cell death, with no effect seen on TNF-α or TNF-related apoptosis-inducing ligand-induced apoptosis. TLCK treatment had no effect on Fas expression levels on the cell surface, and neither promoted death-inducing signaling complex formation nor decreased expression levels of cellular inhibitors of apoptosis (FLICE inhibitory protein, X chromosome-linked inhibitor of apoptosis, and Bcl-2). Activation of the Fas-mediated apoptotic pathway by anti-Fas Ab is accompanied by aggregation of Fas molecules to form oligomers that are stable to boiling in SDS and β-ME. Fas aggregation is often considered to be required for Fas-mediated apoptosis. However, sensitization of cells to Fas-mediated apoptosis by TLCK or other agents (cycloheximide, protein kinase C inhibitors) causes less Fas aggregation during the apoptotic process compared with that in nonsensitized cells. These results show that Fas aggregation and Fas-mediated apoptosis are not directly correlated and may even be inversely correlated.
IL-1 Enhances T Cell-Dependent Antibody Production Through Induction of CD40 Ligand and OX40 on T Cells
Authors: Nakae Susumu , Asano Masahide , Horai Reiko , Sakaguchi Nobuo , Iwakura Yoichiro
Published: April 2014

IL-1 is a proinflammatory cytokine that plays pleiotropic roles in host defense mechanisms. We investigated the role of IL-1 in the humoral immune response using gene-targeted mice. Ab production against SRBC was significantly reduced in IL-1α/β-deficient (IL-1−/−) mice and enhanced in IL-1R antagonist−/− mice. The intrinsic functions of T, B, and APCs were normal in IL-1−/− mice. However, we showed that IL-1−/− APCs did not fully activate DO11.10 T cells, while IL-1R antagonist −/− APCs enhanced the reaction, indicating that IL-1 promotes T cell priming through T-APC interaction. The function of IL-1 was CD28-CD80/CD86 independent. We found that CD40 ligand and OX40 expression on T cells was affected by the mutation, and the reduced Ag-specific B cell response in IL-1−/− mice was recovered by the treatment with agonistic anti-CD40 mAb both in vitro and in vivo. These observations indicate that IL-1 enhances T cell-dependent Ab production by augmenting CD40 ligand and OX40 expression on T cells.
Cutting Edge: Leukocyte Receptor Complex-Encoded Immunomodulatory Receptors Show Differing Specificity for Alternative HLA-B27 Structures
Authors: Allen Rachel L. , Raine Tim , Haude Anja , Trowsdale John , Wilson Michael J.
Published: April 2014

We studied recognition of the disease-associated HLA-B27 allele by immunomodulatory receptors encoded within the leukocyte receptor complex. HLA class I are ligands for members of the killer Ig receptor (KIR) and Ig-like transcript (ILT)/LIR/LILR families (the new LILR nomenclature is described at www. gene.ucl.ac.uk/nomenclature/genefamily/lilr.html). Members of these families bound HLA-B27 in both classical and β2 microglobulin-independent forms. Classical complexes bound ILT2, ILT4, and LIR6 transfectants but not ILT1, ILT3, or ILT5. A free H chain form of HLA-B27 bound ILT4 and LIR6. Both forms of HLA-B27 bound KIR3DL1 transfectants. HLA-B27 free H chain bound CD14+ cells in PBL from healthy controls, consistent with ILT4 expression on monocytes. Alternative recognition of different forms of HLA-B27 by KIR or ILT could influence their immunomodulatory function and may imply a role in inflammatory disease.