bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytokine Coexpression During Human Th1/Th2 Cell Differentiation: Direct Evidence for Coordinated Expression of Th2 Cytokines
Authors: Cousins David J. , Lee Tak H. , Staynov Dontcho Z.
Published: April 2014

We have developed an in vitro differentiation assay in which human naive CD4+ cells are driven toward either the Th1 or Th2 phenotype. We have examined the interrelationships among the expression of IL-2, IL-4, IL-5, IL-10, IL-13, GM-CSF, and IFN-γ in individual cells using intracellular cytokine staining at various times during the differentiation process. We provide direct evidence that the Th2 cytokines IL-4, IL-5, and IL-13, unlike the other cytokines, are regulated by a coordinated mechanism. We also show that IL-10 is expressed by a different subset of cells that is prevalent at early stages of Th2 differentiation, but then diminishes. Additionally we demonstrate that while naive cells can express IL-2 upon activation, they cannot express GM-CSF. Commitment to GM-CSF expression occurs during differentiation in a Th1/Th2 subset-independent manner. Furthermore, we have examined the levels of GATA3, c-Maf, T-bet, and Ets-related molecule during human Th1/Th2 differentiation and suggest that differences in the levels of these critical transcription factors are responsible for commitment toward the Th1 or Th2 lineage.
Identification of Multiple Novel Epididymis-Specific β-Defensin Isoforms in Humans and Mice
Authors: Yamaguchi Yasuhiro , Nagase Takahide , Makita Ryosuke , Fukuhara Shigetomo , Tomita Tetsuji , Tominaga Takashi , Kurihara Hiroki , Ouchi Yasuyoshi
Published: April 2014

Defensins comprise a family of cationic antimicrobial peptides that are characterized by the presence of six conserved cysteine residues. We identified two novel human β-defensin (hBD) isoforms by mining the public human genomic sequences. The predicted peptides conserve the six-cysteine motif identical with hBD-4, termed hBD-5 and hBD-6. We also evaluated the characteristics of the mouse homologs of hBD-5, hBD-6, and HE2β1, termed mouse β-defensin (mBD)-12, mBD-11, and mouse EP2e (mEP2e). The mBD-12 synthetic peptide showed salt-dependent antimicrobial activity. We demonstrate the epididymis-specific expression pattern of hBD-5, hBD-6, mBD-11, mBD-12, and mEP2e. In situ hybridization revealed mBD-11, mBD-12, and mEP2e expression in the columnar epithelium of the caput epididymis, contrasting with the predominant expression of mBD-3 in the capsule or septum of the whole epididymis. In addition, the regional specificity of mBD-11, mBD-12, and mEP2e was somewhat overlapping, but not identical, in the caput epididymis, suggesting that specific regulation may work for each member of the β-defensin family. Our findings indicated that multiple β-defensin isoforms specifically and cooperatively contribute to the innate immunity of the urogenital system.
IL-18 Regulates Intestinal Mastocytosis and Th2 Cytokine Production Independently of IFN-γ DuringTrichinella spiralisInfection
Authors: Helmby Helena , Grencis Richard K.
Published: April 2014

Expulsion of the gastrointestinal nematode Trichinella spiralis is associated with pronounced mastocytosis mediated by a Th2-type response involving IL-4, IL-10, and IL-13. Here we demonstrate that IL-18 is a key negative regulator of protective immune responses against T. spiralis in vivo. IL-18 knockout mice are highly resistant to T. spiralis infection, expel the worms rapidly and subsequently develop low levels of encysted muscle larvae. The increased speed of expulsion is correlated with high numbers of mucosal mast cells and an increase in IL-13 and IL-10 secretion. When normal mice were treated with rIL-18 in vivo, worm expulsion was notably delayed, and the development of mastocytosis and Th2 cytokine production was significantly reduced. The treatment had no effect on intestinal eosinophilia or goblet cell hyperplasia but specifically inhibited the development of mastocytosis. Addition of rIL-18 to in vitro cultures of bone marrow-derived mast cells resulted in a significant reduction in cell yields as well as in the number of IL-4-secreting mast cells. In vivo treatment of T. spiralis-infected IFN-γ knockout mice with rIL-18 demonstrated that the inhibitory effect of IL-18 on mastocytosis and Th2 cytokine secretion is independent of IFN-γ. Hence, IL-18 plays a significant biological role as a negative regulator of intestinal mast cell responses and may promote the survival of intestinal parasites in vivo.
Subset-Specific Reductions in Lung Lymphocyte Accumulation Following Intratracheal Antigen Challenge in Endothelial Selectin-Deficient Mice
Authors: Curtis Jeffrey L. , Sonstein Joanne , Craig Ronald A. , Todt Jill C. , Knibbs Randall N. , Polak Timothy , Bullard Daniel C. , Stoolman Lloyd M.
Published: April 2014

We previously demonstrated induction and expression of CD62E and CD62P in the lungs of mice primed and then challenged with intratracheal (i.t.) SRBC. The current study examined accumulation of endogenous lymphocytes in the lungs of endothelial E- and P-selectin-deficient (E−P−) mice after i.t. SRBC challenge. Compared with syngeneic wild-type (wt) mice, E−P− mice showed an 85–95% decrease in CD8+ T cells and B cells in the lungs at both early and late time points. In contrast, CD4+ T cell accumulation was reduced by ∼60% early, but equivalent to wt levels later. Surprisingly, many γδ T cells were found in lungs and blood of E−P− mice but were undetectable in the lungs and blood of wt mice. Absolute numbers of peripheral blood CD4, CD8, and B lymphocytes in E−P− mice equaled or exceeded the levels in wt mice, particularly after challenge. Trafficking studies using αβ T lymphoblasts confirmed that the recruitment of circulating cells after challenge was markedly reduced in E−P− mice. Furthermore, Ag priming occurred normally in both the selectin-deficient and wt mice, because primed lymphocytes from both groups transferred Ag sensitivity into naive wt mice. Lung production of mRNA for six CC and two CXC chemokines after challenge was equivalent by RT-PCR analysis in wt and E−P− mice. Therefore, reduced lung accumulation of αβ T cells and B cells in E−P− mice did not result from reduced delivery of circulating lymphocytes to the lungs, unsuccessful Ag priming, or defective pulmonary chemokine production. Selectin-dependent lymphocyte recruitment into the lungs following i.t.-SRBC challenge is subset specific and time dependent.
Vitamin K-Dependent Protein S Localizing Complement Regulator C4b-Binding Protein to the Surface of Apoptotic Cells
Authors: Webb Joanna H. , Blom Anna M. , Dahlbäck Björn
Published: April 2014

Apoptosis is characterized by a lack of inflammatory reaction in surrounding tissues, suggesting local control of complement activation. During the initial stage of apoptosis, cells expose negatively charged phospholipid phosphatidylserine on their surfaces. The vitamin K-dependent protein S has a high affinity for this type of phospholipid. In human plasma, 60–70% of protein S circulates in complex with C4b-binding protein (C4BP). The reason why protein S and C4BP form a high-affinity complex in plasma is not known. However, C4BP is an important regulator of the classical pathway of the complement system where it acts as a cofactor in degradation of complement protein C4b. Using Jurkat cells as a model system for apoptosis, we now show protein S to bind to apoptotic cells. We further demonstrate protein S-mediated binding of C4BP to apoptotic cells. Binding of the C4BP-protein S complex to apoptotic cells was calcium-dependent and could be blocked with Abs directed against the phospholipid-binding domain in protein S. Annexin V, which binds to exposed phosphatidylserine on the apoptotic cell surface, could inhibit the binding of protein S. The C4BP that was bound via protein S to the apoptotic cells was able to interact with the complement protein C4b, supporting a physiological role of the C4BP/protein S complex in regulation of complement on the surface of apoptotic cells.
Complement Factor B Gene Regulation: Synergistic Effects of TNF-α and IFN-γ in Macrophages
Authors: Huang Yong , Krein Peter M. , Muruve Daniel A. , Winston Brent W.
Published: April 2014

Complement factor B (Bf) plays an important role in activating the alternative complement pathway. The inflammatory cytokines, in particular TNF-α and IFN-γ, are critical in the regulation of Bf gene expression in macrophages. In this study, we investigated the mechanisms of Bf gene regulation by TNF-α and IFN-γ in murine macrophages. Northern analysis revealed that Bf mRNA expression was synergistically up-regulated by TNF-α and IFN-γ in MH-S cells. Truncations of the 5′ Bf promoter identified a region between −556 and −282 bp that mediated TNF-α responsiveness as well as the synergistic effect of TNF-α and IFN-γ on Bf expression. Site-directed mutagenesis of a NF-κB-binding element in this region (−433 to −423 bp) abrogated TNF-α responsiveness and decreased the synergistic effect of TNF-α and IFN-γ on Bf expression. EMSAs revealed nuclear protein binding to this NF-κB cis-binding element on TNF-α stimulation. Supershift analysis revealed that both p50 and p65 proteins contribute to induction of Bf by TNF-α. An I-κB dominant negative mutant blocked Bf induction by TNF-α and reduced the synergistic induction by TNF-α and IFN-γ. In addition, the proteasome inhibitor MG132, which blocks NF-κB induction, blocked TNF-α-induced Bf promoter activity and the synergistic induction of Bf promoter activity by TNF-α and IFN-γ. LPS was found to induce Bf promoter activity through the same NF-κB cis-binding site. These findings suggest that a NF-κB cis-binding site between −433 and −423 bp is required for TNF-α responsiveness and for TNF-α- and IFN-γ-stimulated synergistic responsiveness of the Bf gene.
Pathophysiological Contributions of Fucosyltransferases in Renal Ischemia Reperfusion Injury
Authors: Burne Melissa J. , Rabb Hamid
Published: April 2014

Ischemia reperfusion injury (IRI) is a major cause of delayed graft function. Recent studies have shown that selectins play an important role in IRI. Selectins bind to sialylated and fucosylated sLex receptors, and two enzymes, fucosyltransferase IV (FucT-IV) and VII (FucT-VII), are important in the function of these receptors. We hypothesized that fucosyltransferase (FucT) enzymes were important pathophysiologic mediators of renal IRI. We therefore evaluated renal IRI in mice deficient in FucT-IV, FucT-VII, and both FucT-IV and FucT-VII and compared their renal function, tubular injury, selectin ligand expression, and neutrophil infiltration to those in wild-type control mice. Bilateral 30-min renal IRI was performed, and the results demonstrated that mice deficient in both FucT-IV/FucT-VII were significantly protected from renal IRI at 24 and 48 h compared with wild-type control mice. FucT-IV-deficient mice showed only modest protection from renal injury at 24 h. However, FucT-VII-deficient mice had similar injury as wild-type mice. Histological analysis of kidney tissue postischemia revealed that mice deficient in both FucT-IV and FucT-VII had significantly reduced tubular injury compared with wild-type mice. Selectin ligand expression increased postischemia in wild-type, but not FucT-IV/FucT-VII-deficient, mice. Neutrophil infiltration in postischemic kidneys of FucT-IV/FucT-VII-deficient mice was also attenuated. These data demonstrate that fucosyltransferases are important in the pathogenesis of renal IRI and are potential therapeutic targets.
Blockade of Focal Clustering and Active Conformation in β2-Integrin-Mediated Adhesion of Eosinophils to Intercellular Adhesion Molecule-1 Caused by Transduction of HIV TAT-Dominant Negative Ras
Authors: Myou Shigeharu , Zhu Xiangdong , Boetticher Evan , Myo Saori , Meliton Angelo , Lambertino Anissa , Munoz Nilda M. , Leff Alan R.
Published: April 2014

We transduced dominant negative (dn) HIV TAT-Ras protein into mature human eosinophils to determine the signaling pathways and mechanism involved in integrin-mediated adhesion caused by cytokine, chemokine, and chemoattractant stimulation. Transduction of TAT-dnRas into nondividing eosinophils inhibited endogenous Ras activation and extracellular signal-regulated kinase (ERK) phosphorylation caused by IL-5, eotaxin-1, and fMLP. IL-5, eotaxin-1, or fMLP caused 1) change of Mac-1 to its active conformation and 2) focal clustering of Mac-1 on the eosinophil surface. TAT-dnRas or PD98059, a pharmacological mitogen-activated protein/ERK kinase inhibitor, blocked both focal surface clustering of Mac-1 and the change to active conformational structure of this integrin assessed by the mAb CBRM1/5, which binds the activation epitope. Eosinophil adhesion to the endothelial ligand ICAM-1 was correspondingly blocked by TAT-dnRas and PD98059. As a further control, we used PMA, which activates ERK phosphorylation by postmembrane receptor induction of protein kinase C, a mechanism which bypasses Ras. Neither TAT-dnRas nor PD98059 blocked eosinophil adhesion to ICAM-1, up-regulation of CBRM1/5, or focal surface clustering of Mac-1 caused by PMA. In contrast to β2-integrin adhesion, neither TAT-dnRas nor PD98059 blocked the eosinophil adhesion to VCAM-1. Thus, a substantially different signaling mechanism was identified for β1-integrin adhesion. We conclude that H-Ras-mediated activation of ERK is critical for β2-integrin adhesion and that Ras-protein functions as the common regulator for cytokine-, chemokine-, and G-protein-coupled receptors in human eosinophils.
Eotaxin (CCL11) and Eotaxin-2 (CCL24) Induce Recruitment of Eosinophils, Basophils, Neutrophils, and Macrophages As Well As Features of Early- and Late-Phase Allergic Reactions Following Cutaneous Injection in Human Atopic and Nonatopic Volunteers
Authors: Menzies-Gow Andrew , Ying Sun , Sabroe Ian , Stubbs Victoria L. , Soler Dulce , Williams Timothy J. , Kay A. Barry
Published: April 2014

Eotaxin and eotaxin-2, acting through CCR3, are potent eosinophil chemoattractants both in vitro and in animal models. In this study we examined the capacity of eotaxin and eotaxin-2 to recruit eosinophils and other inflammatory cells in vivo in human atopic and nonatopic skin. Skin biopsies taken after intradermal injection of eotaxin and eotaxin-2 were examined by immunohistochemistry. Allergen- and diluent-challenged sites were used as positive and negative controls. Eotaxin and eotaxin-2 produced a dose- and time-dependent local eosinophilia of comparable intensity in both atopic and nonatopic individuals. This was associated with an acute wheal and flare response at the site of injection and development of a cutaneous late phase reaction in a proportion of subjects. There was an accompanying decrease in mast cell numbers. Both chemokines also induced the accumulation of basophils and an unexpected early infiltration of neutrophils. Macrophages were prominent at the 24-h point. Although there was surface CCR3 expression on neutrophils in whole blood, we were unable to demonstrate any functional neutrophil responses to eotaxin in vitro. Thus, intradermal injection of eotaxin and eotaxin-2 in humans induced infiltration of eosinophils and other inflammatory cells as well as changes consistent with CC chemokine-induced mast cell degranulation.
Mycophenolic Acid Inhibits IL-2-Dependent T Cell Proliferation, But Not IL-2-Dependent Survival and Sensitization to Apoptosis
Authors: Quéméneur Laurence , Flacher Monique , Gerland Luc-Marie , Ffrench Martine , Revillard Jean-Pierre , Bonnefoy-Berard Nathalie
Published: April 2014

Mycophenolic acid (MPA), the active metabolite of the immunosuppressive drug mycophenolate mofetil, is a selective inhibitor of inosine 5′-monophosphate dehydrogenase type II, a de novo purine nucleotide synthesis enzyme expressed in T and B lymphocytes and up-regulated upon cell activation. In this study, we report that the blockade of guanosine nucleotide synthesis by MPA inhibits mitogen-induced proliferation of PBL, an effect fully reversed by addition of guanosine and shared with mizoribine, another inhibitor of inosine 5′-monophosphate dehydrogenase. Because MPA does not inhibit early TCR-mediated activation events, such as CD25 expression and IL-2 synthesis, we investigated how it interferes with cytokine-dependent proliferation and survival. In activated lymphoblasts that are dependent on IL-2 or IL-15 for their proliferation, MPA does not impair signaling events such as of the extracellular signal-regulated kinase 2 and Stat5 phosphorylation, but inhibits down-regulation of the cyclin-dependent kinase inhibitor p27Kip1. Therefore, in activated lymphoblasts, MPA specifically interferes with cytokine-dependent signals that control cell cycle and blocks activated T cells in the mid-G1 phase of the cell cycle. Although it blocks IL-2-mediated proliferation, MPA does not inhibit cell survival and Bcl-xL up-regulation by IL-2 or other cytokines whose receptors share the common γ-chain (CD132). Finally, MPA does not interfere with IL-2-dependent acquisition of susceptibility to CD95-mediated apoptosis and degradation of cellular FLIP. Therefore, MPA has unique functional properties not shared by other immunosuppressive drugs interfering with IL-2R signaling events such as rapamycin and CD25 mAbs.
Interaction of the Estrogen Receptors with the Fas Ligand Promoter in Human Monocytes
Authors: Mor Gil , Sapi Eva , Abrahams Vikki M. , Rutherford Thomas , Song Joon , Hao Xiao-Ying , Muzaffar Saeher , Kohen Fortune
Published: April 2014

The predominance of autoimmune diseases among women suggests that estrogen may modulate immune function. Monocytes and macrophages are important in initiating, maintaining, and resolving inflammatory responses through cell-signaling molecules, which control immune cell survival. One important mechanism of cell survival is mediated by the Fas/Fas ligand (FasL) system. In this study, the link between estrogen, monocytes/macrophages, and the Fas/FasL system was investigated. Estrogen treatment increased FasL expression in monocytes through the binding of the estrogen receptors (ER) to the estrogen recognizing elements and AP-1 motifs present at the FasL promoter. Furthermore, estrogen induced apoptosis in monocytes expressing ERβ, but not in monocyte-differentiated macrophages expressing ERα. The expression of either ERα or ERβ and their response to estrogen in monocytes was found to be dependent on the their stage of cell differentiation. Previously, we have shown that estrogen replacement therapy in postmenopausal women decreased the number of circulating monocytes. In this study, we have characterized the molecular mechanism by which estrogen regulates monocytes homeostasis. These findings indicate that estrogen may regulate immune cell survival through the Fas/FasL system. There is biological relevance to these findings in view of studies showing that accumulation of activated monocytes is involved in the pathogenesis of conditions such as vasculititis, arteriosclerosis, and rheumatoid arthritis.
Cutting Edge: Induced Expression of a RhoA-Specific Guanine Nucleotide Exchange Factor, p190RhoGEF, Following CD40 Stimulation and WEHI 231 B Cell Activation
Authors: Lee Jong Ran , Ha Yun Jung , Kim Hye Jin
Published: April 2014

Stimulation of the B cell surface receptor CD40 induces transcriptional activation and protein expression. To determine which proteins are required for the CD40-mediated B cell activation, we performed a two-dimensional gel electrophoresis of the WEHI 231 B cell lysates. We report in this study the identification of one protein in which the expression was remarkably induced following CD40 stimulation. It was the p190 Rho guanine nucleotide exchange factor (GEF), p190RhoGEF, a recently identified GEF that is specific for RhoA. Overexpression of either p190RhoGEF or RhoA (Q63L), a constitutively active form of RhoA, mimics the effects of CD40 stimulation, such as changes in cellular structure and NF-κB activation. These p190RhoGEF overexpression effects are abrogated when coexpressed with a dominant negative form of RhoA (T19N). We also provide evidence for the CD40-mediated cellular changes that are abrogated in cells that are overexpressed with the dominant negative form of either p190RhoGEF (Y1003A) or RhoA (T19N).
Toll-Like Receptor 2 Ligands as Adjuvants for Human Th1 Responses
Authors: Sieling Peter A. , Chung Woosin , Duong Baochau T. , Godowski Paul J. , Modlin Robert L.
Published: April 2014

Bacterial lipopeptides (bLPs) are increasingly used as adjuvants to activate cell-mediated immune responses to foreign Ags. To explore mechanisms whereby bLPs adjuvant T cell responses, we stimulated human PBMCs with bLPs. We found that bLPs stimulate T cells to proliferate and produce IFN-γ in an accessory cell-dependent manner and in the absence of exogenous protein Ags. The ability of bLPs to stimulate T cell proliferation was Toll-like receptor 2 dependent and required IL-12, interaction with costimulatory molecules, and MHC proteins. Our data suggest that bLPs adjuvant adaptive Th1 responses by enhancing Ag presentation of endogenous peptides.
Regulation of Mouse Mammary Tumor VirusenvTranscriptional Activator Initiated Mammary Tumor Virus Superantigen Transcripts in Lymphomas of SJL/J Mice: Role of Ikaros, Demethylation, and Chromatin Structural Change in the Transcriptional Activation of Mammary Tumor Virus Superantigen
Authors: Thomas Rajan M. , Haleem Kamran , Siddique Abu B. , Simmons William J. , Sen Namita , Zhang Da-Jun , Tsiagbe Vincent K.
Published: April 2014

Mammary tumor virus (Mtv29)-encoded superantigen expressed by SJL/J mouse B cell lymphomas stimulates CD4+Vβ16+ T cells and thereby acquires T cell help necessary for lymphoma growth. Mtv29 mouse mammary tumor virus env transcriptional activator (META) env-controlled Mtv29 superantigen (vSAg29) mRNA transcripts (1.8 kb) are not expressed in normal B or other somatic cells. Real-time PCR-based assays with DNA from normal SJL liver and vSAg29− lymphoma (cNJ101), digested with methylation-sensitive enzymes, showed hypermethylation at AvaI, FspI, HpaII, ThaI, and the distal HgaI sites of the META env, but vSAg29+ lymphoma cells showed significant demethylation at AvaI, HpaII, and the distal HgaI sites. The distal HgaI site that is adjacent to an Ikaros binding site is significantly demethylated in the META env DNA from primary lymphomas. Gel shift assays showed binding of Ikaros to a sequence representing this region in the META env. SJL lymphomas expressed the Ikaros isoform Ik6 that was absent in normal B cells. vSAg29+ cells exhibited increased DNaseI accessibility to chromatin at the vSAg29 initiation site. Treatment of cNJ101 cells with a demethylating agent, 5-azacytidine, and a histone deacetylase inhibitor, trichostatin A, caused hypomethylation at AvaI, HpaII, and distal HgaI sites and led to chromatin structural change at the vSAg29 initiation site, accompanied by the expression of vSAg29 transcripts. This enabled cNJ101 cells to stimulate SJL lymphoma-responsive CD4+Vβ16+ T hybridoma cells. Thus, demethylation at the distal HgaI site of the Mtv29 META env permits vSAg29 expression, which may have an impact on the development of germinal center-derived B cell lymphomas of SJL/J mice.
In Vivo Augmentation of Tumor-Specific CTL Responses by Class I/Peptide Antigen Complexes on Microspheres (Large Multivalent Immunogen)
Authors: Goldberg Jodi , Shrikant Protul , Mescher Matthew F.
Published: April 2014

Tumor membrane Ag immobilized on cell size microspheres (large multivalent immunogen (LMI)) was previously shown to augment tumor-specific CTL activity and reduce tumor growth, and a clinical trial examining this approach is in progress. In the current study, LMI treatment has been examined using adoptive transfer of TCR-transgenic CD8 T cells to visualize Ag-specific cells during the response. OT-I T cells specific for H-2Kb/OVA257–264 were transferred into mice that were then challenged with LMI made by immobilizing H-2Kb/OVA257–264 on microspheres (Kb/OVA257–264-LMI) alone, or along with i.p. challenge with OVA-expressing E.G7 tumor. Kb/OVA257–264-LMI caused significant reduction of tumor growth when administered to E.G7-bearing mice. When administered alone, the Kb/OVA257–264-LMI caused only weak clonal expansion of OT-I cells in the spleen and lymph nodes, although most of the OT-I cells up-regulated expression of CD44 and VLA-4. In contrast, Kb/OVA257–264-LMI administration to E.G7-bearing mice stimulated no detectable expansion of OT-I cells in the spleen and lymph nodes but caused a rapid increase in the number of OT-I cells in the peritoneal cavity, the site of the growing tumor. These results demonstrate the potential for using class I/tumor peptide complexes for immunotherapy. In addition, they suggest a model for the mechanism of CTL augmentation in which recognition of the LMI Ag results in altered trafficking of the tumor-specific CD8 T cells so that they reach the site of a growing tumor more rapidly and in greater numbers, where they may further expand and acquire effector function.
A Gammaherpesvirus G Protein-Coupled Receptor Homologue Is Required for Increased Viral Replication in Response to Chemokines and Efficient Reactivation from Latency
Authors: Lee Bong Joo , Koszinowski Ulrich H. , Sarawar Sally R. , Adler Heiko
Published: April 2014

The open reading frame (ORF) 74 of gamma-2-herpesviruses encodes a G protein-coupled receptor which is highly conserved in members of this subfamily and is homologous to the CXCR2 chemokine receptor. The viral G protein-coupled receptor has been implicated in viral pathogenesis. However, the advantage of such chemokine receptor homologues to the virus is currently unknown. To address this, we constructed ORF74 deletion mutants of a mouse gamma-2-herpesvirus (MHV-68) and examined the effect of the deletion on viral growth and reactivation from latency. Growth of the mutant viruses in NIH 3T3 cells was similar to that of wild-type virus. However, CXC chemokines with ELR motifs, KC, and macrophage-inflammatory protein 2, significantly increased viral replication of the wild-type, but not the mutant viruses, via a pertussis toxin-insensitive, mitogen-activated protein/extracellular signal-regulated kinase and phosphatidylinositol 3-kinase-dependent pathway. IFN-γ-inducible protein 10, a CXC chemokine lacking an ELR motif, was able to reverse the effect of KC on viral replication. The mutant viruses also showed significantly reduced reactivation from latently infected mouse splenocytes. Reinsertion of ORF74 into the mutant virus restored the wild-type phenotype. Utilizing a viral CXCR2 homologue to enhance replication and reactivation from latency represents a novel mechanism by which gammaherpesviruses can subvert the immune response.
Pre-B Cell Antigen Receptor-Mediated Signal Inhibits CD24-Induced Apoptosis in Human Pre-B Cells
Authors: Taguchi Tomoko , Kiyokawa Nobutaka , Mimori Kenichi , Suzuki Toyo , Sekino Takaomi , Nakajima Hideki , Saito Masahiro , Katagiri Yohko U. , Matsuo Nobutake , Matsuo Yoshinobu , Karasuyama Hajime , Fujimoto Junichiro
Published: April 2014

We previously reported that the cross-linking of cluster of differentiation (CD)24 induces apoptosis in Burkitt’s lymphoma cells and that this phenomenon can be enhanced by a B cell Ag receptor (BCR)-mediated signal. In this study, we extend our previous observation and report that CD24 also mediated apoptosis in human precursor-B acute lymphoblastic leukemia cell lines in the pro-B and pre-B stages accompanying activation of multiple caspases. Interestingly, simultaneous cross-linking of pre-BCR clearly inhibited CD24-mediated apoptosis in pre-B cells. We also observed that mitogen-activated protein kinases (MAPKs) were involved in the regulation of this apoptotic process. Pre-BCR cross-linking induced prompt and strong activation of extracellular signal-regulated kinase 1, whereas CD24 cross-linking induced the sustained activation of p38 MAPK, following weak extracellular signal-regulated kinase 1 activation. SC68376, a specific inhibitor of p38 MAPK, inhibited apoptosis induction by CD24 cross-linking, whereas anisomycin, an activator of p38 MAPK, enhanced the apoptosis. In addition, PD98059, a specific inhibitor of MEK-1, enhanced apoptosis induction by CD24 cross-linking and reduced the antiapoptotic effects of pre-BCR cross-linking. Collectively, whether pre-B cells survive or die may be determined by the magnitude of MAPK activation, which is regulated by cell surface molecules. Our findings should be important to understanding the role of CD24-mediated cell signaling in early B cell development.
Cutting Edge: L-Selectin (CD62L) Expression Distinguishes Small Resting Memory CD4+ T Cells That Preferentially Respond to Recall Antigen
Authors: Hengel Richard L. , Thaker Vishakha , Pavlick Mark V. , Metcalf Julia A. , Dennis Glynn , Yang Jun , Lempicki Richard A. , Sereti Irini , Lane H. Clifford
Published: April 2014

Naive CD4+ T cells use L-selectin (CD62L) expression to facilitate immune surveillance. However, the reasons for its expression on a subset of memory CD4+ T cells are unknown. We show that memory CD4+ T cells expressing CD62L were smaller, proliferated well in response to tetanus toxoid, had longer telomeres, and expressed genes and proteins consistent with immune surveillance function. Conversely, memory CD4+ T cells lacking CD62L expression were larger, proliferated poorly in response to tetanus toxoid, had shorter telomeres, and expressed genes and proteins consistent with effector function. These findings suggest that CD62L expression facilitates immune surveillance by programming CD4+ T cell blood and lymph node recirculation, irrespective of naive or memory CD4+ T cell phenotype.
Expression of the Murine CD27 Ligand CD70 In Vitro and In Vivo
Authors: Tesselaar Kiki , Xiao Yanling , Arens Ramon , van Schijndel Gijs M. W. , Schuurhuis Danita H. , Mebius Reina E. , Borst Jannie , van Lier René A. W.
Published: April 2014

The interaction between TNFR family member CD27 and its ligand CD70 promotes lymphocyte expansion and effector cell formation. In humans, control of CD27 function is partly regulated by the restricted expression of CD70. We used newly developed mAbs to characterize murine (m) CD70 expression in vitro and in vivo. On resting lymphocytes and immature dendritic cells (DC), mCD70 is absent. In vitro, Ag receptor triggering induced mCD70 mRNA in T cells, but cell surface protein expression was very low. Activated B cells synthesized much higher levels of mCD70 mRNA than activated T cells and clearly expressed mCD70 at the cell surface. mCD70 cell surface expression could also be induced on the DC line D1 and on in vitro-generated murine DC upon maturation. In lymphoid organs of naive mice, virtually no mCD70-expressing cells were found, with exception of cells in the thymic medulla, which may be epithelial in origin. However, after intranasal infection with influenza virus, lung-infiltrating T cells and T and B cells in draining lymph nodes expressed mCD70 according to immunohistology. In such activated lymphocytes, mCD70 protein is largely retained intracellularly. Plasma membrane expression of mCD70 was only detectable by flow cytometry on a small proportion of lung-infiltrating T cells and peaked at the height of the primary response. Thus, expression of CD70 in the mouse is highly regulated at the transcriptional and posttranslational level. This most likely serves to limit excessive effector cell formation after antigenic stimulation.
Fibroblast Growth Factor-Inducible 14 Mediates Multiple Pathways of TWEAK-Induced Cell Death
Authors: Nakayama Masafumi , Ishidoh Kazumi , Kojima Yuko , Harada Norihiro , Kominami Eiki , Okumura Ko , Yagita Hideo
Published: April 2014

TWEAK, a TNF family member, is produced by IFN-γ-stimulated monocytes and induces multiple pathways of cell death, including caspase-dependent apoptosis, cathepsin B-dependent necrosis, and endogenous TNF-α-mediated cell death, in a cell type-specific manner. However, the TWEAK receptor(s) that mediates these multiple death pathways remains to be identified. Recently, fibroblast growth factor-inducible 14 (Fn14) has been identified to be a TWEAK receptor, which was responsible for TWEAK-induced proliferation of endothelial cells and angiogenesis. Because Fn14 lacks the cytoplasmic death domain, it remains unclear whether Fn14 can also mediate the TWEAK-induced cell death. In this study, we demonstrated that TWEAK could induce apoptotic cell death in Fn14 transfectants. A pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, rather sensitized the Fn14 transfectants to TWEAK-induced cell death by necrosis via reactive oxygen intermediates and cathepsin B-dependent pathway. By using newly generated agonistic anti-Fn14 mAbs, we also observed that Fn14 is constitutively expressed on the cell surface of all TWEAK-sensitive tumor cell lines, and can transmit the multiple death signals. Moreover, an anti-Fn14 mAb that blocks TWEAK-Fn14 interaction could totally abrogate TWEAK binding and TWEAK-induced cell death in all TWEAK-sensitive tumor cell lines. These results revealed that the multiple pathways of TWEAK-induced cell death are solely mediated by Fn14.
Src Homology 2 Domain-Containing Leukocyte Phosphoprotein of 76 kDa and Phospholipase Cγ1 Are Required for NF-κB Activation and Lipid Raft Recruitment of Protein Kinase Cθ Induced by T Cell Costimulation
Authors: Dienz Oliver , Möller Andreas , Strecker Andreas , Stephan Nadja , Krammer Peter H. , Dröge Wulf , Schmitz M. Lienhard
Published: April 2014

The NF-κB activation pathway induced by T cell costimulation uses various molecules including Vav1 and protein kinase C (PKC)θ. Because Vav1 inducibly associates with further proteins including phospholipase C (PLC)γ1 and Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76), we investigated their role for NF-κB activation in Jurkat leukemia T cell lines deficient for expression of these two proteins. Cells lacking SLP-76 or PLCγ1 failed to activate NF-κB in response to T cell costimulation. In contrast, replenishment of SLP-76 or PLCγ1 expression restored CD3/CD28-induced IκB kinase (IKK) activity as well as NF-κB DNA binding and transactivation. PKCθ activated NF-κB in SLP-76- and PLCγ1-deficient cells, showing that PKCθ is acting further downstream. In contrast, Vav1-induced NF-κB activation was normal in SLP-76− cells, but absent in PLCγ1− cells. CD3/CD28-stimulated recruitment of PKCθ and IKKγ to lipid rafts was lost in SLP-76- or PLCγ1-negative cells, while translocation of Vav1 remained unaffected. Accordingly, recruitment of PKCθ to the immunological synapse strictly relied on the presence of SLP-76 and PLCγ1, but synapse translocation of Vav1 identified in this study was independent from both proteins. These results show the importance of SLP-76 and PLCγ1 for NF-κB activation and raft translocation of PKCθ and IKKγ.
Guanine Exchange-Dependent and -Independent Effects of Vav1 on Integrin-Induced T Cell Spreading
Authors: del Pozo Miguel Angel , Schwartz Martin A. , Hu Junru , Kiosses William B. , Altman Amnon , Villalba Martin
Published: April 2014

Vav1 is a 95-kDa member of the Dbl family of guanine exchange factors and a prominent hemopoietic cell-specific protein tyrosine kinase substrate, the involvement of which in cytoskeletal rearrangements has been linked to its ability to activate Rho family small GTPases. β1 integrin ligation by fibronectin induced Vav1 phosphorylation in peripheral blood lymphocytes and in two different T cell lines. Vav1 overexpression led to massive T cell spreading on β1 integrin ligands, and, conversely, two dominant negative mutants blocked integrin-induced spreading. Vav1 and β1 integrin ligation synergistically activated Pak, but not Rac, Cdc42, or c-Jun N-terminal kinase. In addition, Vav1 cooperated with constitutively active V12Rac mutant, but not with V12Cdc42, to induce T cell spreading after integrin occupancy. More importantly, a Vav1 mutant that lacked guanine exchange factor activity still cooperated with V12Rac. In contrast, a point mutation in the SH2 domain of Vav1 abolished this synergistic effect. Therefore, our results suggest a new regulatory effect of Vav1 in T cell spreading, which is independent of its guanine exchange factor activity.
Survival of Mycobacterium tuberculosis in Host Macrophages Involves Resistance to Apoptosis Dependent upon Induction of Antiapoptotic Bcl-2 Family Member Mcl-1
Authors: Sly Laura M. , Hingley-Wilson Suzanne M. , Reiner Neil E. , McMaster W. Robert
Published: April 2014

Mcl-1 protein expression was found to be up-regulated during infection with virulent Mycobacterium tuberculosis strain H37Rv. Mcl-1 induction in THP-1 cells was optimal at a multiplicity of infection of 0.8–1.2 bacilli per macrophage and was independent of opsonin coating of the bacteria. Mcl-1 expression was elevated as early as 4 h, peaked at 5.8-fold above control cells at 24 h, and remained elevated at 48 h after infection. In THP-1 cells, mMcl-1 mRNA was induced by infection with live H37Rv but not with attenuated M. tuberculosis strain H37Ra, heat-killed H37Rv, or latex beads. In THP-1 cells and monocyte-derived macrophages (MDMs), Mcl-1 protein was induced by infection with live H37Rv but not with attenuated M. tuberculosis strain H37Ra, heat-killed H37Rv, or latex beads. Treatment of uninfected, H37Ra-infected, and H37Rv-infected THP-1 cells and MDMs with antisense oligonucleotides to mcl-1 reduced Mcl-1 expression by >84%. This resulted in an increase in apoptosis of both MDMs and THP-1 cells that were infected with H37Rv, but not cells that were uninfected or infected with H37Ra. Increased apoptosis correlated with a decrease in M. tuberculosis CFUs recovered from antisense-treated, H37Rv-infected cells at 4 and 7 days after infection. In contrast, CFU recoveries from sense-treated, H37Rv-infected cells or from antisense- or sense-treated, H37Ra-infected cells were unchanged from controls. Thus, the antiapoptotic effect of the induction of Mcl-1 expression in H37Rv-infected macrophages promotes the survival of virulent M. tuberculosis.
Proteomics and Immunological Analysis of a Novel Shrimp Allergen, Pen m 2
Authors: Yu Chia-Jung , Lin Yu-Fen , Chiang Bor-Luen , Chow Lu-Ping
Published: April 2014

Shellfish are a common cause of adverse food reactions in hypersensitive individuals and shrimp is one of the most frequently reported causes of allergic reactions. A novel allergen from Penaeus monodon, designated Pen m 2, was identified by two-dimensional immunoblotting using sera from subjects with shrimp allergy, followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of the peptide digest. This novel allergen was then cloned and the amino acid sequence deduced from the cDNA sequence. The cloned cDNA encoded a 356-aa protein with an acetylated N terminus at Ala2, identified by postsource decay analysis. Comparison of the Pen m 2 sequence with known protein sequences revealed extensive similarity with arginine kinase (EC 2.7.3.3) from crustaceans. Pen m 2 was purified by anion exchange chromatography and shown to have arginine kinase activity and to react with serum IgE from shrimp allergic patients and induce immediate type skin reactions in sensitized patients. Using Pen m 2-specific antisera and polyclonal sera from shrimp-sensitive subjects in a competitive ELISA inhibition assay, Pen m 2 was identified as a novel cross-reactive Crustacea allergen. This novel allergen could be useful in allergy diagnosis and in the treatment of Crustacea-derived allergic disorders.
Characterization of Lung γδ T Cells Following Intranasal Infection withMycobacterium bovisBacillus Calmette-Guérin
Authors: Dieli Francesco , Ivanyi Juraj , Marsh Philip , Williams Ann , Naylor Irene , Sireci Guido , Caccamo Nadia , Di Sano Caterina , Salerno Alfredo
Published: April 2014

The lungs are considered to have an impaired capacity to contain infection by pathogenic mycobacteria, even in the presence of effective systemic immunity. In an attempt to understand the underlying cellular mechanisms, we characterized the γδ T cell population following intranasal infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG). The peak of γδ T cell expansion at 7 days postinfection preceded the 30 day peak of αβ T cell expansion and bacterial count. The expanded population of γδ T cells in the lungs of BCG-infected mice represents an expansion of the resident Vγ2 T cell subset as well as an influx of Vγ1 and of four different Vδ gene-bearing T cell subsets. The γδ T cells in the lungs of BCG-infected mice secreted IFN-γ following in vitro stimulation with ionomycin and PMA and were cytotoxic against BCG-infected peritoneal macrophages as well as against the uninfected J774 macrophage cell line. The cytotoxicity was selectively blocked by anti-γδ TCR mAb and strontium ions, suggesting a granule-exocytosis killing pathway. Depletion of γδ T cells by injection of specific mAb had no effect on the subsequent developing CD4 T cell response in the lungs of BCG-infected mice, but significantly reduced cytotoxic activity and IFN-γ production by lung CD8 T cells. Thus, γδ T cells in the lungs might help to control mycobacterial infection in the period between innate and classical adaptive immunity and may also play an important regulatory role in the subsequent onset of αβ T lymphocytes.
Lack of C3 Affects Th2 Response Development and the Sequelae of Chemotherapy in Schistosomiasis
Authors: La Flamme Anne Camille , MacDonald Andrew S. , Huxtable Clive R. , Carroll Michael , Pearce Edward J.
Published: April 2014

The role of the third component of complement (C3) during schistosome infection was investigated using mice deficient in C3. While no effect was observed 8 wk after infection on worm development or liver pathology, Ag-specific Th2-associated cytokine production (IL-13, IL-5, IL-6, and IL-10) was significantly reduced, and IFN-γ production was enhanced in the absence of C3. IgG1 and IgE, but not IgG2a or IgM, Ab responses were also significantly impaired in infected C3−/− mice, suggesting that C3 may play a role in IL-4-mediated Th2 response enhancement during schistosome infection. Furthermore, C3-deficient mice could not effectively clear adult worms after praziquantel (PZQ) treatment and suffered increased morbidity due to the overproduction of proinflammatory mediators following drug administration. However, the ischemic liver damage that normally accompanies PZQ administration in infected wild-type mice was substantially reduced in treated C3-deficient mice, probably due to the absence of dead or dying worms in the livers of these animals. Together these results indicate that C3 enhances Th2 responses during schistosome infection, potentiates PZQ-mediated parasite clearance, and reduces chemotherapy-induced proinflammatory mediator production.
Induction of In Vitro Reprogramming by Toll-Like Receptor (TLR)2 and TLR4 Agonists in Murine Macrophages: Effects of TLR “Homotolerance” Versus “Heterotolerance” on NF-κB Signaling Pathway Components
Authors: Dobrovolskaia Marina A. , Medvedev Andrei E. , Thomas Karen E. , Cuesta Natalia , Toshchakov Vladimir , Ren Tianbo , Cody Michael J. , Michalek Suzanne M. , Rice Nancy R. , Vogel Stefanie N.
Published: April 2014

In this study, tolerance induction by preexposure of murine macrophages to Toll-like receptor (TLR)2 and TLR4 agonists was revisited, focusing on the major signaling components associated with NF-κB activation. Pretreatment of macrophages with a pure TLR4 agonist (protein-free Escherichia coli (Ec) LPS) or with TLR2 agonists (Porphyromonas gingivalis LPS or synthetic lipoprotein Pam3Cys) led to suppression of TNF-α secretion, IL-1R-associated kinase-1, and IκB kinase (IKK) kinase activities, c-jun N-terminal kinase, and extracellular signal-regulated kinase phosphorylation, and to suppression of NF-κB DNA binding and transactivation upon challenge with the same agonist (TLR4 or TLR2 “homotolerance,” respectively). Despite inhibited NF-κB DNA binding, increased levels of nuclear NF-κB were detected in agonist-pretreated macrophages. For all the intermediate signaling elements, heterotolerance was weaker than TLR4 or TLR2 homotolerance with the exception of IKK kinase activity. IKK kinase activity was unperturbed in heterotolerance. TNF-α secretion was also suppressed in P. gingivalis LPS-pretreated, Ec LPS-challenged cells, but not vice versa, while Pam3Cys and Ec LPS did not induce a state of cross-tolerance at the level of TNF-α. Experiments designed to elucidate novel mechanisms of NF-κB inhibition in tolerized cells revealed the potential contribution of IκBε and IκBξ inhibitory proteins and the necessity of TLR4 engagement for induction of tolerance to Toll receptor-IL-1R domain-containing adapter protein/MyD88-adapter-like-dependent gene expression. Collectively, these data demonstrate that induction of homotolerance affects a broader spectrum of signaling components than in heterotolerance, with selective modulation of specific elements within the NF-κB signaling pathway.
House Dust Mite Dermatophagoides farinae Augments Proinflammatory Mediator Productions and Accessory Function of Alveolar Macrophages: Implications for Allergic Sensitization and Inflammation
Authors: Chen Chih-Long , Lee Chen-Ting , Liu Yi-Chun , Wang Jiu-Yao , Lei Huan-Yao , Yu Chun-Keung
Published: April 2014

In this study, we examine the effects of Dermatophagoides farinae (Der f), a major source of airborne allergens, on alveolar macrophages (AMs), and we also test its contribution to allergic responses in mice. Der f activated NF-κB of AMs and, unlike OVA or LPS stimulation, up-regulated IL-6, TNF-α, and NO. In addition, it down-regulated antioxidants, but affected neither the expression nor production of IL-12. Der f-stimulated AMs expressed enhanced levels of costimulatory B7 molecules, supported T cell proliferation, and promoted Th2 cell development. The enhanced accessory function was suppressed by blockade mAbs to B7.2, IL-6, and TNF-α and by N-monomethyl-l-arginine, an NO synthase inhibitor, and N-acetylcysteine, a thiol antioxidant, whereas it was augmented by (±)-S-nitroso-N-acetylpenicillamine, an NO donor. Arg-Gly-Asp-Ser peptide and neo-glycoproteins galactose-BSA and mannose-BSA inhibited the Der f-induced IL-6 and TNF-α productions and enhanced accessory function of AMs. Der f was more potent than OVA for inducing pulmonary eosinophilic inflammation, NO, and serum allergen-specific IgG1 Ab production in mice. AMs from Der f-challenged mice expressed enhanced levels of B7 and augmented T cell proliferation ex vivo. In Der f-challenged mice, respiratory syncytial virus infection (5 × 105 pfu; 3 days before Der f instillation) augmented Der f-specific Ab production, whereas dexamethasone (50 mg/kg; 1 h before Der f instillation) diminished the allergic airway inflammation and Ab response. We conclude that AMs are sensitive targets for Der f and that the Der f-induced proinflammatory responses may represent an important mechanism in mediating the development of allergic sensitization and inflammation.