bluuebunny/crossref_metadata_2025 huggingface dataset:
Recombinant Anti-Human HER2/neuIgG3-(GM-CSF) Fusion Protein Retains Antigen Specificity and Cytokine Function and Demonstrates Antitumor Activity
Authors: Dela Cruz Jay S. , Trinh K. Ryan , Morrison Sherie L. , Penichet Manuel L.
Published: April 2014

Anti-HER2/neu therapy of human HER2/neu-expressing malignancies such as breast cancer has shown only partial success in clinical trials. To expand the clinical potential of this approach, we have genetically engineered an anti-HER2/neu IgG3 fusion protein containing GM-CSF. Anti-HER2/neu IgG3-(GM-CSF) expressed in myeloma cells was correctly assembled and secreted. It was able to target HER2/neu-expressing cells and to support growth of a GM-CSF-dependent murine myeloid cell line, FDC-P1. The Ab fusion protein activated J774.2 macrophage cells so that they exhibit an enhanced cytotoxic activity and was comparable to the parental Ab in its ability to effect Ab-dependent cellular cytotoxicity-mediated tumor cell lysis. Pharmacokinetic studies showed that anti-HER2/neu IgG3-(GM-CSF) is stable in the blood. Interestingly, the half-life of anti-HER2/neu IgG3-(GM-CSF) depended on the injected dose with longer in vivo persistence observed at higher doses. Biodistribution studies showed that anti-HER2/neu IgG3-(GM-CSF) is mainly localized in the spleen. In addition, anti-HER2/neu IgG3-(GM-CSF) was able to target the HER2/neu-expressing murine tumor CT26-HER2/neu and enhance the immune response against the targeted Ag HER2/neu. Anti-HER2/neu IgG3-(GM-CSF) is able to enhance both Th1- and Th2-mediated immune responses and treatment with this Ab fusion protein resulted in significant retardation in the growth of s.c. CT26-HER2/neu tumors. Our results suggest that anti-HER2/neu IgG3-(GM-CSF) fusion protein is useful in the treatment of HER2/neu-expressing tumors.
Nonopsonic Phagocytosis of Mycobacterium kansasii by Human Neutrophils Depends on Cholesterol and Is Mediated by CR3 Associated with Glycosylphosphatidylinositol-Anchored Proteins
Authors: Peyron Pascale , Bordier Christine , N′Diaye Elsa-Noah , Maridonneau-Parini Isabelle
Published: April 2014

Receptors involved in the phagocytosis of microorganisms under nonopsonic conditions have been little studied in neutrophils. Complement receptor type 3 (CR3) is a pattern recognition receptor able to internalize zymosan and C3bi-coated particles. We report that Abs directed against CR3 strongly inhibited nonopsonic phagocytosis of Mycobacterium kansasii in human neutrophils. In these cells CR3 has been found associated with several GPI-anchored proteins localized in cholesterol-rich microdomains (rafts) of the plasma membrane. Cholesterol sequestration by nystatin, filipin, or β-cyclodextrin as well as treatment of neutrophils with phosphatidylinositol phospholipase C to remove GPI-anchored proteins from the cell surface markedly inhibited phagocytosis of M. kansasii, without affecting phagocytosis of zymosan or serum-opsonized M. kansasii. Abs directed against several GPI-anchored proteins inhibited phagocytosis of M. kansasii, but not of zymosan. N-acetyl-d-glucosamine, which is known to disrupt interactions between CR3 and GPI proteins, also strongly diminished phagocytosis of these mycobacteria. In conclusion, phagocytosis of M. kansasii involved CR3, GPI-anchored receptors, and cholesterol. In contrast, phagocytosis of zymosan or opsonized particles involved CR3, but not cholesterol or GPI proteins. We propose that CR3, when associated with a GPI protein, relocates in cholesterol-rich domains where M. kansasii are internalized. When CR3 is not associated with a GPI protein, it remains outside of these domains and mediates phagocytosis of zymosan and opsonized particles, but not of M. kansasii.
MHC Class Ib-Restricted CTL Provide Protection Against Primary and SecondaryListeria monocytogenesInfection
Authors: Seaman Michael S. , Wang Chyung-Ru , Forman James
Published: April 2014

Infection of B6 mice with the intracellular pathogen Listeria monocytogenes (LM) results in the activation of CD8+ T cells that respond to Ag presented by both MHC class Ia and class Ib molecules. Enzyme-linked immunospot analysis reveals that these CTL populations expand and contract at different times following a primary sublethal LM infection. Between days 4 and 6 postinfection, class Ib-restricted CTL exhibit a rapid proliferative response that is primarily H2-M3 restricted. The peak response of class Ia-restricted CD8+ T cells occurs a few days later, after the majority of bacteria have been cleared. Although class Ia-restricted CTL exhibit a vigorous recall response to secondary LM infection, we observe limited expansion of class Ib-restricted memory CTL, even in MHC class Ia-deficient mice (B6.Kb−/−Db−/−). Despite this lack of enhanced expansion in vivo, class Ib-restricted memory CTL retain the ability to proliferate and expand when provided with Ag in vitro. Furthermore, we demonstrate that in vivo depletion of CD8+ T cells in LM-immune B6.Kb−/−Db−/− mice severely impairs memory protection. Together, these data demonstrate that class Ib-restricted CTL play an important role in clearing a primary LM infection and generate a memory population capable of providing significant protection against subsequent infection.
IL-5 Up-Regulates Cysteinyl Leukotriene 1 Receptor Expression in HL-60 Cells Differentiated into Eosinophils
Authors: Thivierge Maryse , Doty Micah , Johnson Jeff , Staňková Jana , Rola-Pleszczynski Marek
Published: April 2014

The cysteinyl leukotrienes, leukotriene (LT) C4, LTD4, and LTE4, are lipid mediators that have been implicated in the pathogenesis of several inflammatory processes, including asthma. The human LTD4 receptor, CysLT1R, was recently cloned and characterized. We had previously shown that HL-60 cells differentiated toward the eosinophilic lineage (HL-60/eos) developed specific functional LTD4 receptors. The present work was undertaken to study the potential modulation of CysLT1R expression in HL-60/eos by IL-5, an important regulator of eosinophil function. Here, we report that IL-5 rapidly up-regulates CysLT1R mRNA expression, with consequently enhanced CysLT1R protein expression and function in HL-60/eos. CysLT1R mRNA expression was augmented 2- to 15-fold following treatment with IL-5 (1–20 ng/ml). The effect was seen after 2 h, was maximal by 4 h, and maintained at 8 h. Although CysLT1R mRNA was constitutively expressed in undifferentiated HL-60 cells, its expression was not modulated by IL-5 in the absence of differentiation. Differentiated HL-60/eos cells pretreated with IL-5 (10 ng/ml) for 24 h showed enhanced CysLT1R expression on the cell surface, as assessed by flow cytometry using a polyclonal anti-CysLT1R Ab. They also showed enhanced responsiveness to LTD4, but not to LTB4 or platelet-activating factor, in terms of Ca2+ mobilization, and augmented the chemotactic response to LTD4. Our findings suggest a possible mechanism by which IL-5 can modulate eosinophil functions and particularly their responsiveness to LTD4, and thus contribute to the pathogenesis of asthma and allergic diseases.
Fibrin(ogen)-Induced Expression of ICAM-1 and Chemokines in Human Synovial Fibroblasts
Authors: Liu Xiufang , Piela-Smith Theresa H.
Published: April 2014

It has long been recognized that in most inflamed arthritic joints the coagulation system is activated, leading to the local generation of fibrin, and it has long been hypothesized that the local fibrin deposition promotes inflammation and tissue destruction. However, only recently has the direct effect of fibrin on the inflammatory process been seriously investigated, and specific roles assigned to fibrin or its products as mediators of the inflammatory process. Although fibrin and/or fibrinogen (fibrin(ogen)) is abundantly present in inflamed tissues and joints rich in fibroblastic cells, no significant data on fibrin(ogen)-induced gene expression by fibroblasts have been published. We now demonstrate that coculture of human synovial fibroblasts with fibrin(ogen) results in the up-regulation of ICAM-1 as well as increased production of the chemokines IL-8 and growth-related oncogene-α. Increased ICAM-1 expression was fibrin(ogen) dose-dependent and was demonstrated by ELISA, flow cytometry, and functional adhesion assays. Levels of ICAM-1 induced by fibrin(ogen) were comparable to those that could be induced by cytokine stimulation. Fibrin(ogen) stimulation of ICAM-1 could be suppressed by pyrrolidinedithiocarbamate, an inhibitor of NF-κB activation. Chemokine production was induced by fibrin(ogen) in cell culture supernatants >100-fold as compared with controls. Thus, through its activation of synovial fibroblasts, fibrin(ogen) deposition may promote the recruitment (via chemokines) and retention (via adhesion molecules) of lymphocytes within the arthritic joint.
Transepithelial Neutrophil Migration Is CXCR1 Dependent In Vitro and Is Defective in IL-8 Receptor Knockout Mice
Authors: Godaly Gabriela , Hang Long , Frendéus Björn , Svanborg Catharina
Published: April 2014

Neutrophil migration across infected mucosal surfaces is chemokine dependent, but the role of chemokine receptors has not been investigated. In this study, chemokine receptors were shown to be expressed by epithelial cells lining the urinary tract, and to play an essential role for neutrophil migration across the mucosal barrier. Uroepithelial CXCR1 and CXCR2 expression was detected in human urinary tract biopsies, and in vitro infection of human uroepithelial cell lines caused a dramatic increase in both receptors. As a consequence, there was higher binding of IL-8 to the cells and the IL-8-dependent neutrophil migration across the infected epithelial cell layers was enhanced. Abs to IL-8 or to the CXCR1 receptor inhibited this increase by 60% (p < 0.004), but anti-CXCR2 Abs had no effect, suggesting that CXCR1 was the more essential receptor in this process. Similar observations were made in the mouse urinary tract, where experimental infection stimulated epithelial expression of the murine IL-8 receptor, followed by a rapid flux of neutrophils into the lumen. IL-8 receptor knockout mice, in contrast, failed to express the receptor, their neutrophils were unable to cross the epithelial barrier, and accumulated in massive numbers in the tissues. These results demonstrate that epithelial cells express CXC receptors and that infection increases receptor expression. Furthermore, we show that CXCR1 is required for neutrophil migration across infected epithelial cell layers in vitro, and that the murine IL-8 receptor is needed for neutrophils to cross the infected mucosa of the urinary tract in vivo.
Up-Regulation of IL-17 Is Associated with Bioactive IL-8 Expression inHelicobacter pylori-Infected Human Gastric Mucosa
Authors: Luzza Francesco , Parrello Tiziana , Monteleone Giovanni , Sebkova Ladislava , Romano Marco , Zarrilli Raffaele , Imeneo Maria , Pallone Francesco
Published: April 2014

Helicobacter pylori (Hp)-associated gastritis is characterized by an increased number of acute and chronic inflammatory cells secreting cytokines that contribute to maintain and expand the local inflammation. Locally induced IL-8 is believed to play a major role in the Hp-associated acute inflammatory response. Factors/mechanisms that regulate IL-8 induction are, however, not fully understood. In the present study we investigated whether Hp infection is associated with an increased production of IL-17, a T cell-derived cytokine capable of modulating IL-8 gene expression. We showed that both IL-17 RNA transcripts and protein were expressed at a higher level in the whole gastric mucosal and lamina propria mononuclear cell samples from Hp-infected patients than in those from uninfected subjects. Hp eradication was associated with a marked down-regulation of IL-17 expression. The addition of a neutralizing anti-IL-17 Ab to the gastric lamina propria mononuclear cell cultures resulted in a significant inhibition of IL-8 secretion, indicating that IL-17 contributes to enhance IL-8 in the Hp-colonized gastric mucosa. Consistently, stimulation of MKN 28 cells, a gastric epithelial cell line, with IL-17 increased IL-8 secretion. Finally, conditioned medium from the IL-17-stimulated MKN 28 cell cultures promoted the in vitro polymorphonuclear leukocyte migration. This effect was inhibitable by a neutralizing IL-8 but not IL-17 Ab. Together, these data indicate that biologically active IL-17 production is increased during Hp infection, suggesting the possibility that this cytokine may play an important role in the inflammatory response to the Hp colonization.
Development of Myelin Oligodendrocyte Glycoprotein Autoreactive Transgenic B Lymphocytes: Receptor Editing In Vivo After Encounter of a Self-Antigen Distinct from Myelin Oligodendrocyte Glycoprotein
Authors: Litzenburger Tobias , Blüthmann Horst , Morales Patricia , Pham-Dinh Danielle , Dautigny André , Wekerle Hartmut , Iglesias Antonio
Published: April 2014

We explored mechanisms involved in B cell self-tolerance against brain autoantigens in a double-transgenic mouse model carrying the Ig H-chain (introduced by gene replacement) and/or the L-chain κ (conventional transgenic) of the mAb 8.18C5, specific for the myelin oligodendrocyte glycoprotein (MOG). Previously, we demonstrated that B cells expressing solely the MOG-specific Ig H-chain differentiate without tolerogenic censure. We show now that double-transgenic (THκmog) B cells expressing transgenic Ig H- and L-chains are subjected to receptor editing. We show that in adult mice carrying both MOG-specific Ig H- and L-chains, the frequency of MOG-binding B cells is not higher than in mice expressing solely the transgenic Ig H-chain. In fact, in THκmog double-transgenic mice, the transgenic κmog L-chain was commonly replaced by endogenous L-chains, i.e., by receptor editing. In rearrangement-deficient RAG-2− mice, differentiation of THκmog B cells is blocked at an immature stage (defined by the B220lowIgMlowIgD− phenotype), reflecting interaction of the autoreactive B cells with a local self-determinant. The tolerogenic structure in the bone marrow is not classical MOG, because back-crossing THκmog mice into a MOG-deficient genetic background does not lead to an increase in the proportion of MOG-binding B cells. We propose that an as yet undefined self-Ag distinct from MOG cross-reacts with the THκmog B cell receptor and induces editing of the transgenic κmog L-chain in early immature B cells without affecting the pathogenic potential of the remaining MOG-specific B cells. This phenomenon represents a particular form of chain-specific split tolerance.
Cutting Edge: The Related Molecules CD28 and Inducible Costimulator Deliver Both Unique and Complementary Signals Required for Optimal T Cell Activation
Authors: Gonzalo Jose-Angel , Delaney Tracy , Corcoran Justin , Goodearl Andrew , Gutierrez-Ramos Jose Carlos , Coyle Anthony J.
Published: April 2014

Optimal T cell activation requires engagement of CD28 with its counterligands B7-1 and B7-2. Inducible costimulator (ICOS) is the third member of the CD28/CTLA4 family that binds a B7-like protein, B7RP-1. Administration of ICOS-Ig attenuates T cell expansion following superantigen (SAg) administration, but fails to regulate either peripheral deletion or anergy induction. ICOS-Ig, but not CTLA4-Ig, uniquely regulates SAg-induced TNF-α production, whereas IL-2 secretion is modulated by CTLA4-Ig, but not ICOS-Ig. In contrast, both ICOS and CD28 are required for complete attenuation of IL-4 production. Our data suggest that ICOS and CD28 regulate T cell expansion and that ligation of either CD28 or ICOS can either uniquely regulate cytokine production (IL-2/TNF-α) or synergize for optimal cytokine production (IL-4) after SAg administration.
C-C Chemokine Receptor 4 Expression Defines a Major Subset of Circulating Nonintestinal Memory T Cells of Both Th1 and Th2 Potential
Authors: Andrew David P. , Ruffing Nancy , Kim Chang H. , Miao Wenyan , Heath Heidi , Li You , Murphy Kristine , Campbell James J. , Butcher Eugene C. , Wu Lijun
Published: April 2014

CCR4, a chemokine receptor for macrophage-derived chemokine (MDC) and thymus and activation-regulated chemokine (TARC), has been implicated as a preferential marker for Th2 lymphocytes. Following in vitro polarization protocols, most Th2 lymphocytes express CCR4 and respond to its ligands TARC and MDC, whereas Th1 lymphocytes express CXC chemokine receptor 3 and CCR5 (but not CCR4). We show in this study that CCR4 is a major receptor for MDC and TARC on T lymphocytes, as anti-CCR4 mAbs significantly inhibit the migration of these cells to MDC and TARC. CCR4 is also highly expressed in most single-positive CD4+ thymocytes and on a major fraction of blood nonintestinal (α4β7−) memory CD4 lymphocytes, including almost all skin memory CD4+ cells expressing the cutaneous lymphocyte Ag (CLA), but weakly or not expressed in other subsets in thymus and blood. Interestingly, major fractions of circulating CCR4+ memory CD4 lymphocytes coexpress the Th1-associated receptors CXC chemokine receptor 3 and CCR5, suggesting a potential problem in using these markers for Th1 vs Th2 lymphocyte cells. Moreover, although production of Th2 cytokines in blood T cells is associated with CCR4+ CD4 lymphocytes, significant numbers of freshly isolated circulating CCR4+ memory CD4 lymphocytes (including both CLA+ and CLA− fractions) readily express the Th1 cytokine IFN-γ after short-term stimulation. Our results are consistent with a role for CCR4 as a major trafficking receptor for systemic memory T cells, and indicate that the patterns and regulation of chemokine receptor expression in vivo are more complex than indicated by current in vitro models of Th1 vs Th2 cell generation.
B Cell Receptor-Stimulated Mitochondrial Phospholipase A2 Activation and Resultant Disruption of Mitochondrial Membrane Potential Correlate with the Induction of Apoptosis in WEHI-231 B Cells
Authors: Katz Elad , Deehan Maureen R. , Seatter Sandra , Lord Caroline , Sturrock Roger D. , Harnett Margaret M.
Published: April 2014

Cross-linking of the Ag receptors on the immature B cell lymphoma, WEHI-231, leads to growth arrest and apoptosis. We now show that although commitment to such B cell receptor (BCR)-mediated apoptosis correlates with mitochondrial phospholipase A2 activation, disruption of mitochondrial function, and ATP depletion, it is executed independently of caspase activation. First, we demonstrate a pivotal role for mitochondrial function in determining B cell fate by showing up-regulation of cytosolic phospholipase A2 expression, induction of mitochondrial phospholipase A2 activity, arachidonic acid-mediated collapse of mitochondrial transmembrane inner potential (Δψm), and depletion of cellular ATP under conditions of apoptotic, but not proliferative, signaling via the BCR. Importantly, disruption of Δψm, ATP depletion, and apoptosis can be prevented by rescue signals via CD40 or by Δψm stabilizers such as antimycin or oligomycin. Second, we show that commitment and postmitochondrial execution of BCR-mediated apoptosis are not dependent on caspase activation by demonstrating that such apoptotic signaling does not induce release of cytochrome c from the mitochondria or activation of effector caspases, as evidenced by poly(ADP-ribose) polymerase or Bcl-xL cleavage. Indeed, apoptotic signaling via the BCR in WEHI-231 B cells does not stimulate the activation of caspase-3 and, consistent with this, BCR-mediated disruption of Δψm and commitment to apoptosis take place in the presence of caspase inhibitors. In contrast, BCR signaling induces the postmitochondrial activation of cathepsin B, and resultant apoptosis is blocked by the cathepsin B inhibitor, (23,35)trans-epoxysuccinyl-l-leucylamindo-3-methylbutane ethyl ester (EST) suggesting a key role for this executioner protease in Ag receptor-driven apoptosis of WEHI-231 immature B cells.
Functional Reconstitution and Regulation of IL-18 Activity by the IL-18Rβ Chain
Authors: Hyun Kim Soo , Reznikov Leonid L. , Stuyt Rogier J. L. , Selzman Craig H. , Fantuzzi Giamilia , Hoshino Tomoaki , Young Howard A. , Dinarello Charles A.
Published: April 2014

IL-18 and IL-12 are major IFN-γ-inducing cytokines but the unique synergism of IL-18 and IL-12 remains unclear. In the human NK cell line NKO, IL-18Rα, and IL-18Rβ are expressed constitutively but IL-18 did not induce IFN-γ unless IL-12 was present. COS-1 fibroblasts, which produce the chemokine IL-8 when stimulated by IL-1β or TNF-α, do not respond to IL-18, despite abundant expression of the IL-18Rα chain. COS-1 cells lack expression of the IL-18Rβ chain. The IL-18Rβ cDNA was cloned from a human T-B lymphoblast cDNA library and COS-1 cells were transiently transfected with the IL-18Rβ chain and a luciferase reporter. In transfected COS-1 cells, IL-18 induced IL-8 and luciferase in the absence of IL-12 and independently of IL-1 and TNF. Ab against the IL-18Rα chain, however, prevented IL-18 responsiveness in COS-1 cells transfected with the IL-18Rβ chain, suggesting that both chains be functional. In NKO cells and PBMC, IL-12 increased steady-state mRNA levels of IL-18Rα and IL-18Rβ; the production of IFN-γ corresponded to IL-12-induced IL-18Rα and IL-18Rβ chains. We conclude that functional reconstitution of the IL-18Rβ chain is essential for IL-12-independent proinflammatory activity of IL-18-induced IL-8 in fibroblasts. The synergism of IL-18 plus IL-12 for IFN-γ production is, in part, due to IL-12 up-regulation of both IL-18Rα and IL-18Rβ chains, although postreceptor events likely contribute to IFN-γ production.
Cutting Edge: Functional Interactions Between Toll-Like Receptor (TLR) 2 and TLR1 or TLR6 in Response to Phenol-Soluble Modulin
Authors: Hajjar Adeline M. , O’Mahony D. Shane , Ozinsky Adrian , Underhill David M. , Aderem Alan , Klebanoff Seymour J. , Wilson Christopher B.
Published: April 2014

Toll-like receptor (TLR) 2 and TLR4 play important roles in the early, innate immune response to microbial challenge. TLR2 is preferentially involved in the inflammatory response to lipoteichoic acid, lipopeptides, and glycans from a variety of microbes, whereas TLR4 is essential for a complete response to LPSs. We report here that TLR2 transduces the response to phenol-soluble modulin, a factor secreted by Staphylococcus epidermidis. The TLR2-mediated response to this modulin was enhanced by TLR6 but inhibited by TLR1, indicating a functional interaction between these receptors. We also demonstrate that a response to phenol-soluble modulin mediated by TLR2 and TLR6 was more refractory to inhibition by TLR1 than one mediated by TLR2 alone.
RANTES Potentiates Antigen-Specific Mucosal Immune Responses
Authors: Lillard James W. , Boyaka Prosper N. , Taub Dennis D. , McGhee Jerry R.
Published: April 2014

RANTES is produced by lymphoid and epithelial cells of the mucosa in response to various external stimuli and is chemotactic for lymphocytes. The role of RANTES in adaptive mucosal immunity has not been studied. To better elucidate the role of this chemokine, we have characterized the effects of RANTES on mucosal and systemic immune responses to nasally coadministered OVA. RANTES enhanced Ag-specific serum Ab responses, inducing predominately anti-OVA IgG2a and IgG3 followed by IgG1 and IgG2b subclass Ab responses. RANTES also increased Ag-specific Ab titers in mucosal secretions and these Ab responses were associated with increased numbers of Ab-forming cells, derived from mucosal and systemic compartments. Splenic and mucosally derived CD4+ T cells of RANTES-treated mice displayed higher Ag-specific proliferative responses and IFN-γ, IL-2, IL-5, and IL-6 production than control groups receiving OVA alone. In vitro, RANTES up-regulated the expression of CD28, CD40 ligand, and IL-12R by Ag-activated primary T cells from DO11.10 (OVA-specific TCR-transgenic) mice and by resting T cells in a dose-dependent fashion. These studies suggest that RANTES can enhance mucosal and systemic humoral Ab responses through help provided by Th1- and select Th2-type cytokines as well as through the induction of costimulatory molecule and cytokine receptor expression on T lymphocytes. These effects could serve as a link between the initial innate signals of the host and the adaptive immune system.
Negative Regulation of Expression and Function of FcγRIII by CD3ζ in Murine NK Cells
Authors: Arase Hisashi , Suenaga Tadahiro , Arase Noriko , Kimura Yoshimitsu , Ito Katsuhiko , Shiina Ritsuko , Ohno Hiroshi , Saito Takashi
Published: April 2014

FcγRIII is involved in Ab-dependent cell-mediated cytotoxicity (ADCC) and cytokine production by NK cells. Signaling and expression of FcγRIII are dependent on FcRγ. Although NK cells express not only FcRγ but also CD3ζ, the role of CD3ζ in NK cell function remains unclear. Here, we found that the expression of FcγRIII on NK cells from CD3ζ-deficient mice is unexpectedly up-regulated compared with that on cells from normal mice. Furthermore, ADCC and IFN-γ production upon FcγRIII-cross-linking by NK cells from CD3ζ-deficient mice were also up-regulated. Up-regulation of the surface expression of FcγRIII on CD3ζ-deficient NK cells is not mediated by transcriptional augmentation of either FcγRIII or FcRγ gene because there was no significant difference in the expression of mRNA for FcγRIII and FcRγ. Transfection of CD3ζ into a cell line expressing FcγRIII and FcRγ induced a decrease in the cell surface expression of FcγRIII. These findings reveal a negative regulatory role of CD3ζ in FcγRIII-mediated function of murine NK cells.
The Transient Expression of C-C Chemokine Receptor 8 in Thymus Identifies a Thymocyte Subset Committed to Become CD4+ Single-Positive T Cells
Authors: Kremer Leonor , Carramolino Laura , Goya Íñigo , Zaballos Ángel , Gutiérrez Julio , del Carmen Moreno-Ortiz María , Martínez-A. Carlos , Márquez Gabriel
Published: April 2014

Developing T cells journey through the different thymic microenvironments while receiving signals that eventually will allow some of them to become mature naive T cells exported to the periphery. This maturation can be visualized by the phenotype of the developing cells. CCR8 is a β-chemokine receptor preferentially expressed in the thymus. We have developed 8F4, an anti-mouse CCR8 mAb that is able to neutralize the ligand-induced activation of CCR8, and used it to characterize the CCR8 protein expression in the different thymocyte subsets. Taking into account the intrathymic lineage relationships, our data showed that CCR8 expression in thymus followed two transient waves along T cell maturation. The first one took place in CD4− CD8− double-negative thymocytes, which showed a low CCR8 expression, and the second wave occurred after TCR activation by the Ag-dependent positive selection in CD4+ CD8+ double-positive cells. From that maturation stage, CCR8 expression gradually increased as the CD4+ cell differentiation proceeded, reaching a maximum at the CD4+ CD8− single-positive stage. These CD4+ cells expressing CCR8 were also CD69high CD62Llow thymocytes, suggesting that they still needed to undergo some differentiation step before becoming functionally competent naive T cells ready to be exported from the thymus. Interestingly, no significant amounts of CCR8 protein were detectable in CD4− CD8+ thymocytes. Our data showing a clear regulation of the CCR8 protein in thymus suggest a relevant role for CCR8 in this lymphoid organ, and identify CCR8 as a possible marker of thymocyte subsets recently committed to the CD4+ lineage.
Bi-Directional Activation Between Human Airway Smooth Muscle Cells and T Lymphocytes: Role in Induction of Altered Airway Responsiveness
Authors: Hakonarson Hakon , Kim Cecilia , Whelan Russel , Campbell Donald , Grunstein Michael M.
Published: April 2014

Because both T lymphocyte and airway smooth muscle (ASM) cell activation are events fundamentally implicated in the pathobiology of asthma, this study tested the hypothesis that cooperative intercellular signaling between activated T cells and ASM cells mediates proasthmatic changes in ASM responsiveness. Contrasting the lack of effect of resting human T cells, anti-CD3-activated T cells were found to adhere to the surface of naive human ASM cells, increase ASM CD25 cell surface expression, and induce increased constrictor responsiveness to acetylcholine and impaired relaxation responsiveness to isoproterenol in isolated rabbit ASM tissues. Comparably, exposure of resting T cells to ASM cells prestimulated with IgE immune complexes reciprocally elicited T cell adhesion to ASM cells and up-regulated T cell expression of CD25. Extended studies demonstrated that: 1) ASM cells express mRNAs and proteins for the cell adhesion molecules (CAMs)/costimulatory molecules, CD40, CD40L, CD80, CD86, ICAM-1 (CD54), and LFA-1 (CD11a/CD18); 2) apart from LFA-1, ASM cell surface expression of the latter molecules is up-regulated in the presence of activated T cells; and 3) pretreatment of ASM cells and tissues with mAbs directed either against CD11a or the combination of CD40 and CD86 completely abrogated both the activated T cell-induced changes in expression of the above CAMs/costimulatory molecules in ASM cells and altered ASM tissue responsiveness. Collectively, these observations identify the presence of bi-directional cross-talk between activated T cells and ASM cells that involves coligation of specific CAMs/costimulatory molecules, and this cooperative intercellular signaling mediates the induction of proasthmatic-like changes in ASM responsiveness.
Down-Regulation of p27Kip1 Expression Is Required for Development and Function of T Cells
Authors: Tsukiyama Tadasuke , Ishida Noriko , Shirane Michiko , Minamishima Yohji A. , Hatakeyama Shigetsugu , Kitagawa Masatoshi , Nakayama Keiko , Nakayama Kei-ichi
Published: April 2014

The proliferation of T cells is regulated in a development-dependent manner, but it has been unclear whether proliferation is essential for T cell differentiation. The cyclin-dependent kinase inhibitor p27Kip1 is abundant throughout development in cells of the T cell lineage, with the exception of late stage CD4−CD8− thymocytes and activated mature T cells, both of which show a high rate of proliferation. The role of down-regulation of p27Kip1 expression in T cell development and function has now been investigated by the generation and characterization of three strains of p27 transgenic mice that express the transgene at various levels specifically in the T cell lineage. The numbers of thymocytes at CD4+CD8+, CD4+CD8−, and CD4−CD8+ stages of development as well as those of mature T cells in peripheral lymphoid tissues were reduced in transgenic mice in a manner dependent on the level of p27Kip1 expression. The development of thymocytes in the transgenic strain in which p27Kip1 is most abundant (p27-Tghigh mice) appeared to be blocked at the CD4−CD8−CD25+CD44low stage. Peripheral T cells from p27-Tghigh mice exhibited a reduced ability to proliferate in response to mitogenic stimulation compared with wild-type T cells. Moreover, Ag-induced formation of germinal centers and Ig production were defective in p27-Tghigh mice. These results suggest that down-regulation of p27Kip1 expression is required for the development, proliferation, and immunoresponsiveness of T cells.
Autoantigenic HCgp39 Epitopes Are Presented by the HLA-DM-Dependent Presentation Pathway in Human B Cells
Authors: Patil Namrata S. , Hall Frances C. , Drover Sheila , Spurrell David R. , Bos Ebo , Cope Andrew P. , Sonderstrup Grete , Mellins Elizabeth D.
Published: April 2014

It is hypothesized that autoimmune diseases manifest when tolerance to self-Ags fails. One possible mechanism to break tolerance is presentation of self-Ag in an altered form. Most Ags are presented by APCs via the traditional presentation pathway that includes “epitope editing” by intracellular HLA-DM, a molecule that selects for stable MHC-peptide complexes. We were interested in testing the hypothesis that autoreactive MHC-peptide complexes may reach the cell surface by an alternate pathway without being edited by HLA-DM. We selected a cartilage autoantigen human cartilage glycoprotein 39 to which T cell responses are observed in rheumatoid arthritis (RA) patients and some DR*04 healthy subjects. RA is genetically associated with certain DRB1 alleles, including DRB1*0401 but closely related allele DRB1*0402 is either neutral or mildly protective with respect to RA. We generated human B lymphoblastoid cell line cells expressing DR*0401 or DR*0402 in the presence or absence of intracellular HLA-DM and assessed their ability to present a candidate autoantigen, human cartilage glycoprotein 39. Our results show that the presence of intracellular HLA-DM is critical for presentation of this autoantigen to CD4+ T cell hybridomas generated from DR*04-transgenic mice. Presentation of an autoantigen by the traditional HLA-DM-dependent pathway has implications for Ag presentation events in RA.
Role of Two Conserved Cytoplasmic Threonine Residues (T410 and T412) in CD5 Signaling
Authors: Vilà Josep M. , Calvo Javier , Places Lourdes , Padilla Olga , Arman Mònica , Gimferrer Idoia , Aussel Claude , Vives Jordi , Lozano Francisco
Published: April 2014

CD5 is a transmembrane coreceptor that modulates activation and differentiation signals mediated by the Ag-specific receptor present on both T and B1a lymphocytes. CD5 lacks intrinsic catalytic activity, and its immunomodulatory properties result from intracellular interactions mediated by the CD5 cytoplasmic tail. The nature of these interactions is currently a matter of investigation. Here, we present a selective mutagenesis analysis of two conserved threonine residues (T410 and T412) located at the membrane-proximal cytoplasmic region of CD5. These residues are contained within consensus phosphorylation motifs for protein kinase C and are shown here to be critical for in vivo protein kinase C-mediated phosphorylation of CD5. Functional studies revealed that the integrity of T410 and T412 is also critical for CD5-mediated phosphatidylcholine-specific phospholipase C (PC-PLC) activation and phorbol ester-mediated inhibition of Ab-induced internalization of CD5. These results strongly argue in favor of a role for T410 and T412 in the signaling mediated by CD5.
NK T Cell-Derived IL-10 Is Essential for the Differentiation of Antigen-Specific T Regulatory Cells in Systemic Tolerance
Authors: Sonoda Koh-Hei , Faunce Douglas E. , Taniguchi Masaru , Exley Mark , Balk Steven , Stein-Streilein Joan
Published: April 2014

In a model of systemic tolerance called Anterior Chamber-Associated Immune Deviation (ACAID), the differentiation of the T regulatory (Tr) cells depends on NK T cells and occurs in the spleen. We now show that the CD1d-reactive NK T cell subpopulation, required for development of systemic tolerance, expresses the invariant Vα14Jα281 TCR because Jα281 knockout (KO) mice were unable to generate Ag-specific Tr cells and ACAID. The mechanism for NK T cell-dependent differentiation of Ag-specific Tr cells mediating systemic tolerance was studied by defining the cytokine profiles in heterogeneous and enriched NK T spleen cells. In contrast to there being no differences in most regulatory cytokine mRNAs, both mRNA and protein for IL-10 were increased in splenic NK T cells of anterior chamber (a.c.)-inoculated mice. However, IL-10 mRNA was not increased in spleens after i.v. inoculation. Finally, NK T cells from wild-type (WT) mice, but not from IL-10 KO mice, reconstituted the ACAID inducing ability in Jα281 KO mice. Thus, NK T cell-derived IL-10 is critical for the generation of the Ag-specific Tr cells and systemic tolerance induced to eye-inoculated Ags.
Characterization of Heat Shock Protein 110 and Glucose-Regulated Protein 170 as Cancer Vaccines and the Effect of Fever-Range Hyperthermia on Vaccine Activity
Authors: Wang Xiang-Yang , Kazim Latif , Repasky Elizabeth A. , Subjeck John R.
Published: April 2014

Several studies have confirmed that certain stress proteins can function as potent vaccines against a specific cancer when purified from the same tumor. Recent studies of two long-recognized but unstudied stress proteins, heat shock protein (hsp) 110 and glucose-regulated protein (grp) 170, have shown them to be efficient peptide chain-binding proteins. The present investigation examines the vaccine potential of hsp110 and grp170. First, it is shown that prior vaccination with hsp110 or grp170 purified from methylcholanthrene-induced fibrosarcoma caused complete regression of the tumor. In a second tumor model, hsp110 or grp170 purified from Colon 26 tumors led to a significant growth inhibition of this tumor. In addition, hsp110 or grp170 immunization significantly extended the life span of Colon 26 tumor-bearing mice when applied after tumor transplantation. A tumor-specific cytotoxic T lymphocyte response developed in the mice immunized with tumor-derived hsp110 or grp170. Furthermore, treatments of the mice with bone marrow-derived dendritic cells pulsed with these two proteins from tumor also elicited a strong antitumor response. Last, we showed that mild, fever-like hyperthermic conditions enhance the vaccine efficiency of hsp110 as well as heat shock cognate 70, but not grp170. These studies indicate that hsp110 and grp170 can be used in hsp-based cancer immunotherapy, that Ag-presenting dendritic cells can be used to mediate this therapeutic approach, and that fever-level hyperthermia can significantly enhance the vaccine efficiency of hsps.
Identification and Characterization of T Cell-Stimulating Antigens from Leishmania by CD4 T Cell Expression Cloning
Authors: Probst Peter , Stromberg Erika , Ghalib Hashim W. , Mozel Michelle , Badaro Roberto , Reed Steven G. , Webb John R.
Published: April 2014

Persistent immunity against Leishmania infections in humans is mediated predominantly by CD4+ T cells of the Th1 phenotype. Herein we report the expression cloning of eight Leishmania Ags using parasite-specific T cell lines derived from an immune donor. The Ags identified by this technique include the flagellar proteins α- and β-tubulin, histone H2b, ribosomal protein S4, malate dehydrogenase, and elongation factor 2, as well as two novel parasite proteins. None of these proteins have been previously reported as T cell-stimulating Ags from Leishmania. β-tubulin-specific T cell clones generated against Leishmania major amastigotes responded to Leishmania-infected macrophages and dendritic cells. IFN-γ enzyme-linked immunospot analysis demonstrated the presence of T cells specific for several of these Ags in PBMC from self-healing cutaneous leishmaniasis patients infected with either Leishmania tropica or L. major. The responses elicited by Leishmania histone H2b were particularly striking in terms of frequency of histone-specific T cells in PBMC (1 T cell of 6000 PBMC) as well as the percentage of responding donors (86%, 6 of 7). Ags identified by T cells from immune donors might constitute potential vaccine candidates for leishmaniasis.
Relative Resistance in the Development of T Cell Anergy in CD4+ T Cells from Simian Immunodeficiency Virus Disease-Resistant Sooty Mangabeys
Authors: Bostik Pavel , Mayne Ann E. , Villinger Francois , Greenberg Kenneth P. , Powell Jonathan D. , Ansari Aftab A.
Published: April 2014

Despite high viral loads, T cells from sooty mangabey (SM) monkeys that are naturally infected with SIV but remain clinically asymptomatic, proliferate and demonstrate normal Ag-specific memory recall CD4+ T cell responses. In contrast, CD4+ T cells from rhesus macaques (RM) experimentally infected with SIV lose Ag-specific memory recall responses and develop immunological anergy. To elucidate the mechanisms for these distinct outcomes of lentiviral infection, highly enriched alloreactive CD4+ T cells from humans, RM, and SM were anergized by TCR-only stimulation (signal 1 alone) and subsequently challenged with anti-CD3/anti-CD28 Abs (signals 1 + 2). Whereas alloreactive CD4+T cells from humans and RM became anergized, surprisingly, CD4+ T cells from SM showed marked proliferation and IL-2 synthesis after restimulation. This resistance to undergo anergy was not secondary to a global deficiency in anergy induction of CD4+ T cells from SM since incubation of CD4+ T cells with anti-CD3 alone in the presence of rapamycin readily induced anergy in these cells. The resistance to undergo anergy was reasoned to be due to the ability of CD4+ T cells from SM to synthesize IL-2 when incubated with anti-CD3 alone. Analysis of phosphorylated kinases involved in T cell activation showed that the activation of CD4+ T cells by signal 1 in SM elicited a pattern of response that required both signals 1 + 2 in humans and RM. This function of CD4+ T cells from SM may contribute to the resistance of this species to SIV-induced disease.
Reduced Incidence and Severity of Collagen-Induced Arthritis in Mice Lacking IL-18
Authors: Wei Xiao-qing , Leung Bernard P. , Arthur Helen M. L. , McInnes Iain B. , Liew Foo Y.
Published: April 2014

We have recently reported the presence and a potential proinflammatory role of IL-18 in the synovium of patients with rheumatoid arthritis. To obtain direct evidence that IL-18 plays an influential role in articular inflammation, we investigated the development of collagen-induced arthritis in a strain of mice lacking IL-18 (IL-18−/−) of DBA/1 background. IL-18−/− mice developed markedly reduced incidence of arthritis compared with heterozygous or wild-type mice. Of the IL-18−/− mice that developed arthritis, the severity of the disease was significantly reduced compared with the intact mice. This was accompanied by reduced articular inflammation and destruction evident on histology. IL-18−/− mice also had significantly reduced Ag-specific proliferation and proinflammatory cytokine (IFN-γ, TNF-α, IL-6, and IL-12) production by spleen and lymph node cells in response to bovine type II collagen (CII) in vitro compared with wild-type mice, paralleled in vivo by a significant reduction in serum anti-CII IgG2a Ab level. Treatment with rIL-18 completely reversed the disease of the IL-18−/− mice to that of the wild-type mice. These data directly demonstrate a pivotal role of IL-18 in the development of inflammatory arthritis and suggest that antagonists to IL-18 may have therapeutic potential in rheumatic diseases.
Differential Modulation of Stimulatory and Inhibitory Fcγ Receptors on Human Monocytes by Th1 and Th2 Cytokines
Authors: Pricop Luminita , Redecha Patricia , Teillaud Jean-Luc , Frey Jürgen , Fridman Wolf H. , Sautès-Fridman Catherine , Salmon Jane E.
Published: April 2014

Immune complex-mediated inflammatory responses are initiated by FcγR on phagocytes. We report in this study that an inhibitory receptor, FcγRIIb2, is expressed on circulating human monocytes, and when co-cross-linked with stimulatory FcγR it down-regulates effector function. FcγRIIb2 expression is increased by IL-4 and decreased by IFN-γ, in contrast to the activating receptor, FcγRIIa, which is increased by IFN-γ and decreased by IL-4. Thus, Th1 and Th2 cytokines differentially regulate the opposing FcγR systems, altering the balance of activating and inhibiting FcγR. The detection and cytokine modulation of FcγRIIb2 in human myeloid cells provide evidence of a negative regulator of immune complex-mediated responses in human phagocytes and offer a new approach to limit Ab-triggered inflammation in autoimmune disease.
Analysis of the Roles of ICAM-1 in Neutrophil Transmigration Using a Reconstituted Mammalian Cell Expression Model: Implication of ICAM-1 Cytoplasmic Domain and Rho-Dependent Signaling Pathway
Authors: Sans Emmanuelle , Delachanal Elisabeth , Duperray Alain
Published: April 2014

Interaction between ICAM-1 (CD54) and fibrinogen (fg) has been shown to enhance leukocyte adhesion, but its specific role in the process of migration across endothelial cell junctions remains unclear. To overcome the problem of multiple adhesion receptors found on endothelial cells, we have engineered stable Chinese hamster ovary cell lines expressing ICAM-1 (Chinese hamster ovary ICAM-1). The transfection of ICAM-1 alone in these cells is sufficient to recapitulate the entire process of neutrophil adhesion and transmigration. This phenomenon was mediated by fg-ICAM-1 interactions, as depletion of fg, as well as the use of an Ab that specifically inhibits ICAM-1-fg interaction (2D5), completely abolished the effect of ICAM-1 expression on PMN transmigration. In addition, this ICAM-1-mediated transmigration is clearly dependent on the occurrence of fg-ICAM-1 interactions on the monolayer, and not on neutrophils, as the preincubation of the PMN with the mAb was ineffective. Furthermore, PMN transmigration, but not adhesion, is totally abolished when the ICAM-1 cytoplasmic domain is deleted, indicating that signaling inside the cell is required to mediate the fg-ICAM-1 effect on transmigration. Using a specific inhibitor of the small GTP-binding protein Rho, we have obtained evidence that this signaling cascade is involved. Thus, our results clearly show that ICAM-1 plays a key role in the migration of leukocytes across cell junctions, and indicate that this phenomenon is not a direct consequence of the enhanced adhesion mediated by the expression of ICAM-1.
The Chemokine RANTES Is a Crucial Mediator of the Progression from Acute to Chronic Colitis in the Rat
Authors: Ajuebor Maureen N. , Hogaboam Cory M. , Kunkel Steven L. , Proudfoot Amanda E. I. , Wallace John L.
Published: April 2014

Chemokines have well characterized proinflammatory actions, including the ability to induce extravasation of leukocytes that participate in chronic inflammation. In this study, we evaluated the role of a C-C chemokine, RANTES, in the chronic phase of a rat model of colitis. Colitis was induced by intracolonic administration of trinitrobenzene sulfonic acid. At various timepoints thereafter (2 h to 14 days), colonic tissue levels of several chemokines were measured. Unlike the expression of monocyte chemoattractant protein-1, macrophage inflammatory protein-2, and cytokine-induced neutrophil chemoattractant, the expression of RANTES was significantly elevated during the chronic phase of colitis (≥7 days after induction). Colonic RANTES mRNA expression was also significantly elevated during the chronic phase of colitis. The numbers of macrophages and monocytes in the colonic mucosa increased substantially during the chronic phase, as did expression of two of the receptors (CCR1 and CCR5) to which RANTES is known to bind. Administration on days 7 through 14 after trinitrobenzene sulfonic acid administration of a CCR1/CCR5 receptor antagonist, Met-RANTES, resulted in a significant reduction of both macroscopic and microscopic colonic damage, as well as reducing the recruitment into the colon of monocytes, mast cells, and neutrophils. In some rats, treatment with Met-RANTES resulted in a near-complete resolution of colonic damage and inflammation. These results suggest a crucial role of RANTES in the progression from acute to chronic inflammation in a rat model of colitis.