bluuebunny/crossref_metadata_2025 huggingface dataset:
Cutting Edge: The T Cell Chemoattractant IFN-Inducible Protein 10 Is Essential in Host Defense Against Viral-Induced Neurologic Disease
Authors: Liu Michael T. , Chen Benjamin P. , Oertel Patricia , Buchmeier Michael J. , Armstrong David , Hamilton Thomas A. , Lane Thomas E.
Published: April 2014

The contribution of the T cell chemoattractant chemokine IFN-inducible protein 10 (IP-10) in host defense following viral infection of the CNS was examined. IP-10 is expressed by astrocytes during acute encephalomyelitis in mouse hepatitis virus-infected mice, and the majority of T lymphocytes infiltrating into the CNS expressed the IP-10 receptor CXCR3. Treatment of mice with anti-IP-10 antisera led to increased mortality and delayed viral clearance from the CNS as compared with control mice. Further, administration of anti-IP-10 led to a >70% reduction (p ≤ 0.001) in CD4+ and CD8+ T lymphocyte infiltration into the CNS, which correlated with decreased (p ≤ 0.01) levels of IFN-γ. These data indicate that IP-10 functions as a sentinel molecule in host defense and is essential in the development of a protective Th1 response against viral infection of the CNS.
Cutting Edge: CD7 Delivers a Pro-Apoptotic Signal During Galectin-1-Induced T Cell Death
Authors: Pace Karen E. , Hahn Hejin P. , Pang Mabel , Nguyen Julie T. , Baum Linda G.
Published: April 2014

Galectin-1, an endogenous lectin expressed in lymphoid organs and immune-privileged sites, induces death of human and murine thymocytes and T cells. Galectin-1 binds to several glycoproteins on the T cell surface, including CD7. However, the T cell surface glycoprotein receptors responsible for delivering the galectin-1 death signal have not been identified. We show that CD7 is required for galectin-1-mediated death. This demonstrates a novel function for CD7 as a death trigger and identifies galectin-1/CD7 as a new biologic death signaling pair.
Cutting Edge: Outer Membrane Protein A (OmpA) Binds to and Activates Human Macrophages
Authors: Soulas Caroline , Baussant Thierry , Aubry Jean-Pierre , Delneste Yves , Barillat Nicolas , Caron Gersende , Renno Toufic , Bonnefoy Jean-Yves , Jeannin Pascale
Published: April 2014

Outer membrane protein (Omp)A is highly represented and conserved in the Enterobacteriaceae family. Using a recombinant OmpA from Klebsiella pneumoniae (P40), we have analyzed the interaction between OmpA and macrophages. We report that Alexa488-labeled P40 binds (at 4°C) to murine and human macrophages in a dose-dependent manner and is rapidly internalized (at 37°C). No binding or internalization of the Alexa488-labeled glycophorin A control protein is observed under the same conditions. Furthermore, P40 up-regulates the production of IL-1β, IL-8, IL-10, IL-12, and TNF-α by human macrophages and of NO by the RAW 264.7 murine macrophage cell line. P40 also synergizes with IFN-γ and suboptimal concentrations of LPS to up-regulate the production of these mediators. In conclusion, P40 binds to and activates macrophages. These data suggest that recognition of OmpA by macrophages may be an initiating event in the antibacterial host response.
Stress Renders T Cell Blasts Sensitive to Killing by Activated Syngeneic NK Cells
Authors: Rabinovich Brian A. , Shannon John , Su Ruey-Chyi , Miller Richard G.
Published: April 2014

Exposure of primary T cell blasts to stress in the forms of heat, hydrogen peroxide, or high-density growth conditions resulted in a state of enhanced susceptibility to killing by syngeneic IL-2-activated NK cells or lymphokine-activated killer cells, but not to killing by CTL. Cytotoxicity was perforin mediated and was not due to decreased target expression of total MHC class I. The levels of stress used had little effect on cell viability. For thermal stress, sensitization increased with temperature, required a minimum exposure time, and disappeared when cells were given a long enough recovery time. Our data support a model that predicts that activated NK cells play a role in the immunosurveillance of nontransformed stressed cells in normal animals.
Break of T Cell Ignorance to a Viral Antigen in the Liver Induces Hepatitis
Authors: Voehringer David , Blaser Claudine , Grawitz Andrea Busse , Chisari Francis V. , Buerki Kurt , Pircher Hanspeter
Published: April 2014

To study peripheral tolerance of CD8 T cells to a classically MHC-restricted peptide Ag expressed in hepatocytes, ALB1 transgenic (tg) mice expressing the CTL epitope GP33 of the lymphocytic choriomeningitis virus glycoprotein under control of the mouse albumin promoter were generated. ALB1 mice exclusively expressed the GP33 transgene in the liver and, at a 100- to 1000-fold lower level, in the thymus. TCR-tg mice specific for the GP33 epitope were used to directly follow GP33-specific T cells in vivo. These experiments revealed that 1) thymic expression of the GP33 transgene led to incomplete central deletion of TCR-tg cells; and 2) peripheral TCR-tg cells in ALB1 mice ignored the GP33 transgene expressed in hepatocytes. Ignorance of adoptively transferred TCR-tg cells in ALB1 mice was broken by infection with lymphocytic choriomeningitis virus, leading to induction of hepatitis in ALB1, but not in control, mice. Taken together, we have established a novel model of virus-induced CD8 T cell-mediated autoimmune hepatitis in mice and demonstrate that naive CD8 T cells may ignore Ags expressed in the liver.
Activation Via Multiple Signaling Pathways Induces Down-Regulation of Platelet-Activating Factor Receptors on Human B Lymphocytes
Authors: Zhuang Qianli , Bastien Yolande , Mazer Bruce D.
Published: April 2014

Platelet-activating factor receptor (PAFR) has been identified in B cell lines and primary human B cells, but the regulation of PAFR during B cell activation has not been completely elucidated. In the present study, we have investigated the effects of B cell activation on PAFR binding parameters, PAFR mRNA and PAF-triggered intracellular calcium mobilization. The human B lymphoid cell line LA350 was shown to exhibit high levels of PAFR (48,550 ± 4,310 sites/cell) as determined by radio-ligand binding assay with PAFR antagonist [3H]WEB2086. Treatment with phorbol 12,13-dibutyrate caused a biphasic reduction of PAFR binding. The early phase was inhibited by the protein kinase C inhibitor bisindolylmaleimide I (BIM), whereas the late phase was not blocked by BIM, protein tyrosine kinase inhibitor genistein, or the mitogen-activated protein kinase/extracellular signal-related kinase inhibitor PD98059. However, staurosporine, a broad-spectrum protein kinase inhibitor, completely inhibited the late phase down-regulation. Ionomycin also decreased [3H]WEB2086 binding sites, whereas the combination of PDB and ionomycin induced a greater reduction than either agent alone. Cross-linking of B cell receptor by anti-IgM Ab also induced down-regulation of PAFR, which was abolished by genistein or PD98059, but not by BIM or staurosporine. The decrease in surface PAFR number was closely paralleled by the reduction in PAFR mRNA both in LA350 cells and human tonsillar B cells, and was associated with decreased response to PAF indicated by decreased intracellular calcium mobilization. These data show that multiple signaling pathways are involved in down-regulating PAFR expression during B cell activation and development.
Survival and Homeostatic Proliferation of Naive Peripheral CD4+ T Cells in the Absence of Self Peptide:MHC Complexes
Authors: Clarke Sally R. M. , Rudensky Alexander Y.
Published: April 2014

TCR-self peptide:MHC interactions play a critical role in thymic positive selection, yet relatively little is known of their function in the periphery. It has been suggested that continued contact with selecting MHC molecules is necessary for long-term peripheral maintenance of naive T cells. More recent studies have also demonstrated a role for specific self peptide:MHC complexes in the homeostatic expansion of naive T cells in lymphopenic mice. Our examination of these processes revealed that, whereas self class II MHC molecules do have a modest effect on long-term survival of individual CD4+ T cells, interactions with specific TCR ligands are not required for peripheral naive CD4+ T cell maintenance. In contrast, selective engagement of TCRs by self-peptide:MHC complexes does promote proliferation of CD4+ T cells under severe lymphopenic conditions, and this division is associated with an activation marker phenotype that is different from that induced by antigenic stimulation. Importantly, however, the ability of naive T cells to divide in response to homeostatic stimuli does not appear to be stringently dependent on TCR-self peptide:MHC interactions. Therefore, these results show that the factors regulating survival and homeostatic expansion of naive T cells in the periphery are not identical. In addition, we provide evidence for a novel form of T cell proliferation that can occur independently of TCR signaling and suggest that this reflects another mechanism regulating homeostatic T cell expansion.
Binding Kinetics, Structure-Activity Relationship, and Biotransformation of the Complement Inhibitor Compstatin
Authors: Sahu Arvind , Soulika Athena M. , Morikis Dimitrios , Spruce Lynn , Moore William T. , Lambris John D.
Published: April 2014

We have previously identified a 13-residue cyclic peptide, Compstatin, that binds to complement component C3 and inhibits complement activation. Herein, we describe the binding kinetics, structure-activity relationship, and biotransformation of Compstatin. Biomolecular interaction analysis using surface-plasmon resonance showed that Compstatin bound to native C3 and its fragments C3b and C3c, but not C3d. While binding of Compstatin to native C3 was biphasic, binding to C3b and C3c followed the 1:1 Langmuir binding model; the affinities of Compstatin for C3b and C3c were 22- and 74-fold lower, respectively, than that of native C3. Analysis of Compstatin analogs synthesized for structure-function studies indicated that 1) the 11-membered ring between disulfide-linked Cys2-Cys12 constitutes a minimal structure required for optimal activity; 2) retro-inverso isomerization results in loss of inhibitory activity; and 3) some residues of the type I β-turn segment also interact with C3. In vitro studies of Compstatin in human blood indicated that a major pathway of biotransformation was the removal of Ile1, which could be blocked by N-acetylation of the peptide. These findings indicate that acetylated Compstatin is stable against enzymatic degradation and that the type I β-turn segment is not only critical for preservation of the conformational stability, but also involved in intermolecular recognition.
Apoptosis Induced by TGF-β1 in Burkitt’s Lymphoma Cells Is Caspase 8 Dependent But Is Death Receptor Independent
Authors: Inman Gareth J. , Allday Martin J.
Published: April 2014

TGF-β is a potent inducer of apoptosis in many Burkitt’s lymphoma (BL) cell lines. In this study, we characterize this apoptotic process in the EBV-negative BL41 cell line. Induction of apoptosis was detected as early as 8 h after TGF-β treatment, as assayed by TUNEL and poly(ADP-ribose) polymerase cleavage. FACS analysis demonstrates that this proceeds predominately from the G1, but also from the G2/M phases of the cell cycle. We observed no early detectable changes in the steady-state levels of Bcl-2 and several of its family members after TGF-β treatment. We detected cleavage of caspases 2, 3, 7, 8, and 9 into their active subunits. Consistent with the involvement of these enzymes in TGF-β-mediated apoptosis, the broad spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(Ome)-flouromethylketone (ZVAD-fmk) blocked TGF-β-induced apoptosis and revealed a G1 arrest in treated cells. Use of specific caspase inhibitors revealed that the induction of apoptosis is caspase 8 dependent, but caspase 3 independent. Activation of caspase 8 has been shown to be a critical event in death receptor-mediated apoptosis. However, TGF-β treatment of BL41 cells was found not to affect the cell surface expression of Fas, TNF-R1, DR3, DR4, or DR5, or the steady-state expression levels of Fas ligand, TNF-R1, DR3, DR4, and DR5. Furthermore, blocking experiments indicated that TGF-β-mediated apoptosis is not dependent on Fas ligand, TNF-α, tumor necrosis-like apoptosis-inducing ligand, or TNF-like weak inducer of apoptosis signaling. Therefore, it appears that TGF-β induces apoptosis in BL cell lines via caspase 8 in a death receptor-independent fashion.
Two Clusters of Acidic Amino Acids Near the NH2 Terminus of Complement Component C4 α′-Chain Are Important for C2 Binding
Authors: Pan Qun , Ebanks Roger O. , Isenman David E.
Published: April 2014

Previous work has indicated a role for the NH2-terminal segment of the C3 α′-chain in the binding interactions of C3b with a number of its protein ligands. In particular, we have identified two clusters of acidic residues, namely, E736 and E737 and to a lesser extent D730 and E731, as being important in the binding of C3b to factor B and complement receptor 1 and the binding of iC3b to complement receptor 3. Whereas human C3 and C4 have an overall sequence identity of 29%, over a segment near the NH2 termini of their respective α′-chains the sequence identity is 56% (70% chemical similarity). Given the functional similarity between the C4b-C2 and C3b-B interactions in the respective formation of the classical and alternative pathway C3 convertases, as well as the sequence conservation of two acidic clusters, we hypothesized that residues 744EED and 749DEDD within the NH2-terminal segment of the C4 α′-chain would mediate in part the binding of C2 to C4b. We tested this hypothesis using three independent approaches. Site-directed mutagenesis experiments revealed that replacing subsets of the charged residues by their isosteric amides within either acidic cluster resulted in molecules having reduced C2 binding activity. Moreover, a synthetic peptide (C4 residues 740–756) encompassing the two acidic clusters was a specific inhibitor of the binding of C2 to red cell-associated C4b. Finally, Ab raised against the above peptide was able to block the interaction between red cell-associated C4b and fluid phase C2. Taken together, these results strongly suggest that the NH2-terminal acidic residue-rich segment of C4 α′-chain contributes importantly to the interaction of C4b with C2.
Pigs Express Multiple Forms of Decay-Accelerating Factor (CD55), All of Which Contain Only Three Short Consensus Repeats
Authors: Pérez de la Lastra J. M. , Harris C. L. , Hinchliffe S. J. , Holt D. S. , Rushmere N. K. , Morgan B. Paul
Published: April 2014

We report the cloning of cDNAs encoding multiple isoforms of the pig analogue of human decay-accelerating factor (DAF; CD55). Screening of a pig muscle cDNA library using a human DAF probe identified a single clone that encoded a DAF-like molecule comprising three short consensus repeats (SCR) homologous with the amino-terminal three SCR in human DAF, a serine/threonine-rich (ST) region, and sequence compatible with a transmembrane domain and cytoplasmic tail. Northern blot and RT-PCR analysis showed that pig DAF was expressed in a wide range of tissues. Additional isoforms of DAF were sought using RT-PCR and 3′-rapid amplification of cDNA ends followed by sequencing. Isoforms containing a GPI anchor and with differing lengths of ST region were identified; no isoform containing a fourth SCR was found. Cloning of the GPI-anchored isoform from granulocytes confirmed that it was identical with the original transmembrane isoform through the three SCR and first portion of ST and was derived from a frame shift caused by splicing out 176 bp of sequence. A panel of mAbs was generated and used to analyze the distribution and anchoring of pig DAF in circulating cells. Pig DAF was expressed on all circulating cells and was transmembrane anchored on erythrocytes, but completely or partially GPI anchored on granulocytes and mononuclear cells. The transmembrane isoform of pig DAF was expressed on Chinese hamster ovary cells and was shown to affect regulatory activity for the classical pathway of human complement, but was only marginally active against pig serum.
Mycobacterium tuberculosisInfection in Complement Receptor 3-Deficient Mice
Authors: Hu Chenggang , Mayadas-Norton Tanya , Tanaka Katie , Chan John , Salgame Padmini
Published: April 2014

Complement receptor type 3 (CR3) present on macrophages is used by Mycobacterium tuberculosis as one of its major phagocytic receptors. In this study, we examined the in vivo significance of CR3-mediated phagocytosis on the pathogenesis of disease caused by M. tuberculosis. The outcome of tuberculous infection in mice deficient in the CD11b subunit of CR3 (CR3−/−) on a mixed 129SV and C57BL background and control wild-type counterparts was comparable with respect to survival, bacterial burden, granulomatous lesion development, and cytokine expression in the spleen and lungs. M. tuberculosis infection was also examined in CR3−/− mice on C57BL/6 and BALB/c backgrounds and was found to be similar. In conclusion, our results suggest that in the absence of CR3, M. tuberculosis is able to gain entry into host cells via alternative phagocytic receptors and establish infection. The data also indicate that absence of CR3 does not alter disease course in either the relatively resistant C57BL/6 or the relatively susceptible BALB/c strains of mice.
Expression of the Cytolethal Distending Toxin (Cdt) Operon inActinobacillus actinomycetemcomitans:Evidence That the CdtB Protein Is Responsible for G2 Arrest of the Cell Cycle in Human T Cells
Authors: Shenker Bruce J. , Hoffmaster Roselle H. , McKay Terry L. , Demuth Donald R.
Published: April 2014

We have previously shown that Actinobacillus actinomycetemcomitans produces an immunosuppressive factor that is encoded by the cdtB gene, which is homologous to a family of cytolethal distending toxins (Cdt) expressed by several Gram-negative bacteria. In this study, we report that the cdt locus in A. actinomycetemcomitans is composed of five open reading frames, designated orf1, orf2, cdtA, cdtB, and cdtC. The deduced amino acid sequences of the five open reading frames are highly conserved among A. actinomycetemcomitans strains 652, Y4, 29522, and HK1651. There is also strong homology with the Cdt proteins of Haemophilus ducreyi (87–91%), but only partial homology with that of Campylobacter jejuni and Escherichia coli (29–48%). Analysis of A. actinomycetemcomitans mRNA by RT-PCR suggests that the two small open reading frames upstream of cdtA are coexpressed with cdtA, cdtB, and cdtC. We next utilized a series of plasmids that express various combinations of the cdt genes to determine their requirement for expression of immunoinhibitory activity. Cell extracts of E. coli transformed with each of the plasmids were tested for their capacity to induce G2 arrest in the cell cycle of PHA-activated human T cells. These experiments suggest that expression of cdtB alone is sufficient to induce G2 arrest in human T cells, but do not exclude the possibility that cdtC also contributes to cell cycle arrest. The implications of our results with respect to the function of the individual Cdt proteins are discussed.
γδ-T Cells Are Critical for Survival and Early Proinflammatory Cytokine Gene Expression During MurineKlebsiellaPneumonia
Authors: Moore Thomas A. , Moore Bethany B. , Newstead Michael W. , Standiford Theodore J.
Published: April 2014

Although cells of the innate inflammatory response, such as macrophages and neutrophils, have been extensively studied in the arena of Gram-negative bacterial pneumonia, a role for T cells remains unknown. To study the role of specific T cell populations in bacterial pneumonia, mice deleted of their TCR β- and/or δ-chain were intratracheally inoculated with Klebsiella pneumoniae. γδ T cell knockout mice displayed increased mortality at both early and late time points. In contrast, mice specifically lacking only αβ-T cells were no more susceptible than wild-type mice. Pulmonary bacterial clearance in γδ-T cell knockout mice was unimpaired. Interestingly, these mice displayed increased peripheral blood dissemination. Rapid up-regulation of IFN-γ and TNF-α gene expression, critical during bacterial infections, was markedly impaired in lung and liver tissue from γδ-T cell-deficient mice 24 h postinfection. The increased peripheral blood bacterial dissemination correlated with impaired hepatic bacterial clearance following pulmonary infection and increased hepatic injury as measured by plasma aspartate aminotransferase activity. Combined, these data suggest that mice lacking γδ-T cells have an impaired ability to resolve disseminated bacterial infections subsequent to the initial pulmonary infection. These data indicate that γδ-T cells comprise a critical component of the acute inflammatory response toward extracellular Gram-negative bacterial infections and are vital for the early production of the proinflammatory cytokines IFN-γ and TNF-α.
The Chemokine Macrophage-Inflammatory Protein-1α and Its Receptor CCR1 Control Pulmonary Inflammation and Antiviral Host Defense in Paramyxovirus Infection
Authors: Domachowske Joseph B. , Bonville Cynthia A. , Gao Ji-Liang , Murphy Philip M. , Easton Andrew J. , Rosenberg Helene F.
Published: April 2014

In this work, we explore the responses of specific gene-deleted mice to infection with the paramyxovirus pneumonia virus of mice (PVM). We have shown previously that infection of wild type mice with PVM results in pulmonary neutrophilia and eosinophilia accompanied by local production of macrophage-inflammatory protein-1α (MIP-1α). Here we examine the role of MIP-1α in the pathogenesis of this disease using mice deficient in MIP-1α or its receptor, CCR1. The inflammatory response to PVM in MIP-1α-deficient mice was minimal, with ∼10–60 neutrophils/ml and no eosinophils detected in bronchoalveolar lavage fluid. Higher levels of infectious virus were recovered from lung tissue excised from MIP-1α-deficient than from fully competent mice, suggesting that the inflammatory response limits the rate of virus replication in vivo. PVM infection of CCR1-deficient mice was also associated with attenuated inflammation, with enhanced recovery of infectious virus, and with accelerated mortality. These results suggest that the MIP-1α/CCR1-mediated acute inflammatory response protects mice by delaying the lethal sequelae of infection.
Differential Regulation of Responsiveness to fMLP and C5a Upon Dendritic Cell Maturation: Correlation with Receptor Expression
Authors: Yang De , Chen Qian , Stoll Sabine , Chen Xin , Howard O. M. Zack , Oppenheim Joost J.
Published: April 2014

The trafficking of immature and mature dendritic cells (DCs) to different anatomical sites in vivo is critical for fulfilling their roles in the induction of Ag-specific immune responses. Although this process is complex and regulated by many mediators, the capacity of DCs to migrate is predominantly dependent on the expression of particular chemotactic receptors on the surface of DCs that enable them to move along chemotactic gradients formed by the corresponding chemokines and/or classical chemoattractants. Here we show that immature DCs (iDCs) respond to both fMLP and C5a as determined by chemotaxis and Ca2+ mobilization, whereas mature DCs (mDCs) respond to C5a, but not fMLP. Additionally, iDCs express the receptors for both fMLP and C5a at mRNA and protein levels. Upon maturation of DCs, fMLP receptor expression is almost completely absent, whereas C5a receptor mRNA and protein expression is maintained. Concomitantly, mDCs migrate chemotactically and mobilize intracellular Ca2+ in response to C5a, but not fMLP. Thus the interaction between C5a and its receptor is likely involved in the regulation of trafficking of both iDCs and mDCs, whereas fMLP mobilizes only iDCs. The differential responsiveness to fMLP and C5a of iDCs and mDCs suggests that they play different roles in the initiation of immune responses.
TNF-α-Induced Cyclooxygenase-2 Expression in Human Lung Epithelial Cells: Involvement of the Phospholipase C-γ2, Protein Kinase C-α, Tyrosine Kinase, NF-κB-Inducing Kinase, and I-κB Kinase 1/2 Pathway
Authors: Chen Ching-Chow , Sun Yi-Tao , Chen Jun-Jie , Chiu Kuo-Tung
Published: April 2014

TNF-α induced a dose- and time-dependent increase in cyclooxygenase-2 (COX-2) expression and PGE2 formation in human NCI-H292 epithelial cells. Immunofluorescence staining demonstrated that COX-2 was expressed in cytosol and nuclear envelope. Tyrosine kinase inhibitors (genistein or herbimycin) or phosphoinositide-specific phospholipase C inhibitor (U73122) blocked TNF-α-induced COX-2 expression. TNF-α also stimulated phosphatidylinositol hydrolysis and protein kinase C (PKC) activity, and both were abolished by genistein or U73122. The PKC inhibitor, staurosporine, also inhibited TNF-α-induced response. The 12-O-tetradecanoylphorbol 13-acetate (TPA), a PKC activator, also stimulated COX-2 expression, this effect being inhibited by genistein or herbimycin. NF-κB DNA-protein binding and COX-2 promoter activity were enhanced by TNF-α, and these effects were inhibited by genistein, U73122, staurosporine, or pyrolidine dithiocarbamate. TPA stimulated both NF-κB DNA-protein binding and COX-2 promoter activity, these effects being inhibited by genistein, herbimycin, or pyrolidine dithiocarbamate. The TNF-α-induced, but not the TPA-induced, COX-2 promoter activity was inhibited by phospholipase C-γ2 mutants, and the COX-2 promoter activity induced by either agent was attenuated by dominant-negative mutants of PKC-α, NF-κB-inducing kinase, or I-κB (inhibitory protein that dissociates from NF-κB) kinase (IKK)1 or 2. IKK activity was stimulated by both TNF-α and TPA, and these effects were inhibited by staurosporine or herbimycin. These results suggest that, in NCI-H292 epithelial cells, TNF-α might activate phospholipase C-γ2 via an upstream tyrosine kinase to induce activation of PKC-α and protein tyrosine kinase, resulting in the activation of NF-κB-inducing kinase and IKK1/2, and NF-κB in the COX-2 promoter, then initiation of COX-2 expression and PGE2 release.
TNF-α Associated with Extracellular Matrix Fibronectin Provides a Stop Signal for Chemotactically Migrating T Cells
Authors: Franitza Suzanne , Hershkoviz Rami , Kam Na’aman , Lichtenstein Nurit , Vaday Gayle G. , Alon Ronen , Lider Ofer
Published: April 2014

The migration of T cells into extravascular sites of inflammation is regulated by information derived from the molecular structure of the invaded tissue and from chemokine and cytokine gradients in the context of the extracellular matrix (ECM). Although recent studies have highlighted the role of particular chemoattractants in leukocyte migration, to date little is known about how specific combinations of contextual signals control the migration of leukocytes and their localization at sites of inflammation. Here we studied the interplay between a pleiotropic cytokine, TNF-α, and two prototypic chemoattractants, RANTES and stromal cell-derived factor-1α (SDF-1α), on human CD45RO+ T cells migrating within an ECM-like context. For this purpose, we used a newly constructed three-dimensional gel system designed to follow, in real time, the migration of individual leukocytes along chemotactic gradients in vitro. We found that TNF-α, which binds the ECM protein fibronectin and lacks adhesion- and migration-promoting effects of its own, can act as a proadhesive cytokine on T cells exposed to RANTES and SDF-1α. Furthermore, fibronectin-complexed TNF-α provided anchorage signals to the T cells as they moved directionally along chemoattractive gradients. This effect of TNF-α required an intact TNF-α receptor II subtype on the migrating T cells. The anchoring effect of TNF-α appears to be specific; IL-2, an integrin-activating proadhesive cytokine, does not transmit stoppage signals to T cell migration induced by RANTES. Thus, TNF-α present in the ECM at sites of inflammation may function to anchor T cells recruited to these sites by chemotactic signals.
CCR3-Active Chemokines Promote Rapid Detachment of Eosinophils from VCAM-1 In Vitro
Authors: Tachimoto Hiroshi , Burdick Monica M. , Hudson Sherry A. , Kikuchi Matsuo , Konstantopoulos Konstantinos , Bochner Bruce S.
Published: April 2014

Selective eosinophil recruitment is the result of orchestrated events involving cell adhesion molecules, chemokines, and their receptors. The mechanisms by which chemokines regulate eosinophil adhesion and migration via integrins are not fully understood. In our study, we examined the effect of CCR3-active chemokines on eosinophil adhesion to VCAM-1 and BSA under both static and flow conditions. When eotaxin-2 or other CCR3-active chemokines were added to adherent eosinophils, it induced rapid and sustained eosinophil detachment from VCAM-1 in a concentration-dependent manner. Adhesion was detectably reduced within 3 min and was further reduced at 10–60 min. Simultaneously, eotaxin-2 enhanced eosinophil adhesion to BSA. Preincubation of eosinophils with the CCR3-blocking mAb 7B11 completely prevented chemokine-induced changes in adhesion to VCAM-1 and BSA. Using a different protocol, pretreatment of eosinophils with chemokines for 0–30 min before their use in adhesion assays resulted in inhibition of VCAM-1 adhesion and enhancement of BSA adhesion. By flow cytometry, expression of α4 integrins and a β1 integrin activation epitope on eosinophils was decreased by eotaxin-2. In a flow-based adhesion assay, eotaxin-2 reduced eosinophil accumulation and the strength of attachment to VCAM-1. These results show that eotaxin-2 rapidly reduced α4 integrin function while increasing β2 integrin function. These findings suggest that chemokines facilitate migration of eosinophils by shifting usage away from β1 integrins toward β2 integrins.
Reduction of Inflammatory Cytokines and Prostaglandin E2 by IL-13 Gene Therapy in Rheumatoid Arthritis Synovium
Authors: Woods James M. , Katschke Kenneth J. , Tokuhira Michihide , Kurata Hirokazu , Arai Ken-Ichi , Campbell Phillip L. , Koch Alisa E.
Published: April 2014

The rheumatoid arthritis (RA) joint is characterized by an inflammatory synovial pannus which mediates tissue destruction. IL-13 is a cytokine that inhibits activated monocytes/macrophages from secreting a variety of proinflammatory molecules. The aim of this study was to examine whether gene therapy-delivered IL-13 could reduce the production of key proinflammatory mediators in RA synovial tissue (ST) explants. Adenoviral vectors encoding the genes for human IL-13 (AxCAIL-13) and bacterial β-galactosidase were generated and examined for protein production. Vectors were used to infect RA ST explants and RA synovial fibroblasts, and conditioned medium (CM) was collected at various times for analysis by ELISA and competitive immunoassay. AxCAIL-13 decreased the production of RA ST explant proinflammatory IL-1β by 85% after 24 h. Likewise, TNF-α levels were decreased by 82 and 75% whereas IL-8 levels were reduced 54 and 82% after 24 and 48 h, respectively, in RA ST explant CM. Monocyte chemotactic protein-1 concentrations were decreased by 88% after 72 h in RA ST explant CM. RA ST explant epithelial neutrophil-activating peptide-78 concentrations were decreased 85 and 94% whereas growth-related gene product-α levels were decreased by 77 and 85% at 24 and 48 h, respectively, by AxCAIL-13. Further, IL-13 significantly decreased PGE2 and macrophage inflammatory protein-1α production. These results demonstrate that increased expression of IL-13 via gene therapy may decrease RA-associated inflammation by reducing secretion of proinflammatory cytokines and PGE2.
Type IIA Secretory Phospholipase A2 Up-Regulates Cyclooxygenase-2 and Amplifies Cytokine-Mediated Prostaglandin Production in Human Rheumatoid Synoviocytes
Authors: Bidgood Matthew J. , Jamal Omar S. , Cunningham Anne M. , Brooks Peter M. , Scott Kieran F.
Published: April 2014

Human type IIA secretory phospholipase A2 (sPLA2-IIA) is induced in association with several immune-mediated inflammatory conditions. We have evaluated the effect of sPLA2-IIA on PG production in primary synovial fibroblasts from patients with rheumatoid arthritis (RA). At concentrations found in the synovial fluid of RA patients, exogenously added sPLA2-IIA dose-dependently amplified TNF-α-stimulated PGE2 production by cultured synovial fibroblasts. Enhancement of TNF-α-stimulated PGE2 production in synovial cells was accompanied by increased expression of cyclooxygenase (COX)-2 and cytosolic phospholipase A2 (cPLA2)-α. Blockade of COX-2 enzyme activity with the selective inhibitor NS-398 prevented both TNF-α-stimulated and sPLA2-IIA-amplified PGE2 production without affecting COX-2 protein induction. However, both sPLA2-IIA-amplified PGE2 production and enhanced COX-2 expression were blocked by the sPLA2 inhibitor LY311727. Colocalization studies using triple-labeling immunofluorescence microscopy showed that sPLA2-IIA and cPLA2-α are coexpressed with COX-2 in discrete populations of CD14-positive synovial macrophages and synovial tissue fibroblasts from RA patients. Based on these findings, we propose a model whereby the enhanced expression of sPLA2-IIA by RA synovial cells up-regulates TNF-α-mediated PG production via superinduction of COX-2. Therefore, sPLA2-IIA may be a critical modulator of cytokine-mediated synovial inflammation in RA.
IL-10 Is Induced in the Reperfused Myocardium and May Modulate the Reaction to Injury
Authors: Frangogiannis Nikolaos G. , Mendoza Leonardo H. , Lindsey Merry L. , Ballantyne Christie M. , Michael Lloyd H. , Smith C. Wayne , Entman Mark L.
Published: April 2014

Reperfusion of the ischemic myocardium is associated with a dramatic inflammatory response leading to TNF-α release, IL-6 induction, and subsequent neutrophil-mediated cytotoxic injury. Because inflammation is also an important factor in cardiac repair, we hypothesized the presence of components of the inflammatory reaction with a possible role in suppressing acute injury. Thus, we investigated the role of IL-10, an anti-inflammatory cytokine capable of modulating extracellular matrix biosynthesis, following an experimental canine myocardial infarction. Using our canine model of myocardial ischemia and reperfusion, we demonstrated significant up-regulation of IL-10 mRNA and protein in the ischemic and reperfused myocardium. IL-10 expression was first detected at 5 h and peaked following 96–120 h of reperfusion. In contrast, IL-4 and IL-13, also associated with suppression of acute inflammation and macrophage deactivation, were not expressed. In the ischemic canine heart, CD5-positive lymphocytes were the predominant source of IL-10 in the myocardial infarct. In the absence of reperfusion, no significant induction of IL-10 mRNA was noted. In addition, IL-12, a Th1-related cytokine associated with macrophage activation, was not detected in the ischemic myocardium. In vitro experiments demonstrated late postischemic cardiac-lymph-induced tissue inhibitor of metalloproteinases (TIMP)-1 mRNA expression in isolated canine mononuclear cells. This effect was inhibited when the incubation contained a neutralizing Ab to IL-10. Our findings suggest that lymphocytes infiltrating the ischemic and reperfused myocardium express IL-10 and may have a significant role in healing by modulating mononuclear cell phenotype and inducing TIMP-1 expression.
The Activity of Soluble VCAM-1 in Angiogenesis Stimulated by IL-4 and IL-13
Authors: Fukushi Jun-ichi , Ono Mayumi , Morikawa Wataru , Iwamoto Yukihide , Kuwano Michihiko
Published: April 2014

IL-13 is a multifunctional lymphokine sharing a number of biological properties with IL-4. We previously observed that IL-4 shows angiogenic activities in vitro as well as in vivo. In this study we examined the effect of IL-13 on angiogenesis in vitro and in vivo and also the underlying mechanisms. Human IL-13 significantly stimulated the formation of tube-like structures in collagen gels by human microvascular endothelial cells and bovine aortic endothelial cells by about 3-fold over the controls in the absence of the cytokines. Administration of murine IL-13 led to neovascularization when implanted in the rat cornea. Coadministration of neutralizing mAb to the IL-4R inhibited both tubular morphogenesis in vitro and activation of STAT6 induced by IL-4 or IL-13. Both IL-4 and IL-13 markedly increased mRNA levels of VCAM-1 in vascular endothelial cells, and the production of the soluble form of VCAM-1 was also stimulated in response to IL-4 or IL-13. Administration of anti-VCAM-1 Ab in vitro blocked tubular morphogenesis induced by IL-4 and IL-13. Angiogenesis induced in vivo in rat cornea by IL-4 and IL-13 was also inhibited by Ab against the rat α4 integrin subunit. These findings suggest that angiogenesis dependent on IL-4 and IL-13 is mainly mediated through a soluble VCAM-1/α4 integrin pathway.
The Autoantibodies to α6β4 Integrin of Patients Affected by Ocular Cicatricial Pemphigoid Recognize Predominantly Epitopes Within the Large Cytoplasmic Domain of Human β4
Authors: Bhol Kailash C. , Dans Michael J. , Simmons Raymond K. , Foster C. Stephen , Giancotti Filippo G. , Ahmed A. Razzaque
Published: April 2014

This study was undertaken to characterize the antigenic determinants recognized by the autoantibodies of patients with ocular cicatricial pemphigoid (OCP). OCP is a subepithelial, blistering, autoimmune disease that mainly affects the conjunctiva and other mucous membranes. We previously demonstrated that a cDNA clone, isolated from a keratinocyte expression library by using immunoaffinity-purified OCP autoantibody, encoded the cytoplasmic domain of β4 integrin subunit. Our subsequent studies showed that sera from all the OCP patients that were tested recognize the human β4 integrin subunit. To identify the prevalent epitopes of the anti-β4 autoantibodies of OCP, we have used cell lines transfected with vectors encoding a wild-type β4 subunit, a tailless β4 subunit, or a β4 subunit lacking the extracellular domain. Nontransfected cell lines were used as controls. Lysates from these cell lines were analyzed with OCP sera, IgG fractions from OCP sera, and immunoaffinity-purified OCP autoantibodies. Abs to extracellular and cytoplasmic domains of human β4 integrin were used as positive controls, whereas normal human sera and normal human IgG fractions were used as negative controls. The reactivity of OCP Abs was determined by using immunoblotting, immunoprecipitation, and FACS analysis. The results of this study indicate that OCP sera, OCP IgG fractions, and immunoaffinity-purified OCP autoantibodies react with the intracellular and not the extracellular domain of human β4 integrin subunit. In vitro cell culture experiments demonstrated that OCP autoantibody binds to the cytoplasm of the cells. The relevance of these findings to the pathogenesis of OCP is discussed.
Islet-Specific Expression of IL-10 Promotes Diabetes in Nonobese Diabetic Mice Independent of Fas, Perforin, TNF Receptor-1, and TNF Receptor-2 Molecules
Authors: Balasa Balaji , Van Gunst Kurt , Jung Nadja , Balakrishna Deepika , Santamaria Pere , Hanafusa Toshiaki , Itoh Naoto , Sarvetnick Nora
Published: April 2014

Several death-signaling or death-inducing molecules have been implicated in β cell destruction, including Fas, perforin, and TNFR-1. In this study, we examined the role of each death-signaling molecule in the IL-10-accelerated diabetes of nonobese diabetic (NOD) mice. Groups of IL-10-NOD mice, each deficient in either Fas, perforin, or TNFR-1 molecules, readily developed insulitis, and subsequently succumbed to diabetes with an accelerated kinetics and incidence similar to that observed in their wild-type or heterozygous IL-10-NOD littermates. Similarly, a TNFR-2 deficiency did not block accelerated diabetes in IL-10-NOD mice and spontaneous diabetes in NOD mice. These results demonstrate that pancreatic IL-10 promotes diabetes independent of Fas, perforin, TNFR-1, and TNFR-2 molecules. Subsequently, when cyclophosphamide, a diabetes-inducing agent, was injected into insulitis-free NOD.lpr/lpr mice, none of these mice developed insulitis or diabetes. Our data suggest that cyclophosphamide- but not IL-10-induced diabetes is Fas dependent. Overall, these findings provide evidence that pancreatic expression of IL-10 promotes diabetes independent of the major death pathways and provide impetus for identification of novel death pathways precipitating autoimmune destruction of insulin-producing β cells.
Distribution of DNA Vaccines Determines Their Immunogenicity After Intramuscular Injection in Mice
Authors: Dupuis Marc , Denis-Mize Kimberly , Woo Carolyn , Goldbeck Cheryl , Selby Mark J. , Chen Minchao , Otten Gillis R. , Ulmer Jeffrey B. , Donnelly John J. , Ott Gary , McDonald Donald M.
Published: April 2014

Intramuscular injection of DNA vaccines elicits potent humoral and cellular immune responses in mice. However, DNA vaccines are less efficient in larger animal models and humans. To gain a better understanding of the factors limiting the efficacy of DNA vaccines, we used fluorescence-labeled plasmid DNA in mice to 1) define the macroscopic and microscopic distribution of DNA after injection into the tibialis anterior muscle, 2) characterize cellular uptake and expression of DNA in muscle and draining lymph nodes, and 3) determine the effect of modifying DNA distribution and cellular uptake by volume changes or electroporation on the magnitude of the immune response. Injection of a standard 50-μl dose resulted in the rapid dispersion of labeled DNA throughout the muscle. DNA was internalized within 5 min by muscle cells near the injection site and over several hours by cells that were located along muscle fibers and in the draining lymph nodes. Histochemical staining and analysis of mRNA expression in isolated cells by RT-PCR showed that the transgene was detectably expressed only by muscle cells, despite substantial DNA uptake by non-muscle cells. Reduction of the injection volume to 5 μl resulted in substantially less uptake and expression of DNA by muscle cells, and correspondingly lower immune responses against the transgene product. However, expression and immunogenicity were restored when the 5-μl injection was followed by electroporation in vivo. These findings indicate that distribution and cellular uptake significantly affect the immunogenicity of DNA vaccines.
T Cells from Human Allergen-Induced Late Asthmatic Responses Express IL-12 Receptor β2 Subunit mRNA and Respond to IL-12 In Vitro
Authors: Varga Eva Maria , Wachholz Petra , Nouri-Aria Kayhan T. , Verhoef Adrienne , Corrigan Christopher J. , Till Stephen J. , Durham Stephen R.
Published: April 2014

IL-12 suppresses proallergic Th2-type cytokine production and induces Th1-type cytokine production by peripheral blood T cells from subjects with allergic disease. The objective of the present study was to examine the relevance of these findings to target organ T cell responses in human asthma. Bronchoalveolar lavage (BAL) and PBMC were collected from atopic asthmatics 24 h after fiberoptic allergen challenge of a segmental bronchus. BAL T cells and PBMC were cultured with allergen in the presence of recombinant IL-12 or IFN-γ, and cytokines were measured in culture supernatants after 6 days. IL-5 production by BAL T cells and PBMC was inhibited by IL-12 and, to a lesser extent, by IFN-γ. IL-12 also induced IFN-γ production by BAL T cells and PBMC. The effects of IL-12 nor IFN-γ on IL-5 production could not be reversed by neutralizing anti-IFN-γ or anti-IL-12 mAbs, respectively. Thus, the effect of neither IL-12 nor IFN-γ appeared to be mediated through induction of the other cytokine. In situ hybridization revealed that approximately one-third of BAL T cells expressed mRNA transcripts encoding the IL-12R β2 subunit following allergen challenge. Thus, human T cells obtained from BAL during asthmatic late responses, like T cells in the peripheral circulation, remain susceptible to immunomodulation by IL-12. These findings raise the possibility that IL-12 may hold therapeutic potential in allergic diseases such as asthma.
C8/119S Mutation of Major Mite Allergen Derf-2 Leads to Degenerate Secondary Structure and Molecular Polymerization and Induces Potent and Exclusive Th1 Cell Differentiation
Authors: Korematsu Seigo , Tanaka Yoshimasa , Hosoi Susumu , Koyanagi Satoshi , Yokota Toyokazu , Mikami Bunzo , Minato Nagahiro
Published: April 2014

Hyposensitization therapy for atopic diseases has been conducted for decades but suffered from many problems including anaphylactic reactions. We previously developed a mutant protein of the major mite allergen Derf-2, C8/119S, which showed reduced binding to IgE. The C8/119S mutant was shown to exhibit more efficient hyposensitizing effect than Derf-2 in the animal model of allergic bronchial asthma. In the present study, we indicate that C8/119S exhibits markedly augmented immunogenicity for the proliferation of Derf-2-specific human T cells and T cell clones irrespective of the epitope specificity as compared with Derf-2. Furthermore, C8/119S has induced potent and almost exclusive differentiation of Th1 cells from the peripheral blood of atopic patients in vitro. Neither Ag dosage effect nor absence of B cell-mediated Ag presentation could fully account for these effects. C8/119S has been indicated to lose the characteristic β-barrel structure as judged by circular dichroism spectroscopic analysis and to polymerize solubly in physiological condition. Heating of Derf-2 also caused less stable molecular aggregation, but it hardly affected the secondary structure and failed to induce such a polarity toward the Th1 cell differentiation. These results have indicated that the degenerate secondary structure of C8/119S leading to stable molecular polymerization is primarily responsible for the marked increase in T cell-immunogenicity and the induction of exclusive Th1 cell differentiation in atopic patients. It has been suggested strongly that the recombinant C8/119S protein can provide an effective Ag with the least risk of anaphylaxis for allergen immunotherapy against house dust mite in human.
Constitutive Expression of Functional 4-1BB (CD137) Ligand on Carcinoma Cells
Authors: Salih Helmut R. , Kosowski Steven G. , Haluska Vanessa F. , Starling Gary C. , Loo Deryk T. , Lee Francis , Aruffo Alejandro A. , Trail Pamela A. , Kiener Peter A.
Published: April 2014

Members of the TNF superfamily, including Fas, Fas ligand, and CD40, have been shown to be expressed on tumor cells. In the studies described in this work, we report that another family member, the ligand for 4-1BB (CD137), is expressed on various human carcinoma cell lines, on cells of solid tumors derived from these cell lines, and cells obtained from human tumors. Expression of 4-1BB ligand (4-1BBL) mRNA was detected by both RT-PCR and Northern blot analysis, and expression of 4-1BBL protein was detected by Western blot analysis of whole cell lysates and by FACS analysis of tumor cells and cell lines. Incubation of tumor cells with a 4-1BB-Ig fusion protein led to the production of IL-8 by the cells, demonstrating that the 4-1BBL is functionally active and signals back into the tumor cells. Furthermore, 4-1BBL expressed on the carcinoma cells functioned as a costimulatory molecule for the production of cytokines (most notably IFN-γ) in cocultures of T cells and tumor cells. These findings suggest that 4-1BBL expressed on carcinoma cells may significantly influence the outcome of a T cell-tumor cell interaction.