bluuebunny/crossref_metadata_2025 huggingface dataset:
ATP-Mediated Killing ofMycobacterium bovisBacille Calmette-Guérin Within Human Macrophages Is Calcium Dependent and Associated with the Acidification of Mycobacteria-Containing Phagosomes
Authors: Stober Carmel B. , Lammas David A. , Li Cheuk M. , Kumararatne Dinikantha S. , Lightman Stafford L. , McArdle Craig A.
Published: April 2014

We previously demonstrated that extracellular ATP stimulated macrophage death and mycobacterial killing within Mycobacterium bovis Bacille Calmette-Guérin (BCG)-infected human macrophages. ATP increases the cytosolic Ca2+ concentration in macrophages by mobilizing intracellular Ca2+ via G protein-coupled P2Y receptors, or promoting the influx of extracellular Ca2+ via P2X purinoceptors. The relative contribution of these receptors and Ca2+ sources to ATP-stimulated macrophage death and mycobacterial killing was investigated. We demonstrate that 1) ATP mobilizes Ca2+ in UTP-desensitized macrophages (in Ca2+-free medium) and 2) UTP but not ATP fails to deplete the intracellular Ca2+ store, suggesting that the pharmacological properties of ATP and UTP differ, and that a Ca2+-mobilizing P2Y purinoceptor in addition to the P2Y2 subtype is expressed on human macrophages. ATP and the Ca2+ ionophore, ionomycin, promoted macrophage death and BCG killing, but ionomycin-mediated macrophage death was inhibited whereas BCG killing was largely retained in Ca2+-free medium. Pretreatment of cells with thapsigargin (which depletes inositol (1,4,5)-trisphosphate-mobilizable intracellular stores) or 1,2-bis-(2-aminophenoxy)ethane-N, N, N′,N′-tetraacetic acid acetoxymethyl ester (an intracellular Ca2+ chelator) failed to inhibit ATP-stimulated macrophage death but blocked mycobacterial killing. Using the acidotropic molecular probe, 3-(2,4-dinitroanilino)-3′-amino-N-methyl dipropylamine, it was revealed that ATP stimulation promoted the acidification of BCG-containing phagosomes within human macrophages, and this effect was similarly dependent upon Ca2+ mobilization from intracellular stores. We conclude that the cytotoxic and bactericidal effects of ATP can be uncoupled and that BCG killing is not the inevitable consequence of death of the host macrophage.
Cell Density Regulates Neutrophil IL-8 Synthesis: Role of IL-1 Receptor Antagonist and Soluble TNF Receptors
Authors: Hattar Katja , Fink Ludger , Fietzner Karin , Himmel Barbara , Grimminger Friedrich , Seeger Werner , Sibelius Ulf
Published: April 2014

Although cytokine synthesis in polymorphonuclear leukocytes (PMN) was shown to be modulated by soluble mediators, the impact of microenvironmental conditions has not been elucidated. In this study, we investigated the effect of cell density on cytokine release from human neutrophils. PMN were cultured at various cell densities (10 × 106 PMN/ml; 60 × 106 PMN/ml), and LPS-induced release of cytokines was quantified by ELISA technique. Upon an increase in PMN density, secretion of the CXC chemokine IL-8 was progressively reduced. This effect was paralleled by a decrease in IL-8 mRNA. In contrast, TNF-α and IL-1β rose proportionally with increasing cell density. The inhibition of IL-8 secretion was reproduced by conditioned media of PMN at high cell density, but was not affected by blocking β2 integrin-dependent adhesion. When analyzing the supernatant of LPS-challenged neutrophils, large amounts of soluble TNFRs p55 and p75 (sTNFRI, sTNFRII), and IL-1R antagonist (IL-1RA), rising constantly with the cell density, were detected. Interestingly, combined blocking of the bioactivities of these mediators completely restored neutrophil IL-8 secretion at high cell densities, with the anti-IL-1RA Ab being the more potent agent. Moreover, combined application of exogenous IL-1RA and sTNFRs to 10 × 106 PMN/ml reproduced the suppression of IL-8 generation. We conclude that neutrophil IL-8 synthesis is autoregulated, being suppressed under conditions of high cell density. IL-1RA and sTNFRs, accumulating under these circumstances, seem to be centrally involved in this regulatory mechanism by interfering with the IL-1β- and TNF-α-dependent IL-8 generation. This feedback mechanism may control further neutrophil recruitment and activation in a neutrophil-rich environment, thereby preventing tissue destruction.
Reactive Nitrogen Species Inhibit Alveolar Epithelial Fluid Transport After Hemorrhagic Shock in Rats
Authors: Pittet Jean-François , Lu Le N. , Morris David G. , Modelska Kathrin , Welch William J. , Carey Hannah V. , Roux Jeremie , Matthay Michael A.
Published: April 2014

Our recent experimental work demonstrated that a neutrophil-dependent inflammatory response in the lung prevented the normal up-regulation of alveolar fluid clearance by catecholamines following hemorrhagic shock. In this study, we tested the hypothesis that the release of NO within the airspaces of the lung was responsible for the shock-mediated failure of the alveolar epithelium to respond to catecholamines in rats. Hemorrhagic shock was associated with an inducible NO synthase (iNOS)-dependent increase in the lung production of NO and a failure of the alveolar epithelium to up-regulate vectorial fluid transport in response to β-adrenergic agonists. Inhibition of iNOS restored the normal catecholamine-mediated up-regulation of alveolar liquid clearance. Airspace instillation of dibutyryl cAMP, a stable analog of cAMP, restored the normal fluid transport capacity of the alveolar epithelium after prolonged hemorrhagic shock, whereas direct stimulation of adenyl cyclase by forskolin had no effect. Pretreatment with pyrrolidine dithiocarbamate or sulfasalazine attenuated the iNOS-dependent production of NO in the lung and restored the normal up-regulation of alveolar fluid clearance by catecholamines after prolonged hemorrhagic shock. Based on in vitro studies with an alveolar epithelial cell line, A549 cells, the effect of sulfasalazine appeared to be mediated in part by inhibition of NF-κB activation, and the protective effect was mediated by the inhibition of IκBα protein degradation. In summary, these results provide the first in vivo evidence that NO, released within the airspaces of the lung probably secondary to the NF-κB-dependent activation of iNOS, is a major proximal inflammatory mediator that limits the rate of alveolar epithelial transport after prolonged hemorrhagic shock by directly impairing the function of membrane proteins involved in the β-adrenergic receptor-cAMP signaling pathway in alveolar epithelium.
Role of p190RhoGAP in β2 Integrin Regulation of RhoA in Human Neutrophils
Authors: Dib Karim , Melander Fredrik , Andersson Tommy
Published: April 2014

We found that engagement of β2 integrins on human neutrophils induced activation of RhoA, as indicated by the increased ratio of GTP:GTP + GDP recovered on RhoA and translocation of RhoA to a membrane fraction. The clustering of β2 integrins also induced a time-dependent increase in GDP bound to RhoA, which correlated with β2 integrin-induced activation of p190RhoGAP. The activation of p190RhoGAP was completely blocked by [4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine] (PP1), a selective inhibitor of Src family tyrosine kinases. However, clustering of β2 integrins did not increase the basal tyrosine phosphorylation of p190RhoGAP, nor did it affect the amount of p120RasGAP bound to p190RhoGAP. Instead, the β2 integrin-induced activation of p190RhoGAP was accompanied by increased tyrosine phosphorylation of a p190RhoGAP-associated protein, p120RasGAP, and accumulation of both p120RasGAP and p190RhoGAP in a membrane fraction. PP1 blocked the β2 integrin-induced phosphorylation of p120RasGAP, as well as the translocation of p190RhoGAP and p120RasGAP, but it did not affect the accumulation of RhoA in the membrane fraction. In agreement with the mentioned findings, PP1 also increased the GTP:GTP + GDP ratio recovered on RhoA immunoprecipitated from β2 integrin-stimulated cells. Thus, in neutrophils, β2 integrin-induced activation of p190RhoGAP requires a signal from a Src family tyrosine kinase, but it does not occur via the signaling pathway responsible for activation of RhoA.
Antioxidants Inhibit Indoleamine 2,3-Dioxygenase in IFN-γ-Activated Human Macrophages: Posttranslational Regulation by Pyrrolidine Dithiocarbamate
Authors: Thomas Shane R. , Salahifar Houta , Mashima Ryuichi , Hunt Nicholas H. , Richardson Des R. , Stocker Roland
Published: April 2014

Induction of the heme-containing indoleamine 2,3-dioxygenase (IDO) by IFN-γ is implicated in anti-microbial and pro-inflammatory activities of human macrophages. Antioxidants can modulate the expression of immune and inflammatory genes, and pyrrolidine dithiocarbamate (PDTC) is a frequently used antioxidant to inhibit the transcription factor NF-κB. Here we show that IFN-γ treatment of human monocyte-derived macrophages (hMDMs) increased the proportion of oxidized glutathione. PDTC attenuated this increase and inhibited IDO activity, although it increased IDO protein expression and did not affect IDO mRNA expression and enzyme activity directly. Other antioxidants, 2-ME, ebselen, and t-butyl hydroquinone, inhibited IDO protein expression. Similar to PDTC, the heme biosynthesis inhibitor succinylacetone (SA) and the iron-chelator pyridoxal isonicotinoyl hydrazone inhibited cellular IDO activity without affecting protein expression, whereas addition of hemin or the heme precursor δ-aminolevulinic acid increased IDO activity. Also, incubation of IFN-γ-activated hMDM with δ-[14C]-aminolevulinic acid resulted in the incorporation of label into immunoprecipitated IDO, a process inhibited by PDTC and SA. Furthermore, supplementation of lysates from PDTC- or SA-treated hMDM with hemin fully restored IDO activity to control levels, and hemin also reversed the inhibitory action of SA but not PDTC in intact cells. Together these results establish a requirement for de novo heme synthesis for IDO activity in IFN-γ-activated hMDM. They show that, similar to other pro-inflammatory proteins, the activity of IDO is modulated by antioxidants though in the case of PDTC this takes place posttranslationally, in part by limiting the availability of heme for the formation of holo-IDO.
ATP Mediates Calcium Signaling Between Astrocytes and Microglial Cells: Modulation by IFN-γ
Authors: Verderio Claudia , Matteoli Michela
Published: April 2014

Calcium-mediated intercellular communication is a mechanism by which astrocytes communicate with each other and modulate the activity of adjacent cells, including neurons and oligodendrocytes. We have investigated whether microglia, the immune effector cells involved in several diseases of the CNS, are actively involved in this communication network. To address this issue, we analyzed calcium dynamics in fura-2-loaded cocultures of astrocytes and microglia under physiological conditions and in the presence of the inflammatory cytokine IFN-γ. The intracellular calcium increases in astrocytes, occurring spontaneously or as a result of mechanical or bradykinin stimulation, induced the release of ATP, which, in turn, was responsible for triggering a delayed calcium response in microglial cells. Repeated stimulations of microglial cells by astrocyte-released ATP activated P2X7 purinergic receptor on microglial cells and greatly increased membrane permeability, eventually leading to microglial apoptosis. IFN-γ increased ATP release and potentiated the P2X7-mediated cytolytic effect. This is the first study showing that ATP mediates a form of calcium signaling between astrocytes and microglia. This mechanism of intercellular communication may be involved in controlling the number and function of microglial cells under pathophysiologic CNS conditions.
Nonproliferating Bystander CD4+ T Cells Lacking Activation Markers Support HIV Replication During Immune Activation
Authors: Scales David , Ni Houping , Shaheen Farida , Capodici John , Cannon Georgetta , Weissman Drew
Published: April 2014

HIV replicates primarily in lymphoid tissue and immune activation is a major stimulus in vivo. To determine the cells responsible for HIV replication during Ag-driven T cell activation, we used a novel in vitro model employing dendritic cell presentation of superantigen to CD4+ T cells. Dendritic cells and CD4+ T cells are the major constituents of the paracortical region of lymphoid organs, the main site of Ag-specific activation and HIV replication. Unexpectedly, replication occurred in nonproliferating bystander CD4+ T cells that lacked activation markers. In contrast, activated Ag-specific cells were relatively protected from infection, which was associated with CCR5 and CXC chemokine receptor 4 down-regulation. The finding that HIV replication is not restricted to highly activated Ag-specific CD4+ T cells has implications for therapy, efforts to eradicate viral reservoirs, immune control of HIV, and Ag-specific immune defects.
Unique Subpopulations of CD56+ NK and NK-T Peripheral Blood Lymphocytes Identified by Chemokine Receptor Expression Repertoire
Authors: Campbell James J. , Qin Shixin , Unutmaz Derya , Soler Dulce , Murphy Kristine E. , Hodge Martin R. , Wu Lijun , Butcher Eugene C.
Published: April 2014

CD56, an adhesion molecule closely related to neual cell adhesion molecule, is an immunophenotypic marker for several unique populations of PBLs. Although CD56+ cells derive from multiple lymphocyte lineages, they share a role in immunosurveillance and antitumor responses. We have studied the chemokine receptor expression patterns and functional migratory responses of three distinct CD56+ populations from human peripheral blood. NK-T cells were found to differ greatly from NK cells, and CD16+ NK cells from CD16− NK cells. CD16+ NK cells were the predominant population responding to IL-8 and fractalkine, whereas NK-T cells were the predominant population responding to the CCR5 ligand macrophage-inflammatory protein-1β. CD16− NK cells were the only CD56+ population that uniformly expressed trafficking molecules necessary for homing into secondary lymphoid organs through high endothelial venule. These findings describe a diverse population of cells that may have trafficking patterns entirely different from each other, and from other lymphocyte types.
The Role of Adoptively Transferred CD8 T Cells and Host Cells in the Control of the Growth of the EG7 Thymoma: Factors That Determine the Relative Effectiveness and Homing Properties of Tc1 and Tc2 Effectors
Authors: Helmich Brian K. , Dutton Richard W.
Published: April 2014

We had previously examined the factors that regulate the response of OVA-specific TCR-transgenic CD8 T cells to the B16 OVA melanoma, growing as lung metastases. We examine here whether the same parameters operate for EG7, growing intradermally. Tc1 or Tc2 CD8 effector cells from OT-1 mice were injected either mixed with the tumor or i.v. at day 0 or 7. Tc2 were one-fifth to one-tenth as effective as Tc1 when injected with the tumor, in controlling tumor growth, but were only 1/20 to 1/100 injected i.v. Tc1 injected i.v. entered the draining lymph nodes faster than Tc2 and caused a faster accumulation of host cells. Both caused an abrupt termination of host cell entry into lymph nodes and spleen after tumor elimination, but this occurred earlier for Tc1 than for Tc2. Host responses were ineffective in the absence of adoptive transfer but were essential after transfer. Perforin expression in the donor cells plays no role in adoptively transferred Tc1 or Tc2 control of the tumor, and neither IL-4 nor IL5 is needed for Tc1 or Tc2 function. Tc1 cells from mice lacking IFN-γ, however, control tumor growth less well, whereas Tc2 effectors lacking IFN-γ are unaffected. Tc1 from IFN-γ-deficient mice attract fewer host cells to the draining lymph node, whereas Tc1 cells from perforin-deficient donors are unimpaired. We conclude that host cell recruitment is a crucial element in adoptive immunotherapy. The differences between the EG7 and the previous B16 melanoma model are discussed.
A Transcriptional Block in the IL-2 Promoter at the −150 AP-1 Site in Effector CD8+ T Cells
Authors: Finch Rosalynde J. , Fields Patrick E. , Greenberg Philip D.
Published: April 2014

Both CD4+ and CD8+ T cells that produce IL-2 in response to Ag recognition have been isolated. However, most effector CD8+ T cells recovered after exposure to Ag do not produce sufficient IL-2 to sustain growth, and depend on CD4+ T helper cells for this obligate growth factor. IL-2 expression in CD4+ T cells is primarily controlled at the level of transcription, but mechanisms restricting IL-2 production in CD8+ T cells have not been elucidated. To evaluate transcriptional regulation of the IL-2 gene in CD8+ T cells, we stably transfected reporter genes into Ag-specific CD8+ T cell clones. CD28+ CD8+ T cells unable to transcribe the IL-2 gene in response to antigenic stimulation had a block in transactivation of the −150 CD28 response element (CD28RE)/AP-1 site of the IL-2 promoter, but did transactivate the composite NFAT/AP-1 and OCT/AP-1 sites, and a consensus AP-1 motif. Mutation of the nonconsensus −150 AP-1 site to a consensus AP-1 site, or insertion of a CD28RE/AP-1 consensus site upstream of the native −150 CD28RE/AP-1 site restored transactivation of the altered promoter. These results suggest that the defect at the −150 site may reflect the absence or inactivity of a required factor rather than repression of the IL-2 promoter.
Molecular Identification of IgE-Dependent Histamine-Releasing Factor as a B Cell Growth Factor
Authors: Kang Hyung Sik , Lee Min Ju , Song Hyunkeun , Han Seung Hyun , Kim Yong Man , Im Joo Young , Choi Inpyo
Published: April 2014

The culture supernatants of LK1 cells, murine erythroleukemia cells, showed B cell-stimulating activity. Purification and NH2-terminal sequence analysis revealed that one of the candidates was murine IgE-dependent histamine-releasing factor (IgE-HRF), which is known to induce histamine from basophils. Recombinant IgE-HRF (rHRF) obtained from Escherichia coli- or 293-transformed embryonal kidney cells was tested for B cell-stimulating activity. Both rHRFs stimulated B cell proliferation in a dose-dependent manner. However, boiling or anti-HRF Ab abolished the B cell stimulatory effects of rHRF. Recombinant HRF showed strong synergistic effects with IL-2, IL-4, and IL-5 for B cell activation, with maximal activity in the presence of anti-CD40 Ab. Recombinant HRF increased MHC class II expression of B cells. It also increased Ig production from B cells. Treatment with polymyxin B, a neutralizing peptide antibiotic of LPS, did not reduce the activity of rHRF. In addition, FACS analysis using PE-conjugated rHRF showed that HRF bound to B cells. Recombinant HRF up-regulated the expression of IL-1 and IL-6 in B cells. In vivo administration of rHRF or the cDNA for rHRF increased total and Ag-specific Ig synthesis. Taken together, these results indicate that HRF stimulates B cell activation and function.
Inhibition of p38 Kinase Reveals a TNF-α-Mediated, Caspase-Dependent, Apoptotic Death Pathway in a Human Myelomonocyte Cell Line
Authors: Varghese Jishy , Chattopadhaya Souvik , Sarin Apurva
Published: April 2014

TNF-α transduces signals of survival or death via its two receptors, R1/p55/p60 and RII/p80/p75. The role of caspases as effectors of cell death is universally accepted, although caspase inhibitors may potentiate TNF cytotoxicity in some instances. In conditions when macromolecular synthesis is blocked, caspases are part of the machinery that executes TNF-triggered apoptotic death in U937, a human myelomonocyte cell line, and in the Jurkat T cell line. However, inhibition of p38 mitogen-activated protein kinase (p38 MAPK) triggered TNF cytotoxicity in U937 cells and murine splenic macrophages, but not the Jurkat cell line. TNF induced expression of the antiapoptotic protein c-IAP2 (cytoplasmic inhibitor of apoptosis protein 2), and was blocked in the presence of a p38 MAPK inhibitor, which also induced caspase-dependent, TNF-mediated apoptosis in U937 cells. Thus, inhibition of p38 MAPK resulted in the activation of caspase 9 and cleavage of the adaptor molecule BH3 interacting domain death agonist, and blocked NF-κB-mediated transactivation, without affecting the nuclear translocation of NF-κB. Collectively, these data show that activation of p38 MAPK is critical to cell survival by TNF in U937 cells, and demonstrate lineage-specific regulation of TNF-triggered signals of activation or apoptosis.
Early TCR αβ Expression Promotes Maturation of T Cells Expressing FcεRIγ Containing TCR/CD3 Complexes
Authors: Petersson Karin , Ivars Fredrik
Published: April 2014

In a previous study we presented data indicating that the expanded population of CD4−CD8− (DN) αβ T cells in TCRα-chain-transgenic mice was partially if not entirely derived from γδ T cell lineage cells. The development of both γδ T cells and DN αβ T cells is poorly understood; therefore, we thought it would be important to identify the immediate precursors of the transgene-induced DN αβ T cells. We have in this report studied the early T cell development in these mice and we show that the transgenic TCRα-chain is expressed by precursor thymocytes already at the CD3−CD4−CD8− (triple negative, TN) CD44+CD25− stage of development. Both by using purified precursor populations in reconstitution experiments and by analyzing fetal thymocyte development, we demonstrated that early TN precursors expressing endogenous TCRβ-chains matured into DN αβ T cells at several stages of development. The genes encoding the γ-chain of the high affinity receptor for IgE (FcεRIγ) and the CD3ζ protein were found to be reciprocally expressed in TN thymocytes such that during development the FcεRIγ expression decreased whereas CD3ζ expression increased. Furthermore, in a fraction of the transgene-induced DN αβ T cells the FcεRIγ protein colocalized with the TCR/CD3 complex. These data suggest that similarly to γδ T cells and NKT cells, precursors expressing the TCR early in the common αβγδ developmental pathway may use the FcεRIγ protein as a signaling component of the TCR/CD3 complex.
Rigidity of Circulating Lymphocytes Is Primarily Conferred by Vimentin Intermediate Filaments
Authors: Brown Martin J. , Hallam John A. , Colucci-Guyon Emma , Shaw Stephen
Published: April 2014

Lymphocytes need rigidity while in circulation, but must abruptly become deformable to undergo transmigration into tissue. Previously, the control of leukocyte deformability has been attributed to microfilaments or microtubules, but the present studies demonstrate the greater importance of vimentin intermediate filaments (IFs). In circulating T lymphocytes, IFs form a distinctive spherical cage that undergoes a rapid condensation into a juxtanuclear aggregate during chemokine-induced polarization. Measurements of the resistance of peripheral blood T lymphocytes to global deformation demonstrate that their rigidity is primarily dependent on intact vimentin filaments. Microtubules, in contrast, are not sufficient to maintain rigidity. Thus, vimentin IFs are a primary source of structural support in circulating human lymphocytes, and their regulated collapse is likely to be an essential element in chemokine-induced transendothelial migration.
Induction of Telomerase Activity During Development of Human Mast Cells from Peripheral Blood CD34+ Cells: Comparisons with Tumor Mast-Cell Lines
Authors: Chaves-Dias Cristina , Hundley Thomas R. , Gilfillan Alasdair M. , Kirshenbaum Arnold S. , Cunha-Melo Jose Renan , Metcalfe Dean D. , Beaven Michael A.
Published: April 2014

To further characterize the development of mast cells from human hemopoietic pluripotent cells we have investigated the expression of telomerase activity in cultured human peripheral blood CD34+ cells, and CD34+/CD117+/CD13+ progenitor mast cells selected therefrom, with the idea that induction of telomerase is associated with clonal expansion of CD34+/CD117+/CD13+ cells. A rapid increase in telomerase activity preceded proliferation of both populations of cells in the presence of stem cell factor and either IL-3 or IL-6. The induction was transient, and telomerase activity declined to basal levels well before the appearance of mature mast cells. Studies with pharmacologic inhibitors suggested that this induction was initially dependent on the p38 mitogen-activated protein kinase and phosphatidylinositol 3′-kinase, but once cell replication was underway telomerase activity, but not cell replication, became resistant to the effects of inhibitors. Tumor mast cell lines, in contrast, expressed persistently high telomerase activity throughout the cell cycle, and this expression was unaffected by inhibitors of all known signaling pathways in mast cells even when cell proliferation was blocked for extended periods. These results suggest that the transient induction of telomerase activity in human progenitor mast cells was initially dependent on growth factor-mediated signals, whereas maintenance of high activity in tumor mast cell lines was not dependent on intracellular signals or cell replication.
Prolonged Antigen Persistence Within Nonterminal Late Endocytic Compartments of Antigen-Specific B Lymphocytes
Authors: Gondré-Lewis Timothy A. , Moquin Amy E. , Drake James R.
Published: April 2014

Although Ag-specific B lymphocytes can process Ag and express peptide-class II complexes as little as 1 h after Ag exposure, it requires 3–5 days for the immune system to develop a population of Ag-specific effector CD4 T lymphocytes to interact with these complexes. Presently, it is unclear how B cells maintain the expression of cell surface antigenic peptide-class II complexes until effector CD4 T lymphocytes become available. Therefore, we investigated B cell receptor (BCR)-mediated Ag processing and presentation by normal B lymphocytes to determine whether these cells have a mechanism to prolong the cell surface expression of peptide-class II complexes derived from the processing of cognate Ag. Interestingly, after transit of early endocytic compartments, internalized Ag-BCR complexes are delivered to nonterminal late endosomes where they persist for a prolonged period of time. In contrast, Ags internalized via fluid phase endocytosis are rapidly delivered to terminal lysosomes and degraded. Moreover, persisting Ag-BCR complexes within nonterminal late endosomes exhibit a higher degree of colocalization with the class II chaperone HLA-DM/H2-M than with the HLA-DM/H2-M regulator HLA-DO/H2-O. Finally, B cells harboring persistent Ag-BCR complexes exhibit prolonged cell surface expression of antigenic peptide-class II complexes. These results demonstrate that B lymphocytes possess a mechanism for prolonging the intracellular persistence of Ag-BCR complexes within nonterminal late endosomes and suggest that this intracellular Ag persistence allows for the prolonged cell surface expression of peptide-class II complexes derived from the processing of specific Ag.
Tetramer-Guided Epitope Mapping: Rapid Identification and Characterization of Immunodominant CD4+ T Cell Epitopes from Complex Antigens
Authors: Novak Erik J. , Liu Andrew W. , Gebe John A. , Falk Ben A. , Nepom Gerald T. , Koelle David M. , Kwok William W.
Published: April 2014

T cell responses to Ags involve recognition of selected peptide epitopes contained within the antigenic protein. In this report, we describe a new approach for direct identification of CD4+ T cell epitopes of complex Ags that uses human class II tetramers to identify reactive cells. With a panel of 60 overlapping peptides covering the entire sequence of the VP16 protein, a major Ag for HSV-2, we generated a panel of class II MHC tetramers loaded with peptide pools that were used to stain peripheral lymphocytes of an HSV-2 infected individual. With this approach, we identified four new DRA1*0101/DRB1*0401- and two DRA1*0101/DRB1*0404-restricted, VP16-specific epitopes. By using tetramers to sort individual cells, we easily obtained a large number of clones specific to these epitopes. Although DRA1*0101/DRB1*0401 and DRA1*0101/DRB1*0404 are structurally very similar, nonoverlapping VP16 epitopes were identified, illustrating high selectivity of individual allele polymorphisms within common MHC variants. This rapid approach to detecting CD4+ T cell epitopes from complex Ags can be applied to any known Ag that gives a T cell response.
Peptide Binding to Active Class II MHC Protein on the Cell Surface
Authors: Vacchino Judith F. , McConnell Harden M.
Published: April 2014

Solution studies have demonstrated the existence of two functionally distinct isomers of empty class II MHC: an active isomer that binds peptide and an inactive isomer that does not. Empty MHC molecules on the surface of APCs can load antigenic peptides directly from the extracellular medium, facilitating the generation of a diverse peptide repertoire for T cell presentation. In this report, we examine I-Ek on the surface of Chinese hamster ovary cells with respect to the active and inactive isomers. As in the case of purified soluble active I-Ek, active I-Ek on the cell surface is unstable, decaying to the inactive form in ∼14 min. Evidence is presented suggesting that at steady state <1% of the total cell surface I-Ek is active and that a significant fraction of these active molecules originates from intracellular pools as well as reactivation of inactive cell surface I-Ek.
Dissociation of Pax-5 from KI and KII Sites During κ-Chain Gene Rearrangement Correlates with Its Association with the Underphosphorylated Form of Retinoblastoma
Authors: Sato Hiromu , Wang Dan , Kudo Akira
Published: April 2014

The KI and KII sites play a crucial role in κ-chain gene rearrangement, which was investigated in mice deficient for these sites. Previously, we found that Pax-5 can bind to the KI and KII sites; however, the function of Pax-5 in κ-chain gene rearrangement has not been investigated. Here, we have used an in vitro culture system in which differentiation from pre-B cells to immature B cells is induced by removing IL-7. We showed that, after the induction of differentiation, Pax-5 dissociated from the KI and KII revealed by EMSA analyses, and this dissociation occurred specifically at the KI and KII sites, but not at the Pax-5 binding site, in the CD19 promoter because of a lower binding affinity of Pax-5 for the KI and KII sites. During differentiation induced by removing IL-7, the underphosphorylated form of retinoblastoma preferentially associated with Pax-5, which caused dissociation of Pax-5 from KI and KII sites. These results suggest that the dissociation of Pax-5 from the KI and KII sites is important in the induction of κ-chain gene rearrangement.
Naturally Processed HLA Class II Peptides Reveal Highly Conserved Immunogenic Flanking Region Sequence Preferences That Reflect Antigen Processing Rather Than Peptide-MHC Interactions
Authors: Godkin Andrew J. , Smith Kathrine J. , Willis Anthony , Tejada-Simon Maria V. , Zhang Jingwu , Elliott Tim , Hill Adrian V. S.
Published: April 2014

MHC class II heterodimers bind peptides 12–20 aa in length. The peptide flanking residues (PFRs) of these ligands extend from a central binding core consisting of nine amino acids. Increasing evidence suggests that the PFRs can alter the immunogenicity of T cell epitopes. We have previously noted that eluted peptide pool sequence data derived from an MHC class II Ag reflect patterns of enrichment not only in the core binding region but also in the PFRs. We sought to distinguish whether these enrichments reflect cellular processes or direct MHC-peptide interactions. Using the multiple sclerosis-associated allele HLA-DR2, pool sequence data from naturally processed ligands were compared with the patterns of enrichment obtained by binding semicombinatorial peptide libraries to empty HLA-DR2 molecules. Naturally processed ligands revealed patterns of enrichment reflecting both the binding motif of HLA-DR2 (position (P)1, aliphatic; P4, bulky hydrophobic; and P6, polar) as well as the nonbound flanking regions, including acidic residues at the N terminus and basic residues at the C terminus. These PFR enrichments were independent of MHC-peptide interactions. Further studies revealed similar patterns in nine other HLA alleles, with the C-terminal basic residues being as highly conserved as the previously described N-terminal prolines of MHC class II ligands. There is evidence that addition of C-terminal basic PFRs to known peptide epitopes is able to enhance both processing as well as T cell activation. Recognition of these allele-transcending patterns in the PFRs may prove useful in epitope identification and vaccine design.
A Novel Interaction Between Type IV Pili ofNeisseria gonorrhoeaeand the Human Complement Regulator C4b-Binding Protein
Authors: Blom Anna M. , Rytkönen Anne , Vasquez Paola , Lindahl Gunnar , Dahlbäck Björn , Jonsson Ann-Beth
Published: April 2014

C4b-binding protein (C4BP) is an important plasma inhibitor of the classical pathway of complement activation. Several bacterial pathogens bind C4BP, which may contribute to their virulence. In the present report we demonstrate that isolated type IV pili from Neisseria gonorrhoeae bind human C4BP in a dose-dependent and saturable manner. C4BP consists of seven identical α-chains and one β-chain linked together with disulfide bridges. We found that pili bind to the α-chain of C4BP, which is composed of eight homologous complement control protein (CCP) domains. From the results of an inhibition assay with C4b and a competition assay in which we tested mutants of C4BP lacking individual CCPs, we concluded that the binding area for pili is localized to CCP1 and CCP2 of the α-chain. The binding between pili and C4BP was abolished at 0.25 M NaCl, implying that it is based mostly on ionic interactions, similarly to what have been observed for C4b-C4BP binding. Furthermore, the N-terminal part of PilC, a structural component of pili, appeared to be responsible for binding of C4BP. Membrane cofactor protein, previously shown to be a receptor for pathogenic N. gonorrhoeae on the surface of epithelial cells, competed with C4BP for binding to pili only at high concentrations, suggesting that different parts of pili are involved in these two interactions. Accordingly, high concentrations of C4BP were required to inhibit binding of N. gonorrhoeae to Chang conjunctiva cells, and no inhibition of binding was observed with cervical epithelial cells.
Distinct Roles for PECAM-1, ICAM-1, and VCAM-1 in Recruitment of Neutrophils and Eosinophils to the Cornea in Ocular Onchocerciasis (River Blindness)
Authors: Kaifi Jussuf T. , Diaconu Eugenia , Pearlman Eric
Published: April 2014

Infiltration of granulocytes into the transparent mammalian cornea can result in loss of corneal clarity and severe visual impairment. Since the cornea is an avascular tissue, recruitment of granulocytes such as neutrophils and eosinophils into the corneal stroma is initiated from peripheral (limbal) vessels. To determine the role of vascular adhesion molecules in this process, expression of platelet endothelial cell adhesion molecule 1 (PECAM-1), ICAM-1, and VCAM-1 on limbal vessels was determined in a murine model of ocular onchocerciasis in which Ags from the parasitic worm Onchocerca volvulus are injected into the corneal stroma. Expression of each of these molecules was elevated after injection of parasite Ags; however, PECAM-1 and ICAM-1 expression remained elevated from 12 h after injection until 7 days, whereas VCAM-1 expression was more transient, with peak expression at 72 h. Subconjunctival injection of Ab to PECAM-1 significantly inhibited neutrophil recruitment to the cornea compared with eyes injected with control Ab (p = 0.012). Consistent with this finding, corneal opacification was significantly diminished (p < 0.0001). There was no significant reduction in eosinophils. Conversely, subconjunctival injection of Ab to ICAM-1 did not impair neutrophil recruitment, but significantly inhibited eosinophil recruitment (p = 0.0032). Injection of Ab to VCAM-1 did not significantly inhibit infiltration of either cell type to the cornea. Taken together, these results demonstrate important regulatory roles for PECAM-1 and ICAM-1 in recruitment of neutrophils and eosinophils, respectively, to the cornea, and may indicate a selective approach to immune intervention.
CD40 Activates NF-κB and c-Jun N-Terminal Kinase and Enhances Chemokine Secretion on Activated Human Hepatic Stellate Cells
Authors: Schwabe Robert F. , Schnabl Bernd , Kweon Young O. , Brenner David A.
Published: April 2014

Activated hepatic stellate cells (HSCs) are the main producers of extracellular matrix in the fibrotic liver and contribute to hepatic inflammation through the secretion of chemokines and the recruitment of leukocytes. This study assesses the function of CD40 on human HSCs. Activated human HSCs express CD40 in culture and in fibrotic liver, as determined by flow cytometry, RT-PCR, and immunohistochemistry. CD40 expression is strongly enhanced by IFN-γ. Stimulation of CD40 with CD40 ligand (CD40L)-transfected baby hamster kidney cells induces NF-κB, as demonstrated by the activation of I-κB kinase (IKK), increased NF-κB DNA binding, and p65 nuclear translocation. CD40-activated IKK also phosphorylates a GST-p65 substrate at serine 536 in the transactivation domain 1. Concomitant with the activation of IKK, CD40L-transfected baby hamster kidney cell treatment strongly activates c-Jun N-terminal kinase. CD40 activation increases the secretion of IL-8 and monocyte chemoattractant protein-1 by HSCs 10- and 2-fold, respectively. Adenovirally delivered dominant negative (dn) IKK2 and TNFR-associated factor 2dn inhibit IKK-mediated GST-I-κB and GST-p65 phosphorylation, NF-κB binding, and IL-8 secretion, whereas IKK1dn and NF-κB-inducing kinase dominant negative do not have inhibitory effects. We conclude that the CD40-CD40L receptor-ligand pair is involved in a cross-talk between HSCs and immune effector cells that contributes to the perpetuation of HSC activation in liver fibrosis through TNFR-associated factor 2- and IKK2-dependent pathways.
Accelerated Nephrotoxic Nephritis Is Exacerbated in C1q-Deficient Mice
Authors: Robson Michael G. , Cook H. Terence , Botto Marina , Taylor Philip R. , Busso Nathalie , Salvi Roberto , Pusey Charles D. , Walport Mark J. , Davies Kevin A.
Published: April 2014

C1q deficiency strongly predisposes to the development of systemic lupus erythematosus in humans and mice. We used the model of accelerated nephrotoxic nephritis in C1q-deficient mice to explore the mechanisms behind these associations. C1q-deficient mice developed severe glomerular thrombosis within 4 days of induction of disease, whereas wild-type mice developed mild injury. These findings suggest that C1q protects from immune-mediated glomerular injury. This exacerbated thrombosis was also seen in mice triply deficient in C1q, factor B, and C2, excluding a major pathogenic role for the alternative pathway of complement in this phenomenon. However, these mice did not develop elevated creatinine levels. No exacerbation of accelerated nephrotoxic nephritis was observed in mice doubly deficient in factor B and C2, suggesting a protective role for C1q against renal inflammation that is proximal to C2 activation. There were increased murine IgG deposits, neutrophil numbers, and apoptotic cells in the glomeruli of C1q-deficient mice compared with wild-type mice. Renal expression of genes encoding procoagulant proteins was also enhanced in C1q-deficient mice. The increased IgG deposits and apoptotic cells in the glomeruli of C1q-deficient mice suggest that the exacerbation of disease may be due to a defect in the clearance of immune complexes and/or apoptotic cells from their kidneys.
PU.1 and Multiple IFN Regulatory Factor Proteins Synergize to Mediate Transcriptional Activation of the Human IL-1β Gene
Authors: Marecki Sylvia , Riendeau Carrie J. , Liang Michael D. , Fenton Matthew J.
Published: April 2014

Both lymphoid and myeloid cells express two related members of the IFN regulatory factor (IRF) family of transcription factors, specifically IRF-4 and IFN consensus binding protein (ICSBP or IRF-8). We previously reported that macrophages express IRF-4 and in combination with the ETS-like protein PU.1 can synergistically activate a human IL-1β reporter gene. Here we report that this synergy is mediated by a composite PU.1/IRF element located within an upstream enhancer known to confer cytokine- and LPS-inducible expression. In macrophages, synergistic activation of IL-1β reporter gene expression was preferentially mediated by IRF-4, whereas IRF-4 and ICSBP were equally capable of synergizing with PU.1 when coexpressed in fibroblasts. Furthermore, coexpression of IRF-1 and IRF-2 dramatically increased the capacity of both PU.1/IRF-4 and PU.1/ICSBP to induce IL-1β reporter gene expression in fibroblasts. The additional synergy observed with IRF-1 and IRF-2 coexpression is mediated by a region of DNA distinct from either the IL-1β enhancer or promoter. We also assessed the capacity of these transcription factors to activate endogenous IL-1β gene when overexpressed in human embryonic kidney 293 cells. Although ectopic expression of PU.1 alone was sufficient to activate modest levels of IL-1β transcripts, endogenous IL-1β expression was markedly increased following coexpression of additional IRF proteins. Thus, maximal expression of both a human IL-1β reporter gene and the endogenous IL-1β gene was observed in cells that coexpressed PU.1, IRF-4 (or ICSBP), IRF1, and IRF2. Together, our observations suggest that these factors may function together as an enhanceosome.
Bacterial DNA and Lipopolysaccharide Induce Synergistic Production of TNF-α Through a Post-Transcriptional Mechanism
Authors: Gao Jian Jun , Xue Qiao , Papasian Christopher J. , Morrison David C.
Published: April 2014

LPS is well recognized for its potent capacity to activate mouse macrophages to produce TNF-α, an important inflammatory mediator in bacterial infection-related diseases such as septic shock. We demonstrate here that while inducing only low levels of TNF-α alone, DNA from both Gram-negative and Gram-positive bacteria synergizes with subthreshold concentrations of LPS (0.3 ng/ml) to induce TNF-α in the RAW 264.7 macrophage-like cell line. The bacterial DNA effects are mimicked by synthetic CpG-containing oligodeoxynucleotides, but not non-CpG-containing oligodeoxynucleotides. Pretreatment of macrophages with either DNA for 2–8 h inhibits macrophage TNF-α production in responses to DNA/LPS. However, when pretreatment was extended to 24 h, DNA/LPS synergy on TNF-α is further enhanced. RT-PCR analysis indicates that mRNA levels of the TNF-α gene, however, are not synergistically induced by bacterial DNA and LPS. Analyses of the half-life of TNF-α mRNA indicate that TNF-α message has a longer half-life in bacterial DNA- and LPS-treated macrophages than that in bacterial DNA- or LPS-treated macrophages. These findings indicate that the temporally controlled, synergistic induction of TNF-α by bacterial DNA and LPS is not mediated at the transcriptional level. Instead, this synergy may occur via a post-transcriptional mechanism.
Reversing Lipopolysaccharide Toxicity by Ligating the Macrophage Fcγ Receptors
Authors: Gerber Jeffrey S. , Mosser David M.
Published: April 2014

Our laboratory has previously demonstrated that the ligation of phagocytic receptors on macrophages can influence cytokine production. In this study, we examine the cytokine responses to multiple inflammatory stimuli following FcγR ligation. Macrophages were stimulated in vitro with LPS, lipoteichoic acid, CD40 ligand, or low molecular mass hyaluronic acid. All of these stimuli were proinflammatory in character, inducing the production of high levels of IL-12, but only modest amounts of IL-10. The coligation of FcγR along with these stimuli resulted in an anti-inflammatory profile, abrogating IL-12 production and inducing high levels of IL-10. The modulation of these two cytokines occurred by two independent mechanisms. Whereas the abrogation of IL-12 biosynthesis was a property shared by several macrophage receptors, the induction of IL-10 was specific to the FcγR. The biological relevance of these observations was examined in murine models of endotoxemia, in which FcγR ligation induced the rapid production of IL-10 and prevented IL-12 synthesis. Mice could be passively immunized with Abs to LPS to reverse inflammatory cytokine production, and the transfer of macrophages whose FcγR had been ligated could rescue mice from lethal endotoxemia. Thus, the ligation of the macrophage FcγR can be exploited to prevent inappropriate inflammatory cytokine responses.
Blockade of CD40 Ligand Suppresses Chronic Experimental Myasthenia Gravis by Down-Regulation of Th1 Differentiation and Up-Regulation of CTLA-4
Authors: Im Sin-Hyeog , Barchan Dora , Maiti Prasanta Kumar , Fuchs Sara , Souroujon Miriam C.
Published: April 2014

Myasthenia gravis (MG) and experimental autoimmune MG (EAMG) are T cell-dependent Ab-mediated autoimmune disorders, in which the nicotinic acetylcholine receptor (AChR) is the major autoantigen. Th1-type cells and costimulatory factors such as CD40 ligand (CD40L) contribute to disease pathogenesis by producing proinflammatory cytokines and by activating autoreactive B cells. In this study we demonstrate the capacity of CD40L blockade to modulate EAMG, and analyze the mechanism underlying this disease suppression. Anti-CD40L Abs given to rats at the chronic stage of EAMG suppress the clinical progression of the autoimmune process and lead to a decrease in the AChR-specific humoral response and delayed-type hypersensitivity. The cytokine profile of treated rats suggests that the underlying mechanism involves down-regulation of AChR-specific Th1-regulated responses with no significant effect on Th2- and Th3-regulated AChR-specific responses. EAMG suppression is also accompanied by a significant up-regulation of CTLA-4, whereas a series of costimulatory factors remain unchanged. Adoptive transfer of splenocytes from anti-CD40L-treated rats does not protect recipient rats against subsequently induced EAMG. Thus it seems that the suppressed progression of chronic EAMG by anti-CD40L treatment does not induce a switch from Th1 to Th2/Th3 regulation of the AChR-specific immune response and does not induce generation of regulatory cells. The ability of anti-CD40L treatment to suppress ongoing chronic EAMG suggests that blockade of CD40L may serve as a potential approach for the immunotherapy of MG and other Ab-mediated autoimmune diseases.
Western and Chinese Antirheumatic Drug-Induced T Cell Apoptotic DNA Damage Uses Different Caspase Cascades and Is Independent of Fas/Fas Ligand Interaction
Authors: Lai Jenn-Haung , Ho Ling-Jun , Lu Kuo-Cheng , Chang Deh-Ming , Shaio Men-Fang , Han Shou-Hwa
Published: April 2014

Spontaneous or therapeutic induction of T cell apoptosis plays a critical role in establishing transplantation tolerance and maintaining remission of autoimmune diseases. We investigated the mechanisms of apoptosis induced by Chinese and Western antirheumatic drugs (ARDs) in human T cells. We found that hydroxychloroquine, Tripterygium wilfordii hook F, and tetrandrine (Tet), but not methotrexate, at therapeutic concentrations can cause T cell death. In addition, Tet selectively killed T cells, especially activated T cells. Although ARD-induced cytotoxicity was mediated through apoptotic mechanisms, Fas/Fas ligand interaction was not required. We further demonstrated that the processes of phosphatidylserine externalization and DNA damage along the ARD-induced T cell apoptotic pathway could operate independently, and that selective inhibition of DNA damage by caspase inhibitors did not prevent T cells from undergoing cell death. Moreover, we found that Tet- and Tripterygium wilfordii hook F-induced T cell DNA damage required caspase-3 activity, and hydroxychloroquine-induced T cell DNA damage was mediated through a caspase-3- and caspase-8-independent, but Z-Asp-Glu-Val-Asp-fluomethyl ketone-sensitive, signaling pathway. Finally, the observation that ARD-induced activation of caspase-3 in both Fas-sensitive and Fas-resistant Jurkat T cells indicates that Fas/Fas ligand interaction plays no role in ARD-induced T cell apoptosis. Our observations provide new information about the complex apoptotic mechanisms of ARDs, and have implications for combining Western and Chinese ARDs that have different immunomodulatory mechanisms in the therapy of autoimmune diseases and transplantation rejection.