bluuebunny/crossref_metadata_2025 huggingface dataset:
Infection of B Cell-Deficient Mice with CDC 1551, a Clinical Isolate ofMycobacterium tuberculosis: Delay in Dissemination and Development of Lung Pathology
Authors: Bosio Catharine M. , Gardner Donald , Elkins Karen L.
Published: April 2014

Long-term survival of mice infected with Mycobacterium tuberculosis is dependent upon IFN-γ and T cells, but events in early phases of the immune response are not well understood. In this study, we describe a role for B cells during early immune responses to infection with a clinical isolate of M. tuberculosis (CDC 1551). Following a low-dose infection with M. tuberculosis CDC 1551, similar numbers of bacteria were detected in the lungs of both B cell knockout (IgH 6−, BKO) and C57BL/6J (wild-type) mice. However, despite comparable bacterial loads in the lungs, less severe pulmonary granuloma formation and delayed dissemination of bacteria from lungs to peripheral organs were observed in BKO mice. BKO mice reconstituted with naive B cells, but not those given M. tuberculosis-specific Abs, before infection developed pulmonary granulomas and dissemination patterns similar to wild-type animals. Further analysis of lung cell populations revealed greater numbers of lymphocytes, especially CD8+ T cells, macrophages, and neutrophils in wild-type and reconstituted mice than in BKO mice. Thus, less severe lesion formation and delayed dissemination of bacteria found in BKO mice were dependent on B cells, not Abs, and were associated with altered cellular infiltrate to the lungs. These observations demonstrate an important, previously unappreciated, role for B cells during early immune responses to M. tuberculosis infections.
A Filarial Nematode-Secreted Product Signals Dendritic Cells to Acquire a Phenotype That Drives Development of Th2 Cells
Authors: Whelan Michael , Harnett Margaret M. , Houston Katrina M. , Patel Vanshree , Harnett William , Rigley Kevin P.
Published: April 2014

Although exogeneous “danger” signals such as LPS can activate APC to produce a Th1 response, the nature of events initiating a Th2 response is controversial. We now show that pathogen-derived products have the capacity to induce bone marrow-derived dendritic cell cultures to acquire a phenotype that promotes the differentiation of naive CD4+ T cells toward either a Th1 or Th2 phenotype. Thus, LPS-matured dendritic cells (DC1) promote a Th1 response (increased generation of IFN-γ and reduced production of IL-4) by Ag-stimulated CD4+ T cells from the DO.11.10 transgenic mouse expressing a TCR specific for an OVA peptide (OVA323–339). In contrast, a phosphorylcholine-containing glycoprotein, ES-62, secreted by the filarial nematode, Acanthocheilonema viteae, which generates a Th2 Ab response in vivo, is found to induce the maturation of dendritic cells (DC2) with the capacity to induce Th2 responses (increased IL-4 and decreased IFN-γ). In addition, we show that the switch to either Th1 or Th2 responses is not effected by differential regulation through CD80 or CD86 and that a Th2 response is achieved in the presence of IL-12.
Neutrophil-Induced Changes in the Biomechanical Properties of Endothelial Cells: Roles of ICAM-1 and Reactive Oxygen Species
Authors: Wang Qin , Doerschuk Claire M.
Published: April 2014

This study evaluated the changes in the biomechanical properties of endothelial cells (ECs) induced by neutrophil adhesion and the roles of ICAM-1 and reactive oxygen species (ROS) in modulating these changes. Neutrophil adherence to 24-h TNF-α-activated pulmonary microvascular ECs induced an increase in the apparent stiffness of ECs within 2 min, measured with magnetic twisting cytometry. An anti-ICAM-1 Ab blocked the EC stiffening response without inhibiting neutrophil adherence. Moreover, cross-linking ICAM-1 mimicked the stiffening response induced by neutrophils. The neutrophil-induced increase in the apparent stiffness of ECs was inhibited with 1% DMSO (a hydroxyl radical scavenger), allopurinol (a xanthine oxidase inhibitor), or deferoxamine (an iron chelator), suggesting that ROS may be involved in mediating the EC stiffening response. The cellular sources of ROS were determined by measuring the oxidation of dichlorofluorescein. Neutrophil adherence to TNF-α-activated ECs induced ROS production only in ECs, and not in neutrophils. This ROS production in ECs was completely prevented by the anti-ICAM-1 Ab and partially inhibited by allopurinol. These results suggest that ICAM-1-mediated signaling events during neutrophil adherence may activate xanthine oxidase, which in turn mediates the ROS production in ECs that leads to stiffening. ROS generated in ECs on neutrophil adherence appear to mediate cytoskeletal remodeling, which may modulate subsequent inflammatory responses.
Peroxisome Proliferator-Activated Receptor-γ Activators Inhibit IFN-γ-Induced Expression of the T Cell-Active CXC Chemokines IP-10, Mig, and I-TAC in Human Endothelial Cells
Authors: Marx Nikolaus , Mach François , Sauty Alain , Leung Josephine H. , Sarafi Mindy N. , Ransohoff Richard M. , Libby Peter , Plutzky Jorge , Luster Andrew D.
Published: April 2014

Peroxisome proliferator-activated receptor-γ (PPARγ), a member of the nuclear hormone receptor superfamily originally shown to play an important role in adipocyte differentiation and glucose homeostasis, is now known to regulate inflammatory responses. Given the importance of endothelial cell (EC)-derived chemokines in regulating leukocyte function and trafficking, we studied the effects of PPARγ ligands on the expression of chemokines induced in ECs by the Th1 cytokine IFN-γ. Treatment of ECs with PPARγ activators significantly inhibited IFN-γ-induced mRNA and protein expression of the CXC chemokines IFN-inducible protein of 10 kDa (IP-10), monokine induced by IFN-γ (Mig), and IFN-inducible T-cell α-chemoattractant (I-TAC), whereas expression of the CC chemokine monocyte chemoattractant protein-1 was not altered. PPARγ activators decreased IFN-inducible protein of 10 kDa promoter activity and inhibited protein binding to the two NF-κB sites but not to the IFN-stimulated response element ISRE site. Furthermore, PPARγ ligands inhibited the release of chemotactic activity for CXC chemokine receptor 3 (CXCR3)-transfected lymphocytes from IFN-γ-stimulated ECs. These data suggest that anti-diabetic PPARγ activators might attenuate the recruitment of activated T cells at sites of Th1-mediated inflammation.
Phagocytosis of Nonapoptotic Cells Dying by Caspase- Independent Mechanisms
Authors: Hirt Ulrich A. , Gantner Florian , Leist Marcel
Published: April 2014

Caspase activation, exposure of phosphatidylserine (PS) on the outer surface of the plasma membrane, and rapid phagocytic removal of dying cells are key features of apoptosis. Nonapoptotic/necrotic modes of death occur independent of caspase activation, but the role of phagocytosis is largely unknown. To address this issue, we studied phagocytosis by human monocyte-derived macrophages (HMDM) and rat microglial cells. Target cells (Jurkat) were stimulated by several different methods that all caused caspase-independent death. First, we induced necrosis by combining toxins with ATP-depleting agents. Under these conditions, neither PS was exposed nor were such cells phagocytosed before their death. However, once the plasma membrane integrity was lost, the dead cells were rapidly and efficiently engulfed by HMDM. Next, we triggered Jurkat cell death with staurosporine in the presence of the pan-caspase inhibitor zVAD-fmk. Under these conditions, death occurred by delayed necrosis and without exposure of PS. Nevertheless, such lethally challenged cells were phagocytosed before the loss of membrane integrity. Finally, we triggered Ca2+ influx in Jurkat cells with an ionophore, or in neurons by glutamate receptor stimulation, respectively. In both models, PS was exposed on the cell surface. Ca2+-stressed cells were phagocytosed starting at 30 min after stimulation. Protein kinase C inhibitors prevented Ca2+-mediated PS exposure and phagocytosis. Essentially, similar phagocytosis data were obtained for all models with HMDM and microglia. We conclude that also cells dying nonapoptotically and independent of caspase activation may be recognized and removed before, or very quickly after, membrane lysis.
Differential Mitogen-Activated Protein Kinase Stimulation by Fcγ Receptor IIa and Fcγ Receptor IIIb Determines the Activation Phenotype of Human Neutrophils
Authors: Coxon Patricia Y. , Rane Madhavi J. , Powell David W. , Klein Jon B. , McLeish Kenneth R.
Published: April 2014

FcγRs mediate immune complex-induced tissue injury. The hypothesis that FcγRIIa and FcγRIIIb control neutrophil responses by activating mitogen-activated protein kinases was examined. Homotypic and heterotypic cross-linking of FcγRIIa and/or FcγRIIIb resulted in a rapid, transient increase in ERK and p38 activity, with maximal stimulation between 1 and 3 min. FcγRIIa and FcγRIIIb stimulated distinct patterns of ERK and p38 activity, and heterotypic cross-linking failed to stimulate synergistic activation of either ERK or p38 activity. Both FcγRIIa and FcγRIIIb required activation of a nonreceptor tyrosine kinase and phosphatidylinositol 3-kinase for stimulation of ERK and p38. Inhibition of ERK activation with PD98059 enhanced H2O2 production stimulated by homotypic and heterotypic FcγR cross-linking. Inhibition of p38 with SB203580 attenuated H2O2 production stimulated by FcγRIIIb or heterotypic cross-linking, but had no effect on FcγRIIa-stimulated H2O2 production. On the other hand, PD98059 inhibited actin polymerization stimulated by FcγR cross-linking, while SB203580 had no effect. Inhibition of actin polymerization with cytochalasin D enhanced p38 activity stimulated by either FcγRIIa or FcγRIIIb, but cytochalasin D only enhanced H2O2 production stimulated by FcγRIIIb. Our data indicate that FcγRIIa and FcγRIIIb independently activate ERK and p38. The two receptors demonstrate different efficacies for ERK and p38 activation, and they do not act cooperatively. ERK and p38 provide stimulatory and inhibitory signals for neutrophil responses to immune complexes. In addition, these data indicate that actin reorganization may play a role in mediating p38-dependent activation of respiratory burst upon stimulation of FcγRIIIb in neutrophils.
Release of Calcium from Inositol 1,4,5-Trisphosphate Receptor-Regulated Stores by HIV-1 Tat Regulates TNF-α Production in Human Macrophages
Authors: Mayne Michael , Holden Clark P. , Nath Avindra , Geiger Jonathan D.
Published: April 2014

HIV-1 protein Tat is neurotoxic and increases macrophage and microglia production of TNF-α, a cytopathic cytokine linked to the neuropathogenesis of HIV dementia. Others have shown that intracellular calcium regulates TNF-α production in macrophages, and we have shown that Tat releases calcium from inositol 1,4,5-trisphosphate (IP3) receptor-regulated stores in neurons and astrocytes. Accordingly, we tested the hypothesis that Tat-induced TNF-α production was dependent on the release of intracellular calcium from IP3-regulated calcium stores in primary macrophages. We found that Tat transiently and dose-dependently increased levels of intracellular calcium and that this increase was blocked by xestospongin C, pertussis toxin, and by phospholipase C and type 1 protein kinase C inhibitors but not by protein kinase A or phospholipase A2 inhibitors. Xestospongin C, BAPTA-AM, U73122, and bisindolylmalemide significantly inhibited Tat-induced TNF-α production. These results demonstrate that in macrophages, Tat-induced release of calcium from IP3-sensitive intracellular stores and activation of nonconventional PKC isoforms play an important role in Tat-induced TNF-α production.
Lymphocyte Migration Through Monolayers of Endothelial Cell Lines Involves VCAM-1 Signaling Via Endothelial Cell NADPH Oxidase
Authors: Matheny Heather E. , Deem Tracy L. , Cook-Mills Joan M.
Published: April 2014

Lymphocytes migrate from the blood across endothelial cells to reach foreign substances sequestered in peripheral lymphoid organs and inflammatory sites. To study intracellular signaling in endothelial cells during lymphocyte migration, we used murine endothelial cell lines that promote lymphocyte migration and constitutively express VCAM-1. The maximum rate of resting splenic lymphocyte migration across monolayers of the endothelial cells occurred at 0–24 h. This migration was inhibited by anti-VCAM-1 or anti-α4 integrin, suggesting that VCAM-1 adhesion was required for migration. To determine whether signals within the endothelial cells were required for migration, irreversible inhibitors of signal transduction molecules were used to pretreat the endothelial cell lines. Inhibitors of NADPH oxidase activity (diphenyleneiodonium and apocynin) blocked migration >65% without affecting adhesion. Because NADPH oxidase catalyzes the production of reactive oxygen species (ROS), we examined whether ROS were required for migration. Scavengers of ROS inhibited migration without affecting adhesion. Furthermore, VCAM-1 ligand binding stimulated NADPH oxidase-dependent production of ROS by the endothelial cells lines and primary endothelial cell cultures. Finally, VCAM-1 ligand binding induced an apocynin-inhibitable actin restructuring in the endothelial cell lines at the location of the lymphocyte or anti-VCAM-1-coated bead, suggesting that an NADPH oxidase-dependent endothelial cell shape change was required for lymphocyte migration. In summary, VCAM-1 signaled the activation of endothelial cell NADPH oxidase, which was required for lymphocyte migration. This suggests that endothelial cells are not only a scaffold for lymphocyte adhesion, but play an active role in promoting lymphocyte migration.
Characterization of Human γ-Tryptases, Novel Members of the Chromosome 16p Mast Cell Tryptase and Prostasin Gene Families
Authors: Caughey George H. , Raymond Wilfred W. , Blount John L. , Hau Leola W.-T. , Pallaoro Michele , Wolters Paul J. , Verghese George M.
Published: April 2014

Previously, this laboratory identified clusters of α-, β-, and mast cell protease-7-like tryptase genes on human chromosome 16p13.3. The present work characterizes adjacent genes encoding novel serine proteases, termed γ-tryptases, and generates a refined map of the multitryptase locus. Each γ gene lies between an α1H Ca2+ channel gene (CACNA1H) and a βII- or βIII-tryptase gene and is ∼30 kb from polymorphic minisatellite MS205. The tryptase locus also contains at least four tryptase-like pseudogenes, including mastin, a gene expressed in dogs but not in humans. Genomic DNA blotting results suggest that γI- and γII-tryptases are alleles at the same site. βII- and βIII-tryptases appear to be alleles at a neighboring site, and αII- and βI-tryptases appear to be alleles at a third site. γ-Tryptases are transcribed in lung, intestine, and in several other tissues and in a mast cell line (HMC-1) that also expresses γ-tryptase protein. Immunohistochemical analysis suggests that γ-tryptase is expressed by airway mast cells. γ-Tryptase catalytic domains are ∼48% identical with those of known mast cell tryptases and possess mouse homologues. We predict that γ-tryptases are glycosylated oligomers with tryptic substrate specificity and a distinct mode of activation. A feature not found in described tryptases is a C-terminal hydrophobic domain, which may be a membrane anchor. Although the catalytic domains contain tryptase-like features, the hydrophobic segment and intron-exon organization are more closely related to another recently described protease, prostasin. In summary, this work describes γ-tryptases, which are novel members of chromosome 16p tryptase/prostasin gene families. Their unique features suggest possibly novel functions.
Prolonged Elevation of IL-1 inPseudomonas aeruginosaOcular Infection Regulates Macrophage-Inflammatory Protein-2 Production, Polymorphonuclear Neutrophil Persistence, and Corneal Perforation
Authors: Rudner Xiaowen L. , Kernacki Karen A. , Barrett Ronald P. , Hazlett Linda D.
Published: April 2014

The kinetics of IL-1 (α and β) production after Pseudomonas aeruginosa corneal infection was examined in susceptible (cornea perforates) C57BL/6J (B6) and resistant (cornea heals) BALB/cByJ (BALB/c) mice. IL-1α and -1β (mRNA and protein) were elevated in both mouse strains, and levels peaked at 1 day postinfection (p.i.). Significantly greater amounts of IL-1 protein were detected in B6 vs BALB/c mice at 1 and 3 days p.i. At 5 days p.i., IL-1α and -1β (mRNA and protein) remained elevated in B6, but began to decline in BALB/c mice. To test the significance of elevated IL-1 in B6 mice, a polyclonal neutralizing Ab against IL-1β was used to treat infected B6 mice. A combination of subconjunctival and i.p. administration of IL-1β polyclonal Ab significantly reduced corneal disease. The reduction in disease severity in infected B6 mice was accompanied by a reduction in corneal polymorphonuclear neutrophil number, bacterial load, and macrophage inflammatory protein-2 mRNA and protein levels. These data provide evidence that IL-1 is an important contributor to P. aeruginosa corneal infection. At least one mechanism by which prolonged and/or elevated IL-1 expression contributes to irreversible corneal tissue destruction appears to be by increasing macrophage inflammatory protein-2 production, resulting in a prolonged stimulation of polymorphonuclear neutrophil influx into cornea. In contrast, a timely down-regulation of IL-1 appears consistent with an inflammatory response that is sufficient to clear the bacterial infection with less corneal damage.
Recurrent Respiratory Syncytial Virus Infections in Allergen-Sensitized Mice Lead to Persistent Airway Inflammation and Hyperresponsiveness
Authors: Matsuse Hiroto , Behera Aruna K. , Kumar Mukesh , Rabb Hamid , Lockey Richard F. , Mohapatra Shyam S.
Published: April 2014

Respiratory syncytial virus (RSV) infection is considered a risk factor for bronchial asthma; however, the synergy between allergen sensitization and RSV infection in the development of pulmonary inflammation and asthma has been controversial. In this study the effects of primary and recurrent RSV infection on allergic asthma were examined in a group of control, RSV-infected, Dermatophagoides farinae (Df) allergen-sensitized, and Df allergen-sensitized plus RSV-infected BALB/c mice. Primary RSV infection in Df-sensitized mice transiently increases airway responsiveness, which is accompanied by increases in eosinophilic infiltration, the expression of ICAM-1, and macrophage inflammatory protein-1α (MIP-1α) in the lung tissue. A secondary RSV infection persistently enhances airway responsiveness in Df-sensitized mice, with a concomitant increase in MIP-1α and RSV Ag load in lung tissues. Bulk cultures of thoracic lymph node mononuclear cells demonstrate that acute RSV infection augments both Th1- and Th2-like cytokines, whereas secondary and tertiary infections shift the cytokine profile in favor of the Th2-like cytokine response in Df-sensitized mice. The elevated total serum IgE level in the Df-sensitized mice persists following only RSV reinfection. Thus, recurrent RSV infections in Df-sensitized mice augment the synthesis of Th2-like cytokines, total serum IgE Abs, and MIP-1α, which are responsible for persistent airway inflammation and hyperresponsiveness, both of which are characteristics of asthma.
Increased CD40 Expression on Muscle Cells of Polymyositis and Dermatomyositis: Role of CD40-CD40 Ligand Interaction in IL-6, IL-8, IL-15, and Monocyte Chemoattractant Protein-1 Production
Authors: Sugiura Tomoko , Kawaguchi Yasushi , Harigai Masayoshi , Takagi Kae , Ohta Shuji , Fukasawa Chikako , Hara Masako , Kamatani Naoyuki
Published: April 2014

In polymyositis (PM)/dermatomyositis (DM), T cells infiltrate the muscle tissues and interact with muscle cells via cell surface molecules. Recently, myoblasts have been reported to express CD40, but little is known about the role of CD40 in myoblasts. In the present study we examined the expression and involvement of CD40 and CD40 ligand (CD40L) in the interaction between muscle cells and T cells in PM/DM. Immunohistochemical staining revealed that CD40 was expressed on muscle cells in five of five PM and four of five DM patients, and that infiltrating mononuclear cells (MNCs) expressed CD40L in all cases of PM/DM. These CD40L-expressing MNCs were primarily CD4+ T cells. IFN-γ, which is known to induce CD40 expression on various types of cells, was also expressed on the MNCs in four of the PM and four of the DM patients. Although cultured human myoblasts (SkMC 2859) did not express CD40 constitutively, IFN-γ induced CD40 expression in a dose-dependent manner. To clarify the functional roles of CD40-mediated signals, the effects of a trimeric form of recombinant human CD40L on cytokine production were studied in SkMC 2859 that were prestimulated with IFN-γ to express CD40. Recombinant human CD40L markedly increased the production of IL-6, IL-8, IL-15, and monocyte chemoattractant protein-1 of SkMC 2859. The expression of these humoral factors in muscle cells of PM and DM was demonstrated by immunohistochemistry. These results suggest that interaction between T cells and muscle cells via the CD40-CD40L system contributes to the immunopathogenesis of PM/DM by augmenting inflammation via cytokine production by the muscle cells.
Human TNF Can Induce Nonspecific Inflammatory and Human Immune-Mediated Microvascular Injury of Pig Skin Xenografts in Immunodeficient Mouse Hosts
Authors: Kirkiles-Smith Nancy C. , Tereb Denis A. , Kim Richard W. , McNiff Jennifer M. , Schechner Jeffrey S. , Lorber Marc I. , Pober Jordan S. , Tellides George
Published: April 2014

TNF activates endothelial cells to express cell surface molecules that are necessary to recruit a local infiltrate of leukocytes. Because the actions of this proinflammatory cytokine are not species restricted, we investigated whether human TNF can up-regulate porcine endothelial adhesion molecules to elicit human T cell infiltration and damage of pig skin xenografts in a chimeric immunodeficient mouse model. We have previously demonstrated the vigorous rejection of human skin allografts and the absence of injury to porcine skin xenografts in human PBMC-SCID/beige mice. Intradermal administration of human TNF at high doses (600 or 2000 ng) caused nonspecific inflammatory damage of pig skin grafts, whereas low concentrations of TNF (60 or 200 ng) resulted in human PBMC-dependent injury of porcine endothelial cells. There was a strong correlation among pig skin xenograft damage, human T cell infiltration, and the TNF-induced up-regulation of swine MHC class I and class II molecules, VCAM-1, and, in particular, the de novo expression of porcine E-selectin. The microvascular damage and leukocytic infiltration elicited by TNF were enhanced by porcine IFN-γ, suggesting that xenografts may be less prone to cytokine-mediated injury due to the species-restricted effects of recipient IFN-γ. Our results indicate that maintenance of a quiescent endothelium, which does not express E-selectin or other activation-dependent adhesion molecules, is important in preventing human anti-porcine T cell xenoresponses in vivo and that TNF signaling molecules and TNF-responsive gene products are appropriate therapeutic targets to protect against human T cell-mediated rejection of pig xenografts.
CD40-Ligated Dendritic Cells Effectively Expand Melanoma-Specific CD8+ CTLs and CD4+ IFN-γ-Producing T Cells from Tumor-Infiltrating Lymphocytes
Authors: Terheyden Patrick , Straten Per thor , Bröcker Eva-B. , Kämpgen Eckhart , Becker Jürgen C.
Published: April 2014

Professional APC, notably dendritic cells (DC), are necessary for stimulation and expansion of naive T cells. By means of murine models, the interaction between CD40 on DC and its ligand CD154 has been recognized as an important element for conditioning of DC to prime and expand CTL. We translated these findings into the human system, scrutinizing the ability of DC to initiate clonal expansion of single T cells. DC generated under completely autologous conditions from peripheral blood monocytes were cocultured at a rate of 0.3 cell/well with melanoma-infiltrating T cells; this procedure guaranteed that either a CD4+ or a CD8+ cell interacted with the DC, thus avoiding the contact of more than one T cell to the DC. In the absence of further stimulation, this cloning protocol yielded almost exclusively CD4+ T cell clones that predominantly exhibited a Th2 phenotype. However, cross-linking of CD40 on DC resulted in the induction of IFN-γ-producing Th1 CD4+ T cell clones. In addition, CD40-activated DC were capable of expanding CD8+ CTL clones. The ratio of CD4 to CD8 T cell clones corresponded to the ratio present in the initial tumor-infiltrating lymphocyte preparation. The CTL clones efficiently lysed autologous tumor cells whereas autologous fibroblasts or MHC-mismatched melanoma cells were not killed. Our findings support the critical role of CD40/CD154 interactions for the induction of cellular immune responses.
Cutting Edge: A Novel Function for the SLAP-130/FYB Adapter Protein in β1 Integrin Signaling and T Lymphocyte Migration
Authors: Hunter Anne J. , Ottoson Nadine , Boerth Nancy , Koretzky Gary A. , Shimizu Yoji
Published: April 2014

The role of integrin-mediated signaling events in T cell function remains incompletely characterized. We report here that α4β1 integrin stimulation of H9 T cells and normal human T cell blasts results in rapid and transient tyrosine phosphorylation of the adapter protein, SH2 domain-containing 76-kDa protein (SLP-76)-associated phosphoprotein of 130 kDa (SLAP-130)/FYB at levels comparable to those observed following TCR stimulation. Stimulation of T cells via the α4β1 integrin enhances the association of tyrosine phosphorylated SLAP-130/FYB with the SH2 domain of the src tyrosine kinase p59fyn. Activation of normal T cells, but not H9 T cells, via α4β1 leads to tyrosine phosphorylation of SLP-76 as well as SLAP-130/FYB. Overexpression of SLAP-130/FYB in normal T cells enhances T cell migration through fibronectin-coated filters in response to the chemokine stromal cell-derived factor (SDF)-1α. These results identify SLAP-130/FYB as a new tyrosine phosphorylated substrate in β1 integrin signaling and suggest a novel function for SLAP-130/FYB in regulating T lymphocyte motility.
Functional Consequences of the Developmental Arrest and Follicular Exclusion of Anti-Double-Stranded DNA B Cells
Authors: Mandik-Nayak Laura , Seo Su-jean , Eaton-Bassiri Ashlyn , Allman David , Hardy Richard R. , Erikson Jan
Published: April 2014

Anti-dsDNA B cells are actively tolerized in nonautoimmune BALB/c mice, as manifested by their developmental arrest, follicular exclusion, and rapid turnover rate. Previously, we have documented changes in the maturation status and follicular localization of anti-dsDNA B cells in autoimmune-prone MRL (+/+ and lpr/lpr) mice. To determine whether these differences in developmental status and follicular localization affect the functional capacity of anti-dsDNA B cells, we have now compared their in vivo life spans and their responses to in vitro stimuli. Our study shows that although anti-dsDNA B cells from both BALB/c and MRL-+/+ mice are localized to the T/B interface, only those in BALB/c mice have a rapid turnover rate. Therefore, the immature status and not the exclusion from the B cell follicle correlates with a shortened life span. Interestingly, apoptotic anti-dsDNA B cells were not detected at the T/B interface in BALB/c mice, suggesting that they are not dying there. This study also demonstrates that anti-dsDNA B cells, regardless of maturation status or follicular localization, are able to proliferate and up-regulate the costimulatory molecule B7-2 in response to CD40 ligand and IL-4. Therefore, one of the critical in vivo differences between anti-dsDNA B cells in BALB/c and MRL-+/+ mice compared with MRL-lpr/lpr mice may be the availability of T cell help.
c-Src Mediates Mitogenic Signals and Associates with Cytoskeletal Proteins upon Vascular Endothelial Growth Factor Stimulation in Kaposi’s Sarcoma Cells
Authors: Munshi Neru , Groopman Jerome E. , Gill Parkash S. , Ganju Ramesh K.
Published: April 2014

Vascular endothelial growth factor (VEGF) appears to be a critical cytokine modulating the growth and spread of Kaposi’s sarcoma (KS). Furthermore, infection with the KS herpes virus results in up-regulation of VEGF and triggering of VEGF receptor activation. The molecular mechanisms regulating such cytokine-driven proliferation of KS cells are not well characterized. We investigated the role of Src-related tyrosine kinases in VEGF-mediated signaling in model KS 38 tumor cells. VEGF stimulation specifically activated c-Src kinase activity but not that of other related Src kinases such as Lyn, Fyn, or Hck in KS cells. Pyrazolopyrimidine, a selective inhibitor of Src family tyrosine kinases, significantly blocked the VEGF-induced growth of KS cells. Further studies using mutants of c-Src kinase revealed that Src mediates mitogen-activated protein kinase activation induced by VEGF. We also observed that VEGF stimulation resulted in increased tyrosine phosphorylation of the focal adhesion components paxillin and p130cas. Furthermore, VEGF induction enhanced the complex formation between Src kinase and paxillin. Src kinase appears to play an important functional role in VEGF-induced signaling in KS cells and may act to link pathways from the VEGF receptor to mitogen-activated protein kinase and cytoskeletal components, thereby effecting tumor proliferation and migration.
Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase-Activating Polypeptide Inhibit Antigen-Induced Apoptosis of Mature T Lymphocytes by Inhibiting Fas Ligand Expression
Authors: Delgado Mario , Ganea Doina
Published: April 2014

Apoptosis in T and B lymphocytes is a major element controlling the immune response. The Ag-induced cell death (AICD) in T cells is a main mechanism for maintaining peripheral tolerance and for limiting an ongoing immune response. AICD is initiated by Ag re-engagement of the TCR and is mediated through Fas/Fas ligand (FasL) interactions. Vasoactive intestinal peptide (VIP) and the structurally related pituitary adenylate cyclase-activating polypeptide (PACAP) are two multifunctional neuropeptides present in the lymphoid microenvironment that act primarily as anti-inflammatory agents. In the present study we investigated whether VIP and PACAP affect AICD in mature peripheral T cells and T cell hybridomas. VIP and PACAP reduce in a dose-dependent manner anti-CD3-induced apoptosis in Con A/IL-2-preactivated peripheral T cells and the murine T hybridomas 2B4.11 and A1.1. A functional study demonstrates that the inhibition of AICD is achieved through the inhibition of activation-induced FasL expression at protein and mRNA levels. VIP/PACAP-mediated inhibition of both AICD and FasL expression is mediated through the specific receptors VPAC1 and VPAC2. Of obvious biological significance is the fact that VIP and PACAP prevent Ag-induced clonal deletion of CD4+ T cells, but not that of CD8+ T cells. By affecting FasL expression, VIP and PACAP may play a physiological role in both the generation of memory T cells and the inhibition of FasL-mediated T cell cytotoxicity.
CD72 Negatively Regulates Signaling Through the Antigen Receptor of B Cells
Authors: Adachi Takahiro , Wakabayashi Chisato , Nakayama Toshinori , Yakura Hidetaka , Tsubata Takeshi
Published: April 2014

The immunoreceptor tyrosine-based inhibition motif (ITIM) is found in various membrane molecules such as CD22 and the low-affinity Fc receptor for IgG in B cells and the killer cell-inhibitory receptor and Ly-49 in NK cells. Upon tyrosine phosphorylation at the ITIMs, these molecules recruit SH2 domain-containing phosphatases such as SH2-containing tyrosine phosphatase-1 and negatively regulate cell activity. The B cell surface molecule CD72 carries an ITIM and an ITIM-like sequence. We have previously shown that CD72 is phosphorylated and recruits SH2-containing tyrosine phosphatase-1 upon cross-linking of the Ag receptor of B cells (BCR). However, whether CD72 modulates BCR signaling has not yet been elucidated. In this paper we demonstrate that expression of CD72 down-modulates both extracellular signal-related kinase (ERK) activation and Ca2+ mobilization induced by BCR ligation in the mouse B lymphoma line K46μmλ, whereas BCR-mediated ERK activation was not reduced by the ITIM-mutated form of CD72. Moreover, coligation with CD72 with BCR reduces BCR-mediated ERK activation in spleen B cells of normal mice. These results indicate that CD72 negatively regulates BCR signaling. CD72 may play a regulatory role in B cell activation, probably by setting a threshold for BCR signaling.
Induction of CTL Responses by Simultaneous Administration of Liposomal Peptide Vaccine with Anti-CD40 and Anti-CTLA-4 mAb
Authors: Ito Daisuke , Ogasawara Kazumasa , Iwabuchi Kazuya , Inuyama Yukio , Onoé Kazunori
Published: April 2014

Activation of APC via CD40-CD40 ligand pathway induces up-regulation of costimulatory molecules such as B7 and production of IL-12. Interaction between B7 on APC and CD28 on naive T cells is necessary for priming the T cells. On the other hand, interaction between B7 on APC and CTLA-4 on activated T cells transduces a negative regulatory signal to the activated T cells. In the present study, we attempted to generate tumor-specific CTL by s.c. administration of antigenic peptides encapsulated in multilamellar liposomes (liposomal peptide vaccine) with anti-CD40 mAb and/or anti-CTLA-4 mAb. Liposomal OVA257–264 and anti-CD40 mAb or anti-CTLA-4 mAb were administrated to C57BL/6 mice and the splenocytes were cocultured with OVA257–264 for 4 days. The splenic CD8+ T cells showed a significant cytotoxicity against EL4 cells transfected with cDNA of OVA. In addition, administration of both anti-CD40 and anti-CTLA-4 mAb enhanced the CTL responses. Considerable CTL responses were induced in MHC class II deficient mice by the same procedure. This finding indicated that CTL responses could be generated even in the absence of Th cells. When BALB/c mice were immunized with pRL1a peptide that are tumor-associated Ag of RL♂1 leukemia cells using the same procedure, significant CTL responses were induced and prolonged survival of the BALB/c mice was observed following RL♂1 inoculation. These results demonstrate that anti-CD40 mAb and anti-CTLA-4 mAb function as immunomodulators and may be applicable to specific cancer immunotherapy with antitumor peptide vaccine.
Retrovirally Transduced Mouse Dendritic Cells Require CD4+ T Cell Help to Elicit Antitumor Immunity: Implications for the Clinical Use of Dendritic Cells
Authors: Schnell Stefan , Young James W. , Houghton Alan N. , Sadelain Michel
Published: April 2014

Presentation of MHC class I-restricted peptides by dendritic cells (DCs) can elicit vigorous antigen-specific CTL responses in vivo. It is well established, however, that T cell help can augment CTL function, raising the question of how best to present tumor-associated MHC class I epitopes to induce effective tumor immunity. To this end, we have examined the role of MHC class II peptide-complexes present on the immunizing DCs in a murine melanoma model. To present MHC class I- and II-restricted Ags reliably on the same cell, we retrovirally transduced bone marrow-derived DCs with the model Ag OVA encoding well-defined class I- and II-restricted epitopes. The importance of CD4+ T cells activated by the immunizing DCs in this model is demonstrated by the following findings: 1) transduced DCs presenting class I and class II epitopes are more efficient than class I peptide-pulsed DCs; 2) MHC class II-deficient DCs fail to induce tumor protection; 3) CD4+ T cell depletion abolishes induction of tumor protection; and 4) DCs presenting bovine serum Ags are more effective in establishing tumor immunity than DCs cultured in syngeneic serum. When MHC class II-deficient DCs were directly activated via their CD40 receptor, we indeed observed a moderate elevation of OVA-specific CTL activity. However, this increase in CTL activity was not sufficient to induce in vivo tumor rejection. Thus, our results demonstrate the potency of genetically modified DCs that express both MHC class I and II epitopes, but caution against the use of DCs presenting only the former.
CCR9A and CCR9B: Two Receptors for the Chemokine CCL25/TECK/Ckβ-15 That Differ in Their Sensitivities to Ligand
Authors: Yu Cheng-Rong , Peden Keith W. C. , Zaitseva Marina B. , Golding Hana , Farber Joshua M.
Published: April 2014

We isolated cDNAs for a chemokine receptor-related protein having the database designation GPR-9-6. Two classes of cDNAs were identified from mRNAs that arose by alternative splicing and that encode receptors that we refer to as CCR9A and CCR9B. CCR9A is predicted to contain 12 additional amino acids at its N terminus as compared with CCR9B. Cells transfected with cDNAs for CCR9A and CCR9B responded to the chemokine CC chemokine ligand 25 (CCL25)/thymus-expressed chemokine (TECK)/chemokine β-15 (CKβ-15) in assays for both calcium flux and chemotaxis. No other chemokines tested produced responses specific for the cDNA-transfected cells. mRNA for CCR9A/B is expressed predominantly in the thymus, coincident with the expression of CCL25, and highest expression for CCR9A/B among thymocyte subsets was found in CD4+CD8+ cells. mRNAs encoding the A and B forms of the receptor were expressed at a ratio of ∼10:1 in immortalized T cell lines, in PBMC, and in diverse populations of thymocytes. The EC50 of CCL25 for CCR9A was lower than that for CCR9B, and CCR9A was desensitized by doses of CCL25 that failed to silence CCR9B. CCR9 is the first example of a chemokine receptor in which alternative mRNA splicing leads to proteins of differing activities, providing a mechanism for extending the range of concentrations over which a cell can respond to increments in the concentration of ligand. The study of CCR9A and CCR9B should enhance our understanding of the role of the chemokine system in T cell biology, particularly during the stages of thymocyte development.
T Cells Can Induce Somatic Mutation in B Cell Receptor-Engaged BL2 Burkitt’s Lymphoma Cells Independently of CD40-CD40 Ligand Interactions
Authors: Denépoux Stéphane , Fournier Nathalie , Péronne Catherine , Banchereau Jacques , Lebecque Serge
Published: April 2014

The B cell surface trigger(s) and the molecular mechanism(s) of somatic hypermutation remain unknown, partly because of the lack of amendable in vitro models. Recently, however, we reported that upon B cell receptor cross-linking and coculture with activated T cells, the Burkitt’s lymphoma cell line BL2 introduces mutations in its IgVH gene in vitro. We now confirm the relevance of our culture model by establishing that the entire spectrum of somatic mutations observed in vivo, including insertions and deletions, could be found in the DNA of BL2 cells. Additionally, we show that among four human B cell lines, only two with a centroblast-like phenotype can be induced to mutate. Triggering of somatic mutations in BL2 cells requires intimate T-B cell contacts and is independent of CD40-CD40-ligand (CD40L) interactions as shown by 1) the lack of effect of anti-CD40 and/or anti-CD40L blocking Abs on somatic mutation and 2) the ability of a CD40L-deficient T cell clone (isolated from an X-linked hyper-IgM syndrome patient) to induce somatic mutation in B cell receptor-engaged BL2 cells. Thus, our in vitro model reveals that T-B cell membrane interactions through surface molecules different from CD40-CD40L can trigger somatic hypermutation.
Oral Administration of Chitin Down-Regulates Serum IgE Levels and Lung Eosinophilia in the Allergic Mouse
Authors: Shibata Yoshimi , Foster L. Ann , Bradfield John F. , Myrvik Quentin N.
Published: April 2014

Previous studies showed that local macrophages phagocytose nonantigenic chitin particles (1–10 μm polymers of N-acetyl-d-glucosamine) through mannose receptors and produce IL-12, IL-18, and TNF-α. These cytokines lead to the production of IFN-γ by NK cells. To determine whether chitin could down-regulate Th2 responses, chitin was given orally (8 mg/day for 3 days before and 13 days during ragweed allergen immunization) in BALB/c and C57BL/6 mice. These ragweed-immunized mice were given ragweed intratracheally on day 11. Three days after the challenge, the immunized mice with saline (controls) showed increases in serum IgE levels and lung eosinophil numbers. The chitin treatment resulted in decreases of these events in both strains. To dissect the inhibitory mechanisms of Th2 responses, spleen cells (4 × 106 cells/ml) isolated from the ragweed-immunized mice (controls) were cultured in the presence of ragweed and/or chitin for 3 days (recall responses). Ragweed alone stimulated the production of IL-4 (0.6 ng/ml), IL-5 (20 U/ml), and IL-10 (3.2 ng/ml), but not IFN-γ. Ragweed/chitin stimulation resulted in significant decreases of IL-4, IL-5, and IL-10 levels and the production of IFN-γ (48 U/ml). Moreover, spleen cells isolated from the chitin-treated mice showed ragweed-stimulated IFN-γ production (15 U/ml) and significantly lower levels of the Th2 cytokines, suggesting that the immune responses were redirected toward a Th1 response. Collectively, these results indicate that chitin-induced innate immune responses down-regulate Th2-facilitated IgE production and lung eosinophilia in the allergic mouse.
Annexin V Binds to Viable B Cells and Colocalizes with a Marker of Lipid Rafts upon B Cell Receptor Activation
Authors: Dillon Stacey R. , Mancini Marie , Rosen Antony , Schlissel Mark S.
Published: April 2014

Recombinant annexin V (rAnV) has been used to identify apoptotic cells based on its ability to bind phosphatidylserine (PS), a lipid normally restricted to the cytoplasmic face of the plasma membrane, but externalized early during apoptosis. However, this association of rAnV binding and apoptosis is not an obligatory one. We demonstrate that rAnV binds to a large fraction of murine B cells bearing selectable Ag receptors despite the fact that these cells are not apoptotic. Phosphatidylserine, which is uniformly distributed on resting B cells, is mobilized to co-cap with IgM on anti-IgM-treated B cells and to colocalize with GM1, a marker of lipid rafts. Cross-linking PS before anti-IgM treatment sequesters this lipid and alters signaling through IgM. Thus, PS exposed on the majority of B cells in vivo does not reflect early apoptosis, but, instead, plays a role in receptor-mediated signaling events.
Neuronal Fractalkine Expression in HIV-1 Encephalitis: Roles for Macrophage Recruitment and Neuroprotection in the Central Nervous System
Authors: Tong Ning , Perry Seth W. , Zhang Qing , James Harold J. , Guo Huang , Brooks Andrew , Bal Harshawardhan , Kinnear Sandra A. , Fine Steven , Epstein Leon G. , Dairaghi Daniel , Schall Thomas J. , Gendelman Howard E. , Dewhurst Stephen , Sharer Leroy R. , Gelbard Harris A.
Published: April 2014

HIV-1 infection of the brain results in chronic inflammation, contributing to the neuropathogenesis of HIV-1 associated neurologic disease. HIV-1-infected mononuclear phagocytes (MP) present in inflammatory infiltrates produce neurotoxins that mediate inflammation, dysfunction, and neuronal apoptosis. Neurologic disease is correlated with the relative number of MP in and around inflammatory infiltrates and not viral burden. It is unclear whether these cells also play a neuroprotective role. We show that the chemokine, fractalkine (FKN), is markedly up-regulated in neurons and neuropil in brain tissue from pediatric patients with HIV-1 encephalitis (HIVE) compared with those without HIVE, or that were HIV-1 seronegative. FKN receptors are expressed on both neurons and microglia in patients with HIVE. These receptors are localized to cytoplasmic structures which are characterized by a vesicular appearance in neurons which may be in cell-to-cell contact with MPs. FKN colocalizes with glutamate in these neurons. Similar findings are observed in brain tissue from an adult patient with HIVE. FKN is able to potently induce the migration of primary human monocytes across an endothelial cell/primary human fetal astrocyte trans-well bilayer, and is neuroprotective to cultured neurons when coadministered with either the HIV-1 neurotoxin platelet activating factor (PAF) or the regulatory HIV-1 gene product Tat. Thus focal inflammation in brain tissue with HIVE may up-regulate neuronal FKN levels, which in turn may be a neuroimmune modulator recruiting peripheral macrophages into the brain, and in a paracrine fashion protecting glutamatergic neurons.
Cell-Mediated DNA Transport Between Distant Inflammatory Sites Following Intradermal DNA Immunization in the Presence of Adjuvant
Authors: La Cava Antonio , Billetta Rosario , Gaietta Guido , Bonnin Dustan B. , Baird Stephen M. , Albani Salvatore
Published: April 2014

After intradermal genetic immunization, naked DNA is transported from the site of injection to regional lymph nodes. Little is known on how inflammation influences this process and whether DNA is transported beyond local lymph nodes. In the experiments herein reported, we injected naked DNA in the presence of adjuvant to address questions related to 1) the fate of naked DNA in the presence of inflammation; 2) the generation of immune responses to the encoded protein during inflammation; and, more in general, 3) the fate of ingested molecules beyond regional lymph nodes during inflammation. Two sites of inflammation were induced in vivo in mice. Naked DNA was injected in the nape together with adjuvant, and adjuvant only was injected at a distant peritoneal site. Injected DNA, uptaken at the primary dermal site of inflammation, was transported beyond regional lymph nodes to distant organs such as the spleen and to the distant peritoneal site of inflammation. This transport, mediated by CD11b+ cells, was cumulative during chronic inflammation. These results indicate a novel route of transport of DNA beyond regional lymph nodes and may have specific implications for DNA-based immune modulation.
Effects of Liver-Derived Dendritic Cell Progenitors on Th1- and Th2-Like Cytokine Responses In Vitro and In Vivo
Authors: Khanna Ajai , Morelli Adrian E. , Zhong Cuiping , Takayama Takuya , Lu Lina , Thomson Angus W.
Published: April 2014

There is evidence that donor-derived dendritic cells (DC), particularly those at a precursor/immature stage, may play a role in the immune privilege of liver allografts. Underlying mechanisms are poorly understood. We have examined the influence of in vitro generated mouse liver-derived DC progenitors (DCp) on proliferative, cytotoxic, and Th1/Th2 cytokine responses induced in allogeneic T cells. Liver DCp, propagated in GM-CSF from C57B10 mice (H2b), induced only minimal proliferation, and weak cytotoxic responses in allogeneic (C3H; H2k) T cells compared with mature bone marrow (BM)-derived DC. Flow-cytometric analysis of intracellular cytokine staining revealed that mature BM DC, but not liver DCp, elicited CD4+ T cell production of IFN-γ. Intracellular expression of IL-10 was very low in both BM DC- and liver DCp-stimulated CD4+ T cells. Only stimulation by liver DCp was associated with IL-10 secretion in primary MLR. Notably, these liver DCp cocultured with allogeneic T cells stained strongly for IL-10. Following local (s.c.) injection in allogeneic recipients, both BM DC and liver DCp homed to T cell areas of draining lymph nodes and spleen, where they were readily detected by immunohistochemistry up to 2 wk postinjection. Liver DCp induced clusters of IL-10- and IL-4-secreting mononuclear cells, whereas Th2 cytokine-secreting cells were not detected in mice injected with mature BM DC. By contrast, comparatively high numbers of IFN-γ+ cells were induced by BM DC. Modulation of Th2 cytokine production by donor-derived DCp may contribute to the comparative immune privilege of hepatic allografts.