bluuebunny/crossref_metadata_2025 huggingface dataset:
Shear and Time-Dependent Changes in Mac-1, LFA-1, and ICAM-3 Binding Regulate Neutrophil Homotypic Adhesion
Authors: Neelamegham Sriram , Taylor Andrew D. , Shankaran Harish , Smith C. Wayne , Simon Scott I.
Published: April 2014

We examined the relative contributions of LFA-1, Mac-1, and ICAM-3 to homotypic neutrophil adhesion over the time course of formyl peptide stimulation at shear rates ranging from 100 to 800 s−1. Isolated human neutrophils were sheared in a cone-plate viscometer and the kinetics of aggregate formation was measured by flow cytometry. The efficiency of cell adhesion was computed by fitting the aggregate formation rates with a model based on two-body collision theory. Neutrophil homotypic adhesion kinetics varied with shear rate and was most efficient at 800 s−1, where ∼40% of the collisions resulted in adhesion. A panel of blocking Abs to LFA-1, Mac-1, and ICAM-3 was added to assess the relative contributions of these molecules. We report that 1) LFA-1 binds ICAM-3 as its primary ligand supporting homotypic adhesion, although the possibility of other ligands was also detected. 2) Mac-1 binding to an unidentified ligand supports homotypic adhesion with an efficiency comparable to LFA-1 at low shear rates of ∼100 s−1. Above 300 s−1, however, Mac-1 and not LFA-1 were the predominant molecules supporting cell adhesion. This is in contrast to neutrophil adhesion to ICAM-1-transfected cells, where LFA-1 binds with a higher avidity than Mac-1 to ICAM-1. 3) Following stimulation, the capacity of LFA-1 to support aggregate formation decreases with time at a rate ∼3-fold faster than that of Mac-1. The results suggest that the relative contributions of β2 integrins and ICAM-3 to neutrophil adhesion is regulated by the magnitude of fluid shear and time of stimulus over a range of blood flow conditions typical of the venular microcirculation.
Cultured Human Fibroblasts Express Constitutive IL-16 mRNA: Cytokine Induction of Active IL-16 Protein Synthesis Through a Caspase-3-Dependent Mechanism
Authors: Sciaky Daniela , Brazer William , Center David M. , Cruikshank William W. , Smith Terry J.
Published: April 2014

Human fibroblasts can express numerous regulatory molecules that influence immune function. IL-16, a ligand for CD4, is a chemoattractant molecule expressed by lymphocytes, eosinophils, mast cells, and lung epithelium. It appears that the sole target for IL-16 is the CD4-bearing cell. Here we demonstrate that fibroblasts from several tissues can express IL-16 mRNA and protein as well as IL-16-dependent chemoattractant activity. The transcript is expressed abundantly under basal culture conditions as a 2.5-kb band on Northern analysis, similar to that observed in lymphocytes. IL-16 protein and activity are undetectable in fibroblast cultures under these same control conditions. However, when treated with proinflammatory cytokines such as IL-1β, they express very high levels of IL-16 protein and chemoattractant activity, a substantial component of which can be blocked with IL-16-neutralizing Abs. The amount of IL-16 protein released into the medium is 3- to 4-fold greater, on a per cell basis, than that observed in lymphocytes. The induction of IL-16 protein by IL-1β can be attenuated with specific inhibition of caspase-3, which could be detected in IL-1β-treated fibroblasts. IL-1β also induces RANTES mRNA, protein, and activity, and most of the chemoattractant activity released from fibroblasts not derived from IL-16 can be attributed to RANTES. Human fibroblasts appear to be an important source of IL-16 and through expression of this molecule may have key roles in the recruitment of CD4+ cells to sites of inflammation. IL-16 expression and the mechanism involved in its regulation appear to be cell type specific.
Regulation of the RON Receptor Tyrosine Kinase Expression in Macrophages: Blocking the RON Gene Transcription by Endotoxin-Induced Nitric Oxide
Authors: Wang Ming-Hai , Fung Hai-Lin , Chen Yi-Qing
Published: April 2014

Previous studies have shown that activation of the RON receptor tyrosine kinase inhibits inducible NO production in murine peritoneal macrophages. The purpose of this study is to determine whether inflammatory mediators such as LPS, IFN-γ, and TNF-α regulate RON expression. Western blot analysis showed that RON expression is reduced in peritoneal macrophages collected from mice injected with a low dose of LPS. The inhibition was seen as early as 8 h after LPS challenge. Experiments in vitro also demonstrated that the levels of the RON mRNA and protein are diminished in cultured peritoneal macrophages following LPS stimulation. TNF-α plus IFN-γ abrogated macrophage RON expression, although individual cytokines had no significant effect. Because LPS and TNF-α plus IFN-γ induce NO production, we reasoned that NO might be involved in the RON inhibition. Two NO donors, S-nitroglutathione (GSNO) and (±)-S-nitroso-N-acetylpenicillamine (SNAP), directly inhibited macrophage RON expression when added to the cell cultures. Blocking NO production by NO inhibitors like TGF-β prevented the LPS-mediated inhibitory effect. In Raw264.7 cells transiently transfected with a report vector, GSNO or SNAP inhibited the luciferase activities driven by the RON gene promoter. Moreover, GSNO or SNAP inhibited the macrophage-stimulating protein-induced RON phosphorylation and macrophage migration. We concluded from these data that RON expression in macrophages is regulated during inflammation. LPS and TNF-α plus IFN-γ are capable of down-regulating RON expression through induction of NO production. The inhibitory effect of NO is mediated by suppression of the RON gene promoter activities.
Polymorphonuclear Leukocytes Modulate Tissue Factor Production by Mononuclear Cells: Role of Reactive Oxygen Species
Authors: Cadroy Yves , Dupouy Dominique , Boneu Bernard , Plaisancié Henri
Published: April 2014

To determine whether polymorphonuclear leukocytes (PMN) modulate the production of tissue factor (TF) by monocytes, PBMC were incubated with increasing concentrations of PMN. PMN did not express any procoagulant activity. After 20-h cocultures, PMN enhanced or inhibited the TF production of PBMC, and this effect depended on the PMN/PBMC ratio. When the ratio increased from 1/1000 to 1/5, without or with LPS, the TF activity of PBMC increased to peak at 2.5-fold the baseline value (p < 0.01). The TF Ag and TF mRNA also increased. This potentiating effect was mediated by reactive oxygen species (ROS) released by PMN during the coculture; it did not require direct cell contact between PMN and PBMC, it was enhanced when PMN were stimulated by fMLP (a chemotactic peptide), and it was inhibited by two antioxidants, N-acetyl cysteine and pyrrolidine dithiocarbamate. In contrast, when the PMN/PBMC ratio was further increased from 1/2 to 2/1, the PBMC TF activity, Ag, and mRNA decreased and were inhibited compared with those of PBMC cultured alone (p < 0.01). This inhibitory effect required direct cell contact between PMN and PBMC, and it was not due to a PMN-mediated cytotoxicity. To confirm the role of ROS, H2O2 enhanced then inhibited the TF activity of PBMC in a dose-dependent manner, similarly to PMN. Thus, PMN may play an important role in the pathogenesis of thrombosis and atherosclerosis by exerting concentration-dependent regulatory effects on the TF production by PBMC via the release of ROS.
Vav Synergizes with Protein Kinase CΘ to Mediate IL-4 Gene Expression in Response to CD28 Costimulation in T Cells
Authors: Hehner Steffen P. , Li-Weber Min , Giaisi Marco , Dröge Wulf , Krammer Peter H. , Schmitz M. Lienhard
Published: April 2014

The secretion of IL-4, which displays many important immunoregulatory functions, is restricted to cells of the Th2 subtype. In this study, we investigated the early signaling events leading to the activation of IL-4 transcription. Vav, the protein kinase C (PKC) isoform Θ, and the adaptor protein SLP76 (SH2-domain-containing leukocyte protein of 76 kDa), induced transcription from the IL-4 promoter. Vav and PKCΘ synergistically activated human IL-4 promoter transcription and IL-4 mRNA production and were found to be constitutively associated in vivo. CD3/CD28-induced IL-4 transcription was inhibited upon coexpression of dominant negative forms of Vav, the adaptor proteins LAT (linker for activation of T cells) and SLP76, PKCΘ, and components of the pathways leading to the activation of c-Jun N-terminal kinase (mitogen-activated protein kinase kinase 7 (MKK7), mixed lineage kinase 3 (MLK3)) and NF-κB (IκB kinase α and IκB kinase β). The Vav/PKCΘ-mediated synergistic activation of IL-4 transcription was not inhibited by cyclosporin A. Three independent experimental approaches revealed that Vav/PKCΘ-derived signals selectively target the P1 and positive regulatory element (PRE)-I elements contained within the human IL-4 promoter. Vav/PKCΘ strongly activated a luciferase reporter construct controlled by trimerized P1 or PRE-I elements and furthermore stimulated DNA binding of nuclear proteins to the P1 and PRE-I elements. Vav/PKCΘ-induced transcription from the IL-4 promoter was almost completely abrogated by mutation of either the P1 or the PRE-I element within the entire IL-4 promoter.
IL-11 Activates Human Endothelial Cells to Resist Immune-Mediated Injury
Authors: Mahboubi Keyvan , Biedermann Barbara C. , Carroll Joseph M. , Pober Jordan S.
Published: April 2014

IL-11, a gp130-signaling cytokine, is protective in several in vivo models of immune-mediated and inflammatory injury. HUVECs express IL-11 receptor α-chain and gp130. Human IL-11 causes rapid (2–10 min) tyrosine phosphorylation of gp130. IL-11 at 0.1 and 10 ng/ml induces tyrosine phosphorylation of STAT3 and STAT1, respectively, although maximal responses require 50 ng/ml. Phospho-STAT3 and phospho-STAT1 levels peak rapidly (2.5 min) and disappear by 60 min. The p42 and p44 mitogen-activated protein kinases (MAPKs) are phosphorylated in response to 0.3 ng/ml IL-11 with maximal activation at 30 ng/ml IL-11. Phosphorylation of p42 and p44 MAPKs, which can be prevented by a mitogen-activated protein/extracellular signal-related kinase kinase-1 inhibitor, peaks by 15–20 min and largely disappears by 40 min. IL-11 does not activate NF-κB nor does it inhibit NF-κB activation by TNF. Similarly, IL-11 neither induces E-selectin or ICAM-1 nor blocks induction by TNF. Although IL-11 does not alter class I MHC complex molecule expression, pretreatment with 0.5 ng/ml IL-11 partially protects HUVECs against lysis by allospecific class I MHC-restricted cytolytic T lymphocytes or by anti-class I MHC Ab plus heterologous C. IL-11-induced cytoprotection is protein synthesis dependent and may depend on mitogen-activated protein/extracellular signal-related kinase kinase-1. Our results indicate that low (i.e., STAT3- and MAPK-activating) concentrations of IL-11 confer resistance to immune-mediated injury in cultured HUVECs without inhibiting proinflammatory responses.
Migration of Eosinophils Across Endothelial Cell Monolayers: Interactions Among IL-5, Endothelial-Activating Cytokines, and C-C Chemokines
Authors: Shahabuddin Syed , Ponath Paul , Schleimer Robert P.
Published: April 2014

Eosinophils are the predominant cell type recruited in inflammatory reactions in response to allergen challenge. The mechanisms of selective eosinophil recruitment in allergic reactions are not fully elucidated. In this study, the ability of several C-C chemokines to induce transendothelial migration (TEM) of eosinophils in vitro was assessed. Eotaxin, eotaxin-2, monocyte chemotactic protein (MCP)-4, and RANTES induced eosinophil TEM across unstimulated human umbilical vein endothelial cells (HUVEC) in a concentration-dependent manner with the following rank order of potency: eotaxin ≈ eotaxin-2 > MCP-4 ≈ RANTES. The maximal response induced by eotaxin or eotaxin-2 exceeded that of RANTES or MCP-4. Preincubation of eosinophils with anti-CCR3 Ab (7B11) completely blocked eosinophil TEM induced by eotaxin, MCP-4, and RANTES. Activation of endothelial cells with IL-1β or TNF-α induced concentration-dependent migration of eosinophils, which was enhanced synergistically in the presence of eotaxin and RANTES. Anti-CCR3 also inhibited eotaxin-induced eosinophil TEM across TNF-α-stimulated HUVEC. The ability of eosinophil-active cytokines to potentiate eosinophil TEM was assessed by investigating eotaxin or RANTES-induced eosinophil TEM across resting and IL-1β-stimulated HUVEC in the presence or absence of IL-5. The results showed synergy between IL-5 and the chemokines but not between IL-5 and the endothelial activator IL-1β. Our data suggest that eotaxin, eotaxin-2, MCP-4, and RANTES induce eosinophil TEM via CCR3 with varied potency and efficacy. Activation of HUVEC by IL-1β or TNF-α or priming of eosinophils by IL-5 both promote CCR3-dependent migration of eosinophils from the vasculature in conjunction with CCR3-active chemokines.
An Essential Role of Mast Cells in the Development of Airway Hyperresponsiveness in a Murine Asthma Model
Authors: Kobayashi Tetsuto , Miura Toru , Haba Tomoko , Sato Miyuki , Serizawa Isao , Nagai Hiroichi , Ishizaka Kimishige
Published: April 2014

Immunization of BALB/c mice with alum-adsorbed OVA, followed by three bronchoprovocations with aerosolized OVA, resulted in the development of airway hyperresponsiveness (AHR) and allergic inflammation in the lung accompanied by severe infiltration of eosinophils into airways. In this murine asthma model, administration of monoclonal anti-IL-5 Ab before each Ag challenge markedly inhibited airway eosinophilia, but the treatment did not affect the development of AHR. Immunization and aerosol challenges with OVA following the same protocol failed to induce AHR in the mast cell-deficient W/Wv mice, but induced AHR in their congenic littermates, i.e., WBB6F1 (+/+) mice. No significant difference was found between the W/Wv mice and +/+ mice with respect to the IgE and IgG1 anti-OVA Ab responses and to the airway eosinophilia after Ag provocations. It was also found that reconstitution of W/Wv mice with bone marrow-derived mast cells cultured from normal littermates restored the capacity of developing Ag-induced AHR, indicating that lack of mast cells was responsible for the failure of W/Wv mice to develop Ag-induced AHR under the experimental conditions. However, the OVA-immunized W/Wv mice developed AHR by increasing the frequency and Ag dose of bronchoprovocations. The results suggested that AHR could be developed by two distinct cellular mechanisms. One would go through mast cell activation and the other is IgE/mast cell independent but an eosinophil/IL-5-dependent mechanism.
Induction of Functional IL-8 Receptors by IL-4 and IL-13 in Human Monocytes
Authors: Bonecchi Raffaella , Facchetti Fabio , Dusi Stefano , Luini Walter , Lissandrini Daniele , Simmelink Marleen , Locati Massimo , Bernasconi Sergio , Allavena Paola , Brandt Ernst , Rossi Filippo , Mantovani Alberto , Sozzani Silvano
Published: April 2014

IL-8 and related Glu-Leu-Arg (ELR+) CXC chemokines are potent chemoattractants for neutrophils but not for monocytes. IL-13 and IL-4 strongly increased CXCR1 and CXCR2 chemokine receptor expression in human monocytes, macrophages, and dendritic cells. The effect was receptor- and cell type-selective, in that CCRs were not increased and no augmentation was seen in neutrophils. The effect was rapid, starting at 4 h, and concentration dependent (EC50 = 6.2 and 8.3 ng/ml for CXCR1 and CXCR2, respectively) and caused by new transcriptional activity. IL-13/IL-4-treated monocytes showed increased CXCR1 and CXCR2 membrane expression. IL-8 and related ELR+ chemokines were potent and effective chemotactic agents for IL-13/IL-4-treated monocytes, but not for untreated mononuclear phagocytes, with activity comparable to that of reference monocyte attractants, such as MCP-1. In the same cells, IL-8 also caused superoxide release. Macrophages and dendritic cells present in biopsies from Omenn’s syndrome and atopic dermatitis patients, two Th2 skewed pathologies, expressed IL-8 receptors by immunohistochemistry. These results show that IL-13 and IL-4 convert IL-8 and related ELR+ chemokines, prototypic neutrophil attractants, into monocyte chemotactic agonists, by up-regulating receptor expression. Therefore, IL-8 and related chemokines may contribute to the accumulation and positioning of mononuclear phagocytes in Th2-dominated responses.
G551D Cystic Fibrosis Mice Exhibit Abnormal Regulation of Inflammation in Lungs and Macrophages
Authors: Thomas Gordon R. , Costelloe Elaine A. , Lunn Dominic P. , Stacey Katryn J. , Delaney Steven J. , Passey Robert , McGlinn Edwina C. , McMorran Brendan J. , Ahadizadeh Azita , Geczy Carolyn L. , Wainwright Brandon J. , Hume David A.
Published: April 2014

The major cause of death in cystic fibrosis (CF) is chronic lung disease associated with persistent infection by the bacterium, Pseudomonas aeruginosa. S100A8, an S-100 calcium-binding protein with chemotactic activity, is constitutively expressed in the lungs and serum of CF patients. Levels of S100A8 mRNA were found to be three to four times higher in the lungs of mice carrying the G551D mutation in CF transmembrane conductance regulator compared with littermate controls. Intravenous injection of bacterial LPS induced S100A8 mRNA in the lung to a greater extent in G551D mice than in wild-type littermates. Localization of S100A8 mRNA and protein in the lung indicate that it is a marker for neutrophil accumulation. Bone marrow-derived macrophages from G551D mice were shown to also exhibit hypersensitivity to LPS, measured by induction of TNF-α. These results provide evidence that the pathology of CF relates to abnormal regulation of the immune system.
Alveolar Macrophages Bind and Phagocytose Allergen- Containing Pollen Starch Granules Via C-Type Lectin and Integrin Receptors: Implications for Airway Inflammatory Disease
Authors: Currie Andrew J. , Stewart Geoffrey A. , McWilliam Andrew S.
Published: April 2014

Recent studies suggest that IgE-independent mechanisms of airway inflammation contribute significantly to the pathophysiology of allergic airway inflammatory diseases such as asthma. Such mechanisms may involve direct interactions between inhaled allergens and cells of the respiratory tract such as macrophages, dendritic cells, and epithelial cells. In this study, we investigated receptor-mediated interactions occurring between alveolar macrophages and allergen-containing pollen starch granules (PSG). We report here that PSG are released from a range of grass species and are rapidly bound and phagocytosed by alveolar macrophages. Human monocyte-derived dendritic cells also bound PSG but no internalization was observed. Phagocytosis of PSG was dependent on Mg2+ and Ca2+ and was inhibited by neo-glycoproteins such as galactose-BSA and N-acetylgalactose-BSA. Partial inhibition of phagocytosis was also seen with the Arg-Gly-Asp-Ser (RGDS) motif and with an anti-CD18 mAb (OX42). The combination of both neo-glycoprotein and anti-CD18 achieved the greatest degree of inhibition (>90%). Together, these data suggest a role for both C-type lectins and β2-integrins in the binding and internalization of PSG. The consequences of this interaction included a rapid up-regulation of inducible NO synthase mRNA and subsequent release of NO by alveolar macrophages. Thus, receptor-mediated recognition of inhaled allergenic particles by alveolar macrophages may represent a potential mechanism for modulating the inflammatory response associated with allergic airway diseases such as asthma.
Granuloctye-Macrophage Colony-Stimulating Factor as an Autocrine Survival Factor for Mature Normal and Malignant B Lymphocytes
Authors: Harris Robert J. , Pettitt Andrew R. , Schmutz Caroline , Sherrington Paul D. , Zuzel Mirko , Cawley John C. , Griffiths Stephen D.
Published: April 2014

The role of GM-CSF in B cell (patho)physiology is unclear. Although B cells can respond to GM-CSF, there is controversy concerning the extent to which various resting and activated B cell types can themselves produce this cytokine, and the possibility that it can function in an autocrine fashion has not previously been considered. The aim of the present study was to address these issues using hairy cells (HCs) and chronic lymphocytic leukemia cells, two intrinsically activated mature malignant B cell types (with activation being more uniform and more pronounced in HCs). Normal B cells were used for comparison. Using a number of techniques, we demonstrated the constitutive production of GM-CSF by all three cell types and showed that the cytokine was biologically active. GM-CSF mRNA and protein were increased after cell activation by PMA, and constitutive production of the cytokine was highest in HCs, suggesting that the level of GM-CSF production is influenced by cell activation. Because GM-CSF is known to be antiapoptotic for myeloid cells, we used blocking anti-GM-CSF Abs to examine the contribution of autocrinely produced cytokine to cell survival. The Abs produced marked reduction in the in vitro survival of HCs, chronic lymphocytic leukemia cells, and normal B cells by promoting apoptosis. Taken together, these findings suggest that, in combination with other known rescue factors, autocrinely produced GM-CSF may contribute to normal and malignant B cell survival in vivo.
Activation of the IL-4 STAT Pathway in Rheumatoid Synovium
Authors: Müller-Ladner Ulf , Judex Martin , Ballhorn Wibke , Kullmann Frank , Distler Oliver , Schlottmann Klaus , Gay Renate E. , Schölmerich Jürgen , Gay Steffen
Published: April 2014

STATs act as second messenger after binding of a signaling molecule to its receptor. IL-4 STAT is directly involved in the IL-4-dependent gene transcription in the nucleus. We examined the expression and activation of IL-4 STAT and its related kinase Jak-1 in rheumatoid synovium. Rheumatoid arthritis (RA) synovial frozen sections of patients with short-term (<1 year) and long-term disease (>2 years) were examined using in situ hybridization and immunohistochemistry. IL-4 STAT mRNA could be detected in synovium of patients with short-term and long-term RA. The most intensive expression of IL-4 STAT mRNA could be seen in follicular inflammatory infiltrates. In the synovial lining, both fibroblasts and macrophages expressed IL-4 STAT mRNA. IL-4 STAT and Jak-1 protein was expressed by synoviocytes, and up-regulation could be induced after stimulation with IL-4. Activation of IL-4 STAT was reflected by phosphorylation of IL-4 STAT. The results indicate that IL-4 STAT is involved in key pathomechanisms in RA synovium and that IL-4 STAT-dependent pathways operate in early and late stages of the disease and presumably contribute to inhibitory immune mechanisms in RA synovium.
Tumor-Specific CD4+ T Lymphocytes from Cancer Patients Are Required for Optimal Induction of Cytotoxic T Cells Against the Autologous Tumor
Authors: Baxevanis Constantin N. , Voutsas Ioannis F. , Tsitsilonis Ourania E. , Gritzapis Angelos D. , Sotiriadou Roula , Papamichail Michael
Published: April 2014

This study focuses on the specific CD4+ T cell requirement for optimal induction of cytotoxicity against MHC class II negative autologous tumors (AuTu) collected from patients with various types of cancer at advanced stages. CD4+ T cells were induced in cultures of cancer patients’ malignant effusion-associated mononuclear cells with irradiated AuTu (mixed lymphocyte tumor cultures (MLTC)) in the presence of recombinant IL-2 and recombinant IL-7. Tumor-specific CD4+ T cells did not directly recognize the AuTu cells, but there was an MHC class II-restricted cross-priming by autologous dendritic cells (DCs), used as APC. CD8+ CTL, also induced during the MLTC, lysed specifically AuTu cells or DCs pulsed with AuTu peptide extracts (acid wash extracts (AWE)) in an MHC class I-restricted manner. Removal of CD4+ T cells or DCs from the MLTC drastically reduced the CD8+ CTL-mediated cytotoxic response against the AuTu. AWE-pulsed DCs preincubated with autologous CD4+ T cells were able, in the absence of CD4+ T cells, to stimulate CD8+ T cells to lyse autologous tumor targets. Such activated CD8+ T cells produced IL-2, IFN-γ, TNF-α, and GM-CSF. The process of the activation of AWE-pulsed DCs by CD4+ T cells could be inhibited with anti-CD40 ligand mAb. Moreover, the role of CD4+ T cells in activating AWE-pulsed DCs was undertaken by anti-CD40 mAb. Our data demonstrate for the first time in patients with metastatic cancer the essential role of CD4+ Th cell-activated DCs for optimal CD8+ T cell-mediated killing of autologous tumors and provide the basis for the design of novel protocols in cellular adoptive immunotherapy of cancer, utilizing synthetic peptides capable of inducing T cell help in vivo.
Identification of a CD8 T Cell That Can Independently Mediate Autoimmune Diabetes Development in the Complete Absence of CD4 T Cell Helper Functions
Authors: Graser Robert T. , DiLorenzo Teresa P. , Wang Fuming , Christianson Gregory J. , Chapman Harold D. , Roopenian Derry C. , Nathenson Stanley G. , Serreze David V.
Published: April 2014

Previous work has indicated that an important component for the initiation of autoimmune insulin-dependent diabetes mellitus (IDDM) in the NOD mouse model entails MHC class I-restricted CD8 T cell responses against pancreatic β cell Ags. However, unless previously activated in vitro, such CD8 T cells have previously been thought to require helper functions provided by MHC class II-restricted CD4 T cells to exert their full diabetogenic effects. In this study, we show that IDDM development is greatly accelerated in a stock of NOD mice expressing TCR transgenes derived from a MHC class I-restricted CD8 T cell clone (designated AI4) previously found to contribute to the earliest preclinical stages of pancreatic β cell destruction. Importantly, these TCR transgenic NOD mice (designated NOD.AI4αβ Tg) continued to develop IDDM at a greatly accelerated rate when residual CD4 helper T cells were eliminated by introduction of the scid mutation or a functionally inactivated CD4 allele. In a previously described stock of NOD mice expressing TCR transgenes derived from another MHC class I-restricted β cell autoreactive T cell clone, IDDM development was retarded by elimination of residual CD4 T cells. Hence, there is variability in the helper dependence of CD8 T cells contributing to the development of autoimmune IDDM. The AI4 clonotype represents the first CD8 T cell with a demonstrated ability to progress from a naive to functionally activated state and rapidly mediate autoimmune IDDM development in the complete absence of CD4 T cell helper functions.
Protection of Nonobese Diabetic Mice from Diabetes by Gene(s) Closely Linked to IFN-γ Receptor Loci
Authors: Kanagawa Osami , Xu Guan , Tevaarwerk Amye , Vaupel Barbara A.
Published: April 2014

Nonobese diabetic (NOD) mice carrying a segment of chromosome flanking the disrupted IFN-γ receptor gene from original 129 ES cells are resistant to development of diabetes. However, extended backcrossing of this mouse line to the NOD mouse resulted in a segregation of the IFN-γR-deficient genotype from the diabetes-resistant phenotype. These results indicate that the protection of NOD mice from the development of diabetes is not directly linked to the defective IFN-γ receptor gene but, rather, is influenced by the presence of a diabetes-resistant gene(s) closely linked to the IFN-γR loci derived from the 129 mouse strain.
Reduced Chemokine and Chemokine Receptor Expression in Spinal Cords of TCR BV8S2 Transgenic Mice Protected Against Experimental Autoimmune Encephalomyelitis with BV8S2 Protein
Authors: Matejuk Agata , Vandenbark Arthur A. , Burrows Gregory G. , Bebo Bruce F. , Offner Halina
Published: April 2014

The perivascular transmigration and accumulation of macrophages and T lymphocytes in the CNS of mice with experimental autoimmune encephalomyelitis (EAE) may be partly regulated by low m.w. chemotactic cytokines. Using the RNase protection assay and ELISA, we quantified expression of chemokines and chemokine receptors in the spinal cord (SC), brain, and lymph nodes of BV8S2 transgenic mice that developed or were protected from EAE by vaccination with BV8S2 protein. In paralyzed control mice, the SC had increased cellular infiltration and strong expression of the chemokines RANTES, IFN-inducible 10-kDa protein, and monocyte chemoattractant protein-1 and the cognate chemokine receptors CCR1, CCR2, and CCR5, with lower expression of macrophage-inflammatory protein (MIP)-1α, MIP-1β, and MIP-2; whereas brain had less infiltration and a lower expression of a different pattern of chemokines and receptors. In TCR-protected mice, there was a decrease in the number of inflammatory cells in both SC and brain. In SC, the reduced cellular infiltrate afforded by TCR vaccination was commensurate with profoundly reduced expression of chemokines and their cognate chemokine receptors. In brain, however, TCR vaccination did not produce significant changes in chemokine expression but resulted in an increased expression of CCR3 and CCR4 usually associated with Th2 cells. In contrast to CNS, lymph nodes of protected mice had a significant increase in expression of MIP-2 and MIP-1β but no change in expression of chemokine receptors. These results demonstrate that TCR vaccination results in selective reduction of inflammatory chemokines and chemokine receptors in SC, the target organ most affected during EAE.
Distinct Requirements for C-C Chemokine and IL-2 Production by Naive, Previously Activated, and Anergic T Cells
Authors: Lerner Cara G. , Horton Maureen R. , Schwartz Ronald H. , Powell Jonathan D.
Published: April 2014

Ag presented by activated APCs promote immunogenic responses whereas Ag presented by resting APCs leads to tolerance. In such a model, the regulation of cytokine release by the presence or absence of costimulation might potentially play a critical role in dictating the ultimate outcome of Ag recognition. C-C chemokines are a structurally defined family of chemoattractants that have diverse effects on inflammation. We were interested in determining the activation requirements for chemokine production by CD4+ T cells. Our data demonstrate for T cell clones and previously activated T cells from TCR-transgenic mice that stimulation with anti-TCR alone results in the production of copious amounts of macrophage-inflammatory protein-1α (MIP-1α) and other C-C chemokines, and that addition of anti-CD28 gives very little augmentation. Furthermore, MIP-1α production is nearly equivalent from both anergic and nonanergic cells. For naive T cells, anti-CD3 stimulation alone led to as much MIP-1α production as Ag + APC stimulation. The addition of costimulation gave a 3–10-fold enhancement, but this was 70-fold less than the effect of costimulation on IL-2 production. Thus, although C-C chemokines play a broad role in influencing inflammation, their production by signal 1 alone makes them unlikely to play a critical role in the decision between a tolerogenic and an immunogenic response. Furthermore, the production of MIP-1α by anergic T cells, as well as following signal 1 alone, raises the possibility that in vivo this chemokine serves to recruit activated T cells to become tolerant.
Repeated Intratracheal Challenge with Particulate Antigen Modulates Murine Lung Cytokines
Authors: Todt Jill , Sonstein Joanne , Polak Timothy , Seitzman Gerami D. , Hu Bin , Curtis Jeffrey L.
Published: April 2014

When lungs of experimental animals are repeatedly challenged with Ag, pulmonary inflammation wanes via unknown mechanisms. We hypothesized that changes in the balance of lung cytokines are responsible for immune down-regulation to repeated Ag challenge. We used intratracheal (IT) challenge of primed C57BL/6 mice with SRBC and on various days after single (1IT) or triple (3IT) challenge counted lung inflammatory cells and measured whole-lung cytokine mRNA and protein concentrations using RT-PCR and ELISA. We found that lung lymphocyte numbers and parenchymal lung inflammation decreased significantly at days 6 and 9 after final Ag challenge in 3IT mice compared with 1IT mice. Lungs of 3IT mice showed the following changes in relative mRNA expression: an earlier peak in IL-10, decreased IL-1β, and a change from a Th2 response in 1IT mice to a Th1 response in 3IT mice (with pronounced increases in IL-12, IL-18, and IFN-γ and decreased IL-4, IL-13, and IL-5). Similar types of changes were seen in whole-lung protein concentrations for TNF-α, IL-10, IL-12 p40, IFN-γ, and IL-4. Additionally, mRNA expression of the endothelial selectins CD62E and CD62P decreased and lung lymphocyte apoptosis increased in the 3IT group. Thus, physiologic down-regulation of the pulmonary immune response to repeated Ag exposure is characterized by increased anti- and decreased proinflammatory cytokines that accompanies Th1 polarization. Similar mechanisms may act to minimize chronic lung inflammation in the majority of normal humans who do not develop progressive lung pathology when repeatedly exposed to inhaled or aspirated environmental Ags.
Fractalkine-Mediated Endothelial Cell Injury by NK Cells
Authors: Yoneda Osamu , Imai Toshio , Goda Seiji , Inoue Hiroshi , Yamauchi Akira , Okazaki Toshio , Imai Hisao , Yoshie Osamu , Bloom Eda T. , Domae Naochika , Umehara Hisanori
Published: April 2014

Endothelial cells (ECs) are primary targets of immunological attack, and their injury can lead to vasculopathy and organ dysfunction in vascular leak syndrome and in rejection of allografts or xenografts. A newly identified CX3C-chemokine, fractalkine, expressed on activated ECs plays an important role in leukocyte adhesion and migration. In this study we examined the functional roles of fractalkine on NK cell activity and NK cell-mediated endothelial cell injury. Freshly separated NK cells expressed the fractalkine receptor (CX3CR1) determined by FACS analysis and efficiently adhered to immobilized full-length fractalkine, but not to the truncated forms of the chemokine domain or mucin domain, suggesting that fractalkine functions as an adhesion molecule on the interaction between NK cells and ECs. Soluble fractalkine enhanced NK cell cytolytic activity against K562 target cells in a dose- and time-dependent manner. This enhancement correlated well with increased granular exocytosis from NK cells, which was completely inhibited by the G protein inhibitor, pertussis toxin. Transfection of fractalkine cDNA into ECV304 cells or HUVECs resulted in increased adhesion of NK cells and susceptibility to NK cell-mediated cytolysis compared with control transfection. Moreover, both enhanced adhesion and susceptibility of fractalkine-transfected cells were markedly suppressed by soluble fractalkine or anti-CX3CR1 Ab. Our results suggest that fractalkine plays an important role not only in the binding of NK cells to endothelial cells, but also in NK cell-mediated endothelium damage, which may result in vascular injury.
Sulfhydryl Regulation of L-Selectin Shedding: Phenylarsine Oxide Promotes Activation-Independent L-Selectin Shedding from Leukocytes
Authors: Bennett Teresa A. , Edwards Bruce S. , Sklar Larry A. , Rogelj Snezna
Published: April 2014

The L-selectin adhesion molecule mediates leukocyte recruitment to inflammatory sites and lymphocyte trafficking through the peripheral lymph nodes. In response to leukocyte activation, L-selectin is proteolytically released from the cell surface, disabling leukocytes from the subsequent L-selectin-dependent interactions. We have found that L-selectin shedding is sensitive to sulfhydryl chemistry; it is promoted by thiol-oxidizing or -blocking reagents and inhibited by reducing reagents. Phenylarsine oxide (PAO), a trivalent arsenical that interacts with vicinal dithiols, is most potent in inducing rapid shedding of L-selectin from isolated neutrophils, eosinophils, and lymphocytes as well as from neutrophils in whole blood. PAO does not cause cell activation, nor does it interfere with integrin function or alter the expression of several other cell surface molecules at the low concentrations that induce L-selectin shedding. PAO is not required to enter the cell to induce L-selectin shedding. TAPI-2 ((N-{d,l-[2-(hydroxyaminocarbonyl)-methyl]-4-methylpentanoyl}-l-3-(tert-butyl)-alanyl-l-alanine, 2-aminoethyl amide), which has previously been shown to inhibit the activation-dependent L-selectin shedding, is also capable of inhibiting PAO-induced L-selectin shedding. We hypothesize that PAO-induced L-selectin shedding involves a regulatory molecule, such as protein disulfide isomerase (PDI), an enzyme that plays a role in the formation and rearrangement of disulfide bonds, contains PAO-binding, vicinal dithiol-active sites, and is expressed on the neutrophil surface. Cell surface expression of PDI, L-selectin shedding induced by PDI-blocking Abs and by bacitracin, a known inhibitor of PDI activity, and direct binding of PDI to PAO, provide supporting evidence for this hypothesis.
Dendritic Cells Infected with a Vaccinia Vector Carrying the Human gp100 Gene Simultaneously Present Multiple Specificities and Elicit High-Affinity T Cells Reactive to Multiple Epitopes and Restricted by HLA-A2 and -A3
Authors: Yang Sixun , Kittlesen David , Slingluff Craig L. , Vervaert Carol E. , Seigler Hilliard F. , Darrow Timothy L.
Published: April 2014

To investigate the ability of human dendritic cells (DC) to process and present multiple epitopes from the gp100 melanoma tumor-associated Ags (TAA), DC from melanoma patients expressing HLA-A2 and HLA-A3 were pulsed with gp100-derived peptides G9154, G9209, or G9280 or were infected with a vaccinia vector (Vac-Pmel/gp100) containing the gene for gp100 and used to elicit CTL from autologous PBL. CTL were also generated after stimulation of PBL with autologous tumor. CTL induced with autologous tumor stimulation demonstrated HLA-A2-restricted, gp100-specific lysis of autologous and allogeneic tumors and no lysis of HLA-A3-expressing, gp100+ target cells. CTL generated by G9154, G9209, or G9280 peptide-pulsed, DC-lysed, HLA-A2-matched EBV transformed B cells pulsed with the corresponding peptide. CTL generated by Vac-Pmel/gp100-infected DC (DC/Pmel) lysed HLA-A2- or HLA-A3-matched B cell lines pulsed with the HLA-A2-restricted G9154, G9209, or G9280 or with the HLA-A3-restricted G917 peptide derived from gp100. Furthermore, these DC/Pmel-induced CTL demonstrated potent cytotoxicity against allogeneic HLA-A2- or HLA-A3-matched gp100+ melanoma cells and autologous tumor. We conclude that DC-expressing TAA present multiple gp100 epitopes in the context of multiple HLA class I-restricting alleles and elicit CTL that recognize multiple gp100-derived peptides in the context of multiple HLA class I alleles. The data suggest that for tumor immunotherapy, genetically modified DC that express an entire TAA may present the full array of possible CTL epitopes in the context of all possible HLA alleles and may be superior to DC pulsed with limited numbers of defined peptides.
Cytokine-Stimulated, But Not HIV-Infected, Human Monocyte-Derived Macrophages Produce Neurotoxic Levels of l-Cysteine
Authors: Yeh Michael W. , Kaul Marcus , Zheng Jialin , Nottet Hans S. L. M. , Thylin Michael , Gendelman Howard E. , Lipton Stuart A.
Published: April 2014

Approximately one-quarter of individuals with AIDS develop neuropathological symptoms that are attributable to infection of the brain with HIV. The cognitive manifestations have been termed HIV-associated dementia. The mechanisms underlying HIV-associated neuronal injury are incompletely understood, but various studies have confirmed the release of neurotoxins by macrophages/microglia infected with HIV-1 or stimulated by viral proteins, including the envelope glycoprotein gp120. In the present study, we investigated the possibility that l-cysteine, a neurotoxin acting at the N-methyl-d-aspartate subtype of glutamate receptor, could contribute to HIV-associated neuronal injury. Picomolar concentrations of gp120 were found to stimulate cysteine release from human monocyte-derived macrophages (hMDM) in amounts sufficient to injure cultured rat cerebrocortical neurons. TNF-α and IL-1β, known to be increased in HIV-encephalitic brains, as well as a cellular product of cytokine stimulation, ceramide, were also shown to induce release of cysteine from hMDM in a dose-dependent manner. A TNF-α-neutralizing Ab and an IL-1βR antagonist partially blocked gp120-induced cysteine release, suggesting that these cytokines may mediate the actions of gp120. Interestingly, hMDM infected with HIV-1 produced significantly less cysteine than uninfected cells following stimulation with TNF-α. Our findings imply that cysteine may play a role in the pathogenesis of neuronal injury in HIV-associated dementia due to its release from immune-activated macrophages but not virus-infected macrophages. Such uninfected cells comprise the vast majority of mononuclear phagocytes (macrophages and microglia) found in HIV-encephalitic brains.
CX3C-Chemokine, Fractalkine-Enhanced Adhesion of THP-1 Cells to Endothelial Cells Through Integrin-Dependent and -Independent Mechanisms
Authors: Goda Seiji , Imai Toshio , Yoshie Osamu , Yoneda Osamu , Inoue Hiroshi , Nagano Yutaka , Okazaki Toshiro , Imai Hisao , Bloom Eda T. , Domae Naochika , Umehara Hisanori
Published: April 2014

Leukocyte adhesion and trafficking at the endothelium requires both cellular adhesion molecules and chemotactic factors. A newly identified CX3C chemokine, fractalkine, expressed on activated endothelial cells, plays an important role in leukocyte adhesion and migration. We examined the functional effects of fractalkine on β1 and β2 integrin-mediated adhesion using a macrophage-like cell line, THP-1 cells. In this study, we report that THP-1 cells express mRNA encoding a receptor for fractalkine, CX3CR1, determined by Northern blotting. Scatchard analysis using fractalkine-SEAP (secreted form of placental alkaline phosphatase) chimeric proteins revealed that THP-1 cells express a single class of CX3CR1 with a dissociation constant of 30 pM and a mean expression of 440 sites per cell. THP-1 cells efficiently adhered, in a fractalkine-dependent manner, to full-length of fractalkine immobilized onto plastic and to the membrane-bound form of fractalkine expressed on ECV304 cells or TNF-α-activated HUVECs. Moreover, soluble-fractalkine enhanced adhesion of THP-1 cells to fibronectin and ICAM-1 in a dose-dependent manner. Pertussis toxin, an inhibitor of Gi, inhibited the fractalkine-mediated enhancement of THP-1 cell adhesion to fibronectin and ICAM-1. Finally, we found that soluble-fractalkine also enhanced adhesion of freshly separated monocytes to fibronectin and ICAM-1. These results indicate that fractalkine may induce firm adhesion between monocytes and endothelial cells not only through an intrinsic adhesion function itself, but also through activation of integrin avidity for their ligands.
Expression of a Functional High-Affinity IgG Receptor, FcγRI, on Human Mast Cells: Up-Regulation by IFN-γ
Authors: Okayama Yoshimichi , Kirshenbaum Arnold S. , Metcalfe Dean D.
Published: April 2014

Biologically relevant activation of human mast cells through Fc receptors is believed to occur primarily through the high-affinity IgE receptor FcεRI. However, the demonstration in animal models that allergic reactions do not necessarily require Ag-specific IgE, nor the presence of a functional IgE receptor, and the clinical occurrence of some allergic reactions in situations where Ag-specific IgE appears to be lacking, led us to examine the hypothesis that human mast cells might express the high-affinity IgG receptor FcγRI and in turn be activated through aggregation of this receptor. We thus first determined by RT-PCR that resting human mast cells exhibit minimal message for FcγRI. We next found that IFN-γ up-regulated the expression of FcγRI. This was confirmed by flow cytometry, where FcγRI expression on human mast cells was increased from ∼2 to 44% by IFN-γ exposure. FcεRI, FcγRII, and FcγRIII expression was not affected. Scatchard plots were consisted with these data where the average binding sites for monomeric IgG1 (Ka = 4–5 × 108 M−1) increased from ∼2,400 to 12,100–17,300 per cell. Aggregation of FcγRI on human mast cells, and only after IFN-γ exposure, led to significant degranulation as evidenced by histamine release (24.5 ± 4.4%): and up-regulation of mRNA expression for specific cytokines including TNF-α, GM-CSF, IL-3 and IL-13. These findings thus suggest another mechanism by which human mast cells may be recruited into the inflammatory processes associated with some immunologic and infectious diseases.
Differential Expression of NK T Cell Vα24JαQ Invariant TCR Chain in the Lesions of Multiple Sclerosis and Chronic Inflammatory Demyelinating Polyneuropathy
Authors: Illés Zsolt , Kondo Takayuki , Newcombe Jia , Oka Nobuyuki , Tabira Takeshi , Yamamura Takashi
Published: April 2014

Human Vα24+ NK T cells are a unique subset of lymphocytes expressing the Vα24JαQ invariant TCR chain. Because they can rapidly produce large amounts of regulatory cytokines, a reduction of NK T cells may lead to the development of certain autoimmune diseases. Using a single-strand conformation polymorphism method, we demonstrate that a great reduction of Vα24JαQ NK T cells in the peripheral blood is an immunological hallmark of multiple sclerosis, whereas it is not appreciable in other autoimmune/inflammatory diseases such as chronic inflammatory demyelinating polyneuropathy. The chronic inflammatory demyelinating polyneuropathy lesions were often found to be infiltrated with Vα24JαQ NK T cells, but multiple sclerosis lesions only rarely expressed the Vα24JαQ TCR. It is therefore possible that the extent of NK T cell alteration may be a critical factor which would define the clinical and pathological features of autoimmune disease. Although the mechanism underlying the NK T cell deletion remains largely unclear, a remarkable contrast between the CNS and peripheral nervous system diseases allows us to speculate a role of tissue-specific elements such as the level of CD1d expression or differences in the CD1d-bound glycolipid.
Secondary Lymphoid Tissue Chemokine Mediates T Cell-Dependent Antitumor Responses In Vivo
Authors: Sharma Sherven , Stolina Marina , Luo Jie , Strieter Robert M. , Burdick Marie , Zhu Li X. , Batra Raj K. , Dubinett Steven M.
Published: April 2014

Secondary lymphoid tissue chemokine (SLC, also referred to as Exodus 2 or 6Ckine) is a recently identified high endothelial-derived CC chemokine. The ability of SLC to chemoattract both Th1 lymphocytes and dendritic cells formed the rationale to evaluate this chemokine in cancer immunotherapy. Intratumoral injection of recombinant SLC evidenced potent antitumor responses and led to complete tumor eradication in 40% of treated mice. SLC-mediated antitumor responses were lymphocyte dependent as evidenced by the fact that this therapy did not alter tumor growth in SCID mice. Studies performed in CD4 and CD8 knockout mice also revealed a requirement for both CD4 and CD8 lymphocyte subsets for SLC-mediated tumor regression. In immunocompetent mice, intratumoral SLC injection led to a significant increase in CD4 and CD8 T lymphocytes and dendritic cells, infiltrating both the tumor and the draining lymph nodes. These cell infiltrates were accompanied by the enhanced elaboration of Th1 cytokines and chemokines monokine induced by IFN-γ and IFN-γ-inducible protein 10 but a concomitant decrease in immunosuppressive cytokines at the tumor site. In response to irradiated autologous tumor, splenic and lymph node-derived cells from SLC-treated tumor-bearing mice secreted significantly more IFN-γ, GM-CSF, and IL-12 and reduced levels of IL-10 than did diluent-treated tumor-bearing mice. After stimulation with irradiated autologous tumor, lymph node-derived lymphocytes from SLC-treated tumor-bearing mice demonstrated enhanced cytolytic capacity, suggesting the generation of systemic immune responses. These findings provide a strong rationale for further evaluation of SLC in tumor immunity and its use in cancer immunotherapy.