sq-23377 and phorbol-12-myristate

Microarray technology has become highly valuable for identifying complex global changes in gene expression patterns. Standard techniques measure changes in total cellular poly(A) mRNA levels. The assumption that changes in gene expression as measured by these techniques are directly and well correlated with changes in rates of new gene synthesis form the basis of attempts to connect coordinated changes in gene expression with shared transcription regulatory elements. Yet systematic attempts at this approach remain difficult to demonstrate convincingly. One reason for this difficulty may result from the intricate convergence of both transcriptional and mRNA turnover events which, together, directly influence steady-state mRNA levels. Recent technical advances have led to the successful scale-up and application of nuclear run-on procedures directly to microarrays. This development has allowed a gene-by-gene comparison between new gene synthesis in the nucleus and measured changes in total cellular polyA mRNA. Results from these studies have begun to challenge the strict interpretation of changes in gene expression measured by conventional microarrays as being closely correlated with changes in mRNA transcription rate, but rather they tend to support the significant expansion of the role played by changes in mRNA stability regulation to standard analyses of gene expression. Gene expression profiles obtained from both polyA mRNA (whole-cell) and nuclear run-on (newly transcribed) RNA across a time course of one hour following the activation of human Jurkat T cells with PMA plus ionomycin revealed that regulation of mRNA stability may account for as much as 50% of all measurements of changes in total cellular polyA mRNA in this system. Stability regulation was inferred by the absence of corresponding regulation of nuclear gene transcription activity for groups of genes strongly regulated at the whole cell level and which were also resistant to inhibition by Actinomycin D pre-treatment. Consistent patterns across the time course were observed for both transcribed and stability-regulated genes. It is proposed that the regulation of mRNA stability in response to external stimuli contributes significantly to observed changes in gene expression as measured by high throughput systems.

Topics: Cell Line, Tumor; Cell Nucleus; Dactinomycin; Gene Expression; Gene Expression Regulation; Humans; Ionomycin; Jurkat Cells; Oligonucleotide Array Sequence Analysis; Phorbol Esters; RNA; RNA, Messenger; Time Factors; Transcription, Genetic

IL-10 is the best known and most studied anti-inflammatory cytokine and, in the last 20 years, it has acquired even greater fame as it has been associated with the regulatory phenotype of B cells. Indeed, although great efforts have been made to find a unique marker, to date IL-10 remains the main way to follow both murine and human regulatory B cells, hence the need of precise and reproducible methods to identify and purify IL-10-producing B cells for both functional and molecular downstream assays. In this chapter, we present our protocols to isolate these cells from the murine spleen and peritoneum and from human peripheral blood. Since the production of IL-10 by B cells is not only a weapon to counteract the adverse effect of pro-inflammatory cytokines but also a response to cellular activation, we focused on those B cells that are prone to IL-10 production and detectable following a short-term stimulation with phorbol-12-myristate-13-acetate, ionomycin, and lipopolysaccharide (murine system) or CpG (human system).

Topics: Animals; B-Lymphocyte Subsets; B-Lymphocytes, Regulatory; Cell Separation; Cytokines; Gene Expression; Humans; Interleukin-10; Ionomycin; Lipopolysaccharides; Lymphocyte Activation; Mice; Phorbol Esters; Spleen; Tetradecanoylphorbol Acetate

The function of lymphocytes is the key to reflect the immune status of hosts. Evaluation of lymphocyte function is a useful tool to monitor the effect of immunosuppressive treatment and predict the prognosis of immune-mediated diseases (e.g., cancer, autoimmune diseases, and infectious diseases). As the lymphocytes have various activities, such as activation, cytotoxicity, and cytokine secretion, it is a challenge to evaluate the function of lymphocytes in clinical practice and the reference intervals (RIs) of lymphocyte function are rarely reported. The present study showed that the secretion of IFN-γ was well correlated with the activation, chemotaxis, and cytotoxicity of CD4

Topics: Adult; Biomarkers; Female; Humans; Interferon-gamma; Ionomycin; Killer Cells, Natural; Lymphocyte Activation; Lymphocytes; Middle Aged; Phorbol Esters; Reproducibility of Results; T-Lymphocytes; Young Adult

We examined the role of PKCs and Ca

Topics: Calcimycin; Calcium; Cell Line, Transformed; Gonadotrophs; Gonadotropin-Releasing Hormone; Ionomycin; Isoenzymes; Models, Biological; p38 Mitogen-Activated Protein Kinases; Peptides; Phorbol Esters; Phosphorylation; Protein Kinase C; Protein Kinase Inhibitors; src-Family Kinases; Time Factors

Natural Killer (NK) cell effector functions are dependent on phosphorylation of the mitogen-activated protein kinases (MAPK) pathway to produce an effective immune response for the clearance of target cells infected with viruses, bacteria or malignantly transformed cells. Intracellular signals activating NK cell cytokine production and cytotoxic activity are propagated through protein phosphorylation of MAPKs including MEK1/2, ERK1/2, p38 and JNK. Reduced NK cell cytotoxic activity is consistently reported in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) patients and intracellular signalling by MAPK in NK cells remains to be investigated. Therefore, the purpose of this paper was to investigate MAPK downstream signalling molecules in NK cell phenotypes from CFS/ME patients.. Flow cytometric protocols were used to measure phosphorylation of the MAPK pathway in CD56(bright)CD16(dim/-) and CD56(dim)CD16(+) NK cells following stimulation with K562 tumour cells or phorbol-12-myristate-13-acetate plus ionomycin. NK cell cytotoxic activity, degranulation, lytic proteins and cytokine production were also measured as markers for CD56(bright)CD16(dim/-) and CD56(dim)CD16(+) NK cell function using flow cytometric protocols.. CFS/ME patients (n = 14) had a significant decrease in ERK1/2 in CD56(dim)CD16(+) NK cells compared to the non-fatigued controls (n = 11) after incubation with K562 cells. CD56(bright)CD16(dim/-) NK cells from CFS/ME patients had a significant increase in MEK1/2 and p38 following incubation with K562 cells.. This is the first study to report significant differences in MAPK intracellular signalling molecules in CD56(dim)CD16(+) and CD56(bright)CD16(dim/-) NK cells from CFS/ME patients. The current results highlight the importance of intracellular signalling through the MAPK pathway for synergistic effector function of CD56(dim)CD16(+) and CD56(bright)CD16(dim/-) NK cells to ensure efficient clearance of target cells. In CFS/ME patients, dysfunctional MAPK signalling may contribute to reduced NK cell cytotoxic activity.

Topics: Antigens, CD; Case-Control Studies; Cytokines; Cytotoxicity, Immunologic; Extracellular Signal-Regulated MAP Kinases; Fatigue Syndrome, Chronic; Humans; Ionomycin; Killer Cells, Natural; Lymphocyte Activation; Mitogen-Activated Protein Kinase Kinases; p38 Mitogen-Activated Protein Kinases; Phenotype; Phorbol Esters; Signal Transduction

Experimental autoimmune encephalomyelitis (EAE) is T-cell-dependent disease of the central nervous system (CNS) of mice. This model resembles multiple sclerosis (MS) in many aspects. Therapies that focus in the modulation of the immune response and cellular infiltration in the CNS present best effects in the clinics. Artesunate (Art) is a semi-synthetic sesquiterpene derivative from artemisinin and has been shown to reduce the clinical signs of autoimmune disease models through mechanisms not yet understood. In this study, we aimed to evaluate whether administration of Art would ameliorate EAE.. C57BL6 mice were immunized with MOG35-55 peptide to induce EAE. At the same time, Art treatment started (3 mg/kg/day via i.p.) for five consecutive days. We found that Art treatment reduced the clinical signs of EAE and that correlated with a reduced infiltration of cells in the CNS. Disease amelioration did not correlate with immunomodulation as recall responses, leukocyte subpopulations, and gene expression analysis were similar among treated and untreated mice. Ultimately, further analysis provided data indicating that a possible mechanism of action for Art is dependent on the cellular migration to the CNS.. Artesunate reduces the severity of EAE by inhibiting migration of pathogenic T cells to the CNS.

Topics: Analysis of Variance; Animals; Anti-Inflammatory Agents; Artemisinins; Artesunate; Brefeldin A; Cell Movement; Central Nervous System; Cytokines; Disease Models, Animal; DNA-Binding Proteins; Encephalomyelitis, Autoimmune, Experimental; Enzyme Inhibitors; Female; Flow Cytometry; Gene Expression Regulation; Ionomycin; Leukocytes; Mice; Mice, Inbred C57BL; Mice, Knockout; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Phorbol Esters

Regulatory B cells (Breg) are a distinct B cell subset with immunoregulatory properties. Pivotal to Breg function is interleukin-10. This study was to investigate the role of IL-10-producing B cell (B10) and its association with Treg and Th17 subsets in immune thrombocytopenia (ITP) patients.. Peripheral blood mononuclear cells from ITP patients and controls were stimulated with PMA, ionomycin, and Brefeldin A. The frequencies of CD19(+)IL-10(+) B cells, CD3(+)CD4(+)IL-17(+) Th17 cells, and CD4(+)CD25(hi)Foxp3(+) Treg cells were analyzed by flow cytometry. The mRNA expression of Foxp3 and RORγt was detected by real-time quantitative PCR.. The number of B10 cells was elevated in ITP patients. After first-line therapies, it remained at high level in patients who achieved complete or partial response but decreased in those who acquired no response. There was a positive correlation between B10 cells and Tregs in ITP both before and after therapies. The ratio of Treg/Th17 decreased in ITP, and it strongly correlated with B10 cells.. The frequency of B10 cells is elevated in ITP and it correlates with both the Tregs counts and the Treg/Th17 ratio. B10 cells to regulate functional T cell subsets might be impaired in patients with ITP.

Topics: Adult; Aged; Antigens, CD19; B-Lymphocytes; Brefeldin A; CD3 Complex; CD4-Positive T-Lymphocytes; Female; Flow Cytometry; Humans; Interleukin-10; Interleukin-17; Interleukin-2 Receptor alpha Subunit; Ionomycin; Leukocytes, Mononuclear; Male; Middle Aged; Phorbol Esters; Polymerase Chain Reaction; Purpura, Thrombocytopenic, Idiopathic; Th17 Cells; Treatment Outcome; Young Adult

Stimulated human basophils exhibit different degranulation patterns with release of mediators and appearance of activation markers such as CD63 and CD203c. Traditionally, released mediators are quantified in the supernatant of activated cells, whereas the expression of activation markers by individual cells is analyzed by flow cytometry. Alternatively, intracellular histamine and its release by basophils and mast cells have been repeatedly studied applying an enzyme-affinity-gold method based on the affinity of the histaminase diamine oxidase for its substrate histamine.. To develop a flow cytometric technique enabling to study histamine release by individual basophils in combination with the expression of activation markers. To elucidate the principles of basophil degranulation on a single cell level.. Intracellular histamine and its release is analyzed flow cytometrically by an enzyme-affinity method using diamine oxidase conjugated to laser-excitable fluorochromes. Phenotyping of cells implied flow cytometric quantification of CD63 and CD203c. Stimuli such as allergen, anti-IgE, N-formyl-met-leu-phe (fMLP), phorbol 12-myristate 13-acetate (PMA), ionomycin and interleukin (IL-)3 are applied to obtain different degranulation profiles.. Stimulation with anti-IgE, allergen, fMLP and PMA±ionomycin induces a rapid release of histamine that can be analyzed flow cytometrically. Analyses on a single cell level reveal that histamine release is not restricted to cells showing significant up-regulation of CD63. Alternatively, up-regulation of CD203c does not per se indicate histamine release. In some patients, priming of cells with IL-3 not only facilitates basophil responsiveness but also implies an increased ability of DAO to label the cells.. This study provides the proof-of-concept that histamine and its release can be studied by multicolor flow cytometry on a single cell level (HistaFlow). Coupling the data to simultaneous phenotyping of activated basophils confirms that histamine release principally results from anaphylactic degranulation and in a lesser extent from piecemeal degranulation.

Topics: Adolescent; Adult; Allergens; Amine Oxidase (Copper-Containing); Antibodies, Anti-Idiotypic; Basophil Degranulation Test; Basophils; Child; Female; Flow Cytometry; Histamine; Histamine Release; Humans; Interleukin-3; Ionomycin; Male; Middle Aged; N-Formylmethionine Leucyl-Phenylalanine; Phorbol Esters; Phosphoric Diester Hydrolases; Pyrophosphatases; Tetraspanin 30; Young Adult

Interleukin-10 (IL-10) mediates its broad anti-inflammatory and immunoregulatory effects through two cell surface receptors by which binding to the IL-10 receptor 1 (IL-10R1) is the initial step that leads to recruitment of IL-10R2 and initiation of the ternary complex signal transduction cascade. The duck IL-10R1 (duIL-10R1) cDNA was obtained by using RT-PCR and 5'RACE. The deduced 574 amino acid protein has an amino acid identity of 62%, 27% and 28% with chicken, mouse and human IL-10R1, respectively. Comparison of the duIL-10R1 cDNA with duck genomic sequences revealed a seven exon-six intron structure of the duck IL-10R1 gene that shares a similar size with the respective exons 1-7 of the chicken and human IL-10R1 genes, but the avian genes are more compact. Promoter analysis identified putative binding sites for regulatory elements such as CCAAT enhancer binding protein-α, specificity protein 1 (Sp1), nuclear factor 1 (NF1), transcriptional regulatory protein Oct-1, nuclear factor (NF) κB and interferon-stimulated gene factor-3 (ISGF-3). A canonical TATA box was absent in proximity of the transcription initiation site, but a CpG island was present. Sequence analysis of the predicted duIL-10R1 protein revealed characteristic features of class-II cytokine receptors (CFR2) family members and a considerable degree of conservation of residues implicated in ligand binding across higher vertebrates. The predicted secondary structure of the duIL-10R1 extracellular domain is compatible with the two-subdomain structure of the human IL-10R1 protein established by its crystal structure. The 3D model structure shows conservation of the positions of conserved contact residues within four of the five ligand-binding loops. Within the cytoplasmic domain, residues implicated in signal transduction were conserved including two redundant peptide motifs GYXXQ essential for recruitment and activation of STAT3. DuIL-10R1 mRNA expression was most abundant in spleen, thymus, peripheral blood mononuclear cells (PBMCs) and lung. Mitogen stimulation of PBMCs transiently increased duIL-10R1 mRNA expression. Our observations suggest significant evolutionary conservation of the IL-10R1 genomic organization, protein structure and receptor function through the JAK/STAT signalling pathway across higher vertebrates.

Topics: Amino Acid Sequence; Animals; Binding Sites; Chromosome Mapping; Cloning, Molecular; Computational Biology; Conserved Sequence; CpG Islands; DNA, Complementary; Ducks; Exons; Humans; Interferon-Stimulated Gene Factor 3; Interleukin-10 Receptor alpha Subunit; Ionomycin; Leukocytes, Mononuclear; Mice; Molecular Sequence Data; NF-kappa B; Phorbol Esters; Phylogeny; Promoter Regions, Genetic; Protein Structure, Secondary; Protein Structure, Tertiary; RNA, Messenger; Sequence Alignment; Signal Transduction; Sp1 Transcription Factor; Transcription, Genetic

The role of the type-2 T helper (Th2) cell-mediated immune response in the immunopathogenesis of atopic dermatitis (AD) is well documented. Whether polarized immunoresponse is confined to antigen-specific T cells or is distributed among all T cell subsets is still controversial. We investigated frequencies of interleukin-2 (IL-2), IL-4, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) producing CD3(+) and CD4(+) T cells in peripheral blood from children with atopic dermatitis and healthy subjects with and without in vitro stimulation. Children with severe AD had a significantly lower percentage of CD4(+) T cells spontaneously expressing IL-4 compared with healthy controls (p <0.01). Polyclonal stimulation significantly increased cytokine production in both AD patients and healthy individuals. Frequencies of CD3(+) and CD4(+) producing IL-2, IL-4, IFN-gamma, and TNF-alpha after in vitro stimulation with phorbol-12-myristate 13-acetate (PMA) + ionomycin were comparable in the AD and control groups. In response to PMA/ionomycin, children with AD and asthma symptoms had a significantly lower percentage of CD3(+) T cells producing TNF-alpha. We failed to demonstrate evidence of an imbalance with respect to type-2 cytokine productions in children with AD. Comparable induction of Th1 and Th2 cytokines in polyclonally stimulated peripheral CD3(+) and CD4(+)T cells from AD patients and controls puts into question the polarized Th2 immune response as a general characteristic of T cells in children with atopic dermatitis.

Topics: Adolescent; Asthma; CD3 Complex; CD4-Positive T-Lymphocytes; Child; Child, Preschool; Cytokines; Dermatitis, Atopic; Flow Cytometry; Humans; Ionomycin; Lymphocyte Count; Phorbol Esters; Severity of Illness Index; Statistics, Nonparametric; T-Lymphocytes

The neuronal form of nitric oxide synthase (nNOS) was generally assumed to be constitutively expressed at a constant level. However, it is now becoming recognized that its expression can be modulated by a number of physiological and pathophysiological conditions. Previously, we reported that nNOS expression is up-regulated after prolonged muscarinic M(1) receptor stimulation. In this work, we report that muscarinic receptor activation signals the up-regulation of nNOS via multiple pathways in N1E-115 mouse neuroblastoma cells. These include protein kinase C (PKC) activation, cytosolic calcium mobilization and NO production. Further characterization showed that the half-life of nNOS is slightly, but significantly, increased in agonist-pretreated cells compared with vehicle-treated control cells. Based on these data, it appears that the level of nNOS expression is modulated in a complex manner by a number of mechanisms that include, but might not be limited to, those described here.

Topics: Analysis of Variance; Animals; Atropine; Blotting, Western; Carbachol; Cell Line, Tumor; Chelating Agents; Cholinergic Agonists; Drug Interactions; Egtazic Acid; Enzyme Inhibitors; Ionomycin; Ionophores; Mice; Muscarinic Antagonists; Nerve Tissue Proteins; Neuroblastoma; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Phorbol 12,13-Dibutyrate; Phorbol Esters; Receptor, Muscarinic M1; Time Factors; Up-Regulation

CD8(+) cytotoxic T lymphocyte (CTL) clones are able to exert both perforin- and Fas-dependent cytotoxicity. We show in the present work that phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002 prevent TCR/CD3-induced functional Fas ligand (FasL) expression, but not perforin-dependent cytotoxicity. The specific inhibitor of classical protein kinase C (PKC) isoforms, Gö6976, completely inhibited perforin-dependent cytotoxicity and only affected slightly TCR/CD3-induced FasL expression, while the opposite was observed using rottlerin, an inhibitor with higher specificity for PKCtheta. To address further the dependence of FasL expression on PI3K, a luciferase reporter controlled by the FasL promoter was used. Reporter gene induction by anti-CD3 mAb was abolished in cells transfected with dominant-negative PI3K (PI3K-DN) and increased in cells transfected with constitutively active PI3K (PI3K*). Transfection with constitutively active mutants (A/E) of PKCepsilon, and especially of PKCtheta, improved anti-CD3 mAb-induced reporter expression and completely abolished inhibition by wortmannin, while transfection with dominant-negative (K/R) PKCtheta prevented the induction of the reporter. Finally, transfection with PKCalpha A/E, but not with PKCtheta A/E, cooperated with ionomycin to induce degranulation in the CTL line 1.3E6SN. Altogether, the results suggest that TCR/CD3-induced FasL gene transcription is controlled by PI3K and PKCtheta activation, while this signaling pathway is not implicated in CTL degranulation, which is rather dependent on the activation of classical PKC isoforms.

Topics: Acetophenones; Androstadienes; Animals; Antibodies, Monoclonal; Benzopyrans; Carbazoles; Cell Degranulation; Cell Line; Chromones; Cycloheximide; Cytotoxicity, Immunologic; Dactinomycin; Fas Ligand Protein; Humans; Indoles; Ionomycin; Isoenzymes; Jurkat Cells; Luciferases; Lymphocyte Activation; Membrane Glycoproteins; Mice; Morpholines; Perforin; Phorbol Esters; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Pore Forming Cytotoxic Proteins; Protein Kinase C; Protein Kinase C-alpha; Protein Kinase C-epsilon; Protein Kinase C-theta; Receptor-CD3 Complex, Antigen, T-Cell; Receptors, Antigen, T-Cell; Signal Transduction; T-Lymphocytes, Cytotoxic; Transfection; Wortmannin

Neonatal CD4(+) T cells express less CD154 protein and mRNA than adult CD4(+) T cells after activation by calcium ionophore and phorbol ester, but the mechanism for this reduced expression and its relevance to the primary immune response remain unclear. We compared expression of CD154 protein and mRNA and CD154 gene promoter activity by purified naive (CD45RA(high)CD45RO(low)) neonatal and adult CD4(+) T cells after activation by calcium ionophore (ionomycin) and phorbol myristate acetate (PMA) treatment or by engagement of alphabeta TCR-CD3 complex. Substantial and consistent reductions in expression by neonatal cells were found in all cases and were paralleled by decreased CD154-dependent activation of a B cell line. CD69 expression by neonatal CD4(+) T cells after alphabeta TCR-CD3 engagement was also reduced compared to adult cells, which suggested that limitations in activation-induced signaling by neonatal CD4(+) T cells occurred at a point upstream of where the signaling pathways leading to CD154 and CD69 expression diverge. Decreased CD154 expression by neonatal cells after alphabeta TCR-CD3 engagement was paralleled by a lower free intracellular calcium concentration, a key event for CD154 gene transcription. Reduced CD154 promoter activity by neonatal cells persisted when proximal signaling events were bypassed using ionomycin and PMA, suggesting an additional and more distal mechanism for decreased transcription. In contrast, CD154 mRNA stability was similar in neonatal and adult cells after either ionomycin and PMA stimulation or engagement of the alphabeta TCR-CD3 complex. We conclude that decreased CD154 production by neonatal CD4(+) T cells is due to limitations in both proximal and distal activation events, which together ultimately limit CD154 gene transcription.

Topics: Adult; Antibodies; Antibodies, Monoclonal; Antigens, CD; Antigens, Differentiation, T-Lymphocyte; B-Lymphocytes; Blotting, Northern; Calcium; CD28 Antigens; CD3 Complex; CD4-Positive T-Lymphocytes; CD40 Ligand; Cell Line, Tumor; Dactinomycin; Flow Cytometry; Gene Expression Regulation, Developmental; Humans; Infant, Newborn; Intercellular Adhesion Molecule-1; Interleukin-2; Ionomycin; Kinetics; Lectins, C-Type; Leukocyte Common Antigens; Luciferases; Lymphocyte Activation; Phorbol Esters; Promoter Regions, Genetic; Receptor-CD3 Complex, Antigen, T-Cell; Receptors, Antigen, T-Cell, alpha-beta; RNA, Messenger; Signal Transduction