sq-23377 and phorbolol-myristate-acetate

Juglone (5-hydroxy-1, 4-naphthoquinone), is a natural phenolic compound that has been shown to relax smooth muscle. Therefore the aim of this study was to determine the effect of juglone on vascular tone using porcine coronary artery (PCA). Segments of PCA, with or without endothelium, were mounted for isometric tension recording in isolated tissue baths and precontracted with the thromboxane A

Topics: Animals; Calcium; Coronary Vessels; Cyclic GMP; Indoles; Ionomycin; Naphthoquinones; Nitric Oxide; Potassium Channel Blockers; Swine; Tetradecanoylphorbol Acetate; Vasoconstriction; Vasodilation; Vasodilator Agents

Expression of programmed death ligand 1 (PD-L1) on the surface of tumor cells and its interaction with programmed cell death protein 1 (PD-1) on tumor-infiltrating lymphocytes suppress anti-tumor immunity. In breast tumors, PD-L1 expression levels are the highest in estrogen receptor-negative, progesterone receptor-negative, and human epidermal growth factor receptor 2-negative (triple-negative) cancers. In this study, we show that a portion of PD-L1 exogenously expressed in several triple-negative breast cancer cell lines, as well as endogenous PD-L1, is proteolytically cleaved by cell surface metalloproteases. The cleavage generates an ~ 37-kDa N-terminal PD-L1 fragment that is released to the media and a C-terminal PD-L1 fragment that remains associated with cells but is efficiently eliminated by lysosomal degradation. We identify ADAM10 and ADAM17, two closely related members of the ADAM family of cell surface metalloproteases, as enzymes mediating PD-L1 cleavage. Notably, treatment of cells with ionomycin, a calcium ionophore and a known activator of ADAM10, or with phorbol 12-myristate 13-acetate, an activator of ADAM17, dramatically increases the release of soluble PD-L1 to the media. We postulate that ADAM10 and/or ADAM17 may contribute to the regulation of the PD-L1/PD-1 pathway and, ultimately, to anti-tumor immunity in triple-negative breast cancer.

Topics: ADAM10 Protein; ADAM17 Protein; Amyloid Precursor Protein Secretases; B7-H1 Antigen; Cell Line, Tumor; Female; Humans; Ionomycin; Lymphocytes, Tumor-Infiltrating; Lysosomes; Membrane Proteins; Programmed Cell Death 1 Receptor; Proteolysis; Signal Transduction; Tetradecanoylphorbol Acetate; Triple Negative Breast Neoplasms

Topics: Animals; Apoptosis; Cell Differentiation; Cell Line, Tumor; CRISPR-Cas Systems; Gene Knockdown Techniques; Genetic Loci; Ionomycin; Lymphocyte Activation; Mice; Proto-Oncogene Proteins c-bcl-2; RNA, Long Noncoding; T-Lymphocytes; Tetradecanoylphorbol Acetate

Cross-sectional studies have shown that exposure to indoor moisture damage and mold may be associated with subclinical inflammation. Our aim was to determine whether early age exposure to moisture damage or mold is prospectively associated with subclinical systemic inflammation or with immune responsiveness in later childhood. Home inspections were performed in children's homes in the first year of life. At age 6 years, subclinical systemic inflammation was measured by serum C-reactive protein (CRP) and blood leukocytes and immune responsiveness by ex vivo production of interleukin 1-beta (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α) in whole blood cultures without stimulation or after 24 hours stimulation with phorbol 12-myristate 13-acetate and ionomycin (PI), lipopolysaccharide (LPS), or peptidoglycan (PPG) in 251-270 children. Moisture damage in child's main living areas in infancy was not significantly associated with elevated levels of CRP or leukocytes at 6 years. In contrast, there was some suggestion for an effect on immune responsiveness, as moisture damage with visible mold was positively associated with LPS-stimulated production of TNF-α and minor moisture damage was inversely associated with PI-stimulated IL-1β. While early life exposure to mold damage may have some influence on later immune responsiveness, it does not seem to increase subclinical systemic inflammation in later life.

Topics: Air Pollutants; Air Pollution, Indoor; C-Reactive Protein; Child; Cytokines; Environmental Exposure; Female; Fungi; Humans; Infant; Inflammation; Interleukin-1beta; Interleukin-6; Ionomycin; Leukocyte Count; Leukocytes; Lipopolysaccharides; Male; Peptidoglycan; Prospective Studies; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

T cell-mediated cellular immunity and humoral immunity are equally important for the prevention of diseases. To assess activation of human and mouse cellular immunity, early activation markers of lymphocytes are often used in flow cytometry targeting expression of CD69 molecules. Response of humoral immunity against infection or vaccination has been well investigated in pigs, but that of cellular immunity has been largely neglected due to lack of direct evaluation tools. Thus, in pig research a proper assay of antibody reacted with porcine CD69 is still unavailable. In the present study, two anti-porcine CD69 mAb-producing mouse hybridomas, 01-14-22-51 (IgG2b-κ) and 01-22-44-102 (IgG2a-κ), both showing fine reactivity with phorbol 12-myristate 13-acetate (PMA) and ionomycin-stimulated porcine peripheral blood lymphocytes in flow cytometry, were established. When porcine peripheral blood lymphocytes were activated with PMA and ionomycin and analyzed by flow cytometry, it was found that both mAbs generated in this study stained about 70% of lymphocytes. In contrast, after an identical procedure, only 5% and 13.5% of lymphocytes were stained with anti-interferon-γ mAb and anti-tumor necrosis factor-α mAb, respectively. These results indicate that evaluation of cellular immunity activation turns more sensitive after using our newly generated mAbs.

Topics: Animals; Antibodies, Monoclonal; Antigens, CD; Antigens, Differentiation, T-Lymphocyte; Flow Cytometry; Humans; Immunity, Cellular; Ionomycin; Lectins, C-Type; Mice; Swine; T-Lymphocytes; Tetradecanoylphorbol Acetate

Iron deficiency is likely the most common nutritional deficiency worldwide; low iron concentrations have been related to alterations in immune system functions; therefore, the aim of this study was to determine the effect of low serum iron (LSI) concentrations on the production of proinflammatory cytokines by peripheral blood leukocytes in 8- to 12-y-old children from a local community.. We obtained 120 blood samples and determined full blood counts and serum iron concentrations. An LSI and a control group, paired by age and sex were established using serum iron <60 μg/dL as the cutoff point. Ferritin and C-reactive protein concentrations were quantified. Serum interferon (IFN)-γ and tumor necrosis factor (TNF)-α concentrations were measured in these groups by enzyme-linked immunosorbent assay. A second blood sample was taken from children in both groups to isolate peripheral blood mononuclear cells (PBMCs) and measure IFN-γ and TNF-α production by unstimulated and lipopolysaccharide/phorbol myristate acetate/ionomycin-stimulated leukocytes in vitro.. Of the participants in the present study, 17.5% (21 children) presented LSI, as well as decreased ferritin concentrations. Differential counts from total blood samples showed a significant increase in leukocyte numbers in the LSI group, along with increased neutrophil frequencies and numbers but decreased lymphocyte frequencies. Decreased serum IFN-γ concentrations and decreased in vitro production of IFN-γ by PBMCs were found in the LSI group.. The results of the present study suggest that low iron levels alter leukocyte subpopulations in circulation and have a detrimental effect on leukocyte production of proinflammatory cytokines after an antigenic challenge.

Topics: Anemia, Iron-Deficiency; Asymptomatic Diseases; Blood Cell Count; C-Reactive Protein; Calcium Ionophores; Child; Child Nutritional Physiological Phenomena; Female; Ferritins; Humans; Interferon-gamma; Ionomycin; Iron; Leukocytes, Mononuclear; Leukocytosis; Lipopolysaccharides; Male; Mexico; Mitogens; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

To investigate the effect of morphine on T helper lymphocyte differentiation and PI3K/AKT pathway mechanism, CD4+ lymphocytes were treated by phorbol-myristate-acetate (25 ng/ml) (PMA) plus ionomycin (1 μg/ml) in the presence of various concentrations of morphine (25, 50, 100, 200 ng/ml) for 4 h. Th1 and Th2 subsets, supernatant cytokines, and PI3K, AKT, and protein kinase C-theta (PKC-θ) levels were detected. The Th1 cell percentage, Th1-derived cytokines, and ratio of Th1/Th2 decreased in the presence of morphine in a concentration-dependent manner. However, Th2 cell percentage kept stable after morphine treatment. The phosphorylation of PI3K and AKT decreased, but the phosphorylation of PKC-θ did not change in the presence of morphine. The decreased percentage of Th1 cells and ratio of Th1/Th2 was recovered by naloxone concentration-dependently. Morphine can inhibit the differentiation of Th1 lymphocytes and decrease the ratio of Th1/Th2 via the pathway of PI3K/AKT. The effect can be inhibited by naloxone.

Topics: Cell Differentiation; Humans; Interferon-gamma; Interleukin-4; Ionomycin; Isoenzymes; Lymphocyte Activation; Morphine; Naloxone; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Kinase C; Protein Kinase C-theta; Proto-Oncogene Proteins c-akt; Tetradecanoylphorbol Acetate; Th1 Cells; Th1-Th2 Balance; Th2 Cells

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by B cell-dependent autoantibody production. Recently, a new B-cell subset was discovered that has a regulatory capacity. The aim of this study was to analyse regulatory B cells (Bregs) in SLE patients.. Peripheral mononuclear blood cells (PBMCs) of 34 SLE patients fulfilling the American College of Rheumatology (ACR) criteria for SLE and 21 healthy controls (HC) were included. PBMCs were stained for CD19, CD24, and CD38 and analysed by flow cytometry. In vitro stimulated PBMCs with CpG and restimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin were investigated for IL-10(+) Bregs .. The percentages of circulating CD19(+)CD24(hi)CD38(hi) cells in HC were not different those in from SLE patients. The percentages of IL-10(+) Bregs were significantly decreased in SLE patients, in particular those with lupus nephritis (LN), compared to HC. The proportion was independent of disease activity.. This is the first study to demonstrate a decrease in IL-10-producing B cells in LN patients compared to HC, reflecting an impaired regulatory function.

Topics: ADP-ribosyl Cyclase 1; Adult; Antigens, CD19; B-Lymphocytes, Regulatory; Calcium Ionophores; Case-Control Studies; CD24 Antigen; Female; Humans; In Vitro Techniques; Interleukin-10; Ionomycin; Lupus Nephritis; Lymphocyte Count; Male; Membrane Glycoproteins; Middle Aged; Oligodeoxyribonucleotides; Tetradecanoylphorbol Acetate; Young Adult

Epigenetic deregulation of genes encoded on the X chromosome as reported for CD40L in lupus could explain the female predominance of autoimmune diseases. We compared CD40L expression on CD4(+) T cells from primary Sjögren's syndrome (pSS) women and healthy controls and investigated DNA methylation patterns of the promoter and enhancer regions of CD40L. The expression of CD40L on activated CD4(+) T cells was higher in patients with pSS than controls after phorbolmyristate acetate and ionomycin activation (P = 0.02). CD40L mRNA level in CD4(+) T cells did not differ between patients with pSS and controls and was similar in both groups in cultures treated with the demethylating agent 5-azacytidine C. Pyrosequencing analysis revealed no significant differences in methylation profiles between patients and controls. Inducible membrane-bound CD40L on CD4(+) T cells is increased in patients with pSS but was not related to epigenetic deregulation by demethylation patterns of the regulatory regions of CD40L.

Topics: Adult; Aged; Aged, 80 and over; Azacitidine; CD40 Ligand; Cells, Cultured; DNA Methylation; Female; Gene Expression; Humans; Ionomycin; Lymphocyte Activation; Membrane Proteins; Middle Aged; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, DNA; Sjogren's Syndrome; T-Lymphocytes; Tetradecanoylphorbol Acetate; Up-Regulation; Young Adult

Tight control of lung liquid (LL) regulation is vital for pulmonary function. The aim of this work was to determine whether PKC activation is involved in the physiological regulation of LL volume in a whole lung preparation. Rat lungs were perfused with a modified Ringer solution, and the lumen was filled with the same solution without glucose. LL volume was measured during a control period and after modulating drugs were administered, and net LL transepithelial movement (J(v)) was calculated. When the PKC activator PMA (10(-5) M) and the Ca(2+) ionophore ionomycin (10(-6) M) were instilled into the lung together, J(v) was significantly reduced (P = 0.03). This reduction was blocked by the PKC inhibitor chelerythrine chloride (10(-6) M; P = 0.56) and by a second PKC inhibitor GF109203X (10(-5) M; P = 0.98). When PMA and ionomycin were added with the β-adrenergic agonist terbutaline, the terbutaline-induced increase in J(v) was abolished. Addition of PMA and ionomycin with the epithelial Na(+) channel (ENaC) blocker amiloride had no additional inhibitory effect. Together, these results suggest that PKC is likely to be involved in LL absorption, and the ability of PMA/ionomycin to block the terbutaline-induced increase in J(v) suggests that the downstream target of PKC is ENaC.

Topics: Amiloride; Animals; Benzophenanthridines; Biological Transport; Cells, Cultured; Electric Impedance; Enzyme Inhibitors; Epithelial Cells; Epithelial Sodium Channels; Indoles; Ionomycin; Isotonic Solutions; Lung; Male; Maleimides; Protein Kinase C; Rats; Rats, Wistar; Ringer's Solution; Signal Transduction; Sodium Channel Blockers; Terbutaline; Tetradecanoylphorbol Acetate

Human and mouse immune system cells are the most frequently used specimens in ELISPOT assays. In an effect to expand the application of ELISPOT assay to other species, we developed matched antibody pairs for ready-to-use kits designed for studying the frequency of equine IFNγ- and IL-4-secreting peripheral blood mononuclear cells (PBMCs). Equine PBMCs were stimulated with either concanavalin A (Con A) or calcium ionomycin mixed with phorbol 12-myristate 13-acetate (CaI + PMA). We found that Con A, in general, had a more profound stimulating effect than CaI + PMA on IL-4 secretion, whereas both stimulatory and inhibitory effects were observed on IFNγ secretion. Our data demonstrate a large dynamic range in IFNγ and IL-4 secretion among different donors, which may reflect animal health and serve as a valuable diagnostic marker.

Topics: Animals; Calcium; Concanavalin A; Enzyme-Linked Immunospot Assay; Horses; Humans; Interferon-gamma; Interleukin-4; Ionomycin; Leukocytes, Mononuclear; Tetradecanoylphorbol Acetate

Induced regulatory T (iTreg) lymphocytes show promise for application in the treatment of allergic, autoimmune and inflammatory disorders. iTreg cells demonstrate advantages over natural Treg (nTreg) cells in terms of increased number of starting population and greater potential to proliferate. Different activation methods to generate iTreg cells result in iTreg cells that are heterogeneous in phenotype and mechanisms of suppression. Therefore it is of interest to explore new techniques to generate iTreg cells and to determine their physiological relevance.. Using phorbol myristate acetate (PMA)/ionomycin and anti-CD3 activation of CD4⁺CD25⁻ cells we generated in vitro functional CD4⁺CD25⁻ iTreg (TregPMA) cells. Functionality of the generated TregPMA cells was tested in vivo in a mouse model of inflammatory bowel disease (IBD) - CD45RB transfer colitis model.. TregPMA cells expressed regulatory markers and proved to ameliorate the disease phenotype in murine CD45RB transfer colitis model. The body weight loss and disease activity scores for TregPMA treated mice were reduced when compared to diseased control group. Histological assessment of colon sections confirmed amelioration of the disease phenotype. Additionally, cytokine analysis showed decreased levels of proinflammatory colonic and plasma IL-6, colonic IL-1 β and higher levels of colonic IL-17 when compared to diseased control group.. This study identifies a new method to generate in vitro iTreg cells (TregPMA cells) which physiological efficacy has been demonstrated in vivo.

Topics: Analysis of Variance; Animals; Body Weight; Calcium Ionophores; CD3 Complex; CD4 Antigens; Colitis; Cytokines; Interleukin-2 Receptor alpha Subunit; Ionomycin; Leukocyte Common Antigens; Lymphocyte Activation; Mice; Mice, Inbred BALB C; T-Lymphocytes, Regulatory; Tetradecanoylphorbol Acetate

In HIV infection there is a paucity of literature about the degree of immune dysfunction to potentially correlate and/or predict disease progression relative to CD4(+) T cells count or viral load. We assessed functional characteristics of memory T cells subsets as potential prognostic markers for changing viral loads and/or disease progression using the SHIV-infected rhesus macaque model. Relative to long-term non-progressors with low/undetectable viral loads, those with chronic plasma viremia, but clinically healthy, exhibited significantly lower numbers and functional impairment of CD4(+) T cells, but not CD8(+) T cells, in terms of IL-2 production by central memory subset in response to PMA and ionomycine (PMA+I) stimulation. Highly viremic animals showed impaired cytokine-production by all T cells subsets. These results suggest that functional impairment of CD4(+) T cells in general, and of central memory subset in particular, may be a potential indicator/predictor of chronic infection with immune dysfunction, which could be assayed relatively easily using non-specific PMA+I stimulation.

Topics: Animals; Biomarkers; CD4-Positive T-Lymphocytes; Chronic Disease; Cytokines; Disease Progression; Humans; Immunologic Memory; Ionomycin; Macaca mulatta; Prognosis; Simian Acquired Immunodeficiency Syndrome; Tetradecanoylphorbol Acetate; Viral Load; Viremia

T-cell response to hepatitis B virus (HBV) is vigorous, polyclonal and multi-specific in patients with acute hepatitis who ultimately clear the virus, whereas it is narrow and inefficient in patients with chronic disease, where inappropriate early activation events could account for viral persistence. We investigated the induction of activation receptors and cytokine production in response to HBcAg and crosslinking of CD28 molecules, in CD4+ cells from a group of chronically infected patients (CIP) and naturally immune subjects (NIS). We demonstrated that CD4+ cells from CIP did not increase levels of CD40L and CD69 following stimulation with HBcAg alone or associated to CD28 crosslinking, in contrast to subjects that resolved the infection (p<0.01). Furthermore, CD4+ cells from CIP produced elevated levels of IL-10 in response to HBcAg. These results suggest that a predominant inhibitory environment may be responsible for altered T cell costimulation, representing a pathogenic mechanism for viral persistence.

Topics: Adult; Antigens, CD; Antigens, Differentiation, T-Lymphocyte; CD28 Antigens; CD4-Positive T-Lymphocytes; CD40 Ligand; Cytokines; Hepatitis B Core Antigens; Hepatitis B, Chronic; Humans; Interleukin-10; Ionomycin; Ionophores; Lectins, C-Type; Lymphocyte Activation; Middle Aged; Tetanus Toxoid; Tetradecanoylphorbol Acetate

Survival and death of lymphocytes are regulated by the balance between pro- and antiapoptotic members of the Bcl-2 family; this is coordinated with the control of cell cycling and differentiation. Bim, a proapoptotic BH3-only member of the Bcl-2 family, can be regulated by MEK/ERK-mediated phosphorylation, which affects its binding to pro-survival Bcl-2 family members and its turnover. We investigated Bim modifications in mouse B and T lymphoid cells after exposure to apoptotic stimuli and during mitogenic activation. Treatment with ionomycin or cytokine withdrawal caused an elevation in Bim(EL), the most abundant Bim isoform. In contrast, in mitogenically stimulated T and B cells, Bim(EL) was rapidly phosphorylated, and its levels declined. Pharmacological inhibitors of MEK/ERK signaling prevented both of these changes in Bim, reduced proliferation, and triggered apoptosis of mitogen-stimulated T and B cells. Loss of Bim prevented this cell killing but did not restore cell cycling. These results show that during mitogenic stimulation of T and B lymphocytes MEK/ERK signaling is critical for two distinct processes, cell survival, mediated (at least in part) through phosphorylation and consequent inhibition of Bim, and cell cycling, which proceeds independently of Bim inactivation.

Topics: Animals; Apoptosis Regulatory Proteins; B-Lymphocyte Subsets; Bcl-2-Like Protein 11; Cell Survival; Cells, Cultured; Extracellular Signal-Regulated MAP Kinases; Immunoglobulin Fab Fragments; Ionomycin; Lymphocyte Activation; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Mitogen-Activated Protein Kinases; Mitogens; Phosphorylation; Proto-Oncogene Proteins; Rats; Rats, Wistar; T-Lymphocyte Subsets; Tetradecanoylphorbol Acetate

The possible immunological relationship between the pattern of Th1/Th2 cytokine production and tuberculin reactivity was assessed in patients with active Mycobacterium tuberculosis infection. The production of the intracellular cytokines interferon (IFN)-gamma and interleukin-4 (IL-4) was measured in CD4(+) and CD8(+) T cells obtained from peripheral blood and bronchoalveolar lavage fluid (BALF) of 20 tuberculin skin-positive patients and compared with the findings recorded in nine tuberculin skin-negative patients with active pulmonary tuberculosis. Upon stimulation with phorbol 12-myristate acetate/ionomycin for 6 h, tuberculin-negative patients had a significantly higher proportion of IFN-gamma-producing CD4(+) T lymphocytes in BALF than in peripheral blood, while both CD4(+) and CD8(+) T-lymphocyte subsets in BALF of tuberculin-positive patients secreted more IFN-gamma than their peripheral blood counterparts. Tuberculin-negative patients had a significantly higher proportion of IFN-gamma-producing CD4(+) T lymphocytes in peripheral blood than tuberculin-positive patients. There was no significant difference in the production of IFN-gamma by BALF CD4(+) T lymphocytes, or by either peripheral blood or BALF CD8(+) T lymphocytes. In two tuberculin-negative patients, peripheral blood CD4(+) T lymphocytes produced IL-4. Study results suggested a higher immune activity in the blood of tuberculin-negative patients, with an increased lymphocyte activity in BALF versus peripheral blood in both patient groups.

Topics: Adult; Bronchoalveolar Lavage Fluid; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Female; Flow Cytometry; Humans; Interferon-gamma; Interleukin-4; Ionomycin; Male; Middle Aged; T-Lymphocytes; Tetradecanoylphorbol Acetate; Tuberculosis, Pulmonary

To investigate the role of local colonic mucosal NK receptor-positive T (NKR(+) T) cells in the regulation of intestinal inflammation, we analyzed the population and function of these cells in ulcerative colitis (UC).. Colonic mucosal tissues were obtained from colonoscopic biopsies of the descending colon from 96 patients with UC (51 endoscopically uninflamed, 45 inflamed) and 18 normal controls. Endoscopic appearance and histologic score at the biopsied site were determined by Matts' classification. A single cell suspension was prepared from each biopsy by collagenase digestion. Two NKR(+) T cell subsets, CD56(+) (CD56(+)CD3(+)) T cells and CD161(+) (CD161(+)CD3(+)) T cells, were detected by flow cytometric analysis. Intracellular cytokine analysis for anti-inflammatory cytokine interleukin-10 (IL-10) was performed by in vitro stimulation with phorbol-myristate-acetate (PMA) and ionomycin.. CD56(+) T cells and CD161(+) T cells are present in the normal human colon and account for 6.7% and 21.3% of all mononuclear cells, respectively. The populations of both CD56(+) T cells and CD161(+) T cells were decreased significantly in the inflamed mucosa of UC. In contrast, the frequency of conventional T cells (CD56(-)CD3(+) cells and CD161(-)CD3(+) cells) was similar among the patient and control groups. The populations of NKR(+) T cells were correlated inversely with the severity of inflammation, which was classified according to the endoscopic and histologic Matts' criteria. Interestingly, approximately 4% of mucosal NKR(+) T cells expressing IL-10 were detected by in vitro stimulation with PMA and ionomycin.. Selective reduction in the population of colonic mucosal NKR(+) T cells may contribute to the development of intestinal inflammation in UC.

Topics: Adult; Aged; Anti-Inflammatory Agents; Antigens, Surface; Azathioprine; CD56 Antigen; Colitis, Ulcerative; Female; Humans; Immunosuppressive Agents; Interleukin-10; Intestinal Mucosa; Ionomycin; Ionophores; Lectins, C-Type; Male; Middle Aged; NK Cell Lectin-Like Receptor Subfamily B; Prednisolone; T-Lymphocyte Subsets; Tetradecanoylphorbol Acetate

CD4+CD25+ regulatory T cells play an important role in peripheral tolerance. Upon T cell receptor (TCR)-mediated activation, the cells fail to proliferate but are induced to have a suppressor function. The intracellular signaling events that lead to their responses have not been elucidated. In this study, we have examined the proximal TCR signaling events in freshly isolated human CD4+CD25+ regulatory T cells after TCR ligation. In contrast to CD4+CD25- T cells, TCR ligation of CD4+CD25+ regulatory T cells by anti-CD3 cross-linking resulted in a lower calcium influx and extracellular signal-regulated kinase 1/2 phosphorylation. Examination of the CD3zeta chain phosphorylation status indicated that CD4+CD25+ regulatory T cells have poor phosphorylation of the protein and consequently, reduced recruitment of zeta-associated protein-70 to the TCR immunoreceptor tyrosine motif. The adaptor protein, Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa, which relays signals to downstream signaling components, also showed reduced phosphorylation, which correlated with reduced VAV guanine nucleotide exchange factors association. Consistent with other findings, the defect is accompanied with impaired actin cap formation, implicating a failure of actin remodeling of the cells. Together, our results demonstrate that CD4+CD25+ regulatory T cells have altered TCR proximal signaling pathways, which could be critical for inducing the distinct behavior of these cells.

Topics: Actins; Humans; Interleukin-2 Receptor alpha Subunit; Ionomycin; Receptors, Antigen, T-Cell; Signal Transduction; T-Lymphocytes, Regulatory; Tetradecanoylphorbol Acetate

MICA is a stress-regulated molecule recognized by the NK cell-activating receptor NKG2D. Previously, we demonstrated that MICA is induced on activated T cells but regulation by mitogenic cytokines and its biological consequences remain unexplored. Here, we show that IL-2, IL-4, and IL-15 but not TNF-alpha or IFN-alpha induced MICA expression in T lymphocytes present in peripheral blood mononuclear cells (PBMCs), as assessed by Western blot. IL-2 effect involved Jak3/STAT5, p38 MAPK, p70(56) kinase, Lck/fyn kinases, and NF-kappaB. MICA expression was also observed in Th1 and Th2 cells. However, surface expression was not detected. T lymphocytes present in PBMCs and isolated CD4+ T lymphocytes stimulated with phorbol-12-myristate-13-acetate and ionomycin also induced MICA expression as assessed by Western blot, but only low levels were expressed at the cell surface. Activated but not resting CD4+ T lymphocytes were lysed by IL-15- or IL-2-stimulated NK cells, and susceptibility was increased when HLA class I molecules were blocked. Also, cytokine-stimulated NK cells produced more IFN-gamma after culture with activated CD4+ T lymphocytes. However, the participation of MICA in these responses, if any, was marginal. Confocal microscopy revealed that MICA is retained mostly inside activated CD4+ T cells. Our results suggest that low surface expression of MICA on activated CD4+ T lymphocytes might be a safeguard mechanism to protect them from NK cells in an inflammatory, virus-infected, or tumor microenvironment, where NK and activated CD4+ T cells are recruited.

Topics: CD4-Positive T-Lymphocytes; Cell Survival; Cytokines; Cytotoxicity, Immunologic; Histocompatibility Antigens Class I; Humans; Ionomycin; Killer Cells, Natural; Lymphocyte Activation; Microscopy, Confocal; Protein Kinases; Tetradecanoylphorbol Acetate; Th1 Cells; Th2 Cells

2-Arachidonoyl-glycerol (2-AG), an endogenous ligand for cannabinoid receptor types 1 and 2 (CB1 and CB2), has previously been demonstrated to modulate immune functions including suppression of interleukin-2 expression and nuclear factor of activated T cells (NFAT) activity. The objective of the present studies was to investigate the effect of 2-AG on interferon-gamma (IFN-gamma) expression and associated upstream signaling events. Pretreatment of splenocytes with 2-AG markedly suppressed phorbol 12-myristate 13-acetate plus calcium ionophore (PMA/Io)-induced IFN-gamma secretion. In addition, 2-AG suppressed IFN-gamma steady-state mRNA expression in a concentration-dependent manner. To unequivocally determine the putative involvement of CB1 and CB2, splenocytes derived from CB1(-/-)/CB2(-/-) knockout mice were used. No difference in the magnitude of IFN-gamma suppression by 2-AG in wild-type versus CB1/CB2 null mice was observed. Time-of-addition studies revealed that 2-AG treatment up to 12 h post-cellular activation resulted in suppression of IFN-gamma, which was consistent with a time course conducted with cyclosporin A, an inhibitor of NFAT activity. Coincidentally, 2-AG perturbed the nuclear translocation of NFAT protein and blocked thapsigargin-induced elevation in intracellular calcium, suggesting that altered calcium regulation might partly explain the suppression of NFAT nuclear translocation and subsequent IFN-gamma production. Indeed, Io partially attenuated the 2-AG-induced suppression of PMA/Io-stimulated IFN-gamma production. Taken together, these data demonstrate that 2-AG suppresses IFN-gamma expression in murine splenocytes in a CB receptor-independent manner and that the mechanism partially involves suppression of intracellular calcium signaling and perturbation of NFAT nuclear translocation.

Topics: Animals; Arachidonic Acids; Calcium; Cannabinoid Receptor Modulators; Cell Nucleus; Cytoplasm; DNA-Binding Proteins; Endocannabinoids; Female; Gene Expression; Glycerides; Interferon-gamma; Ionomycin; Mice; Mice, Knockout; NFATC Transcription Factors; Nuclear Proteins; Phorbol Esters; Protein Transport; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Spleen; Tetradecanoylphorbol Acetate; Transcription Factors

Sphingosine-1-phosphate, a lipid mediator produced by sphingosine kinases, regulates diverse cellular processes, ranging from cell growth and survival to effector functions, such as proinflammatory mediator synthesis. Using human A549 epithelial lung carcinoma cells as a model system, we observed transient upregulation of sphingosine kinase type 1 (SPHK1) enzyme activity upon stimulation with both TNF-alpha or IL-1beta. This transient activation of SPHK1 was found to be required for cytokine-induced COX-2 transcription and PGE2 production, since not only specific siRNA (abolishing both basal and induced SPHK1 enzyme activity), but also a dominant-negative SPHK1 mutant (suppressing induced SPHK1 activity only) both reduced COX-2 and PGE2. Furthermore, TNF-alpha- or IL-1beta-induced transcription of selected cytokines, chemokines, and adhesion molecules (IL-6, RANTES, MCP-1, and VCAM-1) was found to require SPHK1 activation. Suppression of SPHK1 activation led to reduction of cytokine-induced IkappaBalpha phosphorylation and consequently diminished NFkappaB activity due to reduced nuclear translocation of RelA (p65), explaining the dependence of inflammatory mediator production on SPHK1 activation. Inhibition of basal SPHK1 activity by N,N-dimethylsphingosine or by downregulation of its expression using siRNA induced spontaneous apoptosis in A549 cells, an effect that can be explained through interference with constitutive NFkappaB activity in this cell type. In contrast, expression of the dominant-negative mutant did not induce apoptosis. Taken together, these findings demonstrate a role of SPHK1 activation in proinflammatory signalling and of SPHK1 basal activity in survival of A549 lung carcinoma cells.

Topics: Apoptosis; Arachidonic Acid; Cell Line, Tumor; Cell Nucleus; Cell Survival; Chemokine CCL2; Chemokine CCL5; Cyclooxygenase 2; Dinoprostone; Enzyme Inhibitors; Gene Expression; Humans; I-kappa B Proteins; Inflammation Mediators; Interleukin-1; Interleukin-6; Ionomycin; Membrane Proteins; NF-kappa B; NF-kappa B p50 Subunit; NF-KappaB Inhibitor alpha; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Prostaglandin-Endoperoxide Synthases; Protein Precursors; RNA, Small Interfering; Signal Transduction; Sphingosine; Tetradecanoylphorbol Acetate; Transcription Factor RelA; Transfection; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1

CD8(hi+) cytotoxic T lymphocytes (CTL) are major players in immune defense. In addition, they contribute to the maintenance of immune homeostasis. We now describe a hitherto unavailable, but simple assay to determine ex vivo lytic granule-based cytotoxic functions of human CD8(hi+) CTL subgroups in a clinical setting, under target cell free conditions. Ficoll-isolated peripheral blood lymphocytes from 17 healthy volunteers were stimulated either by phorbol 12-myristate 13-acetate (PMA) in combination with ionomycin or by antibody mediated crosslinking of the CD3 molecule on the T cell surface. Using perforin as a marker for lytic granules, the reduction of CTL granules over time intervals up to 120 min was quantified by FACScan flow cytometry. The kinetics of perforin reduction were compared to the kinetics of NA-CBZ-L-lysine-thiobenzyl ester hydrochloride (BLT)-esterase release and of CD63 upregulation. The reduction in the perforin(+) portion of CD8(hi+) CTLs was correlated inversely with BLT-esterase release and CD63 upregulation. At 30 and 120 min after PMA/ionomycin stimulation, 55 +/- 14% and 42 +/- 14%, respectively, of CD8(hi+) CTLs still stained perforin(+) (time point 0 min = 100%). Perforin-granule release induced by CD3-crosslinking occurred as fast within 30 min (55 +/- 17%), but over the 120 min time interval it was not as complete when compared to PMA/ionomycin-stimulated perforin-reduction. Thus, the combination of an established degranulation assay with the power of immuno flow cytometry allows one to investigate the cytotoxic capability of CTL-subtypes and the kinetics of perforin-granule release. In addition, the assay may prove useful in the elucidation of intracellular signaling cascades governing the perforin-granule release process.

Topics: Biological Assay; CD3 Complex; Cell Degranulation; Cytoplasmic Granules; Flow Cytometry; Humans; Ionomycin; Lymphocyte Activation; Membrane Glycoproteins; Perforin; Pore Forming Cytotoxic Proteins; Protein Transport; T-Lymphocytes, Cytotoxic; Tetradecanoylphorbol Acetate

The multiple cellular and soluble elements of the immune system respond in a coordinated way, orchestrated by cytokines, to preserve the integrity of the organism. In this study, we describe a new and unique whole blood method that, with minimal sample manipulation, allows an overall evaluation of immune responses by simultaneously measuring cell activation and cytokine secretion. The identification of cells actively secreting cytokines is based on the stabilization of tumor necrosis factor alpha (TNFalpha) at the cell surface through the use of a specific inhibitor of the TNFalpha-converting enzyme. This inhibitor does not affect the release of cytokines other than TNFalpha and makes it possible to assess, in the same measurement, the phenotype of TNFalpha(+)-secreting cells and quantify multiple secreted cytokines by using a specific and highly sensitive flow cytometry-based bead immunoassay. Upon stimulation of normal peripheral blood samples with either phorbol 12-myristate 13 acetate (PMA) plus ionomycin or lipopolysaccharide (LPS), both the number of TNFalpha+ cells and the amount of secreted cytokines progressively increased, the former becoming detectable first. After stimulation for 3 h with PMA plus ionomycin, cellular responses were associated with surface TNFalpha expression on the majority of CD3+ T cells and secretion of Th1-associated cytokines: interferon gamma, interleukin (IL)-2, and to a lesser extent IL4. In turn, stimulation with LPS induced a response mainly by inflammatory cells. After 4 h of LPS-stimulation, the majority of CD14+ monocytes showed surface TNFalpha expression; in parallel, high amounts of soluble IL1beta, IL6, and IL8 became detectable. Likewise, stimulation of blood samples with cytomegalovirus (CMV) lysates induced viral-specific immune responses detectable in seropositive but not seronegative volunteers; such responses were associated with the detection of increased numbers of TNFalpha+ monocytes, TNFalpha+/CD8+ T cells and TNFalpha+/CD8- T lymphocytes in association with an increased secretion of IFNgamma, IL6 and TNFalpha.

Topics: Adult; Cytokines; Flow Cytometry; Humans; Ionomycin; Leukocytes, Mononuclear; Lipopolysaccharides; Lymphocyte Activation; Sensitivity and Specificity; T-Lymphocyte Subsets; Tetradecanoylphorbol Acetate

PBSC transplant provides 10 times more T cells than BMT However, the incidence and severity of acute GvHD is similar among recipients of both types of transplants. Studies in mouse models suggest that the similar clinical outcome in BMT and PBSCT is due to differences in the lymphokine profiles.. PBMC, PBMC from G-CSF mobilized donors (G-PBMC)and BM mononuclear cells (BM-MC) were analyzed by flow cytometry and ELISA to detect gamma-IFN and IL-4 production. Hematoxylin and eosin staining was used to identify morphology and annexin/propidium-iodide was used for apoptosis assays.. We show decreased production of gamma-interferon (85%) and IL-4 (60%) in G-PBMC when compared with either PBMC or BM-MCT cells on ex vivo assays. Surprisingly, 85% of fresh G-PBMC is composed of low-density granulocytes (LDG), which undergo apoptosis after 48 h in culture. At this same time, gamma-IFN production from G-PBMC T cell was reverted. In vitro, G-CSF converts granulocytes into LDGs, able to inhibit T-cell function by H2O2 production, and not through immune-deviation towards a Th2-type phenotype.. We show that the estimated numbers of Th1 and Th2 cells infused in BMT and PBSCT do not differ significantly. These findings are discussed with reference to the relatively low incidence of acute GvHD in PBSCT shown in the literature. We suggest that these results might depend on the high number of granulocytes and progenitors infused. The potential use of granulocytes as immunosupressive short-term therapy is now being investigated by our group using a mouse experimental model.

Topics: Annexin A5; Antigens, CD; Apoptosis; Bone Marrow Cells; Bone Marrow Transplantation; Catalase; CD3 Complex; Cell Count; Flow Cytometry; Granulocyte Colony-Stimulating Factor; Granulocytes; Hematopoietic Stem Cell Mobilization; Hematopoietic Stem Cell Transplantation; Humans; Hydrogen Peroxide; Interferon-gamma; Interleukin-4; Ionomycin; Leukocytes, Mononuclear; Leukosialin; Lymphocyte Culture Test, Mixed; Neutrophil Activation; Peripheral Blood Stem Cell Transplantation; Sialoglycoproteins; T-Lymphocytes; Tetradecanoylphorbol Acetate; Time Factors

Topics: Adult; Fetal Blood; Humans; Interferon-gamma; Interleukin-4; Ionomycin; Lymphocyte Activation; Monensin; Tetradecanoylphorbol Acetate; Th1 Cells; Th2 Cells