Phorbol Myristate Acetate Stimulation Research Articles

Anti-Allergic Activity of a Platycodon Root Ethanol Extract

Platycodon grandiflorum (Campanulaceae) is used as traditional medicine in Asian countries. In Korean traditional medicine, Platycodon root has been widely used since ancient times as a traditional drug to treat cold, cough and asthma. However, its effects on bone marrow-derived mast cell (BMMC)-mediated allergy and inflammation mechanisms remain unknown. In this study, the biological effect of Platycodon root ethanol extract (PE) was evaluated in BMMC after induction of allergic mediators by phorbol 12-myristate 13-acetate (PMA) plus calcium ionophore A23187 (A23187) stimulation. The effect of PE on the production of several allergic mediators, such as interleukin-6 (IL-6), prostaglandin D2 (PGD2), leukotriene C4 (LTC4), β-Hexosaminidase (β-Hex) and cyclooxygenase-2 (COX-2) protein, was investigated. The results demonstrate that PE inhibits PMA + A23187 induced production of IL-6, PGD2, LTC4, β-Hexosaminidase and COX-2 protein. Taken together, these results indicate that PE has the potential for use in the treatment of allergy.

Protective effects of luteolin against lipopolysaccharide-induced acute lung injury involves inhibition of MEK/ERK and PI3K/Akt pathways in neutrophils

To investigate whether luteolin, the major polyphenolic components of Lonicera japonica, has beneficial effects against lipopolysaccharide (LPS)-induced acute lung injury (ALI) and to determine whether the protective mechanism involves anti-inflammatory effects on neutrophils. ALI was induced with intratracheal instillation of LPS in mice. The level of ALI was determined by measuring the cell count and protein content in bronchoalveolar lavage (BAL) fluid. Neutrophils were stimulated with formyl-Met-Leu-Phe (fMLP) or LPS in vitro. Chemotaxis and superoxide anion generation were measured to evaluate neutrophil activation. The potential involvement of intracellular signaling molecules in regulating neutrophil activation was analyzed by using Western blot. LPS induced ALI in mice, as evidenced with leukocyte infiltration and protein leakage into the lungs. Luteolin attenuated LPS-induced leukocyte infiltration and protein extravasation. In cell studies, luteolin attenuated the fMLP-induced neutrophil chemotaxis and respiratory burst (IC(50) 0.2+/-0.1 micromol/L and 2.2+/-0.8 micromol/L, respectively), but had a negligible effect on superoxide anion generation during phorbol myristate acetate stimulation. Furthermore luteolin effectively blocked MAPK/ERK kinase 1/2 (MEK), extracellular signal-regulated kinase (ERK), and Akt phosphorylation in fMLP- and LPS-stimulated neutrophils. These results indicate that luteolin has beneficial effects against LPS-induced ALI in mice, and the attenuation of neutrophil chemotaxis and respiratory burst by luteolin involves the blockade of MEK-, ERK-, and Akt-related signaling cascades.

S-Glutathionylation Regulates Inflammatory Activities of S100A9

Reactive oxygen species generated by activated neutrophils can cause oxidative stress and tissue damage. S100A8 (A8) and S100A9 (A9), abundant in neutrophil cytoplasm, are exquisitely sensitive to oxidation, which may alter their functions. Murine A8 is a neutrophil chemoattractant, but it suppresses leukocyte transmigration in the microcirculation when S-nitrosylated. Glutathione (GSH) modulates intracellular redox, and S-glutathionylation can protect susceptible proteins from oxidative damage and regulate function. We characterized S-glutathionylation of A9; GSSG and GSNO generated S-glutathionylated A8 (A8-SSG) and A9 (A9-SSG) in vitro, whereas only A9-SSG was detected in cytosol of neutrophils activated with phorbol myristate acetate (PMA) but not with fMLP or opsonized zymosan. S-Glutathionylation exposed more hydrophobic regions in Zn(2+)-bound A9 but did not alter Zn(2+) binding affinity. A9-SSG had reduced capacity to form heterocomplexes with A8, but the arachidonic acid binding capacities of A8/A9 and A8/A9-SSG were similar. A9 and A8/A9 bind endothelial cells; S-glutathionylation reduced binding. We found little effect of A9 or A9-SSG on neutrophil CD11b/CD18 expression or neutrophil adhesion to endothelial cells. However, A9, A9-SSG and A8/A9 promoted neutrophil adhesion to fibronectin but, in the presence of A8, A9-mediated adhesion was abrogated by glutathionylation. S-Glutathionylation of A9 may protect its oxidation to higher oligomers and reduce neutrophil binding to the extracellular matrix. This may regulate the magnitude of neutrophil migration in the extravasculature, and together with the functional changes we reported for S-nitrosylated A8, particular oxidative modifications of these proteins may limit tissue damage in acute inflammation.

Transcription of human resistin gene involves an interaction of Sp1 with peroxisome proliferator-activating receptor gamma (PPARgamma).

BackgroundResistin is a cysteine rich protein, mainly expressed and secreted by circulating human mononuclear cells. While several factors responsible for transcription of mouse resistin gene have been identified, not much is known about the factors responsible for the differential expression of human resistin.Methodology/Principal FindingWe show that the minimal promoter of human resistin lies within ∼80 bp sequence upstream of the transcriptional start site (−240) whereas binding sites for cRel, CCAAT enhancer binding protein α (C/EBP-α), activating transcription factor 2 (ATF-2) and activator protein 1 (AP-1) transcription factors, important for induced expression, are present within sequences up to −619. Specificity Protein 1(Sp1) binding site (−276 to −295) is also present and an interaction of Sp1 with peroxisome proliferator activating receptor gamma (PPARγ) is necessary for constitutive expression in U937 cells. Indeed co-immunoprecipitation assay demonstrated a direct physical interaction of Sp1 with PPARγ in whole cell extracts of U937 cells. Phorbol myristate acetate (PMA) upregulated the expression of resistin mRNA in U937 cells by increasing the recruitment of Sp1, ATF-2 and PPARγ on the resistin gene promoter. Furthermore, PMA stimulation of U937 cells resulted in the disruption of Sp1 and PPARγ interaction. Chromatin immunoprecipitation (ChIP) assay confirmed the recruitment of transcription factors phospho ATF-2, Sp1, Sp3, PPARγ, chromatin modifier histone deacetylase 1 (HDAC1) and the acetylated form of histone H3 but not cRel, C/EBP-α and phospho c-Jun during resistin gene transcription.ConclusionOur findings suggest a complex interplay of Sp1 and PPARγ along with other transcription factors that drives the expression of resistin in human monocytic U937 cells.

Principles and problems of the electrophoretic mobility shift assay

Introduction The electrophoretic mobility shift assay (EMSA) is classically used to detect DNA binding proteins, the tenet of the EMSA is that DNA with protein bound, migrates through a polyacrylamide gel more slowly than the corresponding free unbound DNA. Methods The classical EMSA protocol has 4 major steps: 1) The isolation of proteins from cells. Since the vast majority of active DNA binding proteins are present within the nucleus, a sequential membrane lysis protocol is used which yields purified nuclear protein. 2) Manufacture and radiolabelling of the DNA probe. Phosphorous 32 ( 32P) is attached to the 5′ ends of the DNA probe through use of 32P-γATP as a substrate for T4 polynucleotide kinase. DNA probes can both be purchased or custom made. 3) Purified proteins and radiolabelled DNA probes are co-incubated with an EMSA binding buffer to promote binding of the proteins with the DNA probe. If a supershift EMSA is being carried out, the reaction also contains a selective antibody which when bound to the protein–DNA complexes, causes further retardation within the gel. 4) The DNA–protein complexes are loaded and run on a non-denaturing polyacrylamide gel causing separation of the DNA–protein complexes from the free DNA probes. The polyacrylamide gels are then dried down and analysed via autoradiography. Results As a demonstration of the effectiveness of this protocol, we show that tumour necrosis factor (TNF)α and phorbol 12-myristate 13-acetate (PMA) stimulation of A549 cells, results in a number of DNA–protein complexes being induced when compared to untreated cells. We also demonstrate that these complexes contain the p50 and p65 subunits of NF-κB through utilisation of the EMSA supershift protocol. Discussion We provide detailed troubleshooting hints and tips for this technique and discuss the limitations of the EMSA, as well as a number of EMSA variants and alternative techniques.

Molecular cloning and characterization of interferon regulatory factors 4 and 8 (IRF-4 and IRF-8) in rainbow trout, Oncorhynchus mykiss

Mammalian interferon regulatory factor (IRF)4 (PIP, LSIRF, and ICSAT) and IRF8 (ICSBP) are known to be critical in regulating a spectrum of functional and developmental processes in lymphomyeloid cell lineages either through direct binding to IRF-E motifs in target gene promoters or indirectly by binding to composite motifs recognized by Ets family members, PU.I and Sp.I. Here we report, for the first time in fish, the sequencing and characterization of full-length cDNA homologues of rainbow trout (rt) IRF4 and rtIRF8. The rtIRF4 molecule consists of 1848 bp with a 45 bp 5′ UTR and a predicted 378 bp 3′ UTR translating into a 474 aa protein. RtIRF8 consists of 1951 bp with a 52 bp 5′ UTR and a 564 bp 3′ UTR translating into a 444 aa protein. Each gene possesses a putative DNA binding domain (DBD) containing the tryptophan pentad-repeat domain found in all IRF family members. Both molecules also possess a well conserved IRF association domain (IAD). The presence of these domains along with phylogenetic analysis places the two genes in the IRF4 subfamily. Both genes were detected in a range of trout tissues where IRF8 was the overall predominant transcript. Consistent with mammalian studies, the highest expression levels of IRF4 and IRF8 were observed in the lymphomyeloid-rich fish tissues, spleen, head kidney and gills. IRF8 expression in stimulated trout splenocytes was significantly up-regulated by polyinosinic:polycytidylic acid (poly I:C), trout recombinant (r)IL-15, phorbol 12-myristate 13-acetate (PMA), and phytohaemagglutinin (PHA) treatment whilst remaining refractory towards lipopolysaccharide (LPS) treatment. IRF4 was significantly down-regulated by LPS stimulation and remained refractory towards poly I:C, trout rIL15, and PHA. PMA stimulation elicited a significant upregulation of IRF4 expression. Overall, these data support the premise that these IRFs are likely to play important roles in the functional and developmental processes occurring in fish lymphomyeloid tissues.

Increased generation of reactive oxygen species in pulmonary arterial smooth muscle cells from monocrotaline-treated rats

Rationale: Chronic alveolar hypoxia induces pulmonary hypertension (PH), concomitant with pulmonary vascular remodeling. Reactive oxygen species (ROS) are thought to play a major role in this process. Our recent findings suggest that ROS production by the NADPH-oxidase (NOX) subunits play an important role in this remodeling process. Objectives: We investigated whether similar mechanisms are active for non-hypoxia induced PH, namely monocrotaline (MCT) induced PH in rats. The aim was to examine ROS production and their possible source. Further we adressed the expression of NOX subunits, antioxidative enzymes SOD1, SOD2, and catalase in pulmonary arterial smooth muscle cells (PASMC), isolated from MCT-treated rats. Methods/Results: Comparing PASMC from MCT-treated and non-treated rats by real-time PCR analysis revealed an upregulation of NOX1 and p22phox (n=6). Upregulation of NOX1 was confirmed by western blot. RT-PCR and immunofluorescence showed an increased SOD2 expression after MCT treatment. ROS measurements using ESR spectroscopy showed an increased intracellular production of ROS in total, but also especially superoxide in PASMC after MCT treatment in comparison to healthy controls. Stimulation with the NOX activator phorbol myristate acetate (PMA) induced an additional ROS increase. Results were validated by dihydroethidium staining. Conclusion: Our results show an increased ROS production in PASMC of MCT-treated rats which may contribute to vascular remodeling. Data from 1) enhanced ROS production upon PMA stimulation, 2) increased expression of NOX1 and 3) the increased SOD2 expression suggests a contribution of both, mitochondria and NOX1 as possible sources of ROS release in MCT treated rats.

Electrochemical monitoring of hydrogen peroxide released from leucocytes on horseradish peroxidase redox polymer coated electrode chip

We developed an electrochemical-sensing device for continuous monitoring extracellular hydrogen peroxide (H2O2). The device consists of an indium-tin-oxide electrode coated with osmium-polyvinylpyridine gel polymer containing horseradish peroxidase (Os-HRP) and a poly-dimethyl siloxane well to house the cells on the chip. Granulocyte-like differentiated HL-60 cells were accommodated in the well and stimulated with phorbol 12-myristate 13-acetate (PMA), which triggered the generation of H2O2. The extracellular H2O2 released from the cells was enzymatically reduced at the Os-HRP-modified electrode chip using Os(II) as an electron donor, resulting in reduction current responses by the device. The reduction current increased immediately upon PMA stimulation and this current transient was similar to that obtained by conventional chemiluminescence assays using sodium luminol. Apocynin, an inhibitor of NADPH oxidase activation, eliminated both the electrochemical and chemiluminescence signals. On the other hand, superoxide dismutase (SOD) increased the amperometric signals and catalase (CAT) decreased, whereas SOD decreased luminescence emission and CAT did not. These results were in accordance with the expected reaction mechanism, and strongly indicate that this new electrochemical-sensing device successfully detects extracellular H2O2 production.

Enhancement of glucagon secretion in mouse and human pancreatic alpha cells by protein kinase C (PKC) involves intracellular trafficking of PKCα and PKCδ

Protein kinase C (PKC) regulates exocytosis in various secretory cells. Here we studied intracellular translocation of the PKC isoenzymes PKCalpha and PKCdelta, and investigated how activation of PKC influences glucagon secretion in mouse and human pancreatic alpha cells. Glucagon release from intact islets was measured in static incubations, and the amounts released were determined by RIA. Exocytosis was monitored as increases in membrane capacitance using the patch-clamp technique. The expression of genes encoding PKC isoforms was analysed by real-time PCR. Intracellular PKC distribution was assessed by confocal microscopy. The PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated glucagon secretion from mouse and human islets about fivefold (p < 0.01). This stimulation was abolished by the PKC inhibitor bisindolylmaleimide (BIM). Whereas PMA potentiated exocytosis more than threefold (p < 0.001), BIM inhibited alpha cell exocytosis by 60% (p < 0.05). In mouse islets, the PKC isoenzymes, PKCalpha and PKCbeta1, were highly abundant, while in human islets PKCeta, PKCepsilon and PKCzeta were the dominant variants. PMA stimulation of human alpha cells correlated with the translocation of PKCalpha and PKCdelta from the cytosol to the cell periphery. In the mouse alpha cells, PKCdelta was similarly affected by PMA, whereas PKCalpha was already present at the cell membrane in the absence of PMA. This association of PKCalpha in alpha cells was principally dependent on Ca(2+) influx through the L-type Ca(2+) channel. PKC activation augments glucagon secretion in mouse and human alpha cells. This effect involves translocation of PKCalpha and PKCdelta to the plasma membrane, culminating in increased Ca(2+)-dependent exocytosis. In addition, we demonstrated that PKCalpha translocation and exocytosis exhibit differential Ca(2+) channel dependence.

Expression and release of angiopoietin-1 from human neutrophils: Intracellular mechanisms

We recently demonstrated that Tie2 receptor activation on human neutrophils by both angiopoietins (Ang1 and Ang2) promoted platelet-activating factor synthesis, β2-integrin activation, and cell migration. Herein, we wanted to assess if human neutrophils express angiopoietins and further delineate their mechanisms of release. Employing Reverse transcriptase-polymerase chain reaction, Real time quantitative transcriptase-polymerase chain reaction, FACScan analysis and ELISA approaches, we observed that neutrophils express Ang1 but not Ang2. For each condition, vascular endothelial growth factor (VEGF) detection was performed as positive control. Using nitrogen cavitation, we observed that Ang1 is localized in the cytosolic fraction whereas VEGF is found in β-granules. Treatment of neutrophils with phorbol myristate acetate (PMA), N-Formyl-Met-Leu-Phe (fMLP) and tumor necrosis factor-α (TNF-α) induced VEGF release. Maximal effect was observed with PMA (80 nM) stimulation inducing a complete release of VEGF content (565 ± 100 pg/ml; 6 × 106 neutrophils), corresponding to a 18.9-fold increase as compared to phosphate buffer saline (PBS) treated neutrophils. By contrast, only a treatment with PMA (80 nM) induced Ang1 release. PMA treatment induced also a complete release of Ang1 (661 ± 148 pg/ml; 6 × 106 neutrophils), corresponding to 2.8-fold increase as compared to PBS-treated neutrophils. In both cases, PMA-mediated release of VEGF and Ang1 was nearly maximal by 15 min. Finally, we observed that the induction of Ang1 release was calcium-independent whereas VEGF release was not. These data demonstrate the capacity of human neutrophils to synthesize Ang1, which is stored and released differently as compared to VEGF. These data suggest a different cascade of events regarding the distribution of selected growth factors during inflammation and angiogenesis.

Different NK cell–activating receptors preferentially recruit Rab27a or Munc13-4 to perforin-containing granules for cytotoxicity

AbstractThe autosomal recessive immunodeficiencies Griscelli syndrome type 2 (GS2) and familial hemophagocytic lymphohistiocytosis type 3 (FHL3) are associated with loss-of-function mutations in RAB27A (encoding Rab27a) and UNC13D (encoding Munc13-4). Munc13-4 deficiency abrogates NK-cell release of perforin-containing lytic granules induced by signals for natural and antibody-dependent cellular cytotoxicity. We demonstrate here that these signals fail to induce degranulation in resting NK cells from Rab27a-deficient patients. In resting NK cells from healthy subjects, endogenous Rab27a and Munc13-4 do not colocalize extensively with perforin. However, phorbol 12-myristate 13-acetate and ionomycin stimulation or conjugation to susceptible target cells induced myosin-dependent colocalization of Rab27a and Munc13-4 with perforin. Unexpectedly, individual engagement of receptors leukocyte functional antigen-1, NKG2D, or 2B4 induced colocalization of Rab27a, but not Munc13-4, with perforin. Conversely, engagement of antibody-dependent cellular cytotoxicity receptor CD16 induced colocalization of Munc13-4, but not Rab27a, with perforin. Furthermore, colocalization of Munc13-4 with perforin was Rab27a-dependent. In conclusion, Rab27a or Munc13-4 recruitment to lytic granules is preferentially regulated by different receptor signals, demonstrating that individual target cell ligands regulate discrete molecular events for lytic granule maturation. The data suggest Rab27a facilitates degranulation at an early step yet highlight a reciprocal relationship between Munc13-4 and Rab27a for degranulation.

Identity of the elusive IgM Fc receptor (FcμR) in humans

Although Fc receptors (FcRs) for switched immunoglobulin (Ig) isotypes have been extensively characterized, FcR for IgM (FcμR) has defied identification. By retroviral expression and functional cloning, we have identified a complementary DNA (cDNA) encoding a bona fide FcμR in human B-lineage cDNA libraries. FcμR is defined as a transmembrane sialoglycoprotein of ∼60 kD, which contains an extracellular Ig-like domain homologous to two other IgM-binding receptors (polymeric Ig receptor and Fcα/μR) but exhibits an exclusive Fcμ-binding specificity. The cytoplasmic tail of FcμR contains conserved Ser and Tyr residues, but none of the Tyr residues match the immunoreceptor tyrosine-based activation, inhibitory, or switch motifs. Unlike other FcRs, the major cell types expressing FcμR are adaptive immune cells, including B and T lymphocytes. After antigen-receptor ligation or phorbol myristate acetate stimulation, FcμR expression was up-regulated on B cells but was down-modulated on T cells, suggesting differential regulation of FcμR expression during B and T cell activation. Although this receptor was initially designated as Fas apoptotic inhibitory molecule 3, or TOSO, our results indicate that FcμR per se has no inhibitory activity in Fas-mediated apoptosis and that such inhibition is only achieved when anti-Fas antibody of an IgM but not IgG isotype is used for inducing apoptosis.

Assessment of the health and antioxidant trade-off in gilthead sea bream ( Sparus aurata L.) fed alternative diets with low levels of contaminants

The aim of the present work was to analyze the effect of partial and total replacement of fish oil (FO) by a blend of vegetable oils on the health and antioxidant status of gilthead sea bream ( Sparus aurata L.) fed primarily plant protein-based diets. The study included measurements of feed-borne contaminants, gene expression analyses of detoxifying and antioxidant pathways and measures of antioxidant and innate immune descriptors. Polybrominated diphenyl ethers (PBDEs) were almost undetectable in all diets, and the loading-charges of polychlorinated biphenyls (PCBs), dioxin-like PCBs, organochlorine pesticides (OCs), and polycyclic aromatic hydrocarbons (PAHs) were at trace levels decreasing their concentrations according to the level of FO replacement with vegetable oils (0%, 33%, 66%, and 100%). Hepatic detoxifying pathways were down regulated by FO replacement, and the hepatic transcription of cytochrome P450 1A1 and aryl hydrocarbon receptor 1 was significantly reduced in fish fed the 100% vegetable oil diet. Dietary intervention did not alter the hepatic expression of the recycling glutathione reductase, whereas glutathione peroxidase-1 and phospholipid glutathione peroxidase were either down- or up-regulated by total FO replacement. This suggests that vegetable oils prime the in situ repair of peroxidized phospholipids rather than the increased turnover of membrane phospholipids from the undamaged pool of cytosolic free fatty acids. The hepatic expression of non-enzymatic antioxidants (metallothionein, glucose regulated protein 75) was down regulated in fish fed 66% and 100% vegetable oil diets. Hepatic glutathione levels and total plasma antioxidant capacity were also lowest in fish fed high levels of vegetable oils, but the concurrent increase in the GSH/GSSG ratio was interpreted as an index of reduced oxidative stress. This redox balance agrees with the enhanced respiratory burst of blood leucocytes after phorbol myristate acetate stimulation in fish feed the 100% vegetable oil. Total plasma peroxidases and plasma alternative complement pathway were not affected by dietary treatment, whereas plasma lysozyme was significantly decreased in fish fed the 66% vegetable oil diet. Taken together, the results suggest that the health and the antioxidant status of gilthead sea bream was not damaged by high levels of FO replacement in eco-friendly diets, but both the scavenging and production of reactive oxygen species were modulated in concert by complex and nutritionally mediated readjustments.

Calcium homeostasis and its relationship to superoxide production in blood and milk neutrophils of lactating goats

Polymorphonuclear neutrophils (PMN), which comprise over 70% of the somatic cells in goat milk, are a major cellular component of innate immunity in the goat mammary gland. However, the function of milk PMNs is modified after diapedesis compared to PMNs in blood. As many aspects of PMN activity depend directly on intracellular Ca 2+ concentration ((Ca 2+) i), the present study aimed to determine the changes in Ca 2+ homeostasis of milk PMNs from lactating goats compared to autologous blood PMNs, and to examine the significance of these variations to the immuno-competency of milk PMNs. The intracellular Ca 2+ store of freshly prepared milk cells was estimated from the elevation of (Ca 2+) i after ionomycin treatment, which was found to be significantly less than blood PMNs. Replenishment of the intracellular Ca 2+ store in milk cells after intracellular Ca 2+ depletion by Bapta-AM followed by spiking with 2.5 mM Ca 2+ for 20 min was also compared to that of blood PMNs, showing that after depletion/spiking the intracellular Ca 2+ store in milk cells was much less than blood PMNs. The production of superoxide anion (O 2 −) in vitro in response to (Ca 2+) i-dependent or (Ca 2+) i-independent modulators was used to evaluate the relevance of altered Ca 2+ homeostasis on the immuno-competency of milk cells compared to blood PMNs. The results indicated that milk cells produced similarly low levels of O 2 − as blood PMNs when treated with ionomycin. However, the amount of O 2 − produced by milk cells in response to phorbol 12-myristate 13-acetate (PMA) stimulation, although greater than ionomycin treatment, was significantly less than that of blood PMNs. The capacity for O 2 − production by both cell types in response to PMA reverted to the resting state with use of the protein kinase C (PKC) inhibitor, staurosporine. In conclusion, the current study demonstrated an irreversible shortage of intracellular Ca 2+ in the milk PMNs of lactating goats compared to blood PMNs. It also showed that preliminary O 2 –production, primed by ionomycin treatment, remained unchanged in milk PMNs, despite the shortage in intracellular Ca 2+, but decreased O 2 − production capacity, mediated via the PKC pathway, in milk PMN. It is suggested that the defects in Ca 2+ homeostasis in milk PMNs of lactating goats is partially attributable for the post-diapedesis functionality modifications.

Regulatory cells, cytokine pattern and clinical risk factors for asthma in infants and young children with recurrent wheeze

Several risk factors for asthma have been identified in infants and young children with recurrent wheeze. However, published literature has reported contradictory findings regarding the underlying immunological mechanisms. This study was designed to assess and compare the immunological status during the first 2 years in steroid-naive young children with >or= three episodes of physician-confirmed wheeze (n=50), with and without clinical risk factors for developing subsequent asthma (i.e. parental asthma or a personal history of eczema and/or two of the following: wheezing without colds, a personal history of allergic rhinitis and peripheral blood eosinophilia >4%), with age-matched healthy controls (n=30). Peripheral blood CD4(+)CD25(+) and CD4(+)CD25(high) T cells and their cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), GITR and Foxp3 expression were analysed by flow cytometry. Cytokine (IFN-gamma, TGF-beta and IL-10), CTLA-4 and Foxp3 mRNA expression were evaluated (real-time PCR) after peripheral blood mononuclear cell stimulation with phorbol 12-myristate 13-acetate (PMA) (24 h) and house dust mite (HDM) extracts (7th day). Flow cytometry results showed a significant reduction in the absolute number of CD4(+)CD25(high) and the absolute and percentage numbers of CD4(+)CD25(+)CTLA-4(+) in wheezy children compared with healthy controls. Wheezy children at a high risk of developing asthma had a significantly lower absolute number of CD4(+)CD25(+) (P=0.01) and CD4(+)CD25(high) (P=0.04), compared with those at a low risk. After PMA stimulation, CTLA-4 (P=0.03) and Foxp3 (P=0.02) expression was diminished in wheezy children compared with the healthy children. After HDM stimulation, CTLA-4 (P=0.03) and IFN-gamma (P=0.04) expression was diminished in wheezy children compared with healthy children. High-risk children had lower expression of IFN-gamma (P=0.03) compared with low-risk and healthy children and lower expression of CTLA-4 (P=0.01) compared with healthy children. Although our findings suggest that some immunological parameters are impaired in children with recurrent wheeze, particularly with a high risk for asthma, further studies are needed in order to assess their potential as surrogate predictor factors for asthma in early life.

The Expression of Proinflammatory Cytokines and Intracellular Minerals in Patients with Ankylosing Spondylitis

Objective: Our study aimed to investigate the expression of intracellular proinflammatory cytokines and mineral contents in patients with ankylosing spondylitis (AS). Methods: Serum samples from 40 patients with AS and 19 healthy individuals were collected for this study. Flow cytometry was used to determine the intracellular concentration of interleukin (IL)-1β, interferon (IFN)-γ and tumor necrosis factor (TNF)-α in the CD3–, CD4–, and CD8– bearing T cells. The mass fraction of calcium (Ca(superscript 2+)) and magnesium (Mg(superscript 2+)) in phytohemagglutin (PHA)-stimulated lymphocytes were measured using a spectrofluorometer. Clinical variables, including erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), Bath ankylosing spondylitis disease activity index (BASDAI), Bath ankylosing spondylitis functional index (BASFI), age at onset, uveitis and family history were recorded. Results: Intracellular proinflammatory cytokine (IL-1β, TNF-α, & IFN-γ) expression in CD3+ and CD4+ T cells after phorbol myristate acetate (PMA) stimulation was more significantly increased in AS patients than in healthy controls (p＜0.05). Intracellular Ca(superscript 2+) exhibited a significant correlation to the TNF-α level in stimulated CD8+ T cells in AS patients (r=0.439, p＜0.05), but not in healthy controls. Comparison between an increase in cytokine expression and clinical variables showed that the increase in IFN-γ was significantly less prominent in patients with a lower BASFI score (BASFI＜20 vs. ≧20, p＜0.05) and more prominent in patients with uveitis. The IL-1β increase after stimulation was less prominent in patients with a higher age of onset (＞30 years of age, p＜0.05). Conclusion: The increased expression of intracellular proinflammatory cytokines, including IL-1β, TNF-α and IFN-γ after PMA-stimulation was more pronounced in AS patients than in normal individuals. A significant correlation between intracellular TNF-α level and Ca(superscript 2+) was found in AS patients. Whether the elevation of intracellular Ca(superscript 2+) can enhance intracellular TNF-α expression in AS patients requires further study.

Identification of a minimal cis-element and cognate trans-factor(s) required for induction of Rac2 gene expression during K562 cell differentiation

Rac2 is a Rho family GTPase that is widely expressed in hematopoietic cells and plays a critical role in host defense. This study investigates the mechanisms responsible for increased Rac2 gene expression during myeloid cell differentiation. Treatment of K562 chronic myelogenous leukemia cells with phorbol-12-myristate-13-acetate (PMA) induces megakaryocytic differentiation and Rac2 gene transcription following a lag of 6–12 h. Promoter/luciferase reporter gene assays reveal that a 135 bp cis-element located between − 4223 and − 4008 bp upstream of the Rac2 transcription start site is necessary and sufficient for PMA-induced gene expression. The AP1 transcription factor binds to three cis-elements within the 135 bp Rac2 gene regulatory region both in vitro and in vivo following PMA treatment, and mutagenesis of the AP1 binding sites ablates the PMA responsiveness of the 135 bp Rac2 gene regulatory region. Over-expression of AP1 is sufficient to induce expression of a transiently transfected Rac2 promoter/luciferase plasmid, but not the endogenous Rac2 gene. Induction of AP1 in vitro DNA-binding activity is apparent within 1 h of PMA stimulation. However, AP1 binding to the endogenous Rac2 promoter exhibits a lag of 5–9 h, which correlates with reduced histone H3-Lys9 methylation, increased histone H3 acetylation, and increased nuclease accessibility within the 135 bp Rac2 gene regulatory region. These results demonstrate that PMA induction of Rac2 expression during terminal myeloid differentiation requires the coordinate induction of transcription factors and remodeling of Rac2 gene chromatin structure.

Activation of equine neutrophils by phorbol myristate acetate or N-formyl-methionyl-leucyl-phenylalanine induces a different response in reactive oxygen species production and release of active myeloperoxidase

Neutrophil (PMN) contribution to the acute inflammatory processes may lead to an excessive generation of reactive oxygen metabolites species (ROS) and secretion of granule enzymes. We compared the effects of either phorbol myristate acetate (PMA) or N-formyl-methionyl-leucyl-phenylalanine (fMLP) in combination with a pre-treatment by cytochalasin B (CB) on the production of ROS and the release of total and active myeloperoxidase (MPO) by isolated equine PMNs. The ROS production was assessed by lucigenin dependent chemiluminescence (CL) and ethylene release by α-keto-γ-methylthiobutyric acid (KMB) oxidation. In the supernatant of activated PMNs, total equine MPO was measured by ELISA and active MPO by the SIEFED (Specific Immunologic Extraction Followed by Enzymatic Detection) technique that allows for the study of the interaction of a compound directly with the enzyme. The stimulation of PMNs with CB-fMLP only modestly increased the release of MPO, but more than 70% of released MPO was active. PMA stimulation markedly increased the production of ROS and release of MPO, but more than 95% of released MPO was inactive. When PMNs were pre-incubated with superoxide dismutase (SOD) prior to PMA activation, the lucigenin enhanced CL, which is linked to the superoxide anion ( O 2 −) production, was much more decreased than KMB oxidation, linked to the hydroxyl-like radical production. The addition of SOD prior to the activation of PMNs by PMA also limited the loss of the activity of released MPO. These results confirm the key role of O 2 − generation in the ROS cascade in PMN and reveal its critical role on MPO inactivation.

Using BioTek's Synergy™ HT Reader to Measure Reactive Oxygen Species (ROS) Generation in Stimulated Cells

The investigation of biological agents that can stimulate or inhibit cell growth is of great interest to cellular biologists. One such measure is the formation of Reactive Oxygen Species (ROS) as a result of cell stimulation. While there are numerous methodologies to assess ROS species, the use of fluorescent probes has become the preferred method. Here we describe the measurement of DCF fluorescence using a SynergyTM HT to detect and analyze the production of ROS as a result of cell growth stimulation.

Identification of an Autoinhibitory Mechanism That Restricts C1 Domain-mediated Activation of the Rac-GAP α2-Chimaerin

Chimaerins are a family of GTPase activating proteins (GAPs) for the small G-protein Rac that have gained recent attention due to their important roles in development, cancer, neuritogenesis, and T-cell function. Like protein kinase C isozymes, chimaerins possess a C1 domain capable of binding phorbol esters and the lipid second messenger diacylglycerol (DAG) in vitro. Here we identified an autoinhibitory mechanism in alpha2-chimaerin that restricts access of phorbol esters and DAG, thereby limiting its activation. Although phorbol 12-myristate 13-acetate (PMA) caused limited translocation of wild-type alpha2-chimaerin to the plasma membrane, deletion of either N- or C-terminal regions greatly sensitize alpha2-chimaerin for intracellular redistribution and activation. Based on modeling analysis that revealed an occlusion of the ligand binding site in the alpha2-chimaerin C1 domain, we identified key amino acids that stabilize the inactive conformation. Mutation of these sites renders alpha2-chimaerin hypersensitive to C1 ligands, as reflected by its enhanced ability to translocate in response to PMA and to inhibit Rac activity and cell migration. Notably, in contrast to PMA, epidermal growth factor promotes full translocation of alpha2-chimaerin in a phospholipase C-dependent manner, but not of a C1 domain mutant with reduced affinity for DAG (P216A-alpha2-chimaerin). Therefore, DAG generation and binding to the C1 domain are required but not sufficient for epidermal growth factor-induced alpha2-chimaerin membrane association. Our studies suggest a role for DAG in anchoring rather than activation of alpha2-chimaerin. Like other DAG/phorbol ester receptors, including protein kinase C isozymes, alpha2-chimaerin is subject to autoinhibition by intramolecular contacts, suggesting a highly regulated mechanism for the activation of this Rac-GAP.