Journal: Critical Care

Article Title: Toll-like receptor 4 in phagocytosis of Escherichia coli by endotoxin-activated human neutrophils in whole blood

doi: 10.1186/cc11767

Figure Lengend Snippet: Influence of antibodies against TLR4, CD14 and CD11b on neutrophil phagocytic activity in whole blood . Control cells without any incentives (C), cells activated by S-LPS (C1) and cells activated by Re-LPS (C2).

Article Snippet: Fluorescein-labeled bioparticles E. coli K12 (Molecular Probes), mouse anti-human TLR4 mAbs HTA125 (IgG2a isotype; Serotec), anti-human CD14 clone UCHM-1 mAbs (IgG2a isotype; Sigma), and anti-human CD11b mAbs clone 44 (IgG1 isotype; Sigma).

Techniques: Activity Assay

Journal: British Journal of Pharmacology

Article Title: The human G protein‐coupled ATP receptor P2Y 11 is a target for anti‐inflammatory strategies

doi: 10.1111/bph.15379

Figure Lengend Snippet: Activation of native P2Y 11 receptors enhances IL‐1 receptor signalling. (a) Phase contrast microscopy of Day‐6 M2 macrophages (magnification: 40×). Primary human monocytes were obtained from peripheral blood mononuclear cells (PBMCs) by magnetic‐activated cell sorting (MACS) using CD14 microbeads. M2 macrophages were differentiated by culturing the isolated monocytes with M‐CSF (50 ng·ml −1 ) for 6 days. (b) The phenotype of differentiated M2 macrophages was determined by flow cytometry and CD163 served as an M2 marker. (c,d) M2 macrophages were treated for 24 h with increasing doses of IL‐1α (c) or IL‐1ß (d) either alone or in combination with P2Y 11 receptor agonist (ATPγS). The antagonist NF340 (10 μM) was used to confirm that agonist‐mediated responses were specific to P2Y 11 receptor stimulation. IL‐6 and IL‐8 were measured in cell culture supernatants. Data are means ± SEM from five independent donors. * P ≤ .05, significantly different as indicated; one‐way ANOVA

Article Snippet: The following antibodies were used for macrophage phenotyping: rabbit polyclonal IgG anti‐human P2Y 11 receptor (Bioss/THP, bs‐12071R‐A488, RRID:AB_2857964), rabbit polyclonal anti‐human P2X1 receptor (Bioss/THP, bs‐12107R‐A647, custom‐conjugation), mouse monoclonal IgG2b anti‐human CD14 (BD Bioscience, clone MφP9, 345,787‐APC, RRID:AB_400509), mouse monoclonal IgG1k anti‐human CD163 (BD Bioscience, clone GHI/61, 556,018‐PE, RRID:AB_2033943), mouse monoclonal IgG2a anti‐human CD284 (TLR4) (BioLegend, clone HTA125, 312,816‐APC, RRID:AB_2562487), goat polyclonal IgG anti‐human IL‐1R1 (R&D Systems/Bio‐techne, FAB269P‐PE, RRID:AB_2124912); mouse monoclonal IgG1 anti‐human P2Y 11 receptor (R&D, clone # 505214‐A647, RRID:AB_2857965) was used to detect human P2Y 11 receptor on glioma cells after gentle cell detachment using Accutase (Sigma‐Aldrich).

Techniques: Activation Assay, Microscopy, FACS, Isolation, Flow Cytometry, Marker, Cell Culture

Journal: British Journal of Pharmacology

Article Title: The human G protein‐coupled ATP receptor P2Y 11 is a target for anti‐inflammatory strategies

doi: 10.1111/bph.15379

Figure Lengend Snippet: Activation of native P2Y 11 receptors enhances IL‐1 receptor signalling. (a) Phase contrast microscopy of Day‐6 M2 macrophages (magnification: 40×). Primary human monocytes were obtained from peripheral blood mononuclear cells (PBMCs) by magnetic‐activated cell sorting (MACS) using CD14 microbeads. M2 macrophages were differentiated by culturing the isolated monocytes with M‐CSF (50 ng·ml −1 ) for 6 days. (b) The phenotype of differentiated M2 macrophages was determined by flow cytometry and CD163 served as an M2 marker. (c,d) M2 macrophages were treated for 24 h with increasing doses of IL‐1α (c) or IL‐1ß (d) either alone or in combination with P2Y 11 receptor agonist (ATPγS). The antagonist NF340 (10 μM) was used to confirm that agonist‐mediated responses were specific to P2Y 11 receptor stimulation. IL‐6 and IL‐8 were measured in cell culture supernatants. Data are means ± SEM from five independent donors. * P ≤ .05, significantly different as indicated; one‐way ANOVA

Article Snippet: The following antibodies were used for macrophage phenotyping: rabbit polyclonal IgG anti‐human P2Y 11 receptor (Bioss/THP, bs‐12071R‐A488, RRID:AB_2857964), rabbit polyclonal anti‐human P2X1 receptor (Bioss/THP, bs‐12107R‐A647, custom‐conjugation), mouse monoclonal IgG2b anti‐human CD14 (BD Bioscience, clone MφP9, 345,787‐APC, RRID:AB_400509), mouse monoclonal IgG1k anti‐human CD163 (BD Bioscience, clone GHI/61, 556,018‐PE, RRID:AB_2033943), mouse monoclonal IgG2a anti‐human CD284 (TLR4) (BioLegend, clone HTA125, 312,816‐APC, RRID:AB_2562487), goat polyclonal IgG anti‐human IL‐1R1 (R&D Systems/Bio‐techne, FAB269P‐PE, RRID:AB_2124912); mouse monoclonal IgG1 anti‐human P2Y 11 receptor (R&D, clone # 505214‐A647, RRID:AB_2857965) was used to detect human P2Y 11 receptor on glioma cells after gentle cell detachment using Accutase (Sigma‐Aldrich).

Techniques: Activation Assay, Microscopy, FACS, Isolation, Flow Cytometry, Marker, Cell Culture

Journal: British Journal of Pharmacology

Article Title: The human G protein‐coupled ATP receptor P2Y 11 is a target for anti‐inflammatory strategies

doi: 10.1111/bph.15379

Figure Lengend Snippet: Ectopic P2Y 11 receptor signalling activates genes related to IL‐1 receptor /TLR4 pathways. (a) Surface expression of P2Y 11 receptors was measured by flow cytometry in the recombinant cell line (P2YR11) and in the P2RY11 knockout control (P2RY11‐KO). Numbers are mean fluorescence intensities (MFIs) of P2Y 11 staining after subtraction of isotype control MFIs. P2RY11‐transfected cells and P2RY11 knockout cells (P2RY11‐KO) were treated for 24 h with ATPγS at 10 μM. IL‐6 and IL‐8 were measured in cell culture supernatants. The antagonist NF340 was used at 10 μM to confirm that agonist‐mediated responses were specific to stimulation of P2Y 11 receptors ( n = 5). * P < .05 significantly different as indicated; one‐way ANOVA. (b) Transcriptional reprogramming in response to P2Y 11 receptor activation was examined using NanoString analysis. P2RY11‐transfected cells and P2RY11‐KO cells were cultured for 6 h and 24 h in the presence of the P2Y 11 receptor agonist ATPγS at 10 μM (three replicates for each condition). Fold change indicates the averaged ratio of expression in agonist‐treated P2RY11 cells to that in agonist‐treated P2RY11‐KO cells (agonist: P2RY11/P2RY11‐KO). P values shown were derived from one‐way ANOVA. n.d., not detectable; n.a., not applicable

Article Snippet: The following antibodies were used for macrophage phenotyping: rabbit polyclonal IgG anti‐human P2Y 11 receptor (Bioss/THP, bs‐12071R‐A488, RRID:AB_2857964), rabbit polyclonal anti‐human P2X1 receptor (Bioss/THP, bs‐12107R‐A647, custom‐conjugation), mouse monoclonal IgG2b anti‐human CD14 (BD Bioscience, clone MφP9, 345,787‐APC, RRID:AB_400509), mouse monoclonal IgG1k anti‐human CD163 (BD Bioscience, clone GHI/61, 556,018‐PE, RRID:AB_2033943), mouse monoclonal IgG2a anti‐human CD284 (TLR4) (BioLegend, clone HTA125, 312,816‐APC, RRID:AB_2562487), goat polyclonal IgG anti‐human IL‐1R1 (R&D Systems/Bio‐techne, FAB269P‐PE, RRID:AB_2124912); mouse monoclonal IgG1 anti‐human P2Y 11 receptor (R&D, clone # 505214‐A647, RRID:AB_2857965) was used to detect human P2Y 11 receptor on glioma cells after gentle cell detachment using Accutase (Sigma‐Aldrich).

Techniques: Expressing, Flow Cytometry, Recombinant, Knock-Out, Fluorescence, Staining, Transfection, Cell Culture, Activation Assay, Derivative Assay

Journal: British Journal of Pharmacology

Article Title: The human G protein‐coupled ATP receptor P2Y 11 is a target for anti‐inflammatory strategies

doi: 10.1111/bph.15379

Figure Lengend Snippet: P2Y 11 receptors preferentially couple to G q and drive cytokine production in an IL‐1 receptor‐dependent manner. (a) IL‐6 and IL‐8, which are targets of IL‐1 receptor signalling, were measured in cell culture supernatants 24 h after stimulation with P2Y 11 receptor agonists (ATPγS and NF546). The antagonist NF340 was used to confirm that agonist‐mediated responses were specific to stimulation of P2Y 11 receptors and inhibitors were used to examine G protein coupling of P2Y 11 receptors (G q : YM‐254890; G s : indirectly via adenylyl cyclase: SQ 22536). * P ≤ .05, significantly different as indicated; one‐way ANOVA. (b) P2RY11 cells were transfected with either control siRNA or IL1R1 siRNA for 72 h, with or without subsequent P2Y 11 stimulation (ATPγS at 30 μM, 24 h). The level of knockdown was quantified by measuring IL‐1R1 protein surface expression by flow cytometry (left panel). Isotype control MFIs were subtracted from each staining and mean values of MFIs were calculated ( n = 5) (right panel). * P ≤ .05, significantly different as indicated; one‐way ANOVA. (c) P2RY11 cells were transfected with either control siRNA or IL1R1 siRNA for 72 h and then treated for 24 h with P2Y 11 agonist (ATPγS) in the presence or absence of either P2Y 11 antagonist NF340 or recombinant human IL‐1 receptor antagonist (IL‐1RA). IL‐6 and IL‐8 were measured in cell culture supernatants. Data are means ± SEM from five independent cell populations. * P ≤ .05, significantly different as indicated; one‐way ANOVA

Article Snippet: The following antibodies were used for macrophage phenotyping: rabbit polyclonal IgG anti‐human P2Y 11 receptor (Bioss/THP, bs‐12071R‐A488, RRID:AB_2857964), rabbit polyclonal anti‐human P2X1 receptor (Bioss/THP, bs‐12107R‐A647, custom‐conjugation), mouse monoclonal IgG2b anti‐human CD14 (BD Bioscience, clone MφP9, 345,787‐APC, RRID:AB_400509), mouse monoclonal IgG1k anti‐human CD163 (BD Bioscience, clone GHI/61, 556,018‐PE, RRID:AB_2033943), mouse monoclonal IgG2a anti‐human CD284 (TLR4) (BioLegend, clone HTA125, 312,816‐APC, RRID:AB_2562487), goat polyclonal IgG anti‐human IL‐1R1 (R&D Systems/Bio‐techne, FAB269P‐PE, RRID:AB_2124912); mouse monoclonal IgG1 anti‐human P2Y 11 receptor (R&D, clone # 505214‐A647, RRID:AB_2857965) was used to detect human P2Y 11 receptor on glioma cells after gentle cell detachment using Accutase (Sigma‐Aldrich).

Techniques: Cell Culture, Transfection, Expressing, Flow Cytometry, Staining, Recombinant

Journal: British Journal of Pharmacology

Article Title: The human G protein‐coupled ATP receptor P2Y 11 is a target for anti‐inflammatory strategies

doi: 10.1111/bph.15379

Figure Lengend Snippet: Ectopic P2Y 11 enhances IL‐1R signalling. (a) P2RY11‐transfected cells and P2RY11 knockout cells (P2RY11‐KO) were treated for 24 h with increasing doses of IL‐1α or IL‐1ß. IL‐6 and IL‐8 were measured in cell culture supernatants. * P ≤ .05; (b) flow cytometric detection of IL‐1R1 on the surface of P2RY11‐transfected cells and P2RY11 knockout cells (P2RY11‐KO) using fluorochrome‐conjugated IL‐1R1 specific antibody and an appropriate isotype control (left panel). Quantification of eight independent stainings at different time points is shown in the right panel. Isotype control MFIs were subtracted from each staining and mean values of MFIs were calculated ( n = 8). * P ≤ .05; (c,d) P2RY11‐transfected cells and P2RY11 knockout cells (P2RY11‐KO) were treated for 24 h with low doses of IL‐1α (c) or IL‐1ß (d) either alone or in combination with P2Y 11 agonist. The antagonist NF340 was used to confirm that agonist‐mediated responses were specific to P2Y 11 stimulation. IL‐6 and IL‐8 were measured in cell culture supernatants. Data shown are means ± SEM from five independent cell populations. * P ≤ .05, significantly different as indicated; one‐way ANOVA

Article Snippet: The following antibodies were used for macrophage phenotyping: rabbit polyclonal IgG anti‐human P2Y 11 receptor (Bioss/THP, bs‐12071R‐A488, RRID:AB_2857964), rabbit polyclonal anti‐human P2X1 receptor (Bioss/THP, bs‐12107R‐A647, custom‐conjugation), mouse monoclonal IgG2b anti‐human CD14 (BD Bioscience, clone MφP9, 345,787‐APC, RRID:AB_400509), mouse monoclonal IgG1k anti‐human CD163 (BD Bioscience, clone GHI/61, 556,018‐PE, RRID:AB_2033943), mouse monoclonal IgG2a anti‐human CD284 (TLR4) (BioLegend, clone HTA125, 312,816‐APC, RRID:AB_2562487), goat polyclonal IgG anti‐human IL‐1R1 (R&D Systems/Bio‐techne, FAB269P‐PE, RRID:AB_2124912); mouse monoclonal IgG1 anti‐human P2Y 11 receptor (R&D, clone # 505214‐A647, RRID:AB_2857965) was used to detect human P2Y 11 receptor on glioma cells after gentle cell detachment using Accutase (Sigma‐Aldrich).

Techniques: Transfection, Knock-Out, Cell Culture, Staining

Journal: British Journal of Pharmacology

Article Title: The human G protein‐coupled ATP receptor P2Y 11 is a target for anti‐inflammatory strategies

doi: 10.1111/bph.15379

Figure Lengend Snippet: Activation of native P2Y 11 receptors enhances IL‐1 receptor signalling. (a) Phase contrast microscopy of Day‐6 M2 macrophages (magnification: 40×). Primary human monocytes were obtained from peripheral blood mononuclear cells (PBMCs) by magnetic‐activated cell sorting (MACS) using CD14 microbeads. M2 macrophages were differentiated by culturing the isolated monocytes with M‐CSF (50 ng·ml −1 ) for 6 days. (b) The phenotype of differentiated M2 macrophages was determined by flow cytometry and CD163 served as an M2 marker. (c,d) M2 macrophages were treated for 24 h with increasing doses of IL‐1α (c) or IL‐1ß (d) either alone or in combination with P2Y 11 receptor agonist (ATPγS). The antagonist NF340 (10 μM) was used to confirm that agonist‐mediated responses were specific to P2Y 11 receptor stimulation. IL‐6 and IL‐8 were measured in cell culture supernatants. Data are means ± SEM from five independent donors. * P ≤ .05, significantly different as indicated; one‐way ANOVA

Article Snippet: The following antibodies were used for macrophage phenotyping: rabbit polyclonal IgG anti‐human P2Y 11 receptor (Bioss/THP, bs‐12071R‐A488, RRID:AB_2857964), rabbit polyclonal anti‐human P2X1 receptor (Bioss/THP, bs‐12107R‐A647, custom‐conjugation), mouse monoclonal IgG2b anti‐human CD14 (BD Bioscience, clone MφP9, 345,787‐APC, RRID:AB_400509), mouse monoclonal IgG1k anti‐human CD163 (BD Bioscience, clone GHI/61, 556,018‐PE, RRID:AB_2033943), mouse monoclonal IgG2a anti‐human CD284 (TLR4) (BioLegend, clone HTA125, 312,816‐APC, RRID:AB_2562487), goat polyclonal IgG anti‐human IL‐1R1 (R&D Systems/Bio‐techne, FAB269P‐PE, RRID:AB_2124912); mouse monoclonal IgG1 anti‐human P2Y 11 receptor (R&D, clone # 505214‐A647, RRID:AB_2857965) was used to detect human P2Y 11 receptor on glioma cells after gentle cell detachment using Accutase (Sigma‐Aldrich).

Techniques: Activation Assay, Microscopy, FACS, Isolation, Flow Cytometry, Marker, Cell Culture

Journal: British Journal of Pharmacology

Article Title: The human G protein‐coupled ATP receptor P2Y 11 is a target for anti‐inflammatory strategies

doi: 10.1111/bph.15379

Figure Lengend Snippet: Activation of P2Y 11 receptors stimulates the release of soluble TNF receptors. (a) P2RY11‐transfected cells were cultured for 24 h with or without P2Y 11 receptor agonist (ATPγS). To confirm that agonist‐mediated cytokine secretion was specific to stimulation of P2Y 11 receptors, the cells were also stimulated with agonist in the presence of the specific P2Y 11 receptor antagonist NF340. Cell culture supernatants were subjected to a human cytokine antibody array. The G‐Series arrays are semiquantitative featuring fluorescent signal detection (a.u., arbitrary units) (left panel). sTNFR1 release was subsequently validated by ELISA (right panel). IL‐6 and IL‐8, which already emerged from the secretome analysis, served as an internal control. (b) M2 macrophages were treated for 24 h with the P2Y 11 receptor agonist (ATPγS). The antagonist NF340 was used to confirm that agonist‐mediated responses were specific to stimulation of P2Y 11 receptors. sTNFR1 and sTNFR2 as well as IL‐8 were measured in cell culture supernatants. Data are means ± SEM from five independent donors. * P ≤ .05, significantly different from control; # P < .05, significantly different from corresponding concentration of ATPγS only; one‐way ANOVA

Article Snippet: The following antibodies were used for macrophage phenotyping: rabbit polyclonal IgG anti‐human P2Y 11 receptor (Bioss/THP, bs‐12071R‐A488, RRID:AB_2857964), rabbit polyclonal anti‐human P2X1 receptor (Bioss/THP, bs‐12107R‐A647, custom‐conjugation), mouse monoclonal IgG2b anti‐human CD14 (BD Bioscience, clone MφP9, 345,787‐APC, RRID:AB_400509), mouse monoclonal IgG1k anti‐human CD163 (BD Bioscience, clone GHI/61, 556,018‐PE, RRID:AB_2033943), mouse monoclonal IgG2a anti‐human CD284 (TLR4) (BioLegend, clone HTA125, 312,816‐APC, RRID:AB_2562487), goat polyclonal IgG anti‐human IL‐1R1 (R&D Systems/Bio‐techne, FAB269P‐PE, RRID:AB_2124912); mouse monoclonal IgG1 anti‐human P2Y 11 receptor (R&D, clone # 505214‐A647, RRID:AB_2857965) was used to detect human P2Y 11 receptor on glioma cells after gentle cell detachment using Accutase (Sigma‐Aldrich).

Techniques: Activation Assay, Transfection, Cell Culture, Ab Array, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: British Journal of Pharmacology

Article Title: The human G protein‐coupled ATP receptor P2Y 11 is a target for anti‐inflammatory strategies

doi: 10.1111/bph.15379

Figure Lengend Snippet: P2Y 11 receptor‐driven release of soluble TNF receptors depends on intracellular cAMP and on TACE/ADAM17: synergistic enhancement by IL‐1α and IL‐1ß (a) M2 macrophages were treated with ATPγS in the presence or absence of increasing concentrations of the phosphodiesterase (PDE) inhibitors IBMX (nonselective) or rolipram (PDE4‐selective). The antagonist NF340 was used to confirm that agonist‐mediated responses were specific to stimulation of P2Y 11 receptors. sTNFR2 was measured in cell culture supernatants. (b) M2 macrophages were treated with ATPγS in the presence or absence of increasing concentrations of the TACE/ADAM17 inhibitors TAPI‐1 and TAPI‐2. The antagonist NF340 was used to confirm that agonist‐mediated responses were specific to stimulation of P2Y 11 receptors. sTNFR2 was measured in cell culture supernatants. (c) M2 macrophages were treated for 24 h with increasing doses of IL‐1α or IL‐1ß either alone or in combination with the P2Y 11 receptor agonist ATPγS (10 μM). The antagonist NF340 (10 μM) was used to confirm that agonist‐mediated responses were specific to stimulation of P2Y 11 receptors. For all graphs, data shown are means ± SEM from five independent donors. * P ≤ .05, significantly different as indicated; one‐way ANOVA

Article Snippet: The following antibodies were used for macrophage phenotyping: rabbit polyclonal IgG anti‐human P2Y 11 receptor (Bioss/THP, bs‐12071R‐A488, RRID:AB_2857964), rabbit polyclonal anti‐human P2X1 receptor (Bioss/THP, bs‐12107R‐A647, custom‐conjugation), mouse monoclonal IgG2b anti‐human CD14 (BD Bioscience, clone MφP9, 345,787‐APC, RRID:AB_400509), mouse monoclonal IgG1k anti‐human CD163 (BD Bioscience, clone GHI/61, 556,018‐PE, RRID:AB_2033943), mouse monoclonal IgG2a anti‐human CD284 (TLR4) (BioLegend, clone HTA125, 312,816‐APC, RRID:AB_2562487), goat polyclonal IgG anti‐human IL‐1R1 (R&D Systems/Bio‐techne, FAB269P‐PE, RRID:AB_2124912); mouse monoclonal IgG1 anti‐human P2Y 11 receptor (R&D, clone # 505214‐A647, RRID:AB_2857965) was used to detect human P2Y 11 receptor on glioma cells after gentle cell detachment using Accutase (Sigma‐Aldrich).

Techniques: Cell Culture

Journal: British Journal of Pharmacology

Article Title: The human G protein‐coupled ATP receptor P2Y 11 is a target for anti‐inflammatory strategies

doi: 10.1111/bph.15379

Figure Lengend Snippet: Activation of P2Y 11 receptors inhibits LPS‐induced TNF‐α production in M2 macrophages. (a) M2 macrophages were treated with a constant dose of lipopolysaccharide (LPS) either alone or in the presence of graded doses of ATPγS. The antagonist NF340 was used to confirm that agonist‐mediated responses were specific to stimulation of P2Y 11 receptors. NF449 was used to examine a potential role of P2X1 receptors. (b,c) M2 macrophages were treated with graded doses of LPS in the presence or absence of a constant dose of ATPγS. IL‐8 and TNF‐α were measured in cell culture supernatants. Data are means ± SEM from six independent donors. * P ≤ .05, significantly different as indicated; one‐way ANOVA

Article Snippet: The following antibodies were used for macrophage phenotyping: rabbit polyclonal IgG anti‐human P2Y 11 receptor (Bioss/THP, bs‐12071R‐A488, RRID:AB_2857964), rabbit polyclonal anti‐human P2X1 receptor (Bioss/THP, bs‐12107R‐A647, custom‐conjugation), mouse monoclonal IgG2b anti‐human CD14 (BD Bioscience, clone MφP9, 345,787‐APC, RRID:AB_400509), mouse monoclonal IgG1k anti‐human CD163 (BD Bioscience, clone GHI/61, 556,018‐PE, RRID:AB_2033943), mouse monoclonal IgG2a anti‐human CD284 (TLR4) (BioLegend, clone HTA125, 312,816‐APC, RRID:AB_2562487), goat polyclonal IgG anti‐human IL‐1R1 (R&D Systems/Bio‐techne, FAB269P‐PE, RRID:AB_2124912); mouse monoclonal IgG1 anti‐human P2Y 11 receptor (R&D, clone # 505214‐A647, RRID:AB_2857965) was used to detect human P2Y 11 receptor on glioma cells after gentle cell detachment using Accutase (Sigma‐Aldrich).

Techniques: Activation Assay, Cell Culture