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Selective and direct activation of human neutrophils but not eosinophils by Toll-like receptor 8  Markus Janke, PhD, Jens Poth, Vera Wimmenauer, Thomas.

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Presentation on theme: "Selective and direct activation of human neutrophils but not eosinophils by Toll-like receptor 8  Markus Janke, PhD, Jens Poth, Vera Wimmenauer, Thomas."— Presentation transcript:

1 Selective and direct activation of human neutrophils but not eosinophils by Toll-like receptor 8 
Markus Janke, PhD, Jens Poth, Vera Wimmenauer, Thomas Giese, MD, Christoph Coch, MD, Winfried Barchet, PhD, Martin Schlee, PhD, Gunther Hartmann, MD  Journal of Allergy and Clinical Immunology  Volume 123, Issue 5, Pages (May 2009) DOI: /j.jaci Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Identification of neutrophils and eosinophils in human whole blood. PMNs were stained with anti-human CD16 mAb. CD16+/FSChigh/SSChigh cells are neutrophils (R2), and CD16−/FSChigh/SSChigh cells are eosinophils (R3; left panels). Sorted cells were stained with May-Grünwald-Giemsa solution and analyzed microscopically (200-fold and 600-fold magnification; right panels, left to right). One of 2 independent experiments is shown. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Activation of whole-blood neutrophils by isRNA. A, Whole blood stimulation with poly-L-arginine–complexed isRNA 9.2s or RNA polyA at 0.5 μg/mL (kinetic; left panel) or at 5.0 to 0.02 μg/mL (dose response; right panel). B, CD62L and CD11b expression on gated neutrophils and eosinophils. The mean ± SEM of 3 independent experiments is shown. ∗P < .03 versus PBS control. MFI, Mean fluorescence intensity. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 IL-8 induction in isRNA-stimulated neutrophils of whole blood. A, Analysis of intracellular IL-8 levels after 12 hours of whole-blood stimulation with poly-L-arginine complexed RNA polyA or isRNA 9.2s at 5.0 to μg/mL. One of 4 independent experiments is shown. B, Intracellular IL-8 expression on neutrophils on stimulation with poly-L-arginine–complexed RNA polyA, isRNA 9.2s (both 0.5 μg/mL), LPS, or R-848. The mean ± SEM of 3 independent experiments is shown. ∗P < .03 versus the PBS control. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 Quantitative analysis of mRNA expression of TLR1 to TLR10 in purified neutrophils. Neutrophils (purity >99%, viability >95%) were incubated with or without 500 U/mL recombinant human IFN-β for 3 hours. Expression of mRNA of TLR1 to TLR10 was determined by means of quantitative real-time PCR. The copy numbers are normalized to the housekeeping genes β-actin and cyclophilin B and are depicted as adjusted transcript number per microliter of cDNA (± SEM of 3 independent donors). Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 Selective direct activation of neutrophils by TLR8 but not TLR7. A, Whole blood was incubated with RNA polyA, isRNA 9.2s, (both 0.5 μg/mL), 3M001, 3M002, R-848, and PBS. Data show the mean ± SEM fluorescence intensity of CD62L and CD11b on neutrophils as a ratio to PBS (n = 3). ∗P < .03, with brefeldin A versus without brefeldin A. MFI, Mean fluorescence intensity. B, Macrophage inflammatory protein (MIP) 1α expression of 4 × 105 neutrophils stimulated with RNA polyA, isRNA 9.2s (both 5 μg/mL), 3M001, 3M002, R-848, LPS, and PBS. The mean ± SEM is shown (n = 3). ∗P < .03 versus PBS. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig 6 Activation of neutrophil function. A, Fold induction ± SEM of neutrophils stimulated with isRNA 9.2s (7 μg/mL), 3M001, 3M002, or fMLP (n = 4; ∗P < .05 vs PBS) and of β-glucuronidase and myeloperoxidase activity (n = 3; ∗P < .05 vs PBS + fMLP boost). B, IL-8 expression of 4 × 105 neutrophils stimulated with 3M002, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)-complexed isRNA polyA, and polyU (7 μg/mL) on a phosphodiester (PD) or PTO. The mean ± SEM is shown (n = 3). ∗P < .05. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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