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Activation of human mast cells through the platelet-activating factor receptor  Naoki Kajiwara, PhD, Tomomi Sasaki, BSc, Peter Bradding, DM, Glenn Cruse,

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Presentation on theme: "Activation of human mast cells through the platelet-activating factor receptor  Naoki Kajiwara, PhD, Tomomi Sasaki, BSc, Peter Bradding, DM, Glenn Cruse,"— Presentation transcript:

1 Activation of human mast cells through the platelet-activating factor receptor 
Naoki Kajiwara, PhD, Tomomi Sasaki, BSc, Peter Bradding, DM, Glenn Cruse, PhD, Hironori Sagara, MD, PhD, Kazumitsu Ohmori, MD, PhD, Hirohisa Saito, MD, PhD, Chisei Ra, MD, PhD, Yoshimichi Okayama, MD, PhD  Journal of Allergy and Clinical Immunology  Volume 125, Issue 5, Pages e6 (May 2010) DOI: /j.jaci Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Mediators released by MCs in response to PAF. Histamine release by lung MCs (A, n = 7), skin MCs (B, n = 3), and PB-derived MCs (C, n = 3) in response to PAF and calcium ionophore A23187 (10−6 mol/L) are shown. PGD2 (D, n = 5), IL-8 (E, n = 3), and PAI-1 (F, n = 3) production by PB-derived MCs in response to PAF and IgE/anti-IgE (α-IgE, 0.3 μg/mL) are also shown. Data shown are means ± SEMs. ∗P < .05 and ∗∗P < .01 compared with vehicle (0.1% chloroform). –, Buffer alone. #n = 3. ND, Not detected. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Expression of PAF-R in PB-derived MCs. A, PAFR mRNA expression was analyzed by means of RT-PCR. B, PAF-R protein expression was analyzed by means of Western blotting. Jurkat cells and PB mononuclear cells were used as negative and positive controls for PAF-R expression, respectively. The data are representative of similar results obtained from 3 independent experiments. C and D, Dual staining of bronchial MCs with anti–PAF-R antibodies (red arrows and red arrowheads indicate tryptase-positive PAF-R–positive cells and tryptase or chymase-negative PAF-R–positive cells, respectively; middle) and anti-tryptase mAb (C, green arrows, left) or anti-chymase mAb (D, green arrows, left). Yellow color indicates merged cells (Fig 2, C, right). The data are representative of similar results obtained from 6 independent experiments with MCs from different donors. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 Ca2+ mobilization through PAF-R in PB-derived MCs in response to PAF. A, Ca2+ mobilization in MCs in response to PAF. B, Effect of the PAF-R antagonist CV-6209 on PAF- or calcium ionophore A23187–induced Ca2+ mobilization. The y-axis and x-axis show intracellular free calcium levels and the incubation time after stimulation, respectively. Arrows indicate the time point of addition of PAF or calcium ionophore A The data are representative of similar results obtained from 3 independent experiments. The vehicle used was 0.1% chloroform. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 PAF-induced histamine release from PB-derived MCs is rapid and inhibited by pretreatments with CV-6209 and PTX. A, Kinetics of histamine release induced by PAF (10−7 mol/L, solid circles) or IgE/anti-IgE (30 μg/mL, open circles; n = 3). B and C, Effects of CV-6209 (n = 6) and PTX (n = 7) on PAF- or IgE/anti-IgE–induced histamine release. Data shown are means ± SEMs. ∗P < .05 and ∗∗P < 0.01 compared with 0 seconds (Fig 4, A). ∗P < .05 and ∗∗P < .01 compared with nontreatment (Fig 4, B and C). Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 Activation of PLCγ1 and PLCβ2 in PB-derived MCs by PAF. A, Effect of U73122 on PAF-induced (solid circles) or IgE/anti-IgE–induced (open circles) histamine release (n = 4). B, Expression of PLCβ and PLCγ in MCs. HeLa cells and HL-60 cells were used as positive controls. (PLCβ1, PLCβ2, and PLCβ3, n = 3; PLCγ1 and PLCγ2, n = 6). C, Membrane translocation of PLCβ2 in response to PAF or IgE/anti-IgE. Anti-Kit was used to verify the membrane protein loading, and anti–heat shock protein (HSP) 90 was used to verify the absence of cytosolic proteins. D, Phosphorylation of PLCγ1 (n = 5) and PLCγ2 (n = 4) in response to PAF or IgE/anti-IgE. Data shown are means ± SEMs. ∗P < .05 and ∗∗P < .01 compared with vehicle (0.1% dimethyl sulfoxide). The data (Fig 5, C and D) were generated by scanning the blots and then normalizing to the response at 0 seconds (n = 3). –, Buffer alone; p-PLCγ, anti–phospho-PLCγ. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig 6 Lentiviral shRNA silencing of PLCγ1 and PLCβ2 reduces FcεRI- and PAF-R-mediated degranulation. A, Assessment for loss of PLCγ1 and PLCβ2 mRNA expression in nontransduced PB-derived MCs, cells transduced with nontargeted shRNA, or lentiviral shRNA constructs for PLCγ1 and PLCβ2. B, Degranulation from nontransduced MCs (open bars), MCs transduced with nontargeted shRNA (hatched bars), or lentiviral shRNA constructs for PLCγ1 (gray bars) and PLCβ2 (solid bars), which were stimulated with PAF (10−7 mol/L), IgE/anti-IgE (30 μg/mL), or calcium ionophore A Data shown are means ± SEMs of 4 independent experiments. ∗P < .05 and ∗∗P < .01. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig 7 Effects of BAPTA/AM and Go6976 on PAF- or IgE/anti-IgE–induced histamine release. A, Effect of BAPTA/AM on PAF-induced (open bars) or IgE/anti-IgE–induced (solid bars) histamine release in the presence or absence of extracellular Ca2+ (n = 6). B, Effect of Go6976 on PAF-induced (solid circles) or IgE/anti-IgE–induced (open circles) histamine release (n = 10). Data shown are means ± SEMs. ∗P < .05 and ∗∗P < .01 compared with vehicle (0.03% dimethyl sulfoxide) in the presence of extracellular Ca2+ (Fig 7, A). ∗P < .05 compared with vehicle (0.1% dimethyl sulfoxide; Fig 7, B). Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Variable response of histamine release from human lung MCs in response to PAF (n = 7). ND, Not determined. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 PAF-R– or FcεRI-mediated cytokine gene expression profiles in human PB-derived MCs. Human MCs were activated with PAF (10−7 mol/L) or IgE/anti-IgE (3 μg/mL) for 1 hour (A) and 4 hours (B). Each experiment was performed with 3 independent donors, and the data are the average of 3 separate analyses. Overlapping sets of PAF- and IgE/anti-IgE–induced cytokine genes are shown. Stimulus-induced cytokine genes were selected if the ratio of the relative expression level between those with and without the stimulus was greater than 4.0. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

11 Ca2+ mobilization through PAF-R in PB-derived MCs in response to PAF
Ca2+ mobilization through PAF-R in PB-derived MCs in response to PAF. A, Ca2+ mobilization in Jurkat cells (n = 3) and PB mononuclear cells (n = 3) in response to PAF. B, Effect of the PAF-R antagonist BN52021 on PAF- or calcium ionophore A23187–induced Ca2+ mobilization (n = 3). Intracellular free calcium levels (y-axis) were monitored with the LSR system. The x-axis shows the incubation time after stimulation. Arrows indicate the time point of addition of PAF or calcium ionophore A The data are representative of similar results obtained from 3 independent experiments with MCs from different donors. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

12 The additive effect of PAF on IgE-dependent histamine release and PGD2 production by MCs. MCs were sensitized with IgE for 16 hours and then washed and challenged with PAF (10−10 to 10−6 mol/L) with (IgE/anti-IgE [+], solid bars) or without (IgE/anti-IgE [−], open bars; 0.3 μg/mL) anti-IgE for 30 minutes for histamine release (A, n = 3) and PGD2 production (B, n = 4). Data shown are means ± SEMs. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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