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Expression of functional leukotriene B4 receptors on human airway smooth muscle cells  Satoko Watanabe, BS, Akira Yamasaki, MD, PhD, Kiyoshi Hashimoto,

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Presentation on theme: "Expression of functional leukotriene B4 receptors on human airway smooth muscle cells  Satoko Watanabe, BS, Akira Yamasaki, MD, PhD, Kiyoshi Hashimoto,"— Presentation transcript:

1 Expression of functional leukotriene B4 receptors on human airway smooth muscle cells 
Satoko Watanabe, BS, Akira Yamasaki, MD, PhD, Kiyoshi Hashimoto, MD, PhD, Yasushi Shigeoka, MD, PhD, Hiroki Chikumi, MD, PhD, Yasuyuki Hasegawa, MD, PhD, Takashi Sumikawa, MD, PhD, Miyako Takata, PhD, Ryota Okazaki, MD, Masanari Watanabe, MD, PhD, Tsuyoshi Yokogawa, BS, Miki Yamamura, MD, Tatsuya Hayabuchi, MD, William T. Gerthoffer, PhD, Andrew J. Halayko, PhD, Eiji Shimizu, MD, PhD  Journal of Allergy and Clinical Immunology  Volume 124, Issue 1, Pages e3 (July 2009) DOI: /j.jaci Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Immunohistochemical staining of human bronchial tissue. Normal bronchial tissue was stained with anti-BLT1 antibody and BLT2 antibody. A, BLT1 staining. B, BLT2 staining. C, Smooth muscle α-actin staining. D, Rabbit IgG negative control. epi, epithelium; sm, smooth muscle. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Expression of LTB4 receptor mRNA in ASM cells in vitro. RT-PCR analysis was performed to detect BLT1 (top) and BLT2 (middle) mRNA in primary human ASM cells and hTERT-ASM cell lines each taken from 5 different donors. Each lane in the gels shown corresponds to samples from different cell cultures. Glyceraldehyde-3-phosphate dehydrogenase (bottom) was used as an internal loading control. Neutrophils obtained from a healthy donor were used as a positive control, and water was used as a negative control. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 Expression of BLT1 and BLT2 proteins in ASM cells. A, Expression of both the nonglycosylated dimer form receptor (∼80 kd) and the glycosylated BLT1 receptor (BLT1R; ∼60 kd). B, Expression of BLT2 receptor (BLT2R). Neutrophils obtained from a healthy donor were used as a positive control. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 Effects of LTB4 on proliferation and migration of ASM cells. A, Results of proliferation assay for the effects of LTB4. Measurements were taken from cells 48 hours after stimulation with 10−8 to 10−13 mol/L LTB4. Stimulation with EGF (10 ng/mL) was used as a positive control. Results are presented as a percentage of negative control. Bars indicate the means ± SEMs from triplicate experiments using 3 cell lines. ∗P < .001 vs control. B, Results of chemokinesis assay for the effects for LTB4. Data are shown as percentage of gold particle–free areas compared with cells incubated in DMEM (control). Bars indicate the means ± SEMs from triplicate experiments using 3 cell lines. ∗P < .001 vs control. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 LTB4 phosphorylates p42/p44 MAPK and Akt1 in ASM cells. Equal amounts of cellular protein (20 μg) were subjected to 10% SDS-PAGE and transferred to PVDF membranes. Blots shown are representative of the 3 different cell lines used in this study. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig 6 BLT1 mediates effects of LTB4 on ASM proliferation and migration via p42/p44 MAPK and PI3 kinase. A, Histogram showing results of proliferation assay 48 hours after LTB4 addition. Results are presented as a percentage of negative control. Bars indicate the means ± SEMs from triplicate experiments using 3 cell lines. ∗P <.001 vs negative control; ∗∗P < .001 vs stimulation with LTB4 (10−8 mol/L). B, Histogram showing results of cumulative cell migration assay after 20 hours of LTB4 exposure. Data are shown as a percentage of gold particle–free areas compared with cells incubated in DMEM only (control). Bars indicate the means ± SEMs from at least 100 cells. ∗P < .001 vs negative control; ∗∗P < .001 vs stimulation with LTB4 (10−8 mol/L). Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Flow cytometry of the surface expression of BLT receptor subtypes on the ASM cells. Representative histograms showing the intensity of cell surface staining for receptor (x-axis) and the number of cells exhibiting different levels of staining (y-axis) are shown. A, BLT1 staining on human hTERT-ASM cells. B, BLT2 staining on human hTERT-ASM cells. Green lines in each histogram show specific immunostaining for BLT receptors, and black lines indicate fluorescence signal obtained from isotype-matched negative controls. FL1-H, fluorescence 1-height; FL-2H, fluorescence 2-height. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Increase of cyclin D1 with LTB4 stimulation of ASM cells
Increase of cyclin D1 with LTB4 stimulation of ASM cells. Western blot shows temporal effects of LTB4 (10−8 mol/L) on cyclin D1 expression on hTERT-ASM cells. Equal amounts of cellular protein (20 μg) were subjected to 10% SDS-PAGE and transferred to PVDF membranes. Cyclin D1 protein (36 kd) was detected by using anti–cyclin D1 antibody. Blot shown is representative of the 3 different cell lines used in this study. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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