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Sphingomyelin and ceramide are physiological ligands for human LMIR3/CD300f, inhibiting FcεRI-mediated mast cell activation  Kumi Izawa, MD, PhD, Masamichi.

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Presentation on theme: "Sphingomyelin and ceramide are physiological ligands for human LMIR3/CD300f, inhibiting FcεRI-mediated mast cell activation  Kumi Izawa, MD, PhD, Masamichi."— Presentation transcript:

1 Sphingomyelin and ceramide are physiological ligands for human LMIR3/CD300f, inhibiting FcεRI-mediated mast cell activation  Kumi Izawa, MD, PhD, Masamichi Isobe, MD, Toshihiro Matsukawa, MD, Shinichi Ito, MD, Akie Maehara, MS, Mariko Takahashi, PhD, Yoshinori Yamanishi, MD, PhD, Ayako Kaitani, PhD, Toshihiko Oki, MD, PhD, Ko Okumura, MD, PhD, Toshio Kitamura, MD, PhD, Jiro Kitaura, MD, PhD  Journal of Allergy and Clinical Immunology  Volume 133, Issue 1, Pages e7 (January 2014) DOI: /j.jaci Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Possible ligands for hLMIR3. A, PCA responses in KitW-sh/W-sh mice harboring LMIR3-deficient BMMCs transduced with mLMIR3-WT, hLMIR3-WT, hLMIR3(Y205F-Y249F-Y284F), or mock (n = 6-7 per group; ± SD). *P < .05. B, The binding of hLMIR3-Fc, Fc, or lysenin to the lipids. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Functional ligands for hLMIR3. A and C, β-Hexosaminidase release (n = 4, ± SD). *P < .05. BMMC transfectants or LAD2 cells treated with liposomes (Fig 2, A) or plated-coated lipids/antibodies (Fig 2, C), respectively. B, Surface receptor expression. D, Confocal imaging. E and F, PCA responses in the transplanted KitW-sh/W-sh mice, pretreated with antibodies (Fig 2, E) or liposomes (Fig 2, F) (n = 5-6 per group, ± SD). *P < .05. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig E1 Representative surface expression levels of FcεRI and c-kit (left panel) or Flag-tagged LMIR3 (right panel) in LMIR3-deficient BMMCs transduced with Flag-tagged mLMIR3-WT, hLMIR3-WT, hLMIR3(Y205F-Y249F-Y284F) mutant, or mock. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig E2 2B4-GFP cells and 2B4-hLMIR3-GFP cells were incubated for 24 hours on plates coated with the anti-hLMIR3 antibody or lipids such as C-24 ceramide, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingomyelin, sphingosylphosphocholine, or vehicle. A total of 20 mg/mL of either the hLMIR3 antibody or the isotype control antibody was added to the culture just after the incubation of the reporter cells on the coated plates. Flow cytometry of GFP expression of the reporter cells. Data are representative of 4 independent experiments. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig E3 The binding of hLMIR3-Fc or Fc to plate-coated lipids was quantified by using ELISA. A total of 10 mg/mL of hLMIR3-Fc or Fc was incubated on plates coated with C-24 ceramide, sphingomyelin, phosphatidylcholine, sphingosylphosphocholine, or vehicle. Data are representative of 4 independent experiments. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig E4 β-Hexosaminidase release (n = 4, ± SD). *P < .05. LAD2 cells stimulated with IgE/antigen plus plated-coated sphingomyelin or ceramide. A total of 2 or 20 μg/mL of either the anti-hLMIR3 antibody or the control antibody was added to the culture just before stimulating with antigens. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig E5 Anti-NP IgE-sensitized LAD2 cells were stimulated with or without 300 ng/mL NP-BSA for 2 minutes on plates coated with C-24 ceramide, sphingomyelin, or vehicle. Immunoprecipitates of cell lysates with the anti-hLMIR3 antibody were immunoblotted with indicated antibodies. Data are representative of 3 independent experiments. Cer, Ceramide; SM, sphingomyelin. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Fig E6 Confocal imaging of mouse ear-skin sections. Ear skin sections of KitWsh/Wsh mice were first stained with lysenin, then with rabbit antilysenin serum, and finally with Alexa Flour 488-conjugated secondary antibody (left panel). Ear skin sections of WT mice were not stained with lysenin, and were stained first with rabbit antilysenin serum and finally with Alexa Flour 488-conjugated secondary antibody (right panel). DAPI, 4'-6-Diamidino-2-phenylindole, dihydrochloride. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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