Journal: Frontiers in Immunology

Article Title: Neuronal substance P-driven MRGPRX2-dependent mast cell degranulation products differentially promote vascular permeability

doi: 10.3389/fimmu.2024.1477072

Figure Lengend Snippet: Binding of compound 48/80 or ciprofloxacin to MRGPRX2 strongly promotes PMC degranulation and murine vascular permeability more than the binding to Mrgprb2. (A) Surface expression levels of FcεRIα and c-Kit (upper panel) and MRGPRX2 (middle panel) and expression levels of tdTomato (lower panel) in the PMCs from WT, Mrgprb2-KO (b2-KO), and MRGPRX2-KI (X2-KI) mice. Control staining is shown in the shaded histograms. (B, D, F) Percentages of surface CD63-positive cells in WT, Mrgprb2-KO, and MRGPRX2-KI PMCs and BMMCs (B) and PMCs (D, F) after treatment with the indicated concentrations of compound 48/80 (B) , 10 μg/mL ciprofloxacin (D) , or 10 μg/mL icatibant (F) . Data are representative of three independent experiments and indicate the mean ± SD. * P < 0.05 and ** P < 0.01. (C, E, G) Quantification of the Evans blue dye that extravasated into the ear skin in WT, Mrgprb2-KO, and MRGPRX2-KI mice after treatment with indicated amounts of compound 48/80 (C) , ciprofloxacin (E) , and 1.75 μg icatibant (G) or phosphate-buffered saline (PBS). n = 5-9; ± SD. * P < 0.05 and ** P < 0.01.

Article Snippet: Compound 48/80 and capsaicin (Sigma-Aldrich, St Louis, MO), SP (Peptide Institute, Inc., Osaka, Japan), Cetirizine (TCI AMERICA, Portland, OR), TY-51469 (MedChemExpress, Monmouth Junction, NJ), RWJ-56110 (Tocris Bioscience, Ellisville, MO), AZ3451, I-191, AMG-517 (Selleck Chemicals LLC), AMG-9810 (Cayman Chemical, Ann Arbor, MI), DAPI (4’,6-diamidino-2-phenylindole) (FujifilmWako, Osaka, Japan), and Piperine (TCI, Tokyo, Japan) were used.

Techniques: Binding Assay, Permeability, Expressing, Control, Staining, Saline

Journal: Frontiers in Immunology

Article Title: Annexin A1 Mimetic Peptide Ac 2-26 Modulates the Function of Murine Colonic and Human Mast Cells

doi: 10.3389/fimmu.2021.689484

Figure Lengend Snippet: Cytokine production by colon explants from C57BL/6 WT and C57BL/6 IL-4 KO mice treated in vivo with 1 mg/kg i.p. Ac 2-26 (+) or PBS (-). ELISA determined the concentration of IL-12 (A) , TNF-α (B) , IL-10 (C) and IL-4 (D) in explants stimulated for 24 h, with 20µg/mL compound 48/80 and with or without 3µM Ac2-26 treatment. Experiments were performed twice with n = 4 animals/group. Values of concentration in pg/mL are expressed as the mean ± S.D, n = 8. Data were analyzed using one-way ANOVA (post Tukey multiple-comparison test). **p <0.01 vs. respective WT group. ## p <0.01 vs. untreated in vivo and ex vivo (-/-) and && p <0.01 vs treated in vivo and not treated ex vivo (-/+).

Article Snippet: Remarkably, our study demonstrated that the AnxA1-derived peptide Ac 2-26 inhibited in vitro the degranulation of HMC-1 cells stimulated with compound 48/80 in FPR-dependent manner, as assessed by blocking the degranulation system using the antagonist BOC-2.

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Concentration Assay, Ex Vivo

Journal: Frontiers in Immunology

Article Title: Annexin A1 Mimetic Peptide Ac 2-26 Modulates the Function of Murine Colonic and Human Mast Cells

doi: 10.3389/fimmu.2021.689484

Figure Lengend Snippet: Effect of peptide Ac2-26 on MC degranulation in colon explants. C57BL/6 WT and C57BL/6 interleukin-4- KO mice were pre-treated with PBS (-) or 3µM Ac2 -26 (+) and stimulated with 20µg/mL compound 48/80 ex vivo for 24 (h) (A) Representative colon sections with examples of intact (arrowheads) and degranulated (arrows) MCs. Staining: Toluidine Blue. Sections: 4μm. Scale bars: 20 μm. (B) Degranulation of MCs was assessed by an enzymatic assay. Experiments were performed twice with n = 4 animals/group. Values of the percentage of β hexosaminidase release are expressed as the mean ± S.D., n = 8. Data were analyzed using one-way ANOVA (post Tukey multiple-comparison test). **p <0.01 compared to respective WT group. ## p <0.01 compared to untreated in vivo and ex vivo (-/-) and && p <0.01 compared to treated and not treated ex vivo (-/+).

Article Snippet: Remarkably, our study demonstrated that the AnxA1-derived peptide Ac 2-26 inhibited in vitro the degranulation of HMC-1 cells stimulated with compound 48/80 in FPR-dependent manner, as assessed by blocking the degranulation system using the antagonist BOC-2.

Techniques: Ex Vivo, Staining, Enzymatic Assay, In Vivo

Journal: Frontiers in Immunology

Article Title: Annexin A1 Mimetic Peptide Ac 2-26 Modulates the Function of Murine Colonic and Human Mast Cells

doi: 10.3389/fimmu.2021.689484

Figure Lengend Snippet: Exogenous AnxA1 (peptide Ac2-26) inhibits HMC-1 cell line degranulation induced by compound 48/80. HMC-1 cells were pre-treated with 3, 5, or 10µM peptide Ac2-26 for 30 min in the absence or presence of FPRs antagonist BOC-2 (5µg/mL). Degranulation was assessed measuring the activity of β-hexosaminidase in culture supernatants. Results are expressed as percentage of enzyme release. All experiments were conducted in triplicate. Data are expressed as the mean ± S.D from four different experiments and analyzed using one-way ANOVA. ***p <0.001 compared to cells treated with 48/80 alone or 48/80 + Ac2-26 (3 or 5 µM); **p <0.01 compared to cells treated with 48/80 + Ac2-26 (10 µM) + BOC-2; ## p <0.01 compared to cells treated with 48/80 alone.

Article Snippet: Remarkably, our study demonstrated that the AnxA1-derived peptide Ac 2-26 inhibited in vitro the degranulation of HMC-1 cells stimulated with compound 48/80 in FPR-dependent manner, as assessed by blocking the degranulation system using the antagonist BOC-2.

Techniques: Activity Assay

Journal: Frontiers in Immunology

Article Title: Annexin A1 Mimetic Peptide Ac 2-26 Modulates the Function of Murine Colonic and Human Mast Cells

doi: 10.3389/fimmu.2021.689484

Figure Lengend Snippet: Exogenous AnxA1 (peptide Ac2-26) did not alter viability of HMC-1 cell line. Cells were pre-treated with 3, 5, or 10µM peptide Ac2-26 for 30 min in the absence or presence of FPRs antagonist BOC-2 (5µg/mL). The cells were then stimulated with 20 µg/mL compound 48/80 for 30 min. Cell viability was assessed by trypan blue exclusion assay and results are expressed as percentage. All experiments were performed in triplicate. Data are expressed as the mean ± S.D from four different experiments.

Article Snippet: Remarkably, our study demonstrated that the AnxA1-derived peptide Ac 2-26 inhibited in vitro the degranulation of HMC-1 cells stimulated with compound 48/80 in FPR-dependent manner, as assessed by blocking the degranulation system using the antagonist BOC-2.

Techniques: Trypan Blue Exclusion Assay