Characterization of a novel human mast cell line that responds to stem cell factor and expresses functional FcεRI Tanya M. Laidlaw, MD, John W. Steinke,

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Presentation transcript:

Characterization of a novel human mast cell line that responds to stem cell factor and expresses functional FcεRI Tanya M. Laidlaw, MD, John W. Steinke, PhD, Adrienne M. Tiñana, MD, Chunli Feng, MD, Wei Xing, MD, PhD, Bing K. Lam, PhD, Sailaja Paruchuri, PhD, Joshua A. Boyce, MD, Larry Borish, MD Journal of Allergy and Clinical Immunology Volume 127, Issue 3, Pages 815-822.e5 (March 2011) DOI: 10.1016/j.jaci.2010.12.1101 Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Morphologic characterization of LUVA cells. Magnifications (×400) of LUVA cells are in the left column, and magnifications of CD34+ derived primary MCs are in the right column. Cells stained with toluidine blue (A and F), tryptase (B and G), chymase (C and H), cathepsin G (D and I), and CPA3 (E and J) are shown. Journal of Allergy and Clinical Immunology 2011 127, 815-822.e5DOI: (10.1016/j.jaci.2010.12.1101) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Cell-surface expression of MC markers. Representative flow cytometric analyses are of the expression of c-kit (A), FcγRII/CD32 (B), and FcγRI/CD64 (C). Basal expression of FcεRIα and upregulation of FcεRIα after incubation with IgE (D) or IL-4 (E) are shown. Each analysis was carried out at least 3 times. Isotype controls are shown in gray. Journal of Allergy and Clinical Immunology 2011 127, 815-822.e5DOI: (10.1016/j.jaci.2010.12.1101) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Mediator release. Quantification of β-hexosaminidase (A), PGD2 (B), TXB2 (C), and MIP-1β (D) levels in cell supernatants are shown. In some experiments the cells were primed for 3 days with IL-4 (10 ng/mL) before activation. Data are shown as means ± SDs (n = 2 or 3 experiments for LUVA cells and n = 1 or 2 experiments for LAD2 cells). Journal of Allergy and Clinical Immunology 2011 127, 815-822.e5DOI: (10.1016/j.jaci.2010.12.1101) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 MC-specific transcript expression. Baseline mRNA transcript expressions of tryptase (TPSAB1), chymase (CMA1), and CPA3 are shown. Each LUVA transcript is expressed as mean ± SEMs (n = 4 experiments), and in parallel are the baseline levels of the same transcripts by LAD2 cells (n = 1). GAPDH, Glyceraldehyde-3-phosphate dehydrogenase. Journal of Allergy and Clinical Immunology 2011 127, 815-822.e5DOI: (10.1016/j.jaci.2010.12.1101) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Cellular proliferation of MCs in the presence or absence of SCF. Cells were stimulated with the indicated doses of SCF for 72 hours. Proliferation rates were determined by means of tritiated thymidine incorporation. Data are shown as means ± SDs (n = 3 experiments). Journal of Allergy and Clinical Immunology 2011 127, 815-822.e5DOI: (10.1016/j.jaci.2010.12.1101) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 Effect of exogenous SCF and imatinib mesylate on apoptosis. LUVA cells were cultured in medium alone (A), with SCF (B), or with SCF and imatinib mesylate (C). Six-week-old primary human cord blood–derived MCs (cbMCs) were cultured with the same conditions (D-F). The percentage of apoptotic cells was measured based on surface expression of Annexin V compared with the isotype control in shaded gray. Each analysis was carried out at least 3 times with similar results. Journal of Allergy and Clinical Immunology 2011 127, 815-822.e5DOI: (10.1016/j.jaci.2010.12.1101) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 7 Activation of the c-kit–signaling pathway in LAD2 and LUVA cells. Cells were starved of SCF for 3 hours and then were stimulated for 10 minutes with SCF (100 ng/mL) or medium alone. A, SDS-PAGE immunoblotting comparing phosphorylated c-kit and total c-kit. B, Phosphorylation of Akt, MEK, p42/44 ERK, and CREB in the same experiment. The data in a second experiment were similar. Journal of Allergy and Clinical Immunology 2011 127, 815-822.e5DOI: (10.1016/j.jaci.2010.12.1101) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Cell-surface expression of additional surface markers Cell-surface expression of additional surface markers. Representative flow cytometric analyses are shown of the expression of the IL-4 receptor α chain/CD124 (A), IL-25 receptor (IL-25R; B), IL-33 receptor (IL-33R; C), CD13 (D), IL-2 receptor α-chain/CD25 (E), FcγRIII/CD16 (F), the thymic stromal lymphopoietin receptor (TSLP-R; G), the IL-3 receptor α chain/CD123 (H), and the IL-9 receptor α chain/CD129 (I). Each analysis was carried out at least 2 times. The isotype control for each antibody is shown in gray. Journal of Allergy and Clinical Immunology 2011 127, 815-822.e5DOI: (10.1016/j.jaci.2010.12.1101) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Additional transcript expression in LUVA cells Additional transcript expression in LUVA cells. The baseline LUVA cell mRNA transcript expression for the IgE high-affinity receptor FCER1A, the SCF receptor KIT, histamine decarboxylase (HDC), LTC4S, PGD synthase (PGDS), microphthalmia-associated transcription factor (MITF), and the leukotriene receptors CYSLTR1 and CYSLTR2 are shown. Data are shown as means ± SEMs (n = 4 experiments). Journal of Allergy and Clinical Immunology 2011 127, 815-822.e5DOI: (10.1016/j.jaci.2010.12.1101) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Growth curve of LUVA cells Growth curve of LUVA cells. LUVA cells were plated at a density of 200,000 cells/mL in StemPro-34 with or without 100 ng/mL SCF in triplicate wells. Data are shown as means ± SEMs (n = 2 experiments). Journal of Allergy and Clinical Immunology 2011 127, 815-822.e5DOI: (10.1016/j.jaci.2010.12.1101) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions