Generation of a Large Number of Connective Tissue Type Mast Cells by Culture of Murine Fetal Skin Cells Nobuo Yamada, Hironori Matsushima, Yutaka Tagaya,

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Generation of a Large Number of Connective Tissue Type Mast Cells by Culture of Murine Fetal Skin Cells Nobuo Yamada, Hironori Matsushima, Yutaka Tagaya, Shinji Shimada, Stephen I. Katz Journal of Investigative Dermatology Volume 121, Issue 6, Pages 1425-1432 (December 2003) DOI: 10.1046/j.1523-1747.2003.12613.x Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 FSMC express FcεRI in a time-dependent manner and degranulate upon cross-linking of FcεRI. x- and y-axes of the histograms represent expression levels of FcεRI as determined by binding of exogenous IgE, and cell numbers, respectively. Day 16 fetal skin cell suspension from C57BL/6 mice was cultured with IL-3 and SCF for 5 d (a), 8 d (b), 11 d (c), 14 d (d), and 28 d (e), and CD45+CD117+ mast cells were gated for the analyses. Bold lines correspond to the fluorescence intensity of cells stained with anti-IgE antibody. Dotted lines correspond to the rat IgG1 isotype control. Fourteen day cultured FSMC and 29 d cultured BMMC from C57BL/6 mice were preincubated in the presence of 0.1 μg anti-DNP IgE per mL for 18 h. After washing four times, the cells were incubated with 10 ng DNP-conjugated HSA per mL for 30 min (f). β-hexosaminidase release was determined in triplicate. The figure is representative of four similar experiments. Journal of Investigative Dermatology 2003 121, 1425-1432DOI: (10.1046/j.1523-1747.2003.12613.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Histamine content of FSMC increases with length of culture. FSMC from C57BL/6 or BALB/C mice were harvested after various times in culture and cellular lysates were obtained by freezing and thawing. Histamine content was determined using an ELISA kit and normalized to the percentage of CD117+ cells by flow cytometry. Journal of Investigative Dermatology 2003 121, 1425-1432DOI: (10.1046/j.1523-1747.2003.12613.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Cytoplasmic granules in FSMC become more electron dense and more densely packed with length of culture. Representative electron micrographs of C57BL/6-derived FSMC after 7 d (a), 14 d (b), and 21 d (c), and BMMC cultured for 30 d (d). Journal of Investigative Dermatology 2003 121, 1425-1432DOI: (10.1046/j.1523-1747.2003.12613.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Demonstration of heparin in FSMC by histochemistry. Fourteen day cultured FSMC (a,b) and 30 d cultured BMMC (c,d) from C57BL/6 mice were stained with alcian blue and safranin red (a,c) or with berberine sulfate (b,d). The red staining in a and the fluorescent staining in b indicate that FSMC contain heparin. Journal of Investigative Dermatology 2003 121, 1425-1432DOI: (10.1046/j.1523-1747.2003.12613.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 FSMC but not BMMC respond to cationic secretagogues. Fourteen day cultured FSMC and 30 d cultured BMMC from C57BL/6 mice were preincubated in the presence or absence of 0.3 μg IgE per mL for 18 h. After washing three times, the cells were incubated with various concentrations of compound 48/80, substance P, and ionomycin, for 15, 20, and 10 min, respectively. Each bar represents mean±SD of the percentages of β-hexosaminidase released as determined in triplicate. The figure is a representative result of three experiments. The p-value was calculated according to the Student's paired t test (*p<0.01; *p<0.05 vs. cells preincubated in the absence of IgE). Journal of Investigative Dermatology 2003 121, 1425-1432DOI: (10.1046/j.1523-1747.2003.12613.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Phenotypic characteristics of FSMC compared with other mast cells. Thirty day cultured BMMC, 14 d cultured FSMC, PMC, CMC from ears, and immature mast cells from day 16 fetal skin of C57BL/6 mice were compared using various phenotypic markers. Each histogram was gated for CD45+ CD117+ mast cells, and the dotted lines correspond to the relevant isotype control. Numbers in each column correspond to MFI. Journal of Investigative Dermatology 2003 121, 1425-1432DOI: (10.1046/j.1523-1747.2003.12613.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 FSMC and BMMC differentially secrete cytokines in response to ionomycin. Eighteen day cultured FSMC and 4 to 5 wk cultured BMMC from C57BL/6 or BALB/C were resuspended and cultured in the presence of 1 μM ionomycin at 1×106 cells per mL for 24 h. Cytokine concentrations in the culture supernatants were determined in duplicate by ELISA. Each bar represents mean±SD of three independent experiments. The p-value was calculated according to the Student's paired t test. Journal of Investigative Dermatology 2003 121, 1425-1432DOI: (10.1046/j.1523-1747.2003.12613.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Increased expression of various MCP by FSMC. RNA purified from 14 d cultured FSMC and 5 wk cultured BMMC from three strains of mice, BALB/C, C57BL/6, and SJL.B6, were subjected to quantitative reverse transcription–PCR using SYBR Green I Dye. The relative gene expression levels were determined in triplicate by differences of cycle threshold numbers as described in Materials and Methods. The figure is a representative result of three similar experiments. Journal of Investigative Dermatology 2003 121, 1425-1432DOI: (10.1046/j.1523-1747.2003.12613.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions