Mast cells express 11β-HSD1.

<p>(A) 11β-HSD1 reductase (conversion of 11-dehydrocorticosterone to corticosterone; Solid lines/black squares) and dehydrogenase (conversion of corticosterone to 11-dehydrocorticosterone; dashed lines/open squares) activities in BMD-MC (>98% pure, assessed by tryptase staining as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054640#pone.0054640-Pemberton1" target="_blank">[24]</a>) are expressed as % conversion of 200 nM substrate by 2×10⁶ cells, over time (h). Values are mean ± SEM of 3 independent assays carried out on pooled BMD-MC. (B) BMD-MC express 11β-HSD1 mRNA (upper panel; 469 bp RT-PCR product with primers 868P and 869P) but not 11β-HSD2 mRNA (lower panel; 400 bp RT-PCR product). Lanes 5 and 6 show RT-PCR products from 2 independent <i>Hsd11b1^+/+</i> BMD-MC RNA samples. Positive controls (+) comprised liver mRNA (lane 4, upper panel) or kidney mRNA (lane 4, lower panel). Negative controls contained BMD-MC RNA but no reverse transcriptase (−, lane 3) or water (lane 2). Lane 1 contains a 100 bp ladder (Promega, Southampton, UK). (C) <i>Hsd11b1^−/−</i> BMD-MC lack 11β-HSD1 activity. 11β-HSD1 reductase activity was measured in <i>Hsd11b1^+/+</i> (+/+) and <i>Hsd11b1^−/−</i> (−/−) BMD-MC. Data are expressed as % conversion of 200 nM 11-dehydrocorticosterone to corticosterone by 2×10⁶ cells in a 10 h assay. Values are means of triplicate assays performed on pooled BMD-MC. ND, not detected. (D) Mast cells transcribe <i>Hsd11b1</i> from the P1 promoter, whereas macrophages use the P2 promoter. Upper panel; RT-PCR products from 3 independent BMD-MC RNA samples showing the 627 bp RT-PCR product from P1 transcripts of 11β-HSD1 (lanes 4–6), but not P2 (predicted product of 647 bp, lanes 7–9) or P3 (predicted product of 542 bp, lanes 10–12). Kidney RNA confirmed transcription from P3 (not shown). Total 11β-HSD1 mRNA was detected using ex2 and 868P primers common to all transcripts (lanes 13–15, 587 bp product). Lower panel; RT-PCR products from BMD macrophage RNA showing the 647 bp RT-PCR P2 product (lane 3), but not the P1 (lane 2) or P3 products (lane 4). Total 11β-HSD1 mRNA (T) was detected using 868P and 869P primers, common to all transcripts (lanes 5, 6; 469 bp product). Lane 1 contains a 1 kb ladder (Invitrogen, Paisley, UK). Lanes marked (0) contain water only and lanes marked (−) show RT-PCR reactions from which the RT was omitted. (E) Flow cytometric staining revealed 11β-HSD1⁺CD117⁺ peritoneal cells. Freshly isolated peritoneal cells (∼5×10⁵ cells per sample) from male C57BL/6 mice were stained with 11β-HSD1 and CD117⁺ antibodies. Total peritoneal cells were first gated according to side scatter (SSC) and forward scatter (FSC) (i), then cells with high granularity, where mast cells lie (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054640#pone.0054640.s001" target="_blank">Figure S1</a>), were assessed for 11β-HSD1 and CD117 staining (ii–vii). Controls included; (ii) high SSC cells alone, (iii) sheep IgG control for 11β-HSD1 antibody, (iv) 11β-HSD1 antibody only, (v) isotype control for CD117 and (vi) CD117 antibody only. Panel (vii) shows cells double stained with 11β-HSD1 and CD117 antibodies.