Fig. 1.
Fig. 1. Mast cells produce IL-25 upon activation via IgE receptors. / (A) Mast cells express IL-25 mRNA upon activation. TH0 cells and TH2 cells were stimulated with plate-bound anti-CD3 antibody at 37°C for one hour. Splenic B cells and M12.4.5 cells were stimulated with LPS (1 μg/mL) for one hour. BMMCs were incubated with anti-DNP IgE for 2 hours and then surface IgE was cross-linked with DNP-HSA for 1 hour. CFTL-15 cells were stimulated with A23187 (500 ng/mL) + PMA (100 ng/mL) for one hour. Total cellular RNA was prepared from these cells, and RT-PCR for IL-25 and β-actin (as a control) mRNA was performed. Preliminary experiments revealed that the peak of IL-25 mRNA expression in anti-CD3–stimulated TH2 cells was one hour after stimulation. Shown are representative data from 5 independent experiments. (B) Cyclosporin A inhibits IL-25 mRNA induction in mast cells. BMMCs were stimulated with the following stimuli for one hour: IL-3 (10 ng/mL), SCF (10 ng/mL), LPS (1 μg/mL), anti-DNP IgE, anti-DNP IgE + DNP-HSA, A23187 (500 ng/mL), PMA (100 ng/mL), and A23187 + PMA. Where indicated, cyclosporin A (CyA) was added at 1 μg/mL. RT-PCR analysis for IL-25 and β-actin mRNA was performed as described in “Study design.” Shown are representative data from 4 independent experiments. (C) Real-time PCR analysis for the effect of cyclosporin A. BMMCs were incubated with anti-DNP IgE alone or anti-DNP IgE + DNP-HSA for one hour in the presence or absence of cyclosporin A (1 μg/mL). Real-time PCR analysis for IL-25 as well as GADPH (as a control) mRNA was performed, and the levels of IL-25 mRNA were normalized to the levels of GADPH mRNA. Data are means ± SD from 4 experiments. *Significantly different from the mean value of control responses (without cyclosporin A); P < .01. (D) Kinetics of IL-25 mRNA expression upon activation. BMMCs were incubated with anti-DNP IgE and then surface IgE was cross-linked with DNP-HSA. At indicated times after IgE cross-linking, total cellular RNA was prepared and RT-PCR for IL-25, TNF-α, and β-actin mRNA was performed. Shown are representative data from 4 independent experiments. (E) Real-time PCR analysis of IL-25 and TNF-α mRNA expression. Similar to panel D, BMMCs were stimulated with anti-DNP IgE + DNP-HAS, and total cellular RNA was prepared at indicated times after stimulation. Real-time PCR analysis for IL-25 (○) and TNF-α (●) as well as for GADPH (as a control) mRNA was performed. The levels of IL-25 or TNF-α mRNA were normalized to the levels of GAPDH mRNA. Data are means ± SD from 4 experiments. (F) Detection of IL-25 at protein levels. COS7 cells were transiently transfected with IL-25 expression vector (pME18S–IL-25) or control vector (pME18S), and these cell lysates as well as recombinant IL-17 (0.5 μg, Endogen, Woburn, MA) were subjected to immunoblotting with rabbit antisera to murine IL-25 or preimmune rabbit serum. (G) Mast cells release IL-25 upon activation. BMMCs were stimulated with and without A23187 + PMA for 3 hours. Culture supernatant was collected by centrifugation, concentrated with Microcon, separated on 12% SDS gel, and blotted with rabbit antisera to IL-25 or with preimmune rabbit serum. Shown is a representative blot from 4 independent experiments.

Mast cells produce IL-25 upon activation via IgE receptors.

(A) Mast cells express IL-25 mRNA upon activation. TH0 cells and TH2 cells were stimulated with plate-bound anti-CD3 antibody at 37°C for one hour. Splenic B cells and M12.4.5 cells were stimulated with LPS (1 μg/mL) for one hour. BMMCs were incubated with anti-DNP IgE for 2 hours and then surface IgE was cross-linked with DNP-HSA for 1 hour. CFTL-15 cells were stimulated with A23187 (500 ng/mL) + PMA (100 ng/mL) for one hour. Total cellular RNA was prepared from these cells, and RT-PCR for IL-25 and β-actin (as a control) mRNA was performed. Preliminary experiments revealed that the peak of IL-25 mRNA expression in anti-CD3–stimulated TH2 cells was one hour after stimulation. Shown are representative data from 5 independent experiments. (B) Cyclosporin A inhibits IL-25 mRNA induction in mast cells. BMMCs were stimulated with the following stimuli for one hour: IL-3 (10 ng/mL), SCF (10 ng/mL), LPS (1 μg/mL), anti-DNP IgE, anti-DNP IgE + DNP-HSA, A23187 (500 ng/mL), PMA (100 ng/mL), and A23187 + PMA. Where indicated, cyclosporin A (CyA) was added at 1 μg/mL. RT-PCR analysis for IL-25 and β-actin mRNA was performed as described in “Study design.” Shown are representative data from 4 independent experiments. (C) Real-time PCR analysis for the effect of cyclosporin A. BMMCs were incubated with anti-DNP IgE alone or anti-DNP IgE + DNP-HSA for one hour in the presence or absence of cyclosporin A (1 μg/mL). Real-time PCR analysis for IL-25 as well as GADPH (as a control) mRNA was performed, and the levels of IL-25 mRNA were normalized to the levels of GADPH mRNA. Data are means ± SD from 4 experiments. *Significantly different from the mean value of control responses (without cyclosporin A); P < .01. (D) Kinetics of IL-25 mRNA expression upon activation. BMMCs were incubated with anti-DNP IgE and then surface IgE was cross-linked with DNP-HSA. At indicated times after IgE cross-linking, total cellular RNA was prepared and RT-PCR for IL-25, TNF-α, and β-actin mRNA was performed. Shown are representative data from 4 independent experiments. (E) Real-time PCR analysis of IL-25 and TNF-α mRNA expression. Similar to panel D, BMMCs were stimulated with anti-DNP IgE + DNP-HAS, and total cellular RNA was prepared at indicated times after stimulation. Real-time PCR analysis for IL-25 (○) and TNF-α (●) as well as for GADPH (as a control) mRNA was performed. The levels of IL-25 or TNF-α mRNA were normalized to the levels of GAPDH mRNA. Data are means ± SD from 4 experiments. (F) Detection of IL-25 at protein levels. COS7 cells were transiently transfected with IL-25 expression vector (pME18S–IL-25) or control vector (pME18S), and these cell lysates as well as recombinant IL-17 (0.5 μg, Endogen, Woburn, MA) were subjected to immunoblotting with rabbit antisera to murine IL-25 or preimmune rabbit serum. (G) Mast cells release IL-25 upon activation. BMMCs were stimulated with and without A23187 + PMA for 3 hours. Culture supernatant was collected by centrifugation, concentrated with Microcon, separated on 12% SDS gel, and blotted with rabbit antisera to IL-25 or with preimmune rabbit serum. Shown is a representative blot from 4 independent experiments.

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