Disturbance of IL-1α-induced down-regulation of IL-18 mRNA expression in EP–/– mice-derived keratinocytes. (A) Wound healing in WT and EP–/– mice. A full-thickness skin excision (6 mm in diameter) was made on the back of WT (open circles; n = 10) and EP–/– (filled circles; n = 10) mice. Values indicate the mean diameter ± SD. (B) Proliferation of primary epidermal keratinocytes from WT (open bars) and EP–/– (filled bars) mice. Keratinocytes (4 × 104 per well in a 96-well plate) were cultured, and cell proliferation at the indicated times was determined by [3H]thymidine incorporation. Data shown in triplicate are the mean ± SD. (C and D) Down-regulation of IL-18 mRNA expression by IL-1α (C) and EP (D) in primary keratinocytes. Keratinocytes (2 × 105 per well in a 24-well plate) were stimulated with 50 ng·ml–1 recombinant mouse IL-1α (C) and 100 ng·ml–1 rmEP (D) for the indicated time, and the expressions were determined by using RT-PCR. Experiments were repeated five times with similar results. (E) Down-regulation of IL-18 expression by EP depends on EGFR. Keratinocytes (2 × 105 per well in a 24-well plate) were preincubated with 2 μM of PD168393 (Calbiochem), 2 μM of AG1296 (Calbiochem), or vehicle for 30 min, followed by incubation with 100 ng·ml–1 rmEP in culture medium for 12 h. The expressions were determined by using RT-PCR.