(A) Skin of C57BL/6 mice was exposed to AFL and excised 24 hours later for side-by-side histological (left) and apoptosis analyses (right). Brown indicates apoptosis signal (*). Scale bar: 100 μm. (B) Skin of C57BL/6 mice was exposed to AFL followed by subcutaneous injection of DRAQ7. Skin was excised 30 minutes later, cryosectioned, and imaged. Dashed curved line: edges of skin MCs. Arrows: skin MCs. Scale bar: 100 μm. (C) C57BL/6 mice were exposed to AFL followed by ID injection of DNase I (2500 units) or BSA (280 μg) or exposed to AFL or sham treatment. Skin was dissected 6 hours later for real-time PCR analysis. (D) Skin of BALB/c mice was exposed to AFL or sham treatment. Skin was dissected 6 and 24 hours later for Western blotting. (E) WT and NLRP3-KO mice were subjected to AFL or sham treatment. Skin was dissected 24 hours later for Western blotting. (F) Relative band intensities of IL-1β to GAPDH were compared. (G) BALB/c mice were subjected to AFL treatment followed by ID injection of DNase I (2500 units) or BSA (280 μg) or subjected to AFL or sham treatment. Thirty minutes later, 10 μg OVA was intradermally injected in all groups. (H) C57BL/6 mice were subjected to AFL or sham treatment followed by ID injection of 10 μg OVA or subjected to AFL treatment followed by ID injection of 10 μg OVA mixed with 10 μg anti–IL-1β antibodies or isotype control. The latter 2 groups were also intraperitoneally injected with 200 μg anti–IL-1β antibodies or isotype control 1 hour before and 18 hours after immunization. (I) WT and NLRP3-KO mice were subjected to AFL or sham treatment followed by ID injection of 10 μg OVA. Serum anti-OVA antibody titer in G–I was measured 3 weeks later. n = 4 (C), n = 4–5 (G–I). One-way ANOVA with Newman-Keuls multiple-comparison test was used to compare differences between groups (C, G, and H). One-tailed Mann-Whitney U test was used to compare differences between groups (F and I). *, P < 0.05; **, P < 0.01; ***, P < 0.001.