LPC aggravated DNFB-induced skin inflammation. (A) Experimental design. We sensitized WT mice with 0.5% DNFB on d0 and d1, and then challenged them with 0.2% DNFB on d5. During the whole period, we injected LPC s.c. into the mice. The extent of CHS is shown as the increase of the ear thickness (ear swelling, Δ), which we calculated by subtracting the ear thickness of the treated mice (DNFB sensitization and DNFB challenge) from that of the control mice (acetone sensitization and DNFB challenge: representing the non-specific irritation). (B) Ear thickness results over time. We pooled data from five independent experiments. The difference of ear swelling between BSA and LPC was statistically significant on d7 and d8. (C) Ear thickness results on d7. We pooled data; each circle represents a single mouse. (D) FACS analysis of the ear skin. The percentages of hematopoietic cells (CD45+, left), macrophages (CD11b+F4/80+, middle) and neutrophils (CD11b+Ly6G+, right) are shown. We pooled data; each circle represents a single mouse. (E) RT-qPCR analysis on the expressions of CXCL1 and CXCL2 in ear skin. We pooled data; each circle represents a single mouse. (F) We treated naïve mice with anti-Ly6G mAb to deplete neutrophils and then sensitized and challenged them with DNFB. The ear thickness results are shown. Data are representative of two independent experiments, and each circle represents a single mouse. Data are presented as the mean ± SD. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.