Figure 2. Challenge of Footpads with E. coli induces a MC-TNF Dependent Increase in DC Numbers.

(A) Analysis of WT footpad, 3 hours after injection of E. coli or saline, for MHC Class II (DCs), CD31 (blood vessel endothelium), and avidin (MCs) by confocal microscopy. Scale bars = 100μM. E = epidermis, D = Dermis, LC = Langerhans cells (arrowheads). The arrow denotes a DC bundle. (B) Total number of CD11c⁺ DCs in single cell suspensions of footpads at the indicated time-points. N=4–5 for each time-point. ^*p<0.05 compared to saline controls. Data was analyzed for statistical significance with one-way ANOVA and Tukey’s post-test. (C) Total number of CD11c⁺ DCs in single cell suspensions of W/W^v, WT-MC or TNF^−/− MC repleted W/W^v footpads, 3 hours after saline or E. coli challenge. N=4 for each sample. ^*p<0.05 compared to saline controls and ^**p<0.05 comparing E. coli challenged WT-MC repleted W/W^v footpad samples to W/W^v and TNF^−/− MC-repleted W/W^v samples. Data was analyzed for statistical significance with one-way ANOVA and Tukey’s post-test. (D) The gating strategy to identify footpad (FP)-DC subsets. The table inset identifies each subset. (E) Total number of each FP-DC subset, regions 1–4, 3 hours after saline or E. coli challenge. N=6. ^*p<0.05 comparing E. coli challenged samples to saline controls within each FP-DC subset. Data was analyzed for statistical significance using unpaired Student’s t-test. Graphical bars represent the mean ±S.D.