CD3 activation causes loss of barrier function and enterocyte apoptosis in IKKβ(EE)^IEC mice. (a) Indicated mouse strains were analyzed 4 and 16 h after anti-CD3 (12.5 µg/mouse) administration. Jejunal sections analyzed at 4 h were stained with either H&E (top) or by in situ TUNEL assay (bottom). Jejunal sections analyzed at 16 h were H&E stained. Representative of four experiments. (b) Rectal temperature of WT (gray diamonds) and IKKβ(EE)^IEC (black squares) mice after anti-CD3 injection. Results are means ± SEM (n = 4). *, P < 0.05 versus WT. Data are representative of three independent experiments. (c) Lysates of small intestinal IECs were prepared 2.5 h after CD3 injection and analyzed (75 µg protein) for TNF content by ELISA. Results are means ± SEM (n = 4). *, P < 0.05 versus WT; # P < 0.05 versus time 0. Data are representative of three independent experiments. (d) RNAs from small intestinal WT or IKKβ(EE)^IEC IECs were prepared 1.5 h after CD3 injection and analyzed in triplicates by Q-RT-PCR for TNF mRNA amounts that were normalized to Hprt mRNA. Results are means ± SEM (n = 3). *, P < 0.05 versus WT; #, P < 0.05 versus time 0. Data are representative of three independent experiments. (e) WT and IKKβ(EE)^IEC enterocytes lysates prepared 40 and 60 min after anti-CD3 injection were analyzed for expression and phosphorylation of the indicated proteins by immunoblotting. Representative of three experiments. (f) Small intestinal enterocyte lysates (75 µg protein) were prepared 90 min after anti-CD3 injection (6.25 µg/mouse) in the absence or presence of RWJ 67657 and analyzed for TNF content by ELISA. Results are means ± SEM (n = 3). *, P < 0.05 versus IKKβ(EE)^IEC. Data are representative of three independent experiments.