Figure 6
Engagement of intercellular adhesion molecule-1 (ICAM-1) in murine intestine in vivo leads to myosin light-chain kinase (MLCK)-dependent increase in intestinal permeability. To induce ICAM-1 expression, mice were injected with a mixture of interferon-γ (IFNγ) and tumor necrosis factor-α (TNFα; 500 ng each, 24 h, intraperitoneally (i.p.)). (a) OCT-frozen segments of mouse intestine were sectioned (7 μm wide) and immunofluorescently stained for ICAM-1 (green) and the tight junction protein Claudin-2 (red). Representative confocal images show apical induction of ICAM-1 expression (white arrow) after IFNγ/TNFα treatment (bottom panel) compared with non-activated tissue (upper panel). Bar = 50 μm. (b) Cartoon depicts the mouse intestinal loop model as described in Methods. (c) In-vivo intestinal epithelial permeability was measured under the specified conditions as described in Methods. ICAM-1 tail peptide (100 μm ml−1) was introduced luminally 30 min before ICAM-1 crosslinking protocols. MLCK inhibitor ML-7 (20 μM) was introduced by i.p. injection 2.5 h before ICAM-1 crosslinking. Increased fluorescein isothiocyanate (FITC)-dextran flux after antibody (Ab) crosslinking of ICAM-1 was prevented with both the addition of ICAM-1 tail peptide and pretreatment with ML-7. N=4 mice per condition, *P<0.05, analysis of variance with Newman–Keuls multiple comparison test.
