Figure 4
Antibody (Ab)-mediated crosslinking of intercellular adhesion molecule-1 (ICAM-1) leads to an myosin light-chain kinase (MLCK)-dependent increase in epithelial permeability. T84 intestinal epithelial cells (IECs) grown on permeable supports were stimulated with interferon-γ (IFNγ) (100 U ml−1, 24 h) to induce ICAM-1 expression. ICAM-1 or control surface protein major histocompatibility complex-1 (MHC-1) were crosslinked by incubation with primary Ab (20 μg ml−1, 60 min) followed by appropriate secondary antibodies (20 μg ml−1, 30 min). TER (a) and flux of fluorescein isothiocyanate (FITC)-dextran (3 kDa) (b) across T84 monolayers before and after ICAM-1 crosslinking were quantified at the indicated time points as an index of epithelial permeability. A pharmacological inhibitor of MLCK, ML-7 (20 μM), was introduced an hour before initiation of the crosslinking protocols. ICAM-1 ligation resulted in decreased TER and increased flux of FITC-dextran, and was dependent on MLCK phosphorylation. (c) Representative western blottings and densitometric analysis (using ImageJ) show increased MLCK phosphorylation after ICAM-1 crosslinking, which was reversed with the addition of ML-7. (d) Confocal microscopy and immunofluorescence labeling were used to examine actin remodeling after crosslinking of apically expressed control receptor MHC-1, and specifically ICAM-1, in the presence or absence of ML-7 (20 μM). Ab-mediated crosslinking of ICAM-1 but not MHC-1 resulted in decreased apical brush border and junctional actin. This effect was reversed in the presence of ML-7. Bar = 50 μm. N=4 independent experiments in triplicates (a,b), *P<0.05, **P<0.01 significantly different from control (a), **P<0.01, ***P<0.01, n.s. not significant, (b,c), analysis of variance with Newman–Keuls multiple comparison test.
