Fig. 4

Localization of ICAM-1 on T84 monolayers and natural human colonic epithelium by immunofluorescence

(A–F) T84 monolayers cultured on permeable supports were stimulated with IFNγ (1000 U/ml, 48 hr), fixed with paraformaldehyde, permeabilized with Triton X-100, followed by labeling with primary antibody as described in Materials and Methods. Primary antibodies included anti-ICAM-1 (R6.5), control normal mouse IgG, and anti-β1 integrin, an epithelial subjunctional adhesion molecule as a basolateral binding control (52). Shown are x-y (en face) and x-z computer reconstructed confocal fluorescence micrographs. (A and B) the x-y (en face) immunofluorescence on the apical surface after staining for ICAM-1 on IFNγ stimulated and control, unstimulated T84 monolayers, respectively. (C and D) the x-z reconstructed immunofluorescence staining pattern for ICAM-1 following IFNγ stimulation (C) or in the absence of IFNγ stimulation (D). (E and F) As controls for basolateral labeling, the immunofluorescence staining patterns for β1 integrin in the mid-zone (x-y) of the monolayer and as an x-z reconstructed image, respectively. As can be seen in Panel B, there is no detectable ICAM-1 immunofluorescence in the absence of IFNγ stimulation. However, there is bright apical ICAM-1 immunofluorescence after IFNγ stimulation (A and C). Treatment of monolayers with normal mouse IgG (not shown) resulted in no detectable immunofluorescence, identical to that shown in Panel D. (G–I) Three-micron-thick frozen sections of human colonic epithelium involved with active inflammatory bowel disease. Sections were air dried, fixed with paraformaldehyde, and fluorescently labeled for ICAM-1 (R6.5, 3 µg/ml). (G and I) The ICAM-1 staining in active Crohn’s disease with focal staining of the apical membrane and sub-epithelial inflammatory cells (to the right of the closed arrows): L, lumen; E, epithelium; closed arrow, the basal pole of the epithelium which abuts the basement membrane. (H) For orientation, the Nomarski image corresponding to that shown in Panel G. In data not shown, there was no significant ICAM-1 staining of epithelium in noninflamed colon sections or with irrelevant IgG. Scale bars in Panels A and G = 20 µ.