TNFα increases anti-ICAM binding but not internalization by endothelial and mesothelioma cells.

(A) FACS analysis using anti-ICAM and anti-TM. TNFα suppresses expression of thrombomodulin (TM, upper panels) by HUVECs and stimulates that of ICAM-1 (lower panels) by HUVEC and REN cells. Dashed line: antibody-free medium; resting (open histogram) or TNFα-challenged (shaded histogram) cells. (B)¹²⁵I–anti-ICAM binding to resting and TNFα-treated HUVEC monolayer. The data are shown as means ± SD, n = 4. (C) Fluorescent micrographs (× 60) of TNFα-stimulated cells incubated with anti-ICAM. The cells were incubated at 4°C or 37°C with anti-ICAM (panels i-viii), antibody-free medium (ix, x) or transferrin (xi, xii). After washing and fixation the cells were sequentially stained with Texas Red secondary antibody, permeabilized, and counterstained with FITC-labeled secondary antibody (yellow, surface-bound anti-ICAM; green, internalized anti-ICAM). On the left, the nonpermeabilized cells were stained with both Texas Red and FITC-labeled antibodies (positive control for surface staining, yellow color). Green color corresponds to the intracellular staining (see panels xi and xii showing staining of HUVECs incubated with fluorescein-labeled transferrin). Insets of subpanels ix and x show phase contrast images in controls. Original magnification, × 60; insets minimized to $19$ of original size.