Figure 3

Confocal microscopy analysis of the ability of sCD11/CD18 complexes to bind intercellular adhesion molecule 1 (ICAM-1) expressed on the human umbilical vein cell line EA.hy926. (A) Illustration of the cellular incubations. In step 1, adherent cells were stimulated with 10 ng/mL tumor necrosis factor-alpha (TNFα), which increased the ICAM-1 expression. In step 2, a source of CD11/CD18 was added (that is, either normal human serum (NHS) or supernatant from synovial fluid mononuclear cell culture). In step 3, biotinylated antibody recognizing ligand-binding activated CD11/CD18 (KIM127) was added followed by addition of fluorochrom-labelled streptavidin for detection using confocal microscopy. Binding of sCD18 to ICAM-1 expressed on EA.hy926 cells incubated with (B) or without (C) TNFα. Red staining indicates the binding of sCD18, further indicated with white arrows. The positions of cell nuclei were located by 4′,6-diamidino-2-phenylindole (DAPI) staining, indicated in blue. The staining was distinctly localized to small foci on the cell membrane on 10% to 15% of the cells. Expression of ICAM-1 on EA.hy926 cells incubated with (D) or without (E) TNFα. ICAM-1 is indicated with a green staining, and cell nuclei are indicated with a blue staining.