Figure 4

Adhesion of mononuclear leukocytes to activated human umbillical vein endothelial cells. (A): HUVEC were incubated with TNF-α for 6 hr and then stained for CD54, CD62E and CD106 by secondary immunofluorescence (FITC-conjugated secondary antibody). CD54, CD62E and CD106 were all induced by TNF-α, and were present in the plasma membrane and around the nuclei (stained blue by DAPI). IgG₁ with an irrelevant specificity (isotype control) did not give staining of the cells. Scale bar, 50 μm. (B): Mononuclear leukocytes from two individuals (black bars: individual 1; white bars: individual 2) were stained with BCECF-AM, given 10 or 100 ng/ml of both MCP-1 and MIP-1α, and then hydrodynamically guided in 500-μm-wide lanes at 10 nl/s (0.75 dyn/cm²) for 10 min over a confluent monolayer of TNF-α-activated HUVEC. HUVEC were washed (15 dyn/cm²), and the number of adherent blood cells was counted. Chemokine-activated mononuclear leukocytes did not adhere to non-activated HUVEC or to the supporting polystyrene slides (not shown). (C): In three different experiments, mononuclear leukocytes from individual 1 were mixed with MCP-1 and MIP-1α (both at 10 ng/ml), and aliquots of this leukocyte-chemokine mixture were applied to TNF-α-activated HUVEC immediately or after a 10, 20, 30 or 40 min preincubation at 37°C. The leukocytes were guided over HUVEC in 200-μm-wide lanes at 4.0 nl/s (0.75 dyn/cm²) for 5 min, and had thus been in contact with the chemokines for 0–5, 10–15, 20–25, 30–35 or 40–45 min, as indicated on the x-axis. HUVEC were washed (15 dyn/cm²), and the number of adherent blood cells was counted (counts are normalized because different counting frames were used). Mononuclear leukocytes activated with chemokines for any of the exposure times did not adhere to non-activated HUVEC (not shown).