Figure 3

hCMEC/D3 cells produced chemokines and membrane ICAM-1 after TWEAK exposure. (A) ELISA analysis of CCL-2, IL-8, Il-6, and IL-10 levels in the supernatants of hCMEC/D3 differentiated cells stimulated with TWEAK or TNF for 24 h or not (CTRL); all samples were analyzed in triplicates. The detection threshold was 16 pg/ml of cytokine. Note the increased secretion of CCL-2, Il-8, and Il-6. (B) Primary HCMECs were stimulated with Fc-TWEAK or its isotype control P1.17 for 24 h or not (CTRL). Supernatants were collected and Il-6, IL-8, and CCL-2; levels were evaluated by ELISA. All samples were analyzed in triplicate. The detection threshold was 16 pg/ml of cytokine. (C) FACS analysis of membrane expression of ICAM-1 and E-selectin in differentiated hCMEC/D3 and HUVEC cells stimulated with TWEAK or TNF for 24 h or not (CTRL). Cells were incubated with anti-human ICAM-1 and E-selectin or isotype control fluorescein-conjugated antibodies. TWEAK induces ICAM-1 labeling at the membrane of hCMEC/D3 cells. In (A) and (B), * p < 0.05 according to Student’s t test.