E-Selectin expression in HUVECs stimulated by aPLAs. HUVECs were preincubated for 24 hours with medium (HUVECs) or with 100 ng/mL of TNF (TNF-pretreated HUVECs), followed by an 8-hour washout with regular medium. (A) Cells were further incubated for 4 hours with 100 ng/mL of TNF, 1 μg/mL of LPS, 10 μg/mL of LTA, or medium alone. (B) Cells were further incubated for 4 hours with 500 μg/mL of aPLAs (n = 19), 100 μg/mL of β2GP1-immunopurified IgGs (n = 2), or 500 μg/mL of control human IgGs. Changes in E-selectin mRNA level were evaluated by qRT-PCR. Unstimulated HUVECs were taken as reference for basal E-selectin expression. Note that the cellular responses to 100 ng/mL of TNF or 1 μg/mL of LPS were similar in the TNF-pretreated cells and in the control cells, which shows that the cells had not become refractory to a subsequent inflammatory stimulus. The E-selectin mRNA level observed for TNF-pretreated HUVEC media conditions corresponded to the remaining effect of the 24-hour TNF stimulation followed by the 8-hour washout. Data are derived from 4 different cell preparations and are expressed as means ± SEM. (C) Cells were incubated for 30 minutes with TLR2- or TLR4-blocking antibodies, followed by a 4-hour incubation with 500 μg/mL of 6 aPLA preparations selected among the highest stimulators, 1 μg/mL of LPS, or 10 μg/mL of LTA for 4 hours. Changes in E-selectin mRNA levels were evaluated by qRT-PCR. The results are expressed as the ratio of the E-selectin response of TNF-pretreated HUVECs incubated with the blocking anti-TLR2 antibodies compared with the E-selectin response of TNF-pretreated HUVECs. Data are derived from 5 different HUVEC preparations, and are expressed as means ± SEM (n = 5).