Induction of tissue factor expression in human monocyte/endothelium cocultures

Abstract

Induction of tissue factor (TF) expression on monocytes and endothelial cells is central to the development of septic coagulopathy. Serum concentrations of endotoxin in septic patients who develop disseminated intravascular coagulation (DIC) do not, however, reach the levels that would directly stimulate TF expression on either monocytes or endothelium. We show, using an in vitro coculture system, that the interaction of monocytes with endothelium induces the expression of significant levels of TF. Unstimulated cocultures of monocytes (2 x 10(4)/well) and endothelial cells (2 x 10(4)/well) produced 35.3 +/- 8.5 mU of PCA/well, representing a 5-fold increase over the combined PCA of each cell type cultured alone (7.1 +/- 1.5 mU, n = 6, P < 0.001). Significant enhancement was also found in the presence of low concentrations of LPS. Induction of TF protein was confirmed by Western blotting. Fixation of monocytes with paraformaldehyde completely abolished TF induction in cocultures, whereas fixation of endothelium had no effect, suggesting that TF induction occurred in monocytes rather than endothelial cells. Induction of TF in cocultures could be further augmented by preincubating the endothelial cells with IFN-gamma. When endothelium was prestimulated with 500 U/ml IFN-gamma there was 142 +/- 11% increase over unstimulated cocultures (n = 5, P < 0.01). TF induction was inhibited by 32 +/- 6% in the presence of anti-ICAM-1 mAb (n = 5, P < 0.01). Our results suggest that monocyte interactions with vascular endothelium, regulated by inflammatory cytokines, and mediated by adhesive ligand binding, leads to the induction of functional monocyte TF protein, which may be responsible for the initiation of DIC in sepsis.

Monocytes Augment Bacterial Species- and Strain-Dependent Induction of Tissue Factor Activity in Bacterium-Infected Human Vascular Endothelial Cells

“…These differences in the activation status of EC, and consequently that of the monocytes bound to these EC, most likely account for the discrepancies in the amount of TFA, the kinetics of TFA induction, and the cell type(s) expressing this TFA that are described in these reports. Consistent, however, with observations by Napoleone et al (30) and Collin et al (14), our present data show that values for TFA expressed by nonstimulated EC or monocytes before coculture were almost negligible but became slightly elevated during 24 h of coculture. In addition, we show that increased adhesion of monocytes to EC stimulated with rIL-1 for 24 h did not generate an increase in TFA during subsequent coculture.…”

“…This, however, awaits further investigation. The ability of monocytes to induce PCA during incubation with cultured human EC has been documented previously by several research groups (14,23,24,30,46). These studies have been performed according to different experimental procedures with confluent or subconfluent cultures of either cytokine-activated or quiescent human EC.…”

Interaction of endothelial microparticles with monocytic cells in vitro induces tissue factor–dependent procoagulant activity

“…32 Cell-cell interactions are known to represent an important mechanism for TF regulation in monocytic cells, as shown by coculture of monocytes with stimulated endothelial cells. 33,34 In a similar way, our results demonstrated that the procoagulant properties of THP-1 cells were amplified by contact with EMPs. Two /mL), or supernatant free of EMP for 15 minutes and 4 hours and then were prepared for assessment of procoagulant activity using clotting assay.…”

Dose-Dependent Modulation of Tissue Factor Protein and Procoagulant Activity in Human Monocyte-Derived Macrophages by Oxidized Low Density Lipoprotein

“…Cell pellets were lysed at room temperature with lysis buffer as described 20) . Samples were then sonicated by 30 icechilled 1-sec medium-intensity bursts with a model 60 Sonic Dismembrator (Fisher Scientific, Pittsburgh, PA).…”