Activation of endothelial cells by endotoxin: direct versus indirect pathways and the role of CD14.

Optimal activation of endothelial cells by nanomolar quantities of endotoxin (lipopolysaccharide, LPS) requires the presence of plasma or serum. We and others have demonstrated that soluble CD14 (sCD14) and LPS binding protein (LBP) were the key plasma proteins mediating endothelial cell responses to LPS. The role of LBP is to transfer LPS to sCD14 and newly formed LPS-sCD14 will in turn activate endothelial cells via an as yet unknown surface receptor. This plasma-dependent pathway of endothelial cells activation is referred as to the direct pathway. However, endothelial cells are in constant contact with whole blood and not only with plasma. In experiments where whole blood was substituted for plasma, we showed that endothelial cells became sensitive to picomolar, rather than nanomolar quantities of LPS. The fact that endothelial cell responses were amplified by the presence of whole blood prompted us to search for the responsible blood cell(s) and mediator(s) for this effect. Blood cell fractionation experiments, experiments with blood from PNH patients and the use of anti-CD14 antibodies pointed to the monocyte as the blood cell responsible for the amplification effect. Moreover, the blood effect could be entirely reproduced by cells from a CD14-expressing cell line, such as calcitriol-differentiated HL-60 cells. Inhibitors to TNF and to IL-1 blocked LPS-induced activation of endothelial cells partially when added separately to whole blood, but abrogated endothelial cell responses when added together. Thus, the whole blood effect begins with LPS activation of monocytes via cell membrane CD14 and results in endothelial cell activation by the effects of TNF and IL-1. The monokine-mediated endothelial cell activation is referred as to the indirect pathway.