The Effects of Selective Complement and CD14 Inhibition on the E-coli-Induced Tissue Factor mRNA Upregulation, Monocyte Tissue Factor Expression, and Tissue Factor Functional Activity in Human Whole Blood

Background : The complement pathway and CD14 play essential roles in inflammation, but little is known about the relative roles of complement and CD14 in E. coli-induced tissue factor (TF) mRNA upregulation, expression by monocytes, and functional activity in human whole blood. Methods : Whole E. coli bacteria were incubated for up to 4 h in human whole blood containing the anticoagulant lepirudin, which does not affect complement activation. TF mRNA levels were analyzed using reverse transcription, quantitative real-time PCR (RT-qPCR), and the expression of TF on the cell surface was analyzed using flow cytometry. Complement was selectively inhibited using the C3 convertase inhibitor compstatin or a C5a receptor antagonist (C5aRa), while CD14 was blocked by an anti-CD14 F(ab')(2) monoclonal antibody. Results : The E. coli-induced TF mRNA upregulation was reduced to virtually background levels by compstatin, whereas anti-CD14 had no effect. Monocyte TF expression and TF activity in plasma microparticles were significantly reduced by C5aRa. Anti-CD14 alone only slightly reduced E. coli-induced monocyte TF expression but showed a modest additive effect when combined with the complement inhibitors. Inhibiting complement and CD14 efficiently reduced the expression of the E. coli-induced cytokines IL-1 beta, IL-6, IL-8, and platelet-derived growth factor bb. Conclusion : Our results indicate that E. coli-induced TF mRNA upregulation is mainly dependent on complement activation, while CD14 plays a modest role in monocyte TF expression and the plasma TF activity in human whole blood.