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-1β, 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.
KeywordsTissue Factor Tissue Factor Expression Median Fluorescence Intensity Tissue Factor Activity Tissue Factor mRNA
The technical assistance of Grethe Bergseth is greatly acknowledged. This work was supported by NIH grants AI-068730 and GM 62134 and grants from Helse Nord RHF.
Conflict of Interest Statement The author Professor J.D. Lambris has submitted several patent applications on complement inhibitors. None of the other authors have conflicts of interest.
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