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

Adv Exp Med Biol. 2013;735:123-36. doi: 10.1007/978-1-4614-4118-2_8.

Abstract

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-1beta, 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 CDI4 plays a modest role in monocyte TF expression and the plasma TF activity in human whole blood.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Anticoagulants / pharmacology
  • Anticoagulants / therapeutic use
  • Complement Inactivating Agents / therapeutic use*
  • Complement System Proteins / metabolism
  • Cytokines / blood
  • Dose-Response Relationship, Drug
  • Enzyme-Linked Immunosorbent Assay
  • Escherichia coli Infections / drug therapy*
  • Escherichia coli Infections / metabolism*
  • Flow Cytometry
  • Hirudins / pharmacology
  • Humans
  • Lipopolysaccharide Receptors / drug effects*
  • Monocytes / drug effects
  • Monocytes / metabolism*
  • RNA, Messenger / biosynthesis*
  • RNA, Messenger / genetics
  • Real-Time Polymerase Chain Reaction
  • Recombinant Proteins / pharmacology
  • Recombinant Proteins / therapeutic use
  • Sepsis / drug therapy
  • Sepsis / microbiology
  • Thromboplastin / biosynthesis*
  • Up-Regulation / drug effects

Substances

  • Anticoagulants
  • Complement Inactivating Agents
  • Cytokines
  • Hirudins
  • Lipopolysaccharide Receptors
  • RNA, Messenger
  • Recombinant Proteins
  • Complement System Proteins
  • Thromboplastin
  • lepirudin