Modulation of Monocyte Function by Activated Protein C, a Natural Anticoagulant

Abstract

Activated protein C is the first effective biological therapy for the treatment of severe sepsis. Although activated protein C is well established as a physiological anticoagulant, emerging data suggest that it also exerts anti-inflammatory and antiapoptotic effects. In this study, we investigated the ability of activated protein C to modulate monocyte apoptosis, inflammation, phagocytosis, and adhesion. Using the immortalized human monocytic cell line THP-1, we demonstrated that activated protein C inhibited camptothecin-induced apoptosis in a dose-dependent manner. The antiapoptotic effect of activated protein C requires its serine protease domain and is dependent on the endothelial cell protein C receptor and protease-activated receptor-1. In primary blood monocytes from healthy individuals, activated protein C inhibited spontaneous apoptosis. With respect to inflammation, activated protein C inhibited the production of TNF, IL-1β, IL-6, and IL-8 by LPS-stimulated THP-1 cells. Activated protein C did not influence the phagocytic internalization of Gram-negative and Gram-positive bioparticles by THP-1 cells or by primary blood monocytes. Activated protein C also did not affect the expression of adhesion molecules by LPS-stimulated blood monocytes nor the ability of monocytes to adhere to LPS-stimulated endothelial cells. We hypothesize that the protective effect of activated protein C in sepsis reflects, in part, its ability to prolong monocyte survival in a manner that selectively inhibits inflammatory cytokine production while maintaining phagocytosis and adherence capabilities, thereby promoting antimicrobial properties while limiting tissue damage.

Activated Protein C Up-Regulates IL-10 and Inhibits Tissue Factor in Blood Monocytes

“…We also observed that recombinant activated protein C increased IL-10 production in LPS-stimulated monocytes at the mRNA and protein levels, and required recombinant activated protein C serine protease activity as well as PAR-1 cleavage. The requirement of PAR-1 for this anti-inflammatory effect of recombinant activated protein C is consistent with previous studies demonstrating that recombinant activated protein C inhibits endothelial and monocyte apoptosis via PAR-1 (12,34,35,47,48). Interestingly, the recombinant activated protein C-mediated up-regulation of IL-10 in monocytes was not significantly affected by blocking recombinant activated protein C-EPCR interaction.…”

“…Human peripheral blood monocytes were isolated from the whole blood of healthy volunteers by MACS as previously described (12). The isolation and use of monocytes from healthy donors was voluntary, imposed minimal risk to the donor, and did not require approval of the Research Ethics Board of the Hamilton Health Sciences.…”

“…Quantification of IL-10 levels in monocyte cultures or plasma samples were determined using either the Human IL-10 ELISA Development kit (Biovision), as per manufacturer's instructions, or the CBA Human Inflammation kit (BD Biosciences) as previously described (12). Monocytes were cultured according to the conditions described above.…”

“…Activated protein C has been shown to exert direct anti-inflammatory effects on monocytes by inhibiting the production of proinflammatory cytokines. For example, in the THP-1 monocytic cell line and in peripheral blood monocytes, activated protein C down-regulates TNF (9 -11), IL-1␤, IL-6, and IL-8 (12), presumably by inhibiting NF-B nuclear translocation (10) and/or down-regulating the transcription of NF-B subunits (13). In vivo studies of animals challenged with endotoxin have shown that activated protein C prevents the production of proinflammatory cytokines (14,15) and inhibits leukocyte accumulation in injured tissues (16).…”

Activated protein C ligation of ApoER2 (LRP8) causes Dab1-dependent signaling in U937 cells

“…Monocytic cell lines, such as the human leukemic monoblast U937 cell line, have been used to study APC's ability to modulate monocyte apoptosis, phagocytosis, inflammation, and tissue factor generation (4,8,(32)(33)(34). To evaluate the potential relevance of ApoER2 for APC cell signaling mechanisms, APCinitiated signaling in U937 cells was assayed based on the hypothesis that APC ligation of ApoER2 would signal similarly to the Reelin signaling pathway wherein ApoER2 ligation by Reelin promotes signaling via phosphorylation of Dab1, which binds to an NPxY sequence of the intracellular C-terminal region of ApoER2 (21)(22)(23)26 Fig.…”