Endothelial protein C receptor is overexpressed in rheumatoid arthritic (RA) synovium and mediates the anti-inflammatory effects of activated protein C in RA monocytes

Objectives

(1) To investigate whether inflammatory synovial tissues from patients with rheumatoid arthritis (RA) express endothelial protein C receptor (EPCR) and (2) to determine the major cell type(s) that EPCR is associated with and whether EPCR functions to mediate the effects of activated protein C (APC) on these cells.

Methods

EPCR, CD68 and PC/APC in synovial tissues were detected by immunostaining and in situ PCR. Monocytes were isolated from peripheral blood of patients with RA and treated with APC, lipopolysaccharide (LPS), and/or EPCR blocking antibody RCR252. Cells and supernatants were collected for RT‐PCR, western blotting, enzyme‐linked immuosorbent assay and chemotaxis assay.Results: EPCR was expressed by both OA and RA synovial tissues but was markedly increased in RA synovium. EPCR was colocalised with PC/APC mostly on CD68 positive cells in synovium. In RA monocytes, APC upregulated EPCR expression and reduced monocyte chemoattractant protein‐1‐induced chemotaxis of monocytes by approximately 50%. APC also completely suppressed LPS‐stimulated NF‐κB activation and attenuated TNF‐α protein by more than 40% in RA monocytes. The inhibitory effects of APC were reversed by RCR252, indicating that EPCR is required.

Conclusions

Our results demonstrate for the first time that EPCR is expressed by synovial tissues, particularly in RA, where it co‐localises with PC/APC on monocytes/macrophages. In addition, APC inhibits the migration and activation of RA monocytes via EPCR. These inhibitory effects on RA monocytes suggest that PC pathway may have a beneficial therapeutic effect in RA.Rheumatoid arthritis (RA) is a chronic autoimmune disease with persistent inflammation of multiple synovial joints, which results in progressive tissue destruction of bone and cartilage.^1,2 It is characterised by the infiltration of inflammatory cells (neutrophils, monocytes and lymphocytes) into the synovial compartment and the production of inflammatory mediators. In RA, monocytes migrate into the synovium to become activated macrophages where they secrete significant amounts of inflammatory cytokines such as interleukin (IL)‐1, tumour necrosis factor (TNF)‐α and proteases, which are important in initiating, propagating and maintaining synovial inflammation.³ Macrophages can also differentiate into dendritic cells and osteoclasts,⁴ the latter being recognised as the key cellular effectors of pathological bone erosion in arthritis.⁵ In rheumatoid synovial sections, most synovial lining cells are highly activated macrophage‐like cells functioning as antigen‐processing and antigen‐presenting cells to T lymphocytes.⁶ Macrophages are critically involved in the pathogenesis of RA, not only by producing a variety of pro‐inflammatory cytokines and chemokines, but also by contributing to the cartilage and bone destruction.Activated protein C (APC) is a 61‐kDa serine protease derived from its vitamin K‐dependent plasma precursor, protein C (PC). Activation of PC occurs on the endothelial cell surface and is triggered by a complex formed between thrombin and thrombomodulin. The conversion to APC is augmented in the presence of its specific receptor, endothelial protein C receptor (EPCR),⁷ which is expressed on the surface of endothelial cells, keratinocytes⁸ and some leucocytes, including eosinophils, neutrophils and monocytes.⁹APC acts as an anticoagulant by neutralising the procoagulant activities of factors Va and VIIIa and inhibiting thrombin generation. In addition, APC exerts significant anti‐inflammatory properties, associated with a decrease in pro‐inflammatory mediators and a reduction of leucocyte recruitment.¹⁰ Many anti‐inflammatory properties of APC are mediated through EPCR, which itself can independently exert anti‐inflammatory effects.^11,12,13 For example, severe EPCR deficiency adversely affects survival and cardiac function of mice subjected to challenge by endotoxin infusion.¹³ Baboons treated with an antibody to block PC binding to EPCR respond lethally to normally sublethal concentrations of E coli and exhibit disseminated intravascular coagulation, intense neutrophil influx into the tissues and elevation of inflammatory cytokines, indicating that EPCR provides a critical step in the host defense against E coli.¹² Over expression of EPCR protects transgenic mice from endotoxin‐induced injury.¹⁴ In addition, recent findings suggest that EPCR is required for embryo survival and development.^15,16,17PC/APC is elevated in RA synovial fluid and synovial joints, where it co‐localises with MMP‐2.¹⁸ However, whether EPCR is present in the inflammatory joint is unknown. The purpose of this study was: (1) to determine whether inflammatory (RA) synovial tissue expresses EPCR and if so whether these levels are higher than non‐inflammatory OA synovial tissue; and (2) to elucidate the major cell type(s) EPCR is associated with and whether it functions to mediate the effects of APC on these cells.