P2X7 regulation of macrophage tissue factor activity and microparticle generation.

Tissue factor (TF) is the high affinity receptor for factor VII (FVII)/VIIa [1]. It is expressed by cells surrounding blood vessels and in certain tissues and is required for hemostasis [2]. During Gram-negative bacterial infection, lipopolysaccharide (LPS) induces TF expression in circulating monocytes [3–5] resulting in local clot formation to immobilize the bacterium and limit the spread of the infection. LPS induction of TF gene expression in human monocytic cells has been studied in detail and requires the activation and nuclear translocation of various transcription factors, including AP-1, NF-κB, and Egr-1 [6–9]. TF expression is also increased during monocyte-macrophage differentiation [10]. However, LPS stimulation of both human monocyte-derived macrophages and mouse macrophages still increases TF expression [10–12]. LPS stimulated human monocytes, human monocytic cell lines, and mouse macrophages also release TF-positive microparticles (MPs) [13–17]. Furthermore, treatment of mice with LPS increases levels of circulating TF-positive MPs, and monocyte-derived TF-positive MPs are increased in human endotoxemia [18, 19]. Interestingly, ATP stimulation of the ligand-gated ion channel P2X7 purinoceptor on human dendritic cells also induces the release of TF-positive MPs [20]. A recent study analyzed the effect of ATP stimulation on TF activity and the release of TF-positive MPs from mouse bone marrow-derived macrophages (BMDMs) [17]. These cells were pretreated with interferon-γ (IFN-γ) and subsequently stimulated with LPS for 4 hours. The study showed that ATP increased cellular TF activity and the release of TF-positive MPs in a P2X7-dependent manner. It was proposed that ATP stimulation induced decryption of TF procoagulant activity [21]. However, the study did not compare the level of ATP-dependent enhancement of cellular TF activity with that observed with LPS alone or with the combination of LPS and ATP. Here, we determined the changes in cellular TF activity and release of TF-positive MPs in IFN-γ pretreated P2rx7−/− and wild-type (WT) mouse BMDMs with or without LPS and/or ATP. All procedures involving mice were approved by the Institutional Animal Care and Use Committee at UNC-Chapel Hill. Femurs were removed from 10–16 week old WT and P2rx7−/− mice [22]. Bones were sterilized in 70% ethanol for 3 minutes and then washed three times with phosphate-buffered saline (PBS). Bone marrow was flushed from bones using a 25 gauge sterile syringe with PBS. Red blood cells were lysed and 5 × 106 cells were plated in 100 mm tissue culture dishes in Dulbecco's Modified Eagle Medium with high glucose (DMEM-H) (Gibco, Carlsbad, CA) containing 10% fetal bovine serum (FBS, Omega Scientific, Tarzana, CA), 1% Pen/Strep (Cellgro, Manassas, VA), and Hepes (Cellgro). The next day, non-adherent cells were removed, pelleted, and re-suspended in DMEM-H containing 10% FBS, 1% Pen/Strep, and 25% L929 conditioned media. Cells were then plated on non-tissue culture treated 100 mm dishes. After 7–10 days BMDMs were collected using ice cold Versene (UNC Culture Facility, Chapel Hill, NC), counted, and re-seeded at a concentration of 5 × 105 cells onto 12-well tissue-culture treated plates (BD Falcon, San Jose CA). The next day, all cells were treated with 100 ng/mL IFN-γ (Peprotech, Rocky Hill, NJ) for 12 hours. Finally, cells were incubated with LPS (1 μg/mL) (Escherichia coli serotype O111:B4, Sigma Alrich, St.Louis, MO) or PBS for 4 hours. After 4 hours, cells were then treated with either ATP (5 mM final concentration, Roche Diagnostics, Indianapolis, IN) or sterile water for an additional 30 minutes. We found that lower concentrations of ATP did not induce the release of MPs from LPS stimulated cells (data not shown). Cell supernatants were collected and frozen at −80°C for later use. Cells were then washed, re-suspended in HBSA buffer (137 mM NaCl, 5.38 mM KCl, 5.55 mM glucose, 10 mM HEPES, 0.1% bovine serum albumin, pH 7.5), gently scraped from the plate, and placed on ice for subsequent analysis. Total factor Xa (FXa) generation was measured by incubating fresh cells with mouse FVIIa (5 nM, Novo Nordisk, Bagsvaerd, Denmark) and FX (150 nM, Haematologic Technologies, Essex Junction, VT) in the presence of either an anti-mouse TF antibody (1H1, 100 μg/mL) or rat IgG control (100 μg/mL, Sigma-Aldrich), followed by the addition of a FXa substrate (4.6 mM, FXa 8595, Centerchem, Norwalk, CT). Cellular TF activity was calculated by subtracting TF-independent FXa generation from the total FXa production. MP TF activity was measured using a two-stage clotting assay as described [18]. We found that LPS induced a dramatic increase in TF expression in WT and P2rx7−/− BMDMs, while ATP treatment alone had no effect on TF activity (Figure 1A). However, ATP enhanced LPS-induced TF activity in WT but not P2rx7−/− cells (Figure 1A). LPS or ATP alone did not increase the release of TF-positive MPs, whereas co-stimulation of WT cells with LPS and ATP induced a robust release of TF-positive MPs into the culture medium (Figure 1B). No TF-positive MPs were generated by P2rx7−/− cells co-stimulated with LPS and ATP (Figure 1B). Next, we determined if P2X7 contributed to the generation of TF-positive MPs in endotoxemic mice. WT and P2rx7−/− mice were injected with LPS (7.5 mg/kg) and levels of MP TF activity measured in the plasma as previously described [18]. No significant difference was detected in levels of circulating TF-positive MPs in WT versus P2rx7−/− mice 6 hours after LPS injection (Figure 1C). Figure 1 Role of P2X7 in cellular TF activity and the generation of TF-positive microparticles in vitro and in vivo Our study supports the conclusion that ATP enhances TF activity in LPS-stimulated macrophages in a P2X7-dependent manner, but importantly demonstrates that this increase in activity is relatively small compared to the increase observed with LPS alone. ATP stimulation of P2X7 in LPS treated cells induced a dramatic increase in TF-positive MPs released into the culture supernatant. However, no significant differences were observed in plasma MP TF activity between endotoxemic WT and P2rx7−/− mice, suggesting that this pathway does not play a major role in the generation of circulating procoagulant TF-positive MPs during endotoxemia.