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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion and conclusion
  7. Acknowledgment
  8. References

Protease-activated receptors (PARs) are stimulated by proteolytic cleavage of their extracellular domain. Coagulation proteases, such as FVIIa, the binary TF-FVIIa complex, free FXa, the ternary TF-FVIIa-FXa complex and thrombin, are able to stimulate PARs. Whereas the role of PARs on platelets is well known, their function in naïve monocytes and peripheral blood mononuclear cells (PBMCs) is largely unknown. This is of interest because PAR-mediated interactions of coagulation proteases with monocytes and PBMCs in diseases with an increased activation of coagulation may promote inflammation. To evaluate PAR-mediated inflammatory reactions in naïve monocytes and PBMCs stimulated with coagulation proteases. For this, PAR expression at protein and RNA level on naïve monocytes and PBMCs was evaluated with flow cytometry and RT-PCR. In addition, cytokine release (IL-1β, IL-6, IL-8, IL-10, TNF-α) in stimulated naïve and PBMC cell cultures was determined. In this study, it is demonstrated that naïve monocytes express all four PARs at the mRNA level, and PAR-1, -3 and -4 at the protein level. Stimulation of naïve monocytes with coagulation proteases did not result in alterations in PAR expression or in the induction of inflammation involved cytokines like interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8, interleukin-10 or tumour necrosis factor-α. In contrast, stimulation of PBMCs with coagulation proteases resulted in thrombin-mediated induction of IL-1β and IL-6 cytokine production and PBMC cell proliferation in a PAR-1-dependent manner. These data demonstrate that naïve monocytes are not triggered by coagulation proteases, whereas thrombin is able to elicit pro-inflammatory events in a PAR-1-dependent manner in PBMCs.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion and conclusion
  7. Acknowledgment
  8. References

The coagulation cascade consists of several serine proteases, including the coagulation proteases Factor VIIa (FVIIa), Factor Xa (FXa) and the main effector protease thrombin [1]. Formation of the tissue factor-factor VIIa (TF-FVIIa) complex is the major physiological trigger for thrombin generation and blood coagulation. The TF-FVIIa complex binds and cleaves the zymogen factor X (FX) to FXa, the active protease. FXa in turn binds its cofactor factor Va, and this prothrombinase complex cleaves prothrombin (FII) to active thrombin (FIIa) the main effector protease [2].

In addition to maintaining normal haemostasis, studies revealed an additional role of coagulation proteases in cell signalling [3]. First, TF as the principal initiator of the coagulation cascade belongs to the cytokine receptor superfamily and recently it becomes of great interests for its ability to prevent apoptosis, to induce cell proliferation and is possible role in cytokine production after its activation by FVIIa [4]. Second, coagulation proteases are able to function as signalling molecules through the activation of specialized G-protein coupled receptors called proteinase-activated receptors (PARs). To date, four PARs have been identified (PAR-1-4) [5-8]. PARs have been detected in numerous cell types including neutrophils, monocytes, macrophages and T cells [9-12].

The unique mechanism whereby serine proteases signal via PARs involves the cleavage of the receptor N-terminal exodomain at a specific site [5]. This cleavage unmasks a new N terminus that subsequently serves as a tethered ligand. The tethered ligand acts as a receptor-activating ligand, resulting in PAR activation.

The role of FVIIa, the binary TF-FVIIa complex, free FXa, the ternary TF-FVIIa-FXa complex and thrombin in PAR-mediated cell signalling has been investigated in different (monocyte) cell lines. In these studies, it was demonstrated that FVIIa, in the presence of TF-expressing cells, as well as the binary TF-FVIIa complex and the combination of soluble TF and FVIIa are able to activate PAR-2 [13-15]. More downstream the coagulation cascade, free FXa and FXa, generated in the TF-initiated coagulation and bound in the ternary TF-FVIIa-FXa complex were found to activate both PAR-1 and PAR-2 [13, 16, 17]. In these studies, it appeared that free FXa and the binary TF-FVIIa complex are much less efficient in PAR activation in comparison with FXa bound in the ternary complex [13]. Finally, thrombin as the main effector protease of the coagulation cascade was found to be able to activate PAR-1, PAR-3, and PAR-4 [18].

In general, activation of PARs with coagulation proteases results in alterations in gene regulation, induction of cell proliferation and cell migration, angiogenesis, and IL-1ß, IL-6, and IL-8 cytokine production [13, 18-21]. Indeed, it is known that coagulating whole blood results in the production of IL-6 and IL-8 and that administration of FVIIa in healthy human subjects results in the release of IL-6 and IL-8 [12].

It is assumed that monocytes and PBMCs play an integral part in both coagulation and inflammation. Furthermore, monocytes express at mRNA level PAR-1 and PAR-3, little PAR-2, and no PAR-4, and at protein level PAR-1, PAR-3 and PAR-4 [10, 12]. Therefore, several of the above-referred studies investigated PAR-mediated cross-talking in monocytes. However, contradicting results have been found, and in most of the above studies, cell lines, or artificially preactivated monocytes and PBMCs or supraphysiological concentrations of coagulation proteases have been used to study the effects of coagulation proteases for potential PAR-mediated inflammatory properties [22].

As such it remains unclear what the immune modulating effects of coagulation proteases are in naïve human monocytes and naïve PBMC cell cultures. This is of interest for diseases, such as systemic infections, rheumatoid arthritis and osteoarthritis, which are associated with an increased activation of coagulation and the presence of physiological concentrations of coagulation proteases, which may contribute to pro- or anti-inflammatory responses in a PAR-dependent manner.

Therefore, in this study, it was investigated whether coagulation proteases (FVIIa, the binary TF-FVIIa complex, the binary TF-FVIIa complex with free FX, free FX, free FXa and thrombin) in physiological concentrations can elicit pro- or anti-inflammatory responses in a PAR-dependent manner in naïve (non-preactivated) human monocytes and PBMCs.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion and conclusion
  7. Acknowledgment
  8. References

Materials

Ficoll-Paque was purchased from Pharmacia (Uppsala, Sweden) and CD14 microbeads from Miltenyi Biotec (Bergisch Gladbach, Germany). Dulbecco's modified Eagle's medium (DMEM) was obtained from Invitrogen (Carlsbad, CA, USA). Heat-inactivated human male AB serum was from Sigma-Aldrich (St. Louis, MO, USA). Allophycocyanin (APC)-conjugated monoclonal mouse anti-human CD14 antibody and APC-conjugated isotype control antibody were from BD Biosciences (Franklin Lakes, NJ, USA). Phycoerythrin (PE)-conjugated monoclonal mouse anti-human PAR-1 (ATAP2) antibody, FITC-conjugated monoclonal mouse anti-human PAR-2 (SAM11) antibody, PE-conjugated monoclonal mouse anti-human PAR-3 (8E8) antibody, and APC-, PE- and FITC-conjugated isotype control antibodies were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). FITC-conjugated polyclonal rabbit anti-human PAR-4 (APR-034-F) antibody was obtained from Alomone Labs (Jerusalem, Israel). PE-conjugated monoclonal mouse anti-human TF (HTF-1) antibody and PE-conjugated isotype control antibody were from BD Biosciences. Recombinant human FVIIa was kindly provided by Novo Nordisk A/S (Maaloev, Denmark). Recombinant human tissue factor (4500L), human factor X (527) and human activated factor X (526) were purchased from American Diagnostica Inc. (Stamford, CT, USA). Human alpha thrombin factor IIa (IHT; activity ≥2700 NIH units/mg) was obtained from Innovative Research (Novi, USA). The activity of the purchased coagulation proteases was tested positive in coagulation assays before use. Purified LPS was purchased from Sigma-Aldrich. PAR-1 antagonist FR171113 was obtained from Tocris Bioscience (Bristol, UK). FR171113 is a highly purified (>98%) specific PAR-1 antagonist which is able to inhibit thrombin-induced platelet aggregation. Interleukin-1β (IL-1β), Interleukin-10 (IL-10) and tumour necrosis factor-alpha (TNF-α) enzyme-linked immunosorbent assay (ELISA) kits were from Invitrogen. Interleukin-6 and IL-8 ELISA kits were obtained from eBioscience (San Diego, CA, USA). All other chemicals were from Sigma-Aldrich.

Isolation and culture of naïve monocytes and PBMCs

Peripheral blood was obtained from five different healthy donors after informed consent (age 37.2 ± 4.9 years; 2 males and 3 females). PBMCs were isolated by Ficoll-Paque (Pharmacia) according to standard procedures. Monocytes were isolated from these PBMCs by positive selection using the autoMACS magnetic cell sorting system (Miltenyi Biotec) according to the manufacturer's instructions. Briefly, PBMCs were incubated with saturating concentrations of CD14 microbeads at 4 °C for 15 min, washed and suspended in PBS containing 2 mm ethylenediaminetetraacetic acid and 0.5% bovine serum albumin (BSA). The cell suspension was then applied to the autoMACS separator using the positive selection programme. The CD14-positive cells were eluted from the magnetic column; a purity of >98% was routinely obtained as confirmed by flow cytometry. Previous studies using mRNA profiling as readout have demonstrated that isolation procedures do not result in relevant activation of the isolated cells.

Naïve PBMCs and naïve CD14+ monocytes were recuperated in culture for 24 h in DMEM supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mm L-glutamine (DMEM+), with 10% heat-inactivated human male AB serum. Naïve PBMCs were cultured in 96-well plate (Nunc) at a concentration of 1 × 105 PBMCs per well according to standard procedures, whereas naïve CD14+ monocytes were cultured in 24-well plate at a concentration of 1 × 106 cells per well. Both naïve PBMCs and naïve CD14+ monocytes were cultured in a cell/tissue incubator under 5% CO2 in air (pH7.4), at 37 °C and 95% humidity. After the 24 h of recuperation, medium was replaced with serum-free DMEM+ with additives according to the experimental conditions, and cells were cultured for an additional 24 h. Experimental conditions included stimulation with FVIIa [25 nm], TF [37 pm] + FVIIa [25 nm], TF [37 pm] + FVIIa [25 nm] + FX [100 nm], FX [100 nm], FXa [10 nm] or Thrombin [300 nm]. These concentrations correspond with a FVIIa dose of 90 μg/kg body weight for FVIIa in case of treatment and known estimated physiological intravascular concentrations of TF [37 pm], FX [135 nm], FXa [13.5 nm] and thrombin [2–300 nm] [[23].]

PAR expression at the RNA level

For reverse transcription–polymerase chain reaction (RT-PCR), RNA was isolated from naïve CD14+ monocytes with RNeasy Mini Kit (Qiagen, Germantown, MD, USA) according to the manufacturer's instructions. RT-PCR was carried out using GeneAmp RNA PCR Core kit (Applied Biosystems, Carlsbad, CA, USA) according to the manufacturer's instructions. DNase-treated RNA samples were reverse transcribed to complement DNA (cDNA) using recombinant Moloney murine leukaemia virus reverse transcriptase. An aliquot of the reaction mixture was then used for PCR amplification using universal PCR master mix and the following primer sequences: PAR-1 (sense) 5′-TACGCCTCTATCTTGCTCATGAC-3′ and PAR-1 (antisense) 5′-TTTGTGGGTCCGAAGCAAAT-3′; PAR-2 (sense) 5′-TGGATGAGTTTTCTGCATCTGTCC-3′ and PAR-2 (antisense) 5′-CGTGATGTTCAGGGCAGGAATG-3′; PAR-3 (sense) 5′-TCCCCTTTTCTGCCTTGGAAG-3′ and PAR-3 (antisense) 5′-AAACTGTTGCCCACACCAGTCCAC-3′; PAR-4 (sense) 5′-AACCTCTATGGTGCCTACGTGC and PAR-4 (antisense) 5′-CCAAGCCCAGCTAATTTTTG-3′; yielding a PCR product with an expected size of 453, 491, 513 and 541 base pair (bp) for PAR-1, PAR-2, PAR-3 and PAR-4, respectively. PAR-1, PAR-2 and PAR-3 were amplified with 35 cycles (94 °C for 30 s, 55 °C for 30 s, 72 °C for 60 s). PAR-4 was amplified with 35 cycles (94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s). Beta-actin (β-actin) was used as positive control using the following primer sequences: β-actin (sense) 5′-CCAAGGCCAACCGCGAGAAGATG-3′ and β-actin (antisense) 5′-AGGGTACATGGTGGTGCCGCCAG-3′; yielding a expected PCR product of 587 bp. Beta-actin was amplified with 35 cycles (94 °C for 60 s, 60 °C for 90 s, 72 °C for 60 s). Negative control was performed for each reaction and included the omission of the reverse transcriptase or the omission of cDNA in the PCR mix. PCR products were resolved on a 1.5% agarose gel for visualization.

Flow Cytometry

Flow cytometry analysis was performed of the freshly isolated naïve CD14+ monocytes and the CD14+ monocytes cultured for 24 h with experimental conditions. Briefly, the freshly isolated naïve CD14+ monocyte cell pellet was washed in PBS containing 1% BSA and 0.1% Na-azide and subsequently used for incubation with fluorochrome-labelled antibodies. The CD14+ monocytes cultured with experimental conditions for 24 h were placed on ice for 1 h. Subsequently, medium with CD14+ monocytes was transferred to 1.5-ml tubes and centrifuged at 900 g for 5 min at room temperature. Supernatants were harvested; the remaining CD14+ cell pellet was washed in PBS containing 1% BSA and 0.1% Na-azide, and centrifuged at 900 g for 5 min at room temperature. After centrifuging, freshly isolated naïve CD14+ monocytes as well as cultured CD14+ monocytes were incubated with APC-conjugated monoclonal mouse anti-human CD14 antibody, PE-conjugated monoclonal mouse anti-human PAR-1 (ATAP2) antibody, FITC-conjugated monoclonal mouse anti-human PAR-2 (SAM11) antibody, PE-conjugated monoclonal mouse anti-human PAR-3 (8E8) antibody, FITC-conjugated polyclonal rabbit anti-human PAR-4 (APR-034-F) antibody, PE-conjugated monoclonal mouse anti-human TF (HTF-1) antibody, and APC-, PE- and FITC-conjugated isotype control antibodies for 30 min at 4 °C in the dark. After a final washing and centrifuging step, cells were fixated in 2% paraformaldehyde. All cells were analysed using the FACS Calibur (BD Biosciences) and FlowJo software (Tree Star Inc., Ashland, OR, USA).

Cytokine assays

For cytokine assays, naïve PBMCs and naïve CD14+ monocytes recuperated for 24 h and subsequently cultured according to the experimental conditions for 24 h were used. Supernatants were harvested, transferred to 1.5 ml tubes, centrifuged at 900 g for 5 min at room temperature and cryopreserved at −80 °C. Cytokine production (IL1-β, IL-6, IL-8, IL-10 and TNF-α) was determined in triplicate. Standard and positive control recovery for each ELISA assay was between 90–110%.

Cell proliferation assay

Cell proliferation was determined by 3H-thymidine incorporation in a subset of naïve PBMCs recuperated for 24 h and subsequently cultured according to the experimental conditions for an additional 24 h. Briefly, for the last 18 h of culture, 20 μl 3H-thymidine (NEN Life Science Products, Amsterdam, The Netherlands) at a concentration of 5μCI/ml was added. 3H-thymidine incorporation was determined by liquid scintillation counting, expressed as counts per minute (CPM) according to standard procedures.

Statistical analysis

For data storage and management, Microsoft Excel (Microsoft, Redmond, WA, USA) was used. Graphic presentation was performed with GraphPad Prism version 5.00 (GraphPad Software, San Diego, CA, USA), and statistical analysis was performed with SPSS version 15.0 (IBM, SPSS, Armonk, NY, USA). Data are shown as median with range unless stated otherwise. Data were analysed by Wilcoxon signed ranks test. Statistical significance was denoted at P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion and conclusion
  7. Acknowledgment
  8. References

Freshly isolated naïve CD14+ monocytes express all PARs at mRNA level

We first investigated the expression of the four PARs at mRNA levels on freshly isolated naïve monocytes. Primers specific for PAR-1, PAR-2 and PAR-3 yielded bands of the expected respective size (Fig. 1). Only a faint band of PAR-4 amplification product was observed. Analysis of monocyte RNA without reverse transcriptase did not lead to amplification of any product, indicating that the PCR products obtained were not due to genomic DNA contamination (data not shown). In all cases, positive control expression of β-actin at mRNA level was found.

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Figure 1. Expression of mRNA's encoding PAR-1, PAR-2, PAR-3, and PAR-4 in freshly isolated naïve CD14+ monocytes. Representative reverse transcriptase-polymerase chain reaction analysis of PAR-1, PAR-2, PAR-3, and PAR-4 gene expression in freshly isolated naïve CD14+ monocytes indicating that monocytes express all four PARs at mRNA level. Equal amounts of total RNA were analyzed. β-actin mRNA served as a positive control for lane loading.

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Freshly isolated naïve CD14+ monocytes express PAR-1, PAR-3, PAR-4, but not PAR-2, nor TF at protein level

We next investigated expression of the four PARs and TF at the protein level on freshly isolated naïve CD14+ monocytes. As an example, freshly isolated naïve CD14+ monocytes showed clear expression of PAR-1, PAR-3 and PAR-4, but not of PAR-2 and TF (Fig. 2). The expression profile is representative for the other individual donors. These results support that PAR-1, PAR-3 and PAR-4 mediated cell signalling in naïve monocytes are possible.

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Figure 2. Freshly isolated naïve monocytes express PAR-1, PAR-3, and PAR-4, but do not express PAR-2 or TF. Naïve monocytes from a representative donor were stained with CD14 (M5E2), PAR-1 (ATAP2), PAR-2 (SAM11), PAR-3 (8E8), PAR-4 (APR-034-F), TF (HTF-1), and isotype-matched control antibodies as described. (A) Gating and selection of isolated CD14 positive monocyte population. (B, D, E) Naïve monocytes express PAR-1, PAR-3, and PAR-4. Flow cytometry staining of autofluorescent (dotted line), matched isotype control (thin solid line), and PAR-1, PAR-3, or PAR-4 (thick solid line). (C, F) naïve monocytes do not express PAR-2 and TF. Flow cytometry staining of autofluorescent (dotted line), isotype control (thin solid line), and PAR-2, or TF (thick solid line). Representative flow cytometry staining for all donors.

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To test whether PAR- and TF expression on naïve CD14+ monocytes changed upon stimulation with possible PAR signalling molecules changed, PAR and TF expressions were evaluated in naïve CD14+ monocytes cultured for 24 h in the presence of FVIIa, the binary TF-FVIIa complex, the binary TF-FVIIa complex with FX, FX, FXa, thrombin and as a positive control LPS. As shown in Figs. 3 and 4, both the percentage positive PAR-1, PAR-3 and PAR-4 expressing naïve monocytes and the mean fluorescence for PAR-1, PAR-3, and PAR-4 were not altered. Percentage positive monocytes for medium conditions were 97% (range 4), 5.84% (range 1.1), and 99.9% (range 0.1), and 3.2% (range 2.86) for PAR-1, PAR-3 and PAR-4, respectively. The median mean fluorescence for medium conditions was 73.5 (range 1), 286.5 (range 97), 183 (range 131) and 38.2 (range 13.4) for PAR-1, PAR-3 and PAR-4, respectively.

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Figure 3. PAR-1, PAR-3, and PAR-4 expression on naïve monocytes is not altered by stimulation with FVIIa, TF + rFVIIa, TF + rFVIIa + FX, FX, FXa, Thrombin, or LPS. Monocytes (106) were cultured for 24 h in the presence of FVIIa [25 nm], TF[37 pm] + FVIIa [25 nm], TF[37 pm] + FVIIa [25 nm] + FX[100 nm], FX[100 nm], FXa [10 nm], Thrombin [300 nm], or LPS[1 ng/ml]. Gated and selected CD14 positive monocytes were stained with PAR-1 (ATAP2), PAR-3 (8E8), PAR-4 (APR-034-F), TF (HTF-1), and isotype-matched control antibodies as described. Illustrative histograms of the percentage positive monocytes for PAR-1(A), PAR-3(B), PAR-4 (C), and TF (D). Data are shown as median with 5–95 percentile from five separate experiments. FVIIa, Factor VIIa; TF, Tissue Factor; FX, Factor X; FXa, activated Factor X; LPS, lipopolysaccharide.

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image

Figure 4. Mean fluorescence of PAR-1, PAR-3, and PAR-4 on naïve monocytes is not altered by FVIIa, TF + rFVIIa, TF + rFVIIa + FX, FX, FXa, Thrombin, or LPS. Monocytes (106) were cultured for 24 h in the presence of FVIIa [25 nm], TF[37 pm] + FVIIa [25 nm], TF[37 pm] + FVIIa [25 nm] + FX[100 nm], FX[100 nm], FXa [10 nm], Thrombin [300 nm], or LPS[1 ng/ml]. Gated and selected CD14 positive monocytes were stained with PAR-1 (ATAP2), PAR-3 (8E8), PAR-4 (APR-034-F), TF (HTF-1), and isotype-matched control antibodies as described. Illustrative histogram of the Mean fluorescence for staining for PAR-1(A), PAR-3(B), PAR-4 (C), and TF (D). Data are shown as median with 5–95 percentile from five separate experiments. FVIIa, Factor VIIa; TF, Tissue Factor; FX, Factor X; FXa, activated Factor X; LPS, lipopolysaccharide.

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Also, TF expression was evaluated on freshly isolated monocytes, and the change in expression upon the different coagulation proteases tested. TF (3.2%; range 2.86) was hardly detectable on the freshly isolated naïve monocytes (Fig. 2E). As expected, LPS induced a statistically significant increase in the percentage TF expression naïve CD14+ monocytes (33.6%; range 58.6; P = 0.008 compared with medium condition; Fig. 3D). The median mean fluorescence for medium condition was 38.2 (range 13.4). LPS induced an increase in mean fluorescent for TF 88 (range 111; nearing statistically significance P = 0.15).

Cytokine release by naïve CD14+ monocytes upon stimulation

FVIIa complex, the binary TF-FVIIa complex with free FX, free FX, free FXa, and thrombin are able to induce PAR-mediated cytokine release in naïve monocytes. Therefore, we tested whether stimulation of naïve CD14+ monocytes with these coagulation proteases resulted in cytokine release. As shown in Fig. 5, FVIIa, the binary TF-FVIIa complex, the binary TF-FVIIa complex with free FX, free FX, free FXa, and thrombin were not able to induce a cytokine release in naïve CD14+ monocytes. In contrast, stimulation of these naïve CD14+ monocytes with LPS as positive control resulted in abundant and statistically significant (P < 0.05) release of IL-1β, IL-6, IL-8, IL-10 and TNF-α cytokines.

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Figure 5. Effects of FVIIa, TF + FVIIa, TF + FVIIa + FX, FX, FXa, Thrombin, and LPS on cytokine production in naïve monocytes. Monocytes (106/ml) were cultured for 24 h in the presence of FVIIa [25 nm], TF[37 pm] + FVIIa [25 nm], TF[37 pm] + FVIIa [25 nm] + FX[100 nm], FX[100 nm], FXa [10 nm], Thrombin [300 nm], or LPS [1 ng/ml]. The culture supernatants were harvested and analyzed for IL-1β (A), IL-6 (B), IL-8 (C), IL-10 (D), and TNF-α (E) by ELISA assay in triplicate. Data are shown as median with 5–95 percentile from five separate experiments. * denotes different for all other conditions, P < 0.05, Wilcoxon signed ranks test. FVIIa, factor FVIIa; TF, Tissue Factor; FX, Factor X; FXa, activated Factor X; LPS, lipopolysaccharide.

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Thrombin-induced IL-1ß and IL-6 cytokine release in naïve PBMCs is PAR-1-dependent

We next investigated whether stimulation of naïve PBMCs with coagulation proteases might induce cytokine release. As shown in Fig. 6, FVIIa, the binary TF-FVIIa complex, the binary TF-FVIIa complex with FX, FX and FXa were not able to induce cytokine releases in naïve PBMCs. In contrast, stimulation of naïve PBMCs with thrombin resulted in a statistically significant release of IL-1β and IL-6 cytokines, but not IL-8, IL-10 and TNF-α. Compared with medium, (10.1 pg/ml; range 18.3) and (5.26 pg/ml; range 3.4) for IL-1β and IL-6, respectively, stimulation of naïve PBMCs with thrombin increased IL-1β (42.5 pg/ml; range 9.2; P = 0.02) and IL-6 (41 pg/ml; range 9; P = 0.02) cytokine levels. Stimulation of PBMCs with LPS as a positive control resulted in abundant and statistically significant release of IL-1β, IL-6, IL-8, IL-10 and TNF-α cytokines (P < 0.05).

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Figure 6. Stimulation of naïve peripheral blood mononuclear cells (PBMC) with thrombin results in IL-1β and IL-6 cytokine release. PBMCs (105) were cultured for 24 h in the presence of FVIIa [25 nm], TF[37 pm] + FVIIa [25 nm], TF[37 pm] + FVIIa [25 nm] + FX [100 nm], FX[100 nm], FXa [10 nm], Thrombin [300 nm], or LPS[1 ng/ml]. The culture supernatants were harvested and analyzed for IL-1β (A), IL-6 (B), IL-8 (C), IL-10 (D), and TNF-α (E) by ELISA assay in triplicate. Data are shown as median with 5–95 percentile from five separate experiments. * denotes different from medium condition.

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As can be seen in Fig. 7, the thrombin-stimulated IL-1β and IL-6 cytokine release in PBMCs was dose-dependently and was completely blocked by PAR-1 antagonist FR171113 [100 μm]. Cytokine levels for thrombin [300 nm] were 42.5 pg/ml (range 9.2) and 41 pg/ml (range 9) for IL-1β and IL-6 respectively. Adding PAR-1 antagonist FR171113 [100 μm] to thrombin [300n] resulted in a statistically significant reduction in release of IL-1β (0.45 pg/ml; range 0.2; P = 0.02) and IL-6 (0.4 pg/ml; range 0.6; P = 0.02). Adding PAR-1 antagonist FR171113 [100 μm] solely to PBMCs did not result in a cytokine release. These results indicate that PAR-1 activation is required for thrombin-induced IL-1β and IL-6 cytokine release in naïve PBMCs.

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Figure 7. PAR-1 dependent release of IL-1β and IL-6 cytokines by thrombin in naïve peripheral blood mononuclear cells (PBMC). PBMCs (105) were cultured for 24 h in the presence of Thrombin [30–300 nm], and Thrombin [300 nm] and PAR-1 antagonist [100 μm]. The culture supernatants were harvested and analyzed for IL-1β (A) and IL-6 (B) by ELISA assay in triplicate. Data are shown as median with 5–95 percentile from five separate experiments. * denotes different from medium condition. # denotes different from Thrombin [300 nm], P < 0.05, Wilcoxon signed ranks test. aPAR1, PAR-1 antagonist FR171113.

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Thrombin induces PAR-1-dependent cell proliferation in naïve PBMCs

Finally, it was assessed whether naïve PBMCs stimulated with FVIIa, the binary TF-FVIIa complex, the binary TF-FVIIa complex with FX, FX, FXa, thrombin, thrombin and PAR-1 antagonist, or LPS influenced PBMC cell proliferation. As shown in Fig. 8A and in line with the findings of the cytokine release experiments, thrombin enhanced PBMC cell proliferation. Stimulation with thrombin (465 CPM; range 435) resulted in a statistically significant increase in PBMC cell proliferation as compared to medium (163 CPM; range 25) (P = 0.02). None of the other coagulation factors were able to induce an increase in PBMC proliferation, whereas LPS as a positive control was effective in stimulating PBMC proliferation. The thrombin-induced PBMC proliferation was dose-dependently and was completely blocked by PAR-1 antagonist FR171113 [100 μm] (41 CPM; range 16) in a statistically significant manner (P = 0.02) (Fig. 8B). Adding PAR-1 antagonist FR171113 [100 μm] solely to PBMCs did not affect cell proliferation. These results indicate besides thrombin-induced cell proliferation in naïve PBMC is PAR-1 dependent.

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Figure 8. Thrombin-induced peripheral blood mononuclear cells (PBMC) proliferation is PAR-1 dependent. PBMCs (105) were cultured for 24 h in the presence of FVIIa [25 nm], TF[37 pm] + FVIIa [25 nm], TF[37 pm] + FVIIa [25 nm] + FX [100 nm], FX[100 nm], FXa [10 nm], Thrombin [30–300 nm], Thrombin [300 nm] and PAR-1 antagonist [100 μm], or LPS [1 ng/ml]. PBMCs were co-cultured in triplicate with 20 μl [3H]-thymidine for the final 18 h. Cell proliferation was measured by [3H]-thymidine incorporation. Thrombin, but none of the other coagulation proteases induced PBMC cell proliferation (A). Thrombin-induced PBMC cell proliferation is PAR-1-dependent (B). Data are shown as median with 5–95 percentile from five separate experiments. * denotes different from medium condition, # denotes different from Thrombin [300 nm], < 0.05, Wilcoxon signed ranks test. FVIIa, factor VIIa; TF, Tissue Factor; FX, Factor X; FXa, activated Factor X; aPAR1, PAR-1 antagonist FR171113; LPS, lipopolysaccharide.

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Discussion and conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion and conclusion
  7. Acknowledgment
  8. References

In this study, using naïve CD14+ monocytes and naïve PBMCs, we demonstrate that monocytes express PAR-1, PAR-2, PAR-3 and PAR-4 at mRNA level, and PAR-1, PAR-3 and PAR-4 at protein level. The data presented herein also show that stimulation of naïve CD14+ with coagulation proteases (FVIIa, the binary TF-FVIIa complex, the binary TF-FVIIa complex with free FX, free FX, free FXa and thrombin) in physiological concentrations did not result in alterations of PAR-1, PAR-3, PAR-4 and TF expression at the protein level. Also, no pro-inflammatory cytokine release is induced. In addition, our study demonstrates that stimulation of naïve PBMCs with coagulation proteases did not resulted in pro-inflammatory cytokine release, except for stimulation of naïve PBMCs with thrombin which resulted in a PAR-1-dependent release of IL-1ß and IL-6 and PBMC cell proliferation.

Cross-talking between coagulation and inflammation mediated by PARs is at present a topic of major interest. Stimulation of different (monocyte) cell lines or artificially preactivated monocytes or PBMCs with coagulation proteases, such as FVIIa, the binary TF-FVIIa complex, FXa and thrombin, resulted in PAR-dependent alterations in gene expression, induction of cell proliferation and cytokine production [3, 12].

To better understand the consequences of cross-talking between coagulation and inflammation in more physiological conditions, we investigated whether coagulation proteases in physiological concentrations were able to elicit pro- or anti-inflammatory responses in a PAR-dependent manner in naïve human monocytes and PBMCs.

First, using purified naïve monocytes, we investigated PAR expression at both mRNA and protein level. Human naïve monocytes were found to express all PARs at mRNA level. Only a faint band of PAR-4 amplification product was observed. At protein level, monocytes expressed PAR-1, PAR-3 and PAR-4. Our findings regarding PAR protein expression are in line with previous work, others also failed to demonstrate PAR-2 protein expression [10, 12]. In contrast, Crilly et al. found PAR-2 expression on monocytes in their study [24, 25]. However, this PAR-2 expression was very limited in healthy humans with a median expression of 0.06%. We are the first to demonstrate PAR-4 expression at mRNA level in monocytes [10, 12]. Li and He [[10].] found PAR-4 protein expression but failed to detect the presence of PAR-4 transcripts due to technical issues. Irrespectively, also in our hands, PAR-4 expression is marginal.

The presence of PAR-1, -3 and -4 at protein level in naïve monocytes suggests that cross-talking between coagulation and inflammation is possible, because PARs are sensitive to protease stimulation. Human PAR-1 can be activated by FXa and thrombin; whereas PAR-2 can be activated by FVIIa, the binary TF-FVIIa complex, FXa and the ternary TF-FVIIa-FXa complex; and PAR-3 and PAR-4 can be activated by thrombin [5-7, 13]. PAR activation is irreversible. Upon activation, PARs are uncoupled from signalling and then internalized and degraded [26, 27].

Therefore, we first investigated whether stimulation of naïve monocytes with the coagulation proteases would alter PAR expression. The percentage monocytes expressing PARs and the MFI of PAR expression did not changed upon stimulation, with the coagulation proteases suggesting that PARs were not activated and internalized [28].

We next investigated whether stimulation of naïve monocytes with coagulation proteases resulted in cytokine production. It is known that coagulating whole blood results in the production of IL-6 and IL-8 [29]. In addition, administration of FVIIa was found to elicit IL-6 and IL-8 release in healthy human subjects [30]. In our study, none of the investigated coagulation proteases induced pro-inflammatory cytokine production by naïve CD14+ monocytes. For FVIIa and the binary TF-FVIIa complex, this seems logic regarding the absence of PAR-2 expression on naïve monocytes. For FXa and thrombin, our findings correspond to previous studies demonstrating that both FXa and thrombin did not promote monocyte IL-1β, IL-6 and TNF-α secretion [31-33].

Thus, although freshly isolated naïve monocytes express PAR-1, PAR-3 and PAR-4 at protein level, our results demonstrate that stimulation with the investigated coagulation proteases does not result in cross-talking with the inflammation cascade leading to pro-inflammatory cytokine production.

To figure out which coagulation protease is responsible for the observed pro-inflammatory cytokine release in coagulating whole blood and upon FVIIa administration in vivo, we next investigated whether stimulation of PBMCs with coagulation proteases resulted in pro-inflammatory cytokine release and proliferation. From the investigated coagulation proteases, only thrombin was found to induce pro-inflammatory effects. Thrombin-induced IL-1β and IL-6 cytokine release and PBMC cell proliferation. This effect clearly appeared to be PAR-1 mediated.

Because isolated CD14+ monocytes did not respond, it could be that the context of PBMC population is necessary to stimulate the monocytes. On the other hand, it is also plausible that other cells within the PBMC population were stimulated by thrombin. Possible candidate cells are monocyte-derived macrophages or T lymphocytes. Simulation of monocyte-derived macrophages with thrombin resulted in the release of IL-1β cytokine in a PAR-1 dependent manner [34-36]. Human T cells were found to express PAR-1, PAR-2 and PAR-3, but not PAR-4 [10]. Stimulation of these T cells with thrombin resulted in a modest but significant increase in IL-6 production. B-cells are unlikely candidates as expression of only PAR-4 has been detected on B-cells in the human liver, but the role of this receptor in B cell function remains unknown [37]. The observed pro-inflammatory effects of thrombin on naïve PBMCs were modest with IL-1β and IL-6 levels below 50 pg/ml. However, correlations of the levels of any cytokine with disease severity do not establish causality, and even with low levels (pg/ml) impressive clinical responses have been reported [38]. Thus, the observed modest increase in cytokine levels in our study is considered of relevance to orchestrates several pathways involved in inflammation and tissue destruction. And in situations with increased activation of coagulation, for example sepsis, the generated thrombin could however potentially induce a larger pro-inflammatory effect.

In conclusion, in this study, we demonstrate that stimulation of naïve monocytes and naïve PBMCs with coagulation proteases in the physiological range in general did not resulted in alterations in PAR expression and/or pro- or anti-inflammatory cytokine production. Only stimulation of PBMCs with thrombin resulted in a modest release of cytokines (IL-1β, IL-6) and the induction of cell proliferation in a PAR-1 dependent manner. These observations indicate that naïve monocytes are not triggered by coagulation proteases and that only thrombin is able to elicit pro-inflammatory events and cell proliferation in a PAR-1-dependent manner in PBMCs. Whether blocking of thrombin in diseases with increased coagulation activation is of therapeutic use needs further study.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion and conclusion
  7. Acknowledgment
  8. References

This study was financially supported by an unrestricted grant of Novo Nordisk. The authors report no other conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion and conclusion
  7. Acknowledgment
  8. References
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