Recombinant human activated protein C ... - Wiley Online Library

research paper

Recombinant human activated protein C upregulates the release of soluble fractalkine from human endothelial cells

Martina Brueckmann,1 Adriane Schulze Nahrup,1 Siegfried Lang,1 Thomas Bertsch,2 Kenji Fukudome,3 Volker Liebe,1 Jens J. Kaden,1 Ursula Hoffmann,1 Martin Borggrefe1 and Guenter Huhle1*

Summary

E-mail: [email protected]

Fractalkine is a unique endothelial cell-derived chemokine that functions both as a chemoattractant and as an adhesion molecule. Recent findings suggest that fractalkine plays an important role in inflammatory diseases by modulating leucocyte endothelial cell interactions. A modulating effect on the immune system in severe sepsis has been suggested for recombinant human activated protein C (rhAPC). However, a little is known about the effect of rhAPC on the endothelial release of soluble fractalkine. The effect of rhAPC (50 ng/ml to 10 lg/ml) and protein C (in equimolar concentrations) on the synthesis of fraktalkine-mRNA and release of soluble protein in human umbilical vein endothelial cells (HUVEC) was determined by reverse transcription-polymerase chain reaction and by an enzyme-linked immunosorbent assay. rhAPC at supra-pharmacological concentrations (1– 10 lg/ml) stimulated fractalkine-messenger RNA-gene transcription and release of soluble fractalkine in a time- and dose-dependent manner, whereas the zymogen protein C was ineffective. As shown by experiments using monoclonal antibodies against the thrombin receptor, protease-activated receptor-1 (PAR-1), PAR-2 and against the endothelial protein C receptor (EPCR), the effect of rhAPC on fractalkine upregulation was mediated by binding to the EPCR-receptor and signalling via PAR-1. These in vitro data demonstrate that induction of fractalkine release is an important response of HUVEC to stimulation with rhAPC and may lead to a better understanding of the molecular pathways involved in the mode of action of rhAPC. Further clinical trials are needed to confirm the in vivo relevance of these data.

heidelberg.de *Guenter Huhle was an employee of Lilly Germany GmbH, Bad Homburg, Germany.

Keywords: activated protein C, drotrecogin alfa (activated), chemokine, fractalkine, endothelium.

1

1st Department of Medicine, Faculty of Clinical

Medicine Mannheim, University of Heidelberg, Mannheim, Germany, 2Institute of Clinical Chemistry and Laboratory Medicine, Clinic Nuremberg, Nuremberg, Germany, and 3

Department of Immunology, Saga Medical

School, Saga, Japan

Received 29 December 2005; accepted for publication 13 February 2006 Correspondence: Martina Brueckmann, 1st Department of Medicine, Faculty of Clinical Medicine Mannheim, University of Heidelberg, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany.

The migration of leucocytes into extravascular tissues is essential for an effective defence against bacterial invasion, immune surveillance and wound repair (Butcher, 1991). Defective adhesive interactions between endothelial cells and leucocytes cause impaired cellular targeting to infected and injured sites. On the other hand, excessive leucocytic activation may be associated with an increased proinflammatory immune response. The endothelium plays an important role in the recruitment and emigration of circulating leucocytes into sites of inflammation by expressing adhesion molecules and by the release of chemotactic factors. Fractalkine (also called CX3CL1) is a unique endothelial-derived

chemokine that exists in two forms, as a membrane-anchored or shed soluble glycoprotein, which originates from extracellular proteolysis by proteases, such as tumour-necrosis factora converting enzyme (TACE; also known as ADAM17) and ADAM10 (Bazan et al, 1997; Garton et al, 2001; Umehara et al, 2004). Consecutively, fractalkine has a dual function, as both adhesion molecule and chemokine: the secreted form of fractalkine has potent chemotactic activity for monocytes and T cells. The interaction of membrane-bound fractalkine with its receptor, CX3CR1, which is present on natural killer (NK) cells, CD14+ monocytes and on some subpopulations of T cells (Imai et al, 1997), can support firm cell adhesion and

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rhAPC upregulates soluble fractalkine transendothelial migration of leucocytes during inflammation (Umehara et al, 2001). The expression of fractalkine can be markedly induced on endothelial cells by inflammatory cytokines, such as tumour-necrosis factor-a and interleukin1. The molecular mechanisms that precisely regulate the expression and release of fractalkine in endothelial cells may be important in many inflammatory diseases, such as atherosclerosis, glomerulonephritis, rheumatoid arthritis and also during severe sepsis. Human activated protein C (APC) constitutes an important natural coagulation inhibitor and key regulator of both coagulation and inflammation (Esmon et al, 1991). Treatment with recombinant human activated protein C [rhAPC, drotrecogin alfa (activated)] in patients with severe sepsis resulted in an improvement of survival and more rapid resolution of cardiovascular and respiratory organ dysfunction (Bernard et al, 2001; Vincent et al, 2003, 2005). Apart from its anticoagulant effects, multiple biological activities, including profibrinolytic, immune-modulating and antiapoptotic properties of APC, have been characterised in non-clinical studies (Joyce & Grinnell, 2002; Brueckmann et al, 2003; Cheng et al, 2003). From in vitro experiments on human microvascular endothelial cells, it has been postulated that APC attenuates thrombin- or cytokine-induced hyperpermeability and promotes endothelial cell survival (Zeng et al, 2004). Moreover, APC can promote proliferation of endothelial cells and induce angiogenesis in a mouse cornea angiogenesis assay (Uchiba et al, 2004). In endothelial cells, many biological responses of APC are mediated by the endothelial protein C receptor (EPCR) (Fukudome & Esmon, 1994) via protease-activated receptor-1 (PAR-1) signalling (Riewald et al, 2002; Riewald & Ruf, 2005). Transcript profiling and flow cytometry of human endothelial cells demonstrated that rhAPC inhibited the expression of adhesion molecules, such as E-selectin and vascular cell adhesion molecule-1 (VCAM-1) (Joyce et al, 2001). Whether rhAPC exerts its beneficial effects on organ function in severe sepsis by regulating fractalkine generation, is unknown up to now. To approach this subject, we set out to determine the in vitro effect of rhAPC on fractalkine-mRNA-expression and fractalkine-release from human umbilical vein endothelial cells (HUVEC). In addition, we investigated receptors possibly being involved in the regulatory effect of rhAPC on fractalkine release from endothelial cells.

Materials and methods Materials RhAPC [drotrecogin alfa (activated)], analytical grade, molecular weight approximately 55 kDa, was provided by Eli Lilly, Indianapolis, IN, USA, as part of a grant. Recombinant hirudin [Lepirudin, Refludan] was provided by Aventis Pharma GmbH (Bad Soden, Germany). Protein C (Ceprotin) was a kind gift of Dr B. Eberspaecher, Baxter

Germany GmbH (Heidelberg, Germany). a-Thrombin was purchased from Calbiochem (Schwalbach, Germany). Endothelial cell growth medium was purchased from PromoCell (Heidelberg, Germany). ATAP2 (sc-13503), a mouse monoclonal IgG1 antibody raised against the human thrombin receptor PAR-1, and SAM 11 (sc-13504), a mouse monoclonal IgG2a antibody raised against the human thrombin receptor PAR-2, were obtained from Santa Cruz Biotechnology, Santa Cruz, CA, USA. The monoclonal anti-EPCR antibody RCR-252, which inhibits the interaction of APC with EPCR, was provided by Prof. K. Fukudome, Saga, Japan. The PAR-1 agonist peptide TFLLRNPNDK was synthesised by Serva (Heidelberg, Germany). The PAR-2 agonist peptide SLIGKV (Cat. no. H-5042) was purchased from Bachem (Weil am Rhein, Germany). Human fractalkine-specific as well as GAPDH-specific primers for reverse transcription followed by polymerase chain reaction (RTPCR) were from Applera, Weiterstadt, Germany.

Cell culture Human umbilical vein endothelial cells were prepared from fresh umbilical cords by collagenase digestion as described by Jaffe et al (1973). Cells in passage 2 and 3 were used in all experiments. Cells were grown in multiwell plates coated with 2% gelatine. Endothelial cell growth medium (Promocell) supplemented with 2% fetal calf serum (FCS), gentamycin (25 lg/ml), amphotericin B (25 ng/ml), hydrocortisone (1 lg/ ml) vascular endothelial growth factor (2 ng/ml), human epidermal growth factor (5 ng/ml) and human fibroblast growth factor (5 ng/ml) was used. Twenty-four hours before starting an experiment this complete growth medium was removed and replaced by the same medium but without FCS, growth factors and hydrocortisone. On the day of the experiment, cells were washed again with medium free of FCS, growth factors and hydrocortisone. Immediately afterwards cells were treated with a single dose of human rhAPC in combination with the thrombin inhibitor lepirudin (5 lg/ml) or with receptor-blocking antibodies for the indicated periods of time. APC concentrations ranged from 50 ng/ml to 10 lg/ ml. When rhAPC was studied in combination with receptorblocking antibodies (RCR-252, ATAP2 or SAM11), rhAPC was given 30 min after the addition of receptor-blocking antibodies. Treatment with rhAPC, receptor-blocking antibodies or receptor agonist peptides at the indicated concentrations did not affect HUVEC viability, as assessed by trypan blue exclusion (>90% viable cells, no difference to untreated controls).

Microarray analysis The effect of rhAPC on tumour-necrosis factor-a (TNF-a)induced transcript levels of fractalkine, TACE (ADAM17) and ADAM10 was screened by using Affymetrix microarrays. Briefly, total RNA from confluent HUVECs was isolated and

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Determination of soluble fractalkine in HUVEC supernatants Endothelial cell culture supernatants were collected after the indicated incubation periods, shock-frozen and stored at )80C until measurement. Soluble fractalkine in HUVEC supernatants was determined by an enzyme-linked immunosorbent assay (Cat. no. DY365; R&D Systems, Wiesbaden, Germany). The immunoassay was performed according to the manufacturers’ protocol.

Reverse transcription-polymerase chain reaction Total RNA from confluent HUVECs was isolated by the use of the Quiagen RNeasy Mini Kit (Quiagen, Hilden, Germany). Three micrograms of total RNA were reverse transcribed and converted to cDNA with oligo(dT) primers. The cDNA was then amplified by PCR. The sequences of the GAPDH primers were as follows (Hicok et al, 1998): sense primer 5¢-ACCACAGTCCATGC CATCAC-3¢ and antisense primer 5¢TCCACCACCCTGTTGCTGTA-3¢. The human fractalkine primer pair was as follows (Sukkar et al, 2004): sense primer 5¢-AACTCGAAA TGGCGGCACCTT-3¢ and antisense primer 5¢-ATGAATTACTACCACAGCTCCG-3¢. 35 cycles of amplification (annealing temperature 62C) were performed for fractalkine and 24 cycles for GAPDH (annealing temperature 55C). PCR products (fractalkine: 887 base pairs, GAPDH: 452 base pairs) were run on a 2% agarose gel and the bands were visualised by ethidium bromide staining and ultraviolet transillumination. The intensity of the bands was quantified by densitometric analysis.

Statistical analysis Fractalkine-levels are presented as mean ± SD or ±SEM, as indicated, of results obtained from at least three independent cell culture experiments. In each experiment, sixfold measurements were performed. Data were compared using the unpaired Student’s t-test for single comparisons. For multiple comparisons, an analysis of variance was performed (anova) followed by an appropriate post-test. Values of P < 0Æ05 (twotailed) were considered statistically significant. The calculations were performed using instat (GraphPad Software, San Diego, CA, USA) and the Statistical Package for the Social Sciences (spss)-software (SPSS Software GmbH, Munich, Germany). 552

Results Microarray analysis Microarrays were performed as screening experiments to assess the effect of rhAPC on basal fractalkine expression, on TNF-aor thrombin-induced transcript levels of fractalkine and on the expression of the proteases TACE (ADAM17) and ADAM10, which can shed soluble fractalkine (Umehara et al, 2004). HUVECs were stimulated with rhAPC (50 ng/ml or 5 lg/ml), TNF-a (0Æ1 ng/ml) or thrombin (1 U/ml) for 6 h. Whereas rhAPC in a concentration of 50 ng/ml was ineffective in modulating fractalkine transcript levels, 5 lg/ml rhAPC significantly upregulated basal as well as thrombin-induced fractalkine-expression. rhAPC did not modulate TNF-ainduced transcript levels of fractalkine and did not change the transcript levels of the proteases TACE (ADAM17) and ADAM10 (data not shown).

rhAPC stimulates fractalkine release from cultured endothelial cells rhAPC (0Æ05–10 lg/ml, which corresponded to 1–200 nmol/l) dose-dependently increased the release of soluble fractalkine in HUVEC supernatants (Fig 1). At the 24-h time-point, 5 lg/ml (100 nmol/l) rhAPC induced the amount of fractalkine in supernatants by approximately 2Æ5-fold when compared with unstimulated controls (5 lg/ml rhAPC: 3956 ± 407 pg/ml versus controls: 1574 ± 308 pg/ml; mean ± SD, n ¼ 3 independent experiments; P < 0Æ001). rhAPC was effective in stimulating fractalkine release in concentrations ranging between 1 and 10 lg/ml. Lower rhAPC concentrations showed no significant effect on fractalkine release. rhAPC (5 lg/ml) induced an upregulation of fractalkine-levels, first seen at 24 h

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converted to cDNA by reverse transcription. Purified cDNA was in vitro transcribed into cRNA in the presence of biotinylated dUTP and dCTP. Fragmented biotinylated cRNA was then hybridised to Affymetrix oligonucleotide array U133 (Affymetrix Ltd, High Wycombe, UK), followed by staining with streptavidin–phycoerythrin. Arrays were scanned and data analysis was performed using genechip 3.1 software. Two independent experiments were performed.

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Fig 1. Effect of recombinant human activated protein C (rhAPC) on fractalkine release from human umbilical vein endothelial cells (HUVEC). rhAPC causes a dose-dependent increase of soluble fractalkine levels in HUVEC supernatants. rhAPC (50 ng/ml to 10 lg/ml) was added to confluent cells, and soluble fractalkine levels were determined by enzyme-linked immunosorbent assay at t ¼ 24 h (mean ± SD; n ¼ 3 independent experiments with n ¼ 6 samples in each experiment; *P < 0Æ001).


rhAPC upregulates soluble fractalkine

rhAPC and thrombin upregulate fractalkine-mRNA expression in HUVEC rhAPC (10 lg/ml) was added to confluent cells, and fractalkine-mRNA levels were determined by semiquantitative RTPCR at t ¼ 8 h. Both rhAPC and thrombin (1 U/ml) induced an upregulation of PDGF-BB-mRNA levels. Figure 3A shows a representative gel of a total of three independent experiments with comparable results. The densitometric analysis of Fig 3A after normalisation to GAPDH-levels is shown in Fig 3B. The rhAPC effect is expressed as the percentage of increase of fractalkine-mRNA band intensity in rhAPC-treated endothelial cells versus untreated controls. At t ¼ 8 h the fractalkinemRNA band intensity in rhAPC treated HUVEC was increased by 76% when compared with controls, whereas thrombin induced an increase of fraktalkine-mRNA band intensity of 144%.

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after the addition of rhAPC and was still evident after an incubation time of 30 h (Fig 2). The upregulatory effect on fractalkine-levels was specific for rhAPC since the zymogen protein C in equimolar concentrations had no effect on fractalkine levels in HUVEC supernatants (Fig 2). To rule out possible thrombin-contamination of the rhAPC preparation (Weber et al, 2003) as a source of fractalkine-upregulation, experiments were conducted using the thrombin-inhibitor lepirudin, which alone had no effect on fractalkine-levels. Recombinant hirudin (lepirudin, 5 lg/ml) did not affect the ability of rhAPC to stimulate fractalkine release indicating that the effect of rhAPC was not because of thrombin-contamination of the rhAPC preparation (data not shown).

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Fig 3. Fractalkine-mRNA expression in response to exposure with recombinant human activated protein C (rhAPC) and thrombin. rhAPC (10 lg/ml) was added to endothelial cells, and fractalkinemRNA levels were determined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) after 8 h of incubation. (A) Representative blot of a total of three independent experiments with comparable results. (B) Densitometric analysis of (A) after normalisation to GAPDH-levels. The percentage of increase of fractalkinemRNA band intensity in rhAPC- and thrombin-treated endothelial cells when compared with untreated controls is shown. Thrombin caused a stronger upregulation of fractalkine-mRNA-levels than rhAPC.

The effect of PAR-1- and PAR-2-agonist peptides on fractalkine release from HUVEC The PAR-1-agonist peptide TFLLRNPNDK (100 lmol/l) and the PAR-2-agonist peptide SLIGKV (100 lmol/l) were used to study the participation of PAR-1 and PAR-2 in the endothelial release of fractalkine (Fig 4). Both the PAR-1- and PAR-2agonists significantly (P < 0Æ01) stimulated the release of fractalkine versus untreated controls. When used in combination, the effect of the PAR-1- and PAR-2-agonist peptides was additive (P < 0Æ001 vs. controls). These data indicate that fractalkine synthesis may be induced upon stimulation of either PAR-1, PAR-2, or a combination of both.

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Time Fig 2. Time-dependent and specific increase of soluble fractalkine by recombinant human activated protein C (rhAPC). Confluent, quiescent monolayers were exposed to rhAPC (5 lg/ml) or the zymogen protein C in equimolar concentrations for 8–30 h. rhAPC enhanced fractalkine release when compared with untreated controls after an incubation time of 24 h. This increase was still evident after 30 h. Protein C had no effect, demonstrating that the upregulation of soluble fractalkine was specific for rhAPC. Data are taken from a representative experiment out of a series of three with similar results (mean ± SEM; *P < 0Æ001).

The effect of EPCR-, PAR-1- and PAR-2-blockage on rhAPC-induced fractalkine release from HUVEC An anti-human EPCR-antibody, RCR-252, was used to study the effect of blocking EPCR on fractalkine release (Fig 5). RCR-252 (20 lg/ml), an antibody that blocks APC binding to human EPCR (Ye et al, 1999), significantly inhibited the rhAPC-induced response in HUVEC (P < 0Æ001). To further study, the role of PAR-1 and PAR-2 in rhAPC-induced fractalkine release, the cleavage blocking anti-human PAR-1antibody ATAP-2 and the cleavage blocking anti-human PAR-


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Fig 4. Soluble fractalkine release in human umbilical vein endothelial cells (HUVEC) is induced via protease-activated receptor-1 (PAR-1)and PAR-2. Both, the PAR-1 agonist peptide TFLLRNPNDK (100 lmol/l) and the PAR-2-agonist peptide SLIGKV (100 lmol/l) significantly stimulated the release of fractalkine versus untreated controls. When used in combination, the effect of the PAR-1- and PAR-2-agonist peptides was additive. These data indicate that fractalkine release may be induced upon stimulation of either PAR-1, PAR-2, or a combination of both. Data are presented as mean ± SD from two representative experiments with n ¼ 6 samples in each experiment; *P < 0Æ01; and **P < 0Æ001 vs. controls).

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release (P < 0Æ001). SAM 11 (20 lg/ml) showed no significant inhibitory effect on fractalkine release, indicating that the PAR-2-receptor is most likely not involved in the observed rhAPC upregulating effect on fractalkine release. When applied without rhAPC, neither RCR-252 and ATAP-2 nor SAM 11 had an effect (data not shown).

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Fig 5. The effect of protease-activated receptor-1 (PAR-1)-, PAR-2and EPCR-blockage on recombinant human activated protein C (rhAPC)-induced fractalkine release. The cleavage blocking antihuman PAR-1-antibody ATAP-2 and the anti-human PAR-2-antibody SAM 11 were incubated with human umbilical vein endothelial cells (HUVEC) 15 min before recombinant human activated protein C (rhAPC) (5 lg/ml) administration. ATAP-2 (20 lg/ml) significantly inhibited the rhAPC-induced fractalkine release at t ¼ 24 h from HUVEC (*P < 0Æ001), whereas SAM 11 (20 lg/ml) showed no significant inhibition. Moreover, RCR-252 (20 lg/ml), an antibody that blocks APC binding to EPCR, significantly reduced the rhAPC-induced fractalkine-response in HUVEC (*P < 0Æ001). These data indicate that both PAR-1 and EPCR are involved in the rhAPC-induced upregulation of fractalkine in endothelial cells. Data are presented as mean ± SD from two representative experiments with n ¼ 6 samples in each experiment. Experiments were performed in the presence of the thrombin inhibitor lepirudin (5 lg/ml), which alone had no effect on fractalkine release.

2-antibody SAM 11 were incubated with HUVEC 15 min before rhAPC administration (5 lg/ml). ATAP-2 (20 lg/ml) completely inhibited the rhAPC-induced effect on fractalkine 554

It has been shown that APC improves the survival of patients with severe sepsis (Bernard et al, 2001; Vincent et al, 2003, 2005) obviously by exerting multiple biological modes of action, such as anticoagulant, profibrinolytic, anti-inflammatory, proliferative activities and possibly also by modulating the responses of the immune system. Recent studies suggested that the protein C pathway induced wound repair by promoting cell proliferation, migration, expression of matrix metalloproteinase-2 and angiogenesis (Xue et al, 2004, 2005). This in vitro work describes a novel mechanism of action that possibly contributes to the observed beneficial effects of APC on the host response to infection. The present study demonstrated that supra-therapeutical concentrations of rhAPC (>1 lg/ml) upregulated endothelial fractalkine-mRNA expression and release of soluble fractalkine. Fractalkine is a chemokine that causes migration of NK cells, cytotoxic T lymphocytes and macrophages to the site of injury. Moreover, membrane-bound fractalkine activates NK cells, leading to increased local cytotoxicity and interferon-c production (Umehara et al, 2004). The upregulatory effect of rhAPC in our study was specific since the zymogen protein C in equimolar concentrations had no effect on fractalkine release. Because rhAPC is generated from protein C by thrombin, endothelial cells to rhAPC were exposed in combination with recombinant hirudin (lepirudin), a highly specific blocker of thrombin. Hirudin did not reduce the rhAPC-mediated upregulation of fractalkine release, excluding the possibility that rhAPC-induced effects were caused by contamination with thrombin (Weber et al, 2003). Our results are in contrast to the data of Joyce et al (2001), who reported a suppression of TNF-a-induced fractalkine expression by rhAPC in microarray screening arrays. In our gene profiling experiments, however, we did not examine a downregulation of TNF-a-induced fractalkine expression. Moreover, we confirmed an upregulating effect of rhAPC on basal and thrombin-induced fractalkine expression by the measurement of mRNA- and protein-levels of fractalkine in a large set of experiments performed on several different preparations of human endothelial cells from umbilical veins.

Soluble fractalkine upregulation involves ECPR and PAR-1 In endothelial cells, most signalling responses of the APCEPCR complex seem to be mediated by PAR-1. APC bound to EPCR is able to activate PAR-1, possibly by facilitating


rhAPC upregulates soluble fractalkine proteolytic activation of PAR-1 (Riewald et al, 2002). Although many PAR-1-triggered responses would be predicted to promote inflammation, some APC-induced PAR-1 dependent mechanisms have been suggested to be protective, which may be of importance in the escalation of sepsis syndrome, such as the inhibition of endothelial cell apoptosis (Mosnier & Griffin, 2003) and the upregulation of monocyte chemoattractant protein-1 (Riewald et al, 2002; Brueckmann et al, 2003). The present study demonstrated that the ability of rhAPC to induce upregulation of fractalkine release was reduced by blocking the EPCR-receptor and it was completely abolished by an anti-PAR-1-antibody. An antibody raised against the PAR-2 cleavage site did not significantly inhibit the upregulating ability of rhAPC on soluble fractalkine levels, indicating that PAR-2 is most likely not involved in the observed rhAPC-effect on fractalkine. These data suggest that APC upregulates the release of soluble fractalkine from HUVEC by binding to EPCR and signalling via PAR-1.

Quantitative differences of APC and thrombin regarding endothelial gene expression Fractalkine expression (Fig 3) serves as an example of the more powerful effects of thrombin on gene expression compared with APC. Many cellular responses to thrombin and APC have been reported to be qualitatively similar, but APC was usually remarkably less potent than thrombin in previous in vitro studies (Riewald & Ruf, 2005). This may be explained be the fact that thrombin causes increased fractalkine-expression not only via PAR-1, but also by crossactivating PAR-2 (Riewald & Ruf, 2005). Moreover, selective activation of different, still unknown intracellular pathways, which are linked to the PAR-1 receptor, may contribute to the observed quantitative differences in fractalkine-expression levels induced by rhAPC and thrombin. One can imagine that low level fractalkine gene induction by rhAPC might be sufficient to stimulate the local host response to infection, but insufficient to promote systemic inflammation, whereas high level chemokine induction by thrombin might lead to excessive accumulation of activated leucocytes, systemic inflammation and vascular injury.

The clinical relevance of the present results obtained at supra-therapeutic rhAPC concentrations must be judged cautiously on the basis of previous experience of extrapolating results from in vitro- and animal rhAPC-sepsis studies to humans. For example, it has been suggested from in vitro examinations that APC exhibits anti-inflammatory properties (Grey et al, 1994; Joyce et al, 2001; Yuksel et al, 2002), whereas clinical trials have definitely shown only the antithrombotic activity of rhAPC, and a strong basis for systemic anti-inflammatory effects is still missing (Dhainaut et al, 2003). These discrepancies of in vitro and in vivo results might be due to the fact that concentrations of APC used in several in vitro studies (Joyce et al, 2001; Riewald et al, 2002; Yuksel et al, 2002) were much higher (around 500 ng/ml to 20 lg/ml, corresponding to 10–400 nmol/l) than plasma levels of rhAPC observed under clinical conditions: the median blood levels achieved with rhAPC treatment (at an infusion rate of 24 lg/kg/h) in patients with severe sepsis were about 45 ng/ml (Macias et al, 2002). In this study, effective concentrations of rhAPC on fractalkine release were above 1 lg/ml. Therefore, the transfer of these in vitro results into in vivo conditions remains difficult and the results of this study cannot explain how rhAPC may act in human sepsis. In conclusion, these in vitro data demonstrate that induction of soluble fractalkine release is an important response of HUVEC to stimulation with rhAPC, and may represent a molecular mechanism by which rhAPC aids in the host response to infection. Further clinical trials are needed to confirm the in vivo relevance of these data.

Acknowledgements This work was supported by a grant of the Faculty of Clinical Medicine Mannheim, University of Heidelberg, Germany, and a research grant by Lilly, Germany. We would like to express our thanks to Claudia Liebetrau and Elke Burmeister for their excellent technical assistance. Oral Presentation at the 18th Annual Congress of the European Society of Intensive Care Medicine, Amsterdam, 2005.

Study limitations

References

The exact mechanisms contributing to an increased level of soluble fractalkine were not determined in this study. An upregulating effect of APC on mRNA-expression of fractalkine was detectable, but we cannot exclude that APC may also influence shedding of the membrane-bound form to generate the soluble form. As determined by microarray analysis, APC did not change mRNA transcript levels of the proteases TACE (ADAM17) and ADAM10, which can shed soluble fractalkine (Garton et al, 2001). However, the activity of these proteases and the amount of membrane-anchored fractalkine were not investigated in this study.

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