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The Journal of Immunology, 2006, 176: 1019-1025.
Copyright © 2006 by The American Association of Immunologists

Endothelial Protein C Receptor-Dependent Inhibition of Migration of Human Lymphocytes by Protein C Involves Epidermal Growth Factor Receptor1

Clemens Feistritzer2,*,{dagger}, Birgit A. Mosheimer2,*, Daniel H. Sturn*, Matthias Riewald{dagger}, Josef R. Patsch* and Christian J. Wiedermann3,*

* Division of General Internal Medicine, Department of Internal Medicine, Medical University of Innsbruck, Innsbruck, Austria; and {dagger} Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The protein C pathway is an important regulator of the blood coagulation system. Protein C may also play a role in inflammatory and immunomodulatory processes. Whether protein C or activated protein C affects lymphocyte migration and possible mechanisms involved was tested. Lymphocyte migration was studied by micropore filter assays. Lymphocytes that were pretreated with protein C (Ceprotin) or activated protein C (Xigris) significantly reduced their migration toward IL-8, RANTES, MCP-1, and substance P, but not toward sphingosine-1-phosphate. The inhibitory effects of protein C or activated protein C were reversed by Abs against endothelial protein C receptor and epidermal growth factor receptor. Evidence for the synthesis of endothelial protein C receptor by lymphocytes is shown by demonstration of receptor mRNA expression and detection of endothelial protein C receptor immunoreactivity on the cells’ surface. Data suggest that an endothelial protein C receptor is expressed by lymphocytes whose activation with protein C or activated protein C arrests directed migration. Exposure of lymphocytes to protein C or activated protein C stimulates phosphorylation of Tyr845 of epidermal growth factor receptor, which may be relevant for cytoprotective effects of the protein C pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The protein C (PC)4 pathway is one of the main modulators of coagulation activation (1, 2). The anticoagulant PC zymogen is converted to the serine protease activated PC by the thrombomodulin-thrombin complex on the phospholipid surface of endothelial cells and monocytes (3, 4). This effect is enhanced in the presence of the endothelial PC receptor (EPCR) (5). Studies have revealed that components of this pathway may also affect inflammatory responses, such as the inhibition of leukocyte adhesion to vascular endothelial cells and leukocyte accumulation in rat lungs (6). Moreover, the anti-inflammatory activity of activated PC may depend on its ability to modulate the secretion of several cytokines, such as TNF-{alpha}, IL-1{beta}, and migration inhibitory factor from endothelial cells and monocytes (3, 7) that were shown to express EPCR (8). In a mouse asthma model, activated PC suppressed the expression of Th2 cytokines and IgE from lymphocytes and thereby inhibited immunological and inflammatory responses (9).

It was previously described that protease-activated receptor (PAR) signaling may play a role in mediating effects of PC (10). In endothelial cells, MCP-1 gene expression was affected through activated PC-induced EPCR-dependent activation of endothelial cell PAR-1 (11). Moreover, the in vitro inhibition of staurosporine-induced apoptosis of endothelial cells by activated PC (12) as well as the neuroprotective effects of activated PC in a murine ischemic stroke model require EPCR and PAR-1 (13, 14). Stimulation of the G protein-coupled PAR-1 by thrombin, the main activator of the PAR-1, leads to a trans activation of the epidermal growth factor receptor (EGFR) via cross-communication of the EGFR and PAR (15, 16, 17, 18). Involvement of EGFR on PC-mediated effects has not yet been reported.

We were therefore interested in the effects of PC and activated PC on PBLs and the involvement of the EPCR and possible coreceptors. We demonstrate that lymphocytes express an EPCR that may take part in the inhibitory effects of PC and activated PC on cell migration. Furthermore, we detected the involvement of EGFR in the PC pathway in human lymphocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

RPMI 1640 with phenol red was purchased from Biological Industries, and endothelial cell growth medium was purchased from Promo Cell. BSA was obtained from Dade Behring. Gelatin, dextran, hirudin, IL-2, anti-actin mAb, rat IgG1 mAb, substance P, MCP-1, penicillin G, and streptomycin were purchased from Sigma-Aldrich. Sphingosine-1-phosphate (S1P) was from BIOMOL. FCS and L-glutamine were obtained from PAA Laboratories, RPMI 1640 without L-methionine and L-glutamine was purchased from Biochrom. Streptavidin-PE was obtained from BD Biosciences. MACS separation columns and microbeads were purchased from Miltenyi Biotec. Cellulose nitrate filters were obtained from Sartorius. Reverse transcriptase was obtained from Invitrogen Life Technologies; hot Star Taq polymerase was purchased from Qiagen. Certified PCR agarose was purchased from Bio-Rad. Protein S was obtained from Enzyme Research Laboratories. The thrombin receptor Ab ATAP2 and PAR-2 Ab (SAM-11) were obtained from Santa Cruz Biotechnology. The PAR-2 agonist (SLIGLV) was purchased from Neosystem. The PAR-1 agonist peptide TFLLRNPNDK was synthesized at The Scripps Research Institute. The Gla Ab and spectrozyme aPC were obtained from American Diagnostica. The biotinylated mouse anti-rat Ab, IgG isotype control, PP1 (4-amino-1-tert-butyl-3-(1'-naphthyl)pyrazolo[3,4-d]pyrimidine), PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), and PP3 (4-amino-7-phenylpyrazol[3,4-d]pyrimidine) were purchased from Calbiochem. The phospho-EGFR (Tyr845) Ab was obtained from Cell Signaling Technology. Anti-EGFR Ab was from obtained Abcam. Anti-EPCR Abs RCR-92 and RCR-252 were a gift from K. Fukudome (Saga Medical School, Saga, Japan). PC (Ceprotin) was purchased from Baxter, and activated PC was human rPC activated by thrombin (Drotrecogin {alpha} activated; Xigris) purchased from Lilly Research Laboratories, unless otherwise stated. All experiments with PC and activated PC were repeated in the presence of hirudin to rule out effects mediated by possible contamination with thrombin.

Preparation of monocytes and lymphocytes

PBMCs were isolated from EDTA blood of healthy volunteers. After Lymphoprep (Nycomed) density gradient centrifugation, PBMCs were collected and washed three times with normal saline. Positive selection of monocytes or lymphocytes was performed by adding MACS colloidal superparamagnetic microbeads conjugated with anti-human CD14 mAbs (monocytes) or anti-human CD3 mAbs (T lymphocytes) to cooled, freshly prepared PBMCs in MACS buffer (PBS with 5 nM EDTA and 0.5% BSA), according to the manufacturer’s instructions. Cells and microbeads were incubated for 15 min at 4°C to 6°C. In the meantime, the separation column was positioned in the MACS magnetic field and washed with MACS buffer at room temperature. The cells were washed with MACS buffer, resuspended, and loaded onto the top of the separation column. The eluent was withdrawn, and after removal of the column from the magnet, trapped monocytes (CD14+) or lymphocytes (CD3+) were eluted with 6-fold amount of cold MACS buffer, centrifuged, and resuspended in medium containing 0.5% BSA. Preparations yielded a purity of ~98% by immunocytochemistry. It is known that IL-2 plays a key role in growth and maturation of lymphocytes (19). In additional experiments, besides using freshly prepared cells, we stimulated lymphocytes with 100 U/ml IL-2 for 24 h before experiments were performed.

For control experiments, Jurkat leukemic T cells were used. Cells were cultured in RPMI 1640 medium containing 100 U/ml penicillin G/streptomycin and 10% FCS at 37°C in 5% CO2.

Human endothelial cell culture

HUVECs from fresh umbilical cords were isolated and grown to confluence in a humidified atmosphere at 37°C. The growth medium was supplemented with 10% FCS. Tissue culture flasks were coated with 0.2% gelatin before seeding of cells. HUVECs of passage 2 were used for RNA extraction.

Lymphocyte migration assay

Migration assays were performed using a modified 48-well Boyden microchemotaxis chamber (NeuroProbe) in which a 5-µm-pore-size cellulose nitrate filter separated the upper and the lower chamber. In various experiments, lymphocyte migration was tested toward PC or activated PC (both 0.1 pg/ml to 10 µg/ml). For deactivation of migration, lymphocytes were preincubated for 30 min at various concentrations of PC or activated PC (0.1 pg/ml to 10 µg/ml). After washing twice in HBSS, lymphocyte migration was assessed toward RANTES, IL-8, MCP-1, substance P, and S1P in the lower wells of the chamber for 90 min at 37°C in a humidified atmosphere (5% CO2).

In some experiments, cells were copreincubated with either function-blocking (RCR-252) or nonblocking (RCR-92) Abs (both rat IgG1 (20); 20 µg/ml) against the EPCR. Incubation with a rat IgG1 Ab served as a control. For blocking the Gla domain of PC cells, coincubation with a Gla Ab (5 µg/ml) was performed. Additionally, to exclude effects of thrombin, cells were pretreated with hirudin (1 U/ml). PC preparations followed by migration toward chemoattractants were performed as described above.

To further elucidate possible involvement of coreceptors and signaling pathways in EPCR-dependent effects on lymphocyte migration, cells were pretreated with either PAR-1 (1 µM) and PAR-2 (100 µM) agonists or Abs (both 10 µg/ml), an anti-EGFR Ab, EGFR tyrosine kinase blockers, and various Src family-selective tyrosine-kinase inhibitors (all 10 µM), and concomitantly with either PC or activated PC, followed by washing and assessment of their migratory response toward IL-8.

After the migration period, the nitrocellulose filters were dehydrated, fixed, and stained with hematoxylin. Migration depth of the cells into the filters was quantified by microscopy, measuring the distance (µm) from the surface of the filter to the leading front of three cells. Data are expressed as a chemotaxis index, which is the ratio between the distance of directed and random migration without attractants of lymphocytes into the nitrocellulose filters.

Apoptosis of lymphocytes

Effects of PC or activated PC on apoptosis (24 h) of lymphocytes were tested using the annexin V/FITC kit (Bender MedSystems). Freshly prepared lymphocytes were incubated in medium, PC (10 µg/ml), or activated PC (10 µg/ml) for 24 h at 37°C in a humidified atmosphere. Staurosporine (1 µg/ml) served as proapoptotic control, and was used with or without PC or activated PC. For quantification, lymphocytes were washed twice in PBS and resuspended in binding buffer (1 x 106 cells/ml); 195 µl of the cells was incubated with 5 µl of annexin V FITC for 10 min at room temperature, washed, and analyzed on a FACS with CellQuest software (BD Biosciences FACScan).

RT-PCR

Total RNA was extracted from 107 cells by using RNA-Bee (Tel-Test). A reverse-transcriptase reaction was performed on 1 µg of RNA using random hexamer primers. To exclude genomic DNA contamination, the transcription reaction was performed in the absence of reverse transcriptase in control experiments. The cDNA product was amplified by 38 cycles of 60 s at 95°C, 60 s at 54°C, and 60 s extension at 72°C using specific primer pairs (MWG Biotec; human EPCR forward, 5'-GGC AGT TTC ATC ATT GCT GG-3', and reverse, 5'-TTG AAC GCC TCA GGT GAT TC-3'). The RT-PCR products (409 bp) were separated in 1.5% (w/v) agarose gels.

FACS analysis of EPCR expression on lymphocytes

A total of 5 x 105 cells was washed twice in Dulbecco’s PBS containing 0.5% BSA and incubated with 150 µg/ml human IgG for 20 min at 4°C. After pelleting, cells were incubated with 10 µg/ml anti-EPCR Ab RCR-252 or the respective isotype-matched control IgG for 30 min at 4°C. After washing, 10 µg/ml biotinylated mouse anti-rat IgG was incubated for another 30 min. Cells were washed twice, and the lymphocytes were subsequently incubated with a 1/25 dilution of streptavidin-PE, washed twice, then immediately analyzed on a FACS with CellQuest software (FACScan; BD Biosciences).

Western blot analysis of EGFR phosphorylation

Cells were incubated with PC and activated PC at various concentrations (10 ng/ml and 1 µg/ml) for 60 min. Cells were lysed in lysis buffer containing 1% Triton X-100. Proteins were separated on 10% SDS polyacrylamide gels and blotted onto polyvinylidene difluoride membranes, which were blocked with 2% phophatase-free milk powder in TBS with 0.1% Tween 20. The Ab against the phospho-EGFR (Tyr845) was then diluted in 0.2% milk powder according to manufacturer’s instructions, and blots were incubated overnight at room temperature. Immunoreactivity was determined using peroxidase-conjugated goat anti-rabbit IgG and Super Signal chemiluminescent substrate (Pierce). Intensity of the Western blot bands was quantified using the Fluor-S MultiImager System and the Quantity One Software (Bio-Rad). Staining with anti-actin served as loading control.

Activation of PC

The amiodolytic activity of generated activated PC was monitored by hydrolysis of the synthetic chromogenic substrate Spectrozyme aPC, as described (21). The ability of human lymphocytes (106 cells/ml) to generate biologically active activated PC was tested in serum-free medium (RPMI 1640/0.5% BSA) with or without adding PC (3 µg/ml; Hematologic Technologies) in the presence and absence of thrombin (1 and 10 nM, respectively). Activation of activated PC by endothelial cells was used as a positive control. The concentration of endogenous generated activated PC in reaction mixtures after an incubation period of 90 and 180 min was determined by reference to results of standard curves, which were prepared by using defined concentrations of exogenous activated PC (Hematologic Technologies). The rate of hydrolysis was monitored at 405 nm at room temperature in a Vmax kinetic plate reader SpectroMAX plus (Molecular Devices).

Statistical methods

Data are expressed as mean ± SEM. Means were compared by the Mann-Whitney U test and Kruskal-Wallis ANOVA. A difference with p < 0.05 was considered to be significant. Statistical analyses were performed using the StatView software package (Abacus Concepts).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Effects of PC and activated PC on lymphocyte migration

Arrest of inflammatory cells at sites of coagulation is an important component of the immune response to injury. Effects of PC and activated PC on lymphocyte motility were therefore tested. Lymphocytes were pretreated at 37°C for 30 min with various concentrations of PC or activated PC to investigate their effects on lymphocyte migration toward different chemoattractants. Both PC and activated PC preparations inhibited migration of lymphocytes toward IL-8 (1 nM) and RANTES (10 ng/ml) in a dose-dependent manner. PC and activated PC maximally inhibited migration at concentrations of 1 µg/ml; the plasma zymogen and the activated protein were similarly active. Moreover, no difference between the unstimulated and IL-2-stimulated lymphocytes in response to PC and activated PC was detectable. Neither PC nor activated PC affected the migration of lymphocytes toward the bioactive lysophospholipid S1P (10 nM) (Fig. 1). Inhibition of lymphocyte chemotaxis by PC and activated PC was also effective toward MCP-1 (10 ng/ml), and the inhibition was pronounced in the IL-2-pretreated cells. Substance P (10 nM)-induced lymphocyte migration was also diminished by PC and activated PC, but to a lesser extent compared with IL-8, RANTES, and MCP-1 (data not shown). Migration of Jurkat T cells toward RANTES (10 ng/ml) was not significantly inhibited by incubation with PC or activated PC.



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  		FIGURE 1. Inhibition of chemokine-induced migration of lymphocytes by PC and activated PC. Unstimulated (A and B) and IL-2-stimulated (C and D) lymphocytes were preincubated with varying PC/activated PC concentrations for 30 min. Then, chemotaxis toward RANTES (10 µg/ml), IL-8 (1 nM), and S1P (10 nM) was tested. Results are given as the mean ± SEM of the migration index, which is the ratio of the distance of migration (µm) toward attractant and the distance toward medium. Mean distance of random migration was 78.0 ± 16.0 µm. *, p < 0.05; Mann-Whitney U test vs medium incubation after multiple group comparison by Kruskal-Wallis test. n = 4.

 
To explore the chemotactic properties of PC and activated PC in the absence of other chemoattractants, freshly prepared lymphocytes were allowed to migrate toward different concentrations of PC or activated PC (1 µg/ml to 1 pg/ml); RANTES (10 ng/ml) was used as a positive control. Neither PC nor activated PC induced a significant migratory response of lymphocytes (data not shown).

Receptor mechanisms in the deactivation of lymphocyte chemotaxis by PC and activated PC

Inhibitory effects of PC and activated PC on migration may be mediated via EPCR on lymphocytes. Therefore, effects of a function-blocking anti-EPCR Ab on lymphocyte chemotaxis were compared with that of a nonblocking anti-EPCR Ab. In the presence of the function-blocking anti-EPCR Ab RCR-252 (20 µg/ml), PC and activated PC failed to inhibit chemotaxis toward RANTES (Fig. 2A) and IL-8 (Fig. 2B). Coincubation of either PC or activated PC with the anti-EPCR Ab RCR-92 (20 µg/ml), which does not block its PC activator function, had no significant effect on the inhibition of chemotaxis.



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  		FIGURE 2. Abrogation of effects of PC and activated PC on chemoattractant-induced migration of lymphocytes through anti-EPCR mAbs. Cells were concomitantly incubated with either PC or activated PC and a function-blocking anti-EPCR Ab RCR-252 or a nonfunction-blocking anti-EPCR Ab RCR-92 for 30 min. Incubation with a rat IgG1 Ab (IgG) served as an isotype control. Then, chemotaxis toward RANTES (A) (10 µg/ml) and IL-8 (B) (1 nM) was tested. Results are given as the mean ± SEM of the migration index, which is the ratio of the distance of migration (µm) toward attractant and the distance toward medium. Mean distance of random migration was 70.0 ± 4.0 µm. *, p < 0.05; Mann-Whitney U test vs incubation with PC or activated PC after multiple group comparison by Kruskal-Wallis test. n = 4.

 
It was previously described that PAR signaling may play a role in mediating effects of PC in endothelial cells (10). Therefore, it was tested whether or not PAR agonists or Abs could influence PC- or activated PC-mediated effects in lymphocytes. Neither treatment of lymphocytes with PC or activated PC in the presence of Abs against PAR-1 (ATAP2, 10 µg/ml) or PAR-2 (SAM-11, 1 µg/ml) nor a PAR-1 agonist (TFLLRNPNDK, 1 µM) or a PAR-2 agonist (SLIGLV, 100 µM) significantly affected directed migration toward IL-8 (1 nM). To exclude the possibility of thrombin, which may be present in contaminating doses, from forming complexes with PC, thrombomodulin and EPCR coincubation with PC or activated PC and hirudin (1U/ml) was also performed. Again, no effect on the inhibitory properties of PC or activated PC on lymphocyte migration of interfering with a thrombin-dependent pathway was seen. Coincubation of PC or activated PC and protein S (10 µg/ml) also had no significant influence on the migration-inhibitory effects of PC or activated PC (Table I).


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  		Table I. Abrogation of effects of PC and activated PC on chemokine-induced migration by Gla Abs and EGFR tyrosine kinase inhibitorsa

 
As the Gla domain of PC has been shown to be important in the PC interaction with EPCR (4), we tested for whether or not blocking the Gla domain could influence PC/activated PC effects. Coincubation with a Gla Ab (5 µg/ml) was able to diminish the migration-inhibitory effects of PC or activated PC (Table I).

Src tyrosine kinases are implicated in multiple signaling pathways, including regulation of cell growth and migration (22). The Src kinase inhibitor PP1 and the inactive analog PP3, which inhibits the EGFR tyrosine kinase, but not the specific PP2 blocker, abolished the effects of PC or activated PC (Table I). Pretreatment with a specific EGFR Ab abolished the inhibitory effects of PC and activated PC on lymphocyte migration (Fig. 3), whereas the IgG2a control Ab was inactive (data not shown).



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  		FIGURE 3. Abrogation of effects of PC and activated PC on IL-8-induced migration of lymphocytes through an anti-EGFR mAb. Cells were concomitantly incubated with either PC or activated PC (both 1 µg/ml) and various concentrations of a function-blocking anti-EGFR Ab for 30 min. Then, chemotaxis toward IL-8 (1 nM) was tested. Migration of lymphocytes after incubation with the EGFR Ab in the absence of PC/activated PC toward IL-8 served as control. Results are given as the mean ± SEM of the migration index, which is the ratio of the distance of migration (µm) toward attractant and the distance toward medium. Mean distance of random migration was 56.5 ± 5.2 µm. *, p < 0.05; Mann-Whitney U test vs PC/activated PC incubation after multiple group comparison by Kruskal-Wallis test. n = 4.

 
Expression of EPCR in lymphocytes

EPCR has been proposed to mediate anti-inflammatory effects of activated PC (23), and its expression in HUVECs and monocytes has been described (8, 24). To determine whether EPCR gene is expressed in lymphocytes, RT-PCR was performed. Data show that EPCR mRNA is expressed in human lymphocytes. EPCR mRNA expression in monocytes and HUVECs served as positive control (Fig. 4A).



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  		FIGURE 4. RT-PCR and FACS analysis of EPCR in lymphocytes. A, EPCR mRNA in lymphocytes, HUVECs, and monocytes. A quantity amounting to 1 µg of total RNA from each sample was reverse transcribed into cDNA and amplified for the EPCR gene using PCR. EPCR is represented by the 409-bp product. PCR without any template served as negative control. FACS analysis of anti-EPCR mAb binding to unstimulated (B) and IL-2-stimulated (C) lymphocytes. Fluorescence analysis used a FACScan flow cytometer, and a histogram of PE fluorescence is shown. Cells were either incubated with isotype-matched control IgG or anti-EPCR mAb and stained with PE-conjugated streptavidin.

 
Presence of EPCR-like immunoreactivity on the surface of lymphocytes was tested by FACS analysis. A significant shift of fluorescence was observed by the anti-EPCR Ab RCR-252, indicative of cell surface presence of EPCR protein (Fig. 4B). Stimulation with IL-2 for 24 h led to a slightly enhanced shift in fluorescence compared with the untreated lymphocytes (Fig. 4C).

In agreement with published data (8), unstimulated Jurkat T cells express only a negligible amount of EPCR, as tested in FACS analysis and RT-PCR. Stimulation with IL-2 slightly increased EPCR expression on the cell surface (data not shown).

Activation of EGFR in lymphocytes

To confirm involvement of EGFR in EPCR-dependent signaling in lymphocytes, Western blot analyses were performed. The Ab used detects endogenous levels of EGFR only when phosphorylated at Tyr845. Phosphorylation of Tyr845 stabilizes the activation loop of the receptor kinase domain and maintains the enzyme in an active state (25, 26). To investigate the phosphorylation status of EGFR after PC or activated PC stimulation, lymphocytes were incubated with different concentrations of PC or activated PC (10 ng/ml and 1 µg/ml) for 1 h. An increase of the active Tyr845-phosphorylated status of EGFR could be detected in response to PC and activated PC (Fig. 5A). This effect could be reversed by coincubation of activated PC with Abs against PAR-1, EPCR, and EGFR, but not against PAR-2 (Fig. 5B).



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  		FIGURE 5. Phosphorylation status of EGFR (Tyr845) in PC- and activated PC-stimulated lymphocytes. Freshly prepared lymphocytes were incubated with PC or activated PC. Cells then were washed twice and lysed. Equal protein concentrations of lysates were loaded onto lanes. A, Phosphorylation status of phospho-EGFR was visualized after incubation with different concentrations of PC and activated PC (10 ng/ml and 1 µg/ml) for 60 min using a mAb against phospho-EGFR Tyr845. B, Phosphorylation of Tyr845 was detected after coincubation of cells with activated PC and Abs against PAR-1, PAR-2, EGFR, and EPCR (all 1 µg/ml). Phospho-EGFR protein is represented by the 175-kDa product.

 
Effects of PC and activated PC on apoptosis of lymphocytes

Effects of PC or activated PC on spontaneous apoptosis of isolated lymphocytes were studied using the annexin V/FITC labeling kit. The number of apoptotic cells was quantified by FACS analysis after 24 h of incubation in serum-free medium, or in the presence of PC (1 µg/ml) or activated PC (1 µg/ml) with or without staurosporine (1 µg/ml; proapoptotic). After 24 h, 27.8 ± 1.8% (n = 5) of lymphocytes underwent apoptosis. Treatment with PC (26.9 ± 3.4%) and activated PC (23.7 ± 3.3%) did not affect spontaneous apoptosis of human lymphocytes. After incubation with the known proapoptotic substance staurosporine, 66.9 ± 9.9% (p < 0.05 vs medium) of the cells were apoptotic. Neither PC (62.1 ± 8.5%) nor activated PC (65.3 ± 10.7%) altered the rate of staurosporine-induced lymphocyte apoptosis (p > 0.1 vs medium).

Generation of activated PC by endothelial cells

Cultured monolayers of human endothelial cells enhance the rate of thrombin-catalyzed PC activation (27). In our assay, based on the amiodolytic activity of generated activated PC, baseline levels measured in the absence of any cells correspond to the activity of 129 ± 20 ng/ml after incubation of PC (3 µg/ml) with 10 nM thrombin for 3 h. In presence of endothelial cells, we detected nearly a 10-fold increase in the generation of biologically active activated PC (1012 ± 150 ng/ml), whereas lymphocytes did not enhance activated PC generation (116 ± 16 ng/ml) using the same conditions. Moreover, the presence of lymphocytes did not affect activated PC activity induced by endothelial cells (848 ± 110 ng/ml). Lower concentrations of thrombin showed a minor generation of activated PC in our assay. In absence of thrombin, activated PC activation could not be detected.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The PC pathway is an important regulator of the blood coagulation system. In addition to its regulatory function in coagulation and fibrinolysis, recent studies suggested that activated PC may also play important roles in inflammatory and immunomodulatory processes directly and independent of other components of the coagulation cascade (6, 28, 29, 30). Our present study implicates expression of EPCR of lymphocytes, which affects PC- and activated PC-induced inhibition of cell migration. These effects could be reversed by an anti-EPCR Ab directed against the PC or activated PC binding site on EPCR. Expression of EPCR in lymphocytes is confirmed by identification of expression of mRNA and detection of cell surface EPCR by FACS analysis. Moreover, PC- and activated PC-mediated effects could be reversed by an Ab against EGFR, and PC or activated PC led to active state phosphorylation of the EGFR.

In addition to its role as an anticoagulant, PC and activated PC have induced anti-inflammatory effects. Treatment of patients with severe sepsis with activated PC may lead to a reduction of mortality (29). The molecular basis for PC’s or activated PC’s anti-inflammatory effects is incompletely understood. We have recently suggested that anti-inflammatory effects of PC may be due to inhibition of migration of inflammatory cells (31, 32). In the present study, it was observed that activated PC inhibits directed migration of lymphocytes in an EPCR-dependent fashion because the inhibitory effects could be reversed with an anti-EPCR Ab (Fig. 2). PC and activated PC were equally effective in reducing lymphocyte chemotaxis. As activation of PC to activated PC by lymphocytes could not be measured or remained under the detection limit of our assay, effects seen can be assumed to be directly mediated by PC. In our experiments, the therapeutic preparations of PC (Ceprotin) and activated rPC (Xigris) were used. As these substances may contain contaminants, a number of control experiments were performed to rule out possible side effects caused by these contaminants. Thrombin is a potential contaminate of PC and activated PC preparations, and experiments in which thrombin was directly inactivated by hirudin were performed. PC and activated PC effects appeared independent of thrombin, as hirudin had no effects on our experiments. Moreover, experiments using specific Abs demonstrated that binding of PC/activated PC to the EPCR mediated the effects seen in our assays. Using therapeutic preparations in our in vitro experiments may lead to a better understanding of the anti-inflammatory mechanisms in vivo.

Migration of lymphocytes was tested toward RANTES, IL-8, MCP-1, substance P, and S1P. PC and activated PC were able to inhibit migration toward IL-8, RANTES, as well as MCP-1, and to a lesser extent toward substance P in a dose-dependent manner, but chemotaxis toward S1P was not affected (Fig. 1). Nearly complete abrogation of stimulated migration was seen at concentrations in the range of 1 µg/ml, which is the amount of PC circulating in animals and humans with normal functioning endothelium (33). Furthermore, these concentrations are in good correlation with the concentrations used in other in vitro assays (10, 11). Diffuse exposure of lymphocytes to circulating PC in the intravascular compartment may prevent lymphocytes from premature activation. PC and activated PC inhibited migration of lymphocytes toward attractants of different classes that differ in their receptor signaling cascades. This observation may indicate that the PC pathway of lymphocytes affects mechanisms central to the process of cell migration rather than chemoattractant or postreceptor signaling. However, neither PC nor activated PC affected the migration of lymphocytes toward S1P. Recent publications have demonstrated a dose-dependent dual role of S1P on the migration of lymphocytes (34). Low concentrations of S1P can induce migration of lymphocytes, whereas higher concentrations diminished chemotaxis toward Exodus-2 and RANTES. In consideration of the previously described cross talk of activated PC and S1P (35, 36), this might be a new concept to clarify effects of PC/activated PC on leukocyte migration.

The ability of activated PC to inhibit thrombin generation has the potential to reduce the proinflammatory activities of thrombin, which are mediated by PAR-1, PAR-3, and PAR-4 (23). PARs mediate thrombin-induced inflammation by generating signals that provide the intracellular, functional link between inflammation at sites of vascular injury, modulating platelet and endothelial cell activation (37, 38, 39). Recently, Riewald et al. (10) demonstrated that PAR-1, the prototypical thrombin receptor, is a target for EPCR-dependent activated PC signaling in endothelial cells and that PAR-1 signaling could account for all activated PC-induced protective genes against sepsis. It was previously published that human lymphocytes constitutively express biologically active PARs, including PAR-1 and PAR-2 (40, 41). We investigated the effect of selective PAR-1 or PAR-2 agonists and Abs against PAR-1 and PAR-2 on lymphocyte chemotaxis. Neither the agonists nor the Abs against PAR-1 and PAR-2 were able to affect PC- or activated PC-induced inhibition of lymphocyte chemotaxis. According to these data, involvement of PAR-1 or PAR-2 in the effects of PC or activated PC on lymphocyte migration is unlikely.

The coagulation and anticoagulation proteases, like factors VII, IX, X, and PC, have a common domain structure with an N-terminal Gla-containing domain that is followed by two domains that are homologous to the epidermal growth factor that provides the coagulation serine proteases with unique properties, such as affinity for certain biological membranes, and also mediates protein-protein interactions (42, 43). In our experiments, Abs against the Gla domain of PC/activated PC abolished the inhibitory effects on lymphocyte migration.

Previous studies have demonstrated that EPCR expression, which plays a critical role in augmenting PC activation by the thrombin-thrombomodulin complex and in modulating the functions of the PC pathway, is not restricted to the endothelium, but has also been detected in monocytes, neutrophils, eosinophils, as well as a number of cancer cell lines (8, 31, 32). Thus, EPCR may also be involved in the lymphocytes’ response to PC and activated PC. A blocking Ab against EPCR was able to diminish effects of PC and activated PC on lymphocyte migration. A control Ab that binds to EPCR, but does not affect EPCR in the activation of PC (20), failed to affect PC- or activated PC-dependent inhibition of migration. This may indicate that ligation of the high affinity binding site of EPCR (27) for PC and activated PC is required for the functional response to occur. Data show for the first time that lymphocytes express the EPCR gene (Fig. 4). Detection of EPCR protein on the cell surface of lymphocytes was also shown by FACS analysis, thus confirming functional and molecular data. Lymphocytes stimulated with IL-2 showed a slightly higher expression of EPCR. However, the response of PC and activated PC on cell migration remained unchanged compared with untreated lymphocytes.

Stimulation of apoptosis has important implications for the resolution of immunologic responses. Inhibition of apoptosis with PC or activated PC such as detected in endothelial cells (28) was not observed in our study, which is in agreement with recent data published for granulocytes (31, 32).

Cross-communication between different signaling systems allows the integration of a great diversity of stimuli that a cell receives under varying physiological situations. The trans activation of EGFR signaling pathways upon stimulation of G protein-coupled receptors, which are critical for mitogenic activity of ligands like endothelin and thrombin, provides evidence for such an interconnected communication network (15, 16, 17, 18). Previous studies revealed that stimulation of rat fibroblasts with thrombin, which primarily activates PAR-1, -3, and -4, leads to a phosphorylation of the EGFR. As the involvement of PAR-1 in anti-inflammatory effects of activated PC is widely discussed and it is known that lymphocytes express PAR-1 and EGFR (41), we tested for effects on EGFR in our settings. In migration experiments, coincubation with an EGFR-blocking Ab or an EGFR tyrosine kinase inhibitor abolished effects of PC and activated PC on human lymphocytes. In Western blot analyses, we could demonstrate a stimulation of the major tyrosine phosphorylation site Tyr845 of EGFR at biologically active concentrations of PC or activated PC. Coincubation with blocking Abs against PAR-1, EPCR, or EGFR diminished the effects of activated PC on phosphorylation of the EGFR. Taken all together in consideration, these results indicate that the EGFR pathway is possibly involved in PC and activated PC signaling, and also that besides the PAR-independent effects on lymphocyte migration, cross talk with PAR-1 may be involved in PC- and activated PC-mediated effects on immune cells.

In conclusion, the results show that PC as well as activated PC inhibit the migratory response of lymphocytes to chemokines in response to potent stimuli via mechanisms that involve EPCR and EGFR of human lymphocytes. This furthermore indicates that modulation of lymphocyte function may be among the effects of the endogenous anticoagulant PC pathway.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Verein zur Förderung von Forschung und Fortbildung in klinischer Kardiologie und Intensivmedizin–Innsbruck. Back

2 C.F. and B.A.M. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Christian J. Wiedermann, Department of Internal Medicine, Medical University of Innsbruck, Anichstrasse 35, A-6020 Innsbruck, Austria. E-mail address: christian.wiedermann{at}uibk.ac.at Back

4 Abbreviations used in this paper: PC, protein C; EGFR, epidermal growth factor receptor; EPCR, endothelial PC receptor; PAR, protease-activated receptor; S1P, sphingosine-1-phosphate. Back

Received for publication December 2, 2004. Accepted for publication November 3, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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