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Originally published online as doi:10.1189/jlb.0907652 on April 24, 2008

Published online before print April 24, 2008
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(Journal of Leukocyte Biology. 2008;84:215-223.)
© 2008 by Society for Leukocyte Biology

Thrombin-induced expression of endothelial CX3CL1 potentiates monocyte CCL2 production and transendothelial migration

Milan Popovic1, Yves Laumonnier1, Ladislav Burysek, Tatiana Syrovets and Thomas Simmet2

Institute of Pharmacology of Natural Products and Clinical Pharmacology, Ulm University, Ulm, Germany

2Correspondence: Institute of Pharmacology of Natural Products and Clinical Pharmacology, Ulm University, Helmholtzstr. 20, D-89081 Ulm, Germany. E-mail: thomas.simmet{at}uni-ulm.de


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ABSTRACT
 
CX3CL1 (fractalkine, neurotactin) is the sole CX3C chemokine. It induces monocyte locomotion in its cleaved form, but in its membrane-anchored form, it also acts as an adhesion molecule. The expression of CX3CL1 is up-regulated in endothelial cells by proinflammatory cytokines such as IL-1 or TNF-{alpha}. Here, we studied the effect of the serine protease thrombin on endothelial CX3CL1 induction and its putative relevance for monocyte function. In HUVEC, thrombin triggered a time- and concentration-dependent expression of CX3CL1 at the mRNA and the protein level as shown by RT-PCR, Western immunoblotting, and flow cytometric analysis. Thrombin induced CX3CL1 by activating protease-activated receptor 1 (PAR1) as demonstrated by the use of PAR1-activating peptide and the PAR1-specific antagonist SCH 79797. The thrombin-induced CX3CL1 expression was NF-{kappa}B-dependent, as shown by EMSA, ELISA, and by inhibition of the NF-{kappa}B signaling pathway by the I{kappa}B kinase inhibitor acety-11-keto-β-boswellic acid or by transient overexpression of a transdominant-negative form of I{kappa}B{alpha}. Upon cocultivation of human monocytes with HUVEC, the thrombin-dependent induction of membrane-anchored CX3CL1 in HUVEC triggered monocyte adhesion and an enhanced release of the MCP-1/CCL2 by monocytes and potentiated the monocyte transendothelial migration. Accordingly, the recombinant extracellular domain of CX3CL1 induced CCL2 release by monocytes. Thus, the thrombin-induced monocyte/endothelial cell cross-talk mediated by increased CX3CL1 expression potentiates the CCL2 chemokine generation that might contribute to the recruitment of monocytes into inflamed areas.

Key Words: Mono-Mac-6 • HUVEC • chemokine • protease-activated receptor 1 • coculture


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INTRODUCTION
 
Leukocyte migration is crucial for inflammation and depends largely on chemokines [1 , 2 ]. Most chemokines are secreted proteins and belong to four subfamillies, based on the relative position of their cysteine residues [2 ]. Fractalkine (CX3CL1, neurotactin) is the sole member of the CX3C chemokine family and unlike other chemokines, exists not only as a secreted glycoprotein but also as a membrane-anchored protein [3 , 4 ]. In its membrane-anchored form, the protein consists of an extracellular domain containing the CX3C chemokine motif and an extended, mucin-like stalk connected to transmembrane and intracellular domains. Secreted CX3CL1 can be generated by cleavage at the membrane-proximal region by TNF-{alpha}-converting enzyme [TACE; a disintegrin and metalloproteinase 17 (ADAM17)] and metalloproteinase ADAM10 [5 , 6 ]. CX3CL1 was regarded as a potent chemotactic molecule, but its role in the regulation of leukocyte adhesion is now also considered a key function [7 ].

CX3CL1 is expressed in a variety of cells such as epithelial cells, dendritic cells, and microglia [4 ]. Increased expression of CX3CL1 has been observed under proinflammatory conditions including vascular diseases [8 , 9 ]. In endothelial cells, cytokines such as IL-1 and TNF-{alpha}, IFN-{gamma}, CD40 ligand, and LPS induce a marked increase in CX3CL1 expression [3 , 10 , 11 ]. Recently, it has been demonstrated that activated protein C induces expression of CX3CL1 in endothelial cells [12 ]. In rat aortic endothelial cells, the cytokine and LPS-induced expression of CX3CL1 proceed via activation of NF-{kappa}B [13 ], yet the mechanisms of the thrombin-induced CX3CL1 expression remain unknown.

Thrombin is not only the key effector of the coagulation cascade but also plays a significant role in inflammatory diseases [14 ]. Thus, besides its crucial role in the generation of fibrin, thrombin is also a potent activator of various cell types. Particularly in endothelial cells, thrombin triggers production of lipid mediators and expression of adhesion molecules such as ICAM-1 and P-selectin [15 , 16 ]. The cellular response to thrombin is mediated through protease-activated receptors (PARs), a family of seven-transmembrane G-protein-coupled receptors activated by proteolytic cleavage of the amino-terminal extracellular domain. Among the four members of the PAR family, PAR1, PAR3, and PAR4 can be activated by thrombin [15 ]. Here, we demonstrate that in HUVEC, thrombin induces the expression of CX3CL1 through PAR1 stimulation and subsequent activation of the NF-{kappa}B pathway. This increased CX3CL1 expression leads to an enhanced, CX3CL1-mediated cross-talk between endothelial cells and monocytes, resulting in a potentiated production of CCL2 and an accelerated monocyte transendothelial migration.


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MATERIALS AND METHODS
 
Cells
Human peripheral monocytes were isolated by Percoll gradient centrifugation as described [17 ]. Preparations with >94% CD14+ cells were used; contaminating cells were lymphocytes (2–6%). Flow cytometry of cells stained additionally with anti-CD41 antibodies did not reveal any platelets associated with monocytes. The monocytic cell line Mono-Mac-6 (DSMZ, Braunschweig, Germany) was grown in RPMI 1640. HUVEC were grown as described [18 ] and used between passages 2 and 6. HUVEC were stimulated with thrombin (Enzyme Research Laboratories, South Bend, IN, USA), PAR1-activating peptide (AP; TFLLRNPNDK; Interactiva, Ulm, Germany), PAR3-AP (TFRGAP), or PAR4-AP (GYPGQV; Bachem, Bubendorf, Switzerland) in serum-free medium for 60 min. The medium was carefully removed, and the cells were kept in fresh medium containing 1% FCS for an additional 2 h for mRNA expression or 11 h for analysis or cocultivation experiments; all experiments followed this activation scheme.

mRNA expression
Total RNA was extracted with Trizol (Invitrogen, Carlsbad, CA, USA), and the CX3CL1 expression was analyzed by RT-PCR [19 ]; GAPDH served as normalization control [20 ]. The identity of the amplification product was verified by automated sequencing (Applied Biosystems, Foster City, CA, USA).

Analysis of protein expression by Western immunoblot and flow cytometry
For Western immunoblots, HUVEC treated with thrombin, PAR1-AP, or TNF-{alpha} (10 ng/ml) for 24 h were analyzed with anti-CX3CL1 mAb (R&D Systems, Minneapolis, MN, USA); actin (antibody from Chemicon International, Temecula, CA, USA) served as loading control. The specific PAR1 antagonist SCH 79797 (10 µM, Tocris, Avonmouth, UK) [21 ] and the I{kappa}B kinase (IKK) inhibitor acety-11-keto-β-boswellic acid (AKβBA; 10 µM) [20 ] were added 20 min prior to stimulation with thrombin (3 U/ml). Soluble CX3CL1 was analyzed by ELISA (R&D Systems).

For flow cytometry, cells treated for 12 h or 24 h were incubated with 10 µg/ml monoclonal anti-CX3CL1, anti-CCL2 (Peprotech, London, UK), or control mouse IgG, PE-conjugated anti-mouse F(ab)2 (Dianova, Hamburg, Germany) and analyzed on a FACScan (BD Biosciences, San Jose, CA, USA). For the analysis of intracellular CCL2, we calculated the mean fluorescence index (MFI) defined as the ratio between mean fluorescence intensity of samples stained with anti-CCL2 antibody and samples stained with control antibody.

Transient transfection
HUVEC were transiently transfected with the pCMV4-I{kappa}B{alpha} super-repressor expression vector encoding a transdominant-negative mutant of I{kappa}B{alpha} (kind gift from Kenneth Marcu, Stony Brook University, Stony Brook, NY, USA) using the jetPEI transfection reagent according to the manufacturer’s instructions (Polyplus, Illkirch, France).

NF-{kappa}B transcription factor assays
Nuclear extracts were prepared from HUVEC stimulated with 3 U/ml thrombin for 30 min and analyzed by EMSA [20 ] and TransAM ELISA for NF-{kappa}B transcription factor (Active Motif, Carlsbad, CA, USA). Briefly, DNA–protein interactions were assayed by incubating 1 µg nuclear extract with a 32P-end labeled, double-stranded, NF-{kappa}B site-specific probe (Promega, Madison, WI, USA) in the presence of 1 µg polydeoxyinosinic:deoxycytidylic acid [poly(dI-dC); GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA] to avoid nonspecific binding in 20 µl binding buffer, pH 7.5 (10 mM Tris-HCl, 4% glycerol, 0.5 mM EDTA, 40 mM NaCl, 0.5 mM DTT, and 1 mM MgCl2), for 30 min. In supershift experiments, nuclear extracts were incubated with p65 mAb or polyclonal antibodies against p52 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 0.5 µg) for 1 h at 4°C before the addition of 32P-end labeled NF-{kappa}B DNA. For competition experiments, nuclear extracts were incubated for 30 min with a 100-fold excess of unlabeled, specific NF-{kappa}B or activator protein 2 oligonucleotides. Activation of p65 and p52 was also determined with the TransAM ELISA in nuclear extracts stimulated with thrombin, 3 U/ml, for 60 min. The data are expressed as amount of transcription factor in stimulated cells compared with unstimulated controls using three to four different nuclear extract preparations [20 ].

Cocultivation and determination of CCL2
HUVEC were stimulated with 3 U/ml thrombin in serum-free medium or left untreated for 60 min. After medium replacement, the cells were kept in culture medium supplemented with 1% FCS for an additional 11 h followed by incubation with human monocytes or Mono-Mac-6 cells for 30 min in serum-free medium. Nonadherent monocytes or monocytic cells were removed, and HUVEC were incubated in medium supplemented with 1% FCS for the next 24 h. To prevent CCL2 release from the cells, 1 µg/ml brefeldin A was added for the last 6 h of incubation before flow cytometric analysis of permeabilized cells. Monocyte adherence was visualized by fluorescence microscopy of monocytes preloaded with the dye PKH26 (2 µM). For CX3CL1 neutralization, HUVEC were treated with CX3CL1 antibodies (5 µg/ml; R&D Systems) for 30 min prior to cocultivation.

Alternatively, freshly isolated human monocytes (0.5x106) resuspended in 200 µl RPMI 1640 containing 1% FCS were added to multiwell plates precoated overnight with the extracellular domain of human recombinant CX3CL1 (R&D Systems); control experiments showed that the recombinant protein stayed firmly attached to the plate. Twenty-four hours later, CCL2 was determined by ELISA (R&D Systems).

Cell migration
Freshly isolated human monocytes resuspended in medium from untreated HUVEC were loaded into the upper compartments of the chemotaxis chambers (Transwell, 5 µm pore size, Costar, Lowell, MA, USA). The media from the various cocultivation experiments of HUVEC/Mono-Mac-6 were loaded into the lower compartments, and the monocytes were allowed to migrate for 90 min. After fixation, cells were stained with H&E. Transmigrated cells were counted in 10 high-power oil immersion fields (1000x), and the chemotactic index was calculated [17 ]; 10 nM fMLP (Sigma-Aldrich, St. Louis, MO, USA) served as standard chemoattractant. For neutralization experiments, the supernatants were pretreated with 10 µg/ml CCL2 neutralizing antibodies (Peprotech) or IgG for 30 min prior to exposure of the monocytes to the media.

For transendothelial migration, HUVEC were seeded into the chemotaxis chambers (5 µm pore size) precoated with 2% gelatin (Sigma-Aldrich); 48 h later, cells were treated with thrombin for 1 h, the medium was changed, and cells were incubated for an additional 11 h. Monocytes were resuspended in serum-free F12K 0.4% BSA, placed onto HUVEC, and allowed to migrate for 4 h. For receptor neutralization, HUVEC were pretreated for 30 min with CX3CL1 or ICAM-1 antibodies (5 µg/ml, BD Biosciences) prior to addition of monocytes. Transmigrated cells were analyzed as described above. Confluency of the endothelial monolayer was assessed by microscopic inspection of stained membranes [22 ].

Statistical analysis
Values shown represent mean ± SEM. Statistical significances were calculated with the Newman-Keuls test for multi-group comparisons.


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RESULTS
 
Thrombin triggers CX3CL1 expression in HUVEC
Stimulation of HUVEC with thrombin for 3 h concentration-dependently induced CX3CL1 mRNA with a markedly increased expression at 1 U/ml and a plateau at 3 U/ml (Fig. 1A ). Kinetic analysis revealed that the CX3CL1 mRNA expression was up-regulated after 3 h of thrombin, 1 U/ml, reaching a maximum after 6 h (Fig. 1A) .


Figure 1
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  		Figure 1. Thrombin induces CX3CL1 expression. (A) Concentration (3 h)- and time-dependent expression of CX3CL1 mRNA in HUVEC stimulated with thrombin; GAPDH, loading control. (B) Western immunoblot of CX3CL1 in HUVEC after 24 h. The specific PAR1 antagonist SCH 79797 (10 µM) was added 20 min before thrombin (3 U/ml). TNF-{alpha} (10 ng/ml)-positive control. Actin-loading control. (C) Flow cytometric analysis of CX3CL1 on HUVEC stimulated with thrombin for 12 h. (Left panel) Unstimulated control cells (control mouse IgG). (Center and right panels) Shaded peaks, Thrombin-treated cells with anti-CX3CL1 antibody; nonshaded peaks, unstimulated cells with anti-CX3CL1 antibody. In all cases, one of at least three experiments is shown.

Stimulation of HUVEC with thrombin for 24 h led to a concentration-dependent increase in the expression of the CX3CL1 protein in whole-cell lysates after 24 h, an effect that was inhibited by the PAR1 antagonist SCH 79797 [21 ] (Fig. 1B) . Similarly, flow cytometry revealed an increased expression of membrane-bound CX3CL1 after stimulation with thrombin for 12 h (Fig. 1C) . Whereas 495 ± 95 pg/ml of the soluble form of CX3CL1 was released into the supernatants of 0.1 x 106 HUVEC stimulated with TNF-{alpha} (10 ng/ml) for 12 h, soluble CX3CL1 remained undetectable after thrombin stimulation (1–10 U/ml) for 12 or 24 h (n=3 each) as measured by ELISA (R&D Systems; data not shown).

To substantiate the role of PAR1 in the thrombin-induced CX3CL1 expression, we stimulated HUVEC with PAR-APs for PAR1, PAR3, and PAR4. Among the APs tested, only PAR1-AP induced a concentration-dependent expression of CX3CL1 mRNA (Fig. 2A ) and protein (Fig. 2B) , respectively.


Figure 2
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  		Figure 2. PAR1-AP induces expression of CX3CL1. (A) mRNA. HUVEC were stimulated with PAR1-AP (TFLLRNPNDK), PAR3-AP, or PAR4-AP (300 µM) in serum-free medium for 1 h and incubated in fresh medium for additional 2 h; GAPDH, loading control. (B) Western immunoblot. HUVEC were stimulated with PAR1-AP as in A yet for 12 h. Actin-loading control. One of at least three experiments is shown. CX3CL1 protein expression was densitometrically quantified and normalized to actin. Results are mean ± SEM of five independent experiments.

In rat endothelial cells, IL-1{alpha}, TNF-{alpha}, and LPS regulate the CX3CL1 expression via NF-{kappa}B [13 ]. Indeed, EMSA clearly showed that thrombin triggers NF-{kappa}B signaling in HUVEC (Fig. 3A ). The formation of a specific complex with extracts from thrombin-stimulated HUVEC was antagonized by competition with an excess of cold NF-{kappa}B but not activator protein 2 probe (Fig. 3A) . Supershift experiments revealed that the NF-{kappa}B/DNA complex contains p65 but not p52 (Fig. 3A , lanes 5 and 6). In addition, the NF-{kappa}B TransAM ELISA confirmed that thrombin induced a rapid activation of p65 but not of p52 in HUVEC (Fig. 3B) .


Figure 3
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  		Figure 3. Thombin-induced CX3CL1 expression is NF-{kappa}B-dependent. (A) EMSA. Nuclear proteins (1 µg) from HUVEC stimulated with 3 U/ml thrombin for 30 min were incubated with 32P-NF-{kappa}B probe in 20 µl buffer containing 1 µg poly(dI-dC). For competition (compet.), an excess of cold oligodeoxynucleotides was used. For supershift, nuclear extracts were preincubated with 0.5 µg of the indicated antibodies before the addition of 32P-NF-{kappa}B DNA. The complexes were resolved by 4% PAGE and detected by phosphor-imaging. ns, Nonspecific signal; AP2, activator protein 2. (B) NF-{kappa}B activation in nuclear extracts from HUVEC treated for 60 min with 3 U/ml thrombin and analyzed with the TransAM ELISA for NF-{kappa}B family transcription factors. (C) Western immunoblot. HUVEC pretreated with AKβBA or the solvent DMSO (Lanes 1 and 2) for 20 min were stimulated with thrombin for 12 h. (D) Expression of I{kappa}B{alpha} super-repressor (I{kappa}B{alpha}SR) impairs CX3CL1 expression. Cells were transfected with 2 µg empty pCMV4 vector or I{kappa}B{alpha}SR expression vector. Twenty-four hours after transfection, the cells were lysed for the analysis of I{kappa}B{alpha}SR by immunoblotting or stimulated with 3 U/ml thrombin for an additional 12 h, followed by immunoblotting for CX3CL1. Each figure shows representative data out of three to four experiments.

To gain further evidence for a role of NF-{kappa}B in the thrombin-induced CX3CL1 induction, we used AKβBA, a pharmacological inhibitor of IKK and downstream NF-{kappa}B signaling [20 , 23 ]. Pretreatment of HUVEC with AKβBA for 20 min concentration-dependently impaired the thrombin-induced CX3CL1 expression (Fig. 3C) . In addition, transient transfection of HUVEC with an expression vector encoding for a mutant of the regulatory protein I{kappa}B{alpha} (S32A, S36A) that blocks its phosphorylation by IKKs and results in cytoplasmic retention of the I{kappa}B{alpha}/NF-{kappa}B complex, even upon stimulation [24 ], blocked the thrombin-induced CX3CL1 induction (Fig. 3D) . Together, these data show that the thrombin/PAR1-mediated induction of CX3CL1 in HUVEC is dependent on the NF-{kappa}B signaling pathway.

CX3CL1 expressed by HUVEC potentiates the release of chemotactic CCL2 during cocultivation with Mono-Mac-6
Direct interaction of endothelial cells with monocytes initiates release of chemokines such as CCL2 [25 ], indicating that these two cell types cross-talk. As membrane-anchored CX3CL1 facilitates monocyte adhesion to the endothelium [26 , 27 ], we investigated the role of the thrombin-induced CX3CL1 expression in this cross-talk. We cocultivated the monocytic cell line Mono-Mac-6, expressing the CX3CL1 receptor CX3CR1 [28 ] and thrombin-prestimulated HUVEC, and determined the CCL2 release in the supernatant. As anticipated, cocultivation of both cell types increased the release of CCL2, which upon cocultivation, was strongly potentiated when HUVEC had been pretreated with thrombin (Fig. 4A ). This increase fully depended on the CX3CL1 expression, as it was abrogated by neutralizing antibody against CX3CL1 (Fig. 4A) , whereas unrelated control IgG had no effect on the level of CCL2. Together, these data show that the thrombin-induced expression of CX3CL1 in HUVEC potentiates the release of CCL2 produced during monocytic/endothelial cell interaction.


Figure 4
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  		Figure 4. Cocultivation of thrombin-preactivated HUVEC with Mono-Mac-6 triggers enhanced release of CCL2 (A), which was from HUVEC alone or cells coincubated with Mono-Mac-6. HUVEC were left untreated or were transiently stimulated with thrombin (3 U/ml) for 1 h and left in fresh medium for additional 11 h. Mono-Mac-6 were added for 30 min. After coincubation, HUVEC were left rested for an additional 24 h. Coincubation took place in the absence or presence of neutralizing antibody against CX3CL1 or control mouse IgG (each 5 µg/ml). CCL2 was analyzed in the supernatants by ELISA. Means ± SEM of three independent experiments are shown. **, P < 0.01. (B) CCL2 released by HUVEC/Mono-Mac-6 coculture induces monocyte chemotaxis. Human monocytes were loaded into the upper compartment of the Transwell chambers, and supernatants from the cocultivation experiments were used as chemoattractants. To ensure that the chemotaxis observed is a result of the released CCL2, neutralizing antibodies against CCL2 (dark gray bar) or IgG (light gray bar; each 10 µg/ml) were added prior to the migration experiment. Values represent the mean chemotactic index ± SEM of four independent experiments. **, P < 0.01.

To explore the biological relevance of the CX3CL1-dependent release of CCL2, we performed chemotactic assays; supernatants from cells cultivated under the indicated experimental conditions were used as chemoattractants. Supernatants from unstimulated, cocultivated HUVEC or thrombin-stimulated HUVEC, which had not been cocultivated with Mono-Mac-6, induced only moderate migration of human monocytes. The extent of the monocyte chemotaxis correlated with the amount of CCL2 release observed during cell culture (Fig. 4A and 4B) . Consistently, the supernatants collected from HUVEC that had been prestimulated with thrombin and cocultivated with Mono-Mac-6 significantly potentiated the chemotactic activity in human peripheral monocytes (Fig. 4B) . For comparison, 10 nM fMLP used as a positive control induced chemotaxis of monocytes with a chemotactic index = 3.8 ± 0.3 (n=4). Consistent with the CCL2 data, addition of neutralizing antibody against CX3CL1 prior to cocultivation of the thrombin-stimulated HUVEC with Mono-Mac-6 or of anti-CCL2 antibody to supernatants from thrombin-stimulated cocultivated cells impaired the chemotactic activity of the supernatants (Fig. 4B) . These data confirm that CX3CL1 potentiates the release of chemotactically active CCL2 by the HUVEC/monocytic cell coculture.

CX3CL1 expressed by HUVEC potentiates the release of CCL2 by primary monocytes
Additional experiments were designed to address the question of which cell type might generate CCL2 when primary monocytes and thrombin-activated HUVEC were cocultured. The adherence of monocytes to thrombin-stimulated HUVEC is largely a result of the thrombin-induced expression of CX3CL1, as it was profoundly inhibited by the anti-CX3CL1 antibody (Fig. 5A ). Flow cytometric analysis of permeabilized cells allowed identification of the cells responsible for the increased production of CCL2. Cocultivation alone induced expression of CCL2 in monocytes (MFI=1.38±0.21; n=4) and HUVEC (MFI=1.60±0.19; n=4; Fig. 5B ). When monocytes were cocultivated with thrombin-pretreated HUVEC instead, this did not further increase CCL2 levels in HUVEC (Fig. 5C , right panels). In contrast, the monocytes exhibited an increased expression of CCL2 when incubated with the thrombin-pretreated HUVEC (Fig. 5C and 5D) . In some experiments, hirudin (100 U/ml) was added to the endothelial cell culture after stimulation with thrombin. Addition of hirudin, a specific thrombin inhibitor, did not affect the CCL2 release by monocytes (not shown), indicating that direct effects of any remaining thrombin on the monocytes can be excluded.


Figure 5
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  		Figure 5. HUVEC preactivated by thrombin potentiate the release of CCL2 by monocytes. (A) Monocyte adherence is increased by pretreatment of HUVEC with thrombin. HUVEC were pretreated with thrombin (3 U/ml) for 1 h and left for an additional 11 h in fresh medium. Primary monocytes, labeled with the fluorescent dye PKH26 were added to HUVEC for 30 min. Monocyte binding was impaired by preincubation of thrombin-stimulated HUVEC with neutralizing antibody against CX3CL1 but not with control mouse IgG (each 5 µg/ml; added 30 min prior to monocytes). Nonadhered monocytes were washed off, the adherent monocytes were detected by fluorescence microscopy, and six high-power fields were counted (100x). **, P < 0.01, versus nonstimulated control; #, P < 0.05, versus thrombin-stimulated control. Results are mean ± SEM of three independent experiments. (B) Cocultivation of HUVEC with monocytes induces a slight increase in CCL2 expression in HUVEC and monocytes. Cells (0.5x106 HUVEC and 1.5x106 monocytes) were cocultured for 30 min, nonadherent monocytes were removed, and incubation continued for another 24 h. Brefeldin A was added for the final 6 h of the incubation. Cells were fixed, permeabilized, stained with IgG or anti-CCL2 antibodies, and analyzed by flow cytometry. FL2-H, Fluorescence 2-height. (C) Increased CCL2 expression by monocytes cocultured with thrombin-pretreated HUVEC, which were pretreated with thrombin for 1 h, washed, and left for an additional 11 h. HUVEC and monocytes were cocultured and analyzed as described in B. Shaded peaks, Thrombin-treated cells with anti-CX3CL1 antibody; nonshaded peaks, unstimulated cells with anti-CX3CL1 antibody. (D) CCL2 expression in monocytes cocultured with thrombin-pretreated HUVEC compared with monocytes cocultured with control HUVEC as quantified by flow cytometry and expressed as MFI ± SEM of four independent experiments. *, P < 0.05. (E) CX3CL1 induces CCL2 release by human peripheral monocytes, which were exposed to the immobilized extracellular domain of CX3CL1 for 24 h, and CCL2 was analyzed by ELISA. Data are mean ± SEM of four independent experiments. *, P < 0.05.

Finally, we analyzed whether CX3CL1 activates monocytes directly and whether it might suffice to induce CCL2 release by monocytes. Indeed, the recombinant extracellular domain of CX3CL1 immobilized in multiwell plates induced a concentration-dependent release of CCL2 by monocytes (Fig. 5E) . Hence, CX3CL1 is sufficient to induce monocyte activation and CCL2 release.

Thrombin-induced CX3CL1 potentiates transendothelial locomotion of primary monocytes
The migration of monocytes through endothelial monolayers was concentration-dependently potentiated by pretreatment of HUVEC with thrombin (Fig. 6 ). Addition of neutralizing antibodies directed against CX3CL1 or ICAM-1 (Fig. 6) but not of antibodies against CCL2 (data not shown) prevented the thrombin-induced increase in monocyte transendothelial locomotion, indicating that CX3CL1 and ICAM-1, expressed by thrombin-stimulated HUVEC, are essential for the observed monocyte migration.


Figure 6
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  		Figure 6. HUVEC preactivated by thrombin potentiate the transendothelial migration of monocytes. HUVEC were seeded in Transwell chambers, pretreated with thrombin for 1 h, and left for additional 11 h. Monocytes (0.15x106) were added to HUVEC and allowed to migrate for 4 h. For neutralization experiments, CX3CL1 or ICAM-1 antibodies (each 5 µg/ml) were added to HUVEC 30 min before addition of the monocytes. Data represent the mean migration index (MI) ± SEM of four independent experiments. *, P < 0.05; **, P < 0.01.


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DISCUSSION
 
CX3CL1, which can exist as a soluble chemokine or as a membrane-anchored cell surface protein, is important for leukocyte adhesion and migration [26 , 27 ]. Several known proinflammatory stimuli, such as IL-1 and TNF-{alpha} [3 , 10 , 11 ] but also activated protein C and the serine protease thrombin, may induce CX3CL1 in endothelial cells [12 ]. Here, we demonstrate that thrombin induces rapid expression of CX3CL1 in HUVEC via PAR1, leading to CCL2 chemokine induction in human monocytes.

Thrombin is known to activate PAR1, PAR3, and PAR4 [15 ]. To clarify the role of the respective receptors for the thrombin-induced CX3CL1 induction, we used receptor-specific PAR-APs. PAR1-AP induced a robust increase in CX3CL1 expression, which could be abolished by the PAR1-specific antagonist SCH 79797 [21 ], implying that thrombin mediates induction of CX3CL1 through PAR1. By contrast, PAR3-AP did not induce CX3CL1, although it increased the cytosolic Ca2+ level in human monocytes (unpublished data), as it had been described by others for vascular smooth muscle cells [29 ]. HUVEC do not express PAR4 [16 ], and PAR4-AP, used as a negative control, failed to mimic the CX3CL1-inducing effects of thrombin. Thus, thrombin induces CX3CL1 in HUVEC via PAR1.

As a result of its dual functional states as a soluble chemokine or as a membrane-anchored cell surface protein, we investigated whether thrombin might elicit release of CX3CL1 into the media of HUVEC cultures. In contrast to TNF-{alpha}-activated HUVEC, no soluble CX3CL1 was detected in supernatants from thrombin-stimulated HUVEC. Similarly, no soluble CX3CL1 was found when endothelial cells were stimulated with IFN-{gamma} [11 ]. Soluble CX3CL1 is generated by proteolytic cleavage of the extracellular domain of the full-length molecule. Indeed, endothelial cells can express constitutively active ADAM10, a metalloprotease capable of cleaving CX3CL1 [6 ]. In addition, in endothelial cells, TNF-{alpha} strongly up-regulates another protease, namely TACE [30 ], which can increase shedding of CX3CL1 from the cell surface [5 ]. Thus, although thrombin was as potent as TNF-{alpha} in inducing CX3CL1 expression, it was only TNF-{alpha} that elicited a detectable release of soluble CX3CL1.

The thrombin-mediated activation of endothelial cells triggers different signaling pathways, including the transcription factor NF-{kappa}B [16 ]. Our analysis of the thrombin-induced NF-{kappa}B activation as well as the blockade of the NF-{kappa}B signaling by pharmacologic inhibition or by the expression of a transdominant-negative form of I{kappa}B{alpha} demonstrates that thrombin-induced NF-{kappa}B activation is indispensable for the expression of CX3CL1 by HUVEC. As cytokines, such as IL-1 or TNF-{alpha}, may also activate NF-{kappa}B signaling, one could speculate about their role as secondary mediators inducing CX3CL1 expression in thrombin-stimulated HUVEC. However, similar to other authors [31 ], we did not find any detectable release of these cytokines into the supernatants of thrombin-activated HUVEC (unpublished data).

Thrombin is known to increase the expression of a number of adhesion molecules such as ICAM-1 on the surface of endothelial cells, thereby supporting adhesion of polymorphonuclear neutrophils and monocytes [16 , 32 ]. The observation that thrombin also increases the amount of CX3CL1 present on the HUVEC surface further emphasizes the crucial role of thrombin as a key regulator of the proadhesive properties of endothelial cells.

CX3CL1 binds cells rapidly, even under flow conditions [33 ]. It is therefore reasonable to postulate that the CX3CL1 expression by thrombin-activated endothelium contributes to monocyte recruitment, as has been shown for HUVEC overexpressing CX3CL1 [26 ]. This phenomenon is functionally complemented by the CX3CL1-mediated release of CCL2 by monocytes. We could induce CCL2 in monocytes by the immobilized extracelluar domain of CX3CL1 in the absence of HUVEC, implying that CX3CL1 is apparently sufficient to elicit monocyte activation.

Locally produced CCL2 has not only important effects on the transendothelial migration of monocytes as a chemoattractant but also by facilitating a firm integrin-dependent monocyte adhesion to endothelial cells [34 ] as well as the rapid expression of CX3CR1 on the monocyte membrane [35 ]. Therefore, the CX3CL1-dependent release of CCL2 by monocytes will result in increased monocyte recruitment and firm binding to the thrombin-exposed endothelium, which could be of particular relevance for cardiovascular diseases. CCL2 is indeed strongly expressed in vascular lesions, and knockout of the CCL2 gene or of its receptor CCR-2 is associated with decreased atherosclerosis in murine models [36 ]. The precise mechanism involved in the CX3CL1-induced CCL2 release remains to be established.

Thrombin-preactivated endothelial cells exhibited not only increased binding of monocytes but also potentiated the monocyte endothelial transmigration, which was inhibited by antibodies against CX3CL1 and ICAM-1, indicating that the adhesion molecules cooperatively induce monocyte adherence and promote transendothelial migration. ICAM-1 is an important integrin ligand involved in leukocyte transendothelial migration, which acts as adhesion molecule as well as a signal transducer in endothelial cells [37 ]. ICAM-1-mediated activation of the signaling pathways in endothelial cells regulates endothelial permeability and is required for the efficient transendothelial migration of leukocytes [37 ]. At variance to ICAM-1, flow chamber experiments suggested that CX3CL1 does not mediate cell rolling but appears to be important for the firm attachment of monocytes to endothelial cells [33 ].

It should be noted that in our experimental settings, the endothelial transmigration of the monocytes is not related to the CX3CL1-mediated CCL2 induction in monocytes. The CCL2 protein expression occurs much later and therefore cannot contribute to the formation of a chemotactic gradient within the chosen observation period. It is well known that activated endothelial cells express adhesion molecules, which are indispensable for the transendothelial migration of monocytes. Thus, the transendothelial migration of mononuclear cells increases approximately eightfold when HUVEC are activated by IL-1β [38 ]. Indeed, binding of monocytes to adhesion molecules such as VCAM-1 and ICAM-1 in TNF-{alpha}-activated HUVEC is required for the expression of the membrane type 1 matrix metalloproteinase that is essential for the endothelial transmigration of human monocytes [39 ]. This raises the interesting possibility that the adherence of monocytes to the thrombin-activated endothelial cells initially enhances their transmigration while inducing at the same time the chemokine CCL2. However, the CCL2 protein would become expressed only once the cells already transmigrated, possibly leading to some kind of transmigration amplification loop.

Our findings point to a critical role of thrombin as a regulator of transendothelial monocyte/leukocyte migration into areas of inflammation. Although our data have been generated in vitro, a number of in vivo studies are in line with our findings. Thus, the direct thrombin inhibitor melagatran reduces lesion size in atherosclerotic mice [40 ], and decreased atherosclerotic lesion formation and macrophage accumulation has been demonstrated in vivo in CX3CR1/apolipoprotein E (apoE) double-knockout mice [41 , 42 ]. Moreover, a recent in vivo study with CX3CL1/CCR2/apoE triple-knockout mice provided evidence for independent roles of CCL2 and CX3CL1 in terms of macrophage accumulation and atherosclerotic lesion formation [43 ]. Consistent with these findings, in apoE-deficient mice, a distinct monocyte subset giving rise to CD11c+ dendritic-like cells has been shown to require the CX3CL1 receptor CX3CR1 to enter atherosclerotic plaques [44 ]. In addition, under physiological conditions, a subset of monocytes was found to patrol the healthy endothelium through long-range crawling using CX3CR1 and LFA-1, a process relevant for rapid tissue invasion during the early inflammatory response [45 ]. Taken together, these in vivo data complement our findings, showing that CX3CL1 along with ICAM-1 is indispensable for the transendothelial migration of monocytes.

In conclusion, our data identify a novel mechanism by which monocytes and endothelial cells cross-talk in the presence of thrombin by expression of the CX3CL1 chemokine in endothelial cells leading to enhanced monocyte recruitment and subsequent induction of the chemokine CCL2 by monocytes. This mechanism is expected to contribute significantly to monocyte recruitment into sites of injury and inflammation.


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ACKNOWLEDGEMENTS
 
This research was supported by the Deutsche Forschungsgemeinschaft.


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FOOTNOTES
 
1 These authors contributed equally to this work. Back

Received September 24, 2007; revised March 20, 2008; accepted March 26, 2008.


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