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Originally published online as doi:10.1189/jlb.0503221 on May 20, 2004

Published online before print May 20, 2004
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(Journal of Leukocyte Biology. 2004;76:388-398.)
© 2004 by Society for Leukocyte Biology

Agonists of proteinase-activated receptor-2 modulate human neutrophil cytokine secretion, expression of cell adhesion molecules, and migration within 3-D collagen lattices

V. M. Shpacovitch*, G. Varga*, A. Strey{dagger}, M. Gunzer*, F. Mooren{ddagger}, J. Buddenkotte*, N. Vergnolle§, C. P. Sommerhoff, S. Grabbe*, V. Gerke{dagger}, B. Homey||, M. Hollenberg§, T. A. Luger* and M. Steinhoff*,1

* Department of Dermatology and Ludwig Boltzmann Institute for Cell- and Immunobiology of the Skin and Institutes of
{dagger} Biochemistry, ZMBE, and
{ddagger} Sport Medicine, University of Muenster, Germany;
Department of Clinical Chemistry & Clinical Biochemistry, University of Munich, Germany;
§ Department of Pharmacology and Therapeutics, University of Calgary, Alberta, Canada; and
|| Department of Dermatology, University of Düsseldorf, Germany

1Correspondence: Department of Dermatology and Ludwig Boltzmann Institute for Immunobiology of the Skin, University of Münster, von-Esmarch-Str. 58, 48149 Münster, Germany. E-mail: msteinho{at}uni-muenster.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Proteinase-activated receptor-2 (PAR2) belongs to a novel subfamily of G-protein-coupled receptors with seven-transmembrane domains. PAR2 can be activated by serine proteases such as trypsin, mast cell tryptase, and allergic or bacterial proteases. This receptor is expressed by various cells and seems to be crucially involved during inflammation and the immune response. As previously reported, human neutrophils express functional PAR2. However, the precise physiological role of PAR2 on human neutrophils and its implication in human diseases remain unclear. We demonstrate that PAR2 agonist-stimulated human neutrophils show significantly enhanced migration in 3-D collagen lattices. PAR2 agonist stimulation also induced down-regulation of L-selectin display and up-regulation of membrane-activated complex-1 very late antigen-4 integrin expression on the neutrophil cell surface. Moreover, PAR2 stimulation results in an increased secretion of the cytokines interleukin (IL)-1ß, IL-8, and IL-6 by human neutrophils. These data indicate that PAR2 plays an important role in human neutrophil activation and may affect key neutrophil functions by regulating cell motility in the extracellular matrix, selectin shedding, and up-regulation of integrin expression and by stimulating the secretion of inflammatory mediators. Thus, PAR2 may represent a potential therapeutic target for the treatment of diseases involving activated neutrophils.

Key Words: proteinases • leukocytes • extracellular matrix • interleukins • G-protein coupled receptors


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Proteinase-activated receptor-2 (PAR2) belongs to a new subfamily of G-protein-coupled receptors with seven putative transmembrane domains. This subfamily has a unique mechanism of activation involving the proteolytic cleavage of the receptor by serine proteases to unmask a new N-terminal sequence, which then acts as a tethered receptor-activating ligand. At present, four PARs are known [1 , 2 ]. A variety of serine proteinases, including trypsin, tryptase, and bacterial-derived enzymes, are capable of activating PAR2 [1 , 3 4 5 6 7 ], which is expressed by various cells involved in inflammatory and immune responses, such as vascular endothelial cells, epithelial cells, T cells, eosinophils, and neutrophils [1 , 2 , 8 , 9 ]. In these cells, activation of PAR2 affects main functions such as proliferation, degranulation, and release of inflammatory mediators [10 11 12 ]. Recent studies shed some light on intracellular signaling pathways in which PAR2 appears to be involved. There are indirect data indicating that PAR2 couples to G-proteins, such as Gq/11 and possibly Go/i [13 ]. Whether PAR2 couples to other G-proteins such as G12 or G13 remains unclear.

Latest works demonstrated an important role of PAR2 for the development of certain inflammatory responses. It has been shown that PAR2 can regulate inflammation via a neurogenic mechanism [6 ]. Additionally, an essential role for PAR2 has been demonstrated for chronic, inflammatory processes such as arthritis [14 ].

Despite several reports concerning the role of PAR2 in epithelial and endothelial function, little is yet known about the role of this receptor in neutrophil function. Recently, the effect of PAR2 activation on leukocyte behavior has been demonstrated in a rat model [15 ]. In this in vivo model, it was shown that PAR2-activating peptides (PAR2-APs) caused a significant increase in leukocyte migration into peritoneal cavity after intraperitoneal injection. Furthermore, PAR2-APs induced a significant increase in leukocyte rolling and adherence [15 ].

Human neutrophils [16 ] and eosinophils [12 ] possess functional PAR2 that can be activated by trypsin or a PAR2-selective agonist peptide (SLIGKV-NH2). In human neutrophils, PAR2 stimulation causes an elevation of intracellular calcium and induces shape changes; this responsiveness of neutrophils to PAR2 activation appears to differ in cells harvested from different donors [16 ]. Like trypsin, the bacterial protease, gingipain-R from Porphyromonas gingivalis, is also able to activate human neutrophil PAR2 [5 ]. However, the precise role of PAR2 for the regulation of human neutrophil function under physiological and pathophysiological conditions remains unclear.

Human neutrophils play a key role as a part of the immune response to microbial infections. One of the main functions of neutrophils is the rapid killing of bacteria and fungi before they are able to spread throughout the body. The main physiological and pathogenic activities of neutrophils are adherence and migration; degranulation and release of inflammatory mediators; and phagocytosis and apoptosis [17 ]. Therefore, the aims of the present study were to investigate the effects of PAR2 agonists on the motility of human neutrophils; the effects of PAR2 agonists on the expression of different cell adhesion molecules by neutrophils; and whether PAR2 agonists modulate the release of various cytokines from human primary neutrophils.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Trypsin (Cat. number T-1426, from bovine pancrease) was obtained from Sigma (Deisenhofen, Germany). Human PAR2-AP with the sequence trans-cinnamoyl-LIGRLO-NH2 (tcAP) and reverse peptide (tcRP: trans-cinnamoyl-OLRGIL-NH2; Dr. Denis McMaster, University of Calgary, Canada) were used as described previously [6 , 18 , 19 ]. The following primary antibodies were used: phycoerythrin (PE)-conjugated monoclonal mouse anti-human CD62L (L-selectin; clone DREG-56) and CD11b/CD18 [membrane-activated complex-1 (Mac-1)] from PharMingen (Hamburg, Germany) and monoclonal mouse anti-human {alpha}2ß1[very late antigen (VLA)-2] and {alpha}4ß1(VLA-4) integrin from Dako Diagnostika GmbH (Hamburg, Germany). Secondary antibodies were: PE-conjugated affinity-purified F(ab')2 goat anti-mouse from Dianova (Hamburg, Germany). The PE-conjugated mouse immunoglobulin G1{kappa} isotype control antibody was received from PharMingen. The following neutralizing antibodies were used: mouse anti-human VLA-2 and VLA-4 from PharMingen; RPMI-1640 cell culture medium without L-glutamine, from BioWhittaker (Aachen, Germany); and other cell culture reagents, from Gibco-Life Technologies (via Invitrogen, Karlsruhe, Germany). Fura-2/AM was from Molecular Probes (via MoBiTec, Göttingen, Germany). Hydroxamic acid-based metalloprotease inhibitor KD-IX-73-4 (Cat. number INH-3850-PI) was purchased from Peptides International (Louisville, KY). Human recombinant interleukin (hrIL)-8 was received from R&D Systems (Wiesbaden-Nordenstadt, Germany). Bovine dermal collagen (Vitrogen) was obtained from Cohesion (Palo Alto, CA). Other reagents and materials were obtained from Sigma, Merck (Darmstadt, Germany), Becton-Dickinson (Heidelberg, Germany), Falcon (via Becton-Dickinson), and Corning (Schiphool, the Netherlands).

Neutrophil isolation
Blood was obtained from healthy adult human volunteers (males and females). Neutrophils were isolated using 71.5% Percoll (Biocoll from Biochrom, Berlin, Germany) or lymphocyte separation medium (PAA Laboratories, Cölbe, Germany), followed by hypotonic shock to remove erythrocytes, as described [20 ]. Neutrophil purity [assessed by differential staining and fluorescein-activated cell sorter (FACS) analysis] was routinely 94–96% (major contaminating cells were eosinophils, <5%; other cells, including monocytes, all together, <1%). The cell viability (trypan blue staining) was >97%, immediately after isolation. Isolated neutrophils were cultured in RPMI-1640 medium, which contained the following components: 2% fetal calf serum (FCS), 1% penicillin/streptomycin, 1% L-glutamine, and 1% non-essential amino acids. After isolation, cells were cultured at 37°C with 5% CO2 for at least 1 h for recovery. For PAR2 stimulation, a receptor-specific PAR2-AP (tcAP) trans-cinnamoyl-LIGRLO-NH2, which is resistant to amino peptidase action, was used at concentrations of 5 x 10–5 M, 1 x 10–4 M, and 2 x 10–4 M. This peptide has been documented previously to be an effective and specific PAR2 agonist in PAR2-expressing cells [19 ], causing effects identical to a tethered ligand agonist such as SLIGRL-NH2 or SLIGKV-NH2. The actions of trans-cinnamoyl-LIGRLO-NH2 were compared with those of the parent-tethered ligand SLIGRL-NH2. The corresponding RPs (tc-OLRGIL-NH2, VKGILS-NH2), which cannot activate PAR2, were used at concentrations of 1 x 10–4 M and 2 x 10–4 M, respectively, and served as negative controls. Trypsin was used at concentrations of 5 x 10–8 M and 10–7 M. After stimulation, cells were immediately used for 3-D collagen assay or were cultured and then used for flow cytometric analysis. Cell culture supernatants were collected and assayed for cytokine content using an enzyme-linked immunosorbent assay (ELISA; see below).

Ca2+mobilization studies
Freshly isolated neutrophils (5x105–1x106) were incubated in a buffer containing 140 mM NaCl, 3 mM KCl, 0.4 mM Na2HPO4, 10 mM HEPES, 5 mM glucose, 1 mM MgCl2, and 1.8 mM CaCl2 with 5–8 µM Fura-2/AM for 30 min at room temperature as described previously [21 ]. Fluorescence was measured in cell suspensions using a cuvette spectrofluorimeter (Deltascan, PTI, NJ) and appropriate software (Deltascan, PTI), as described previously [21 ]. Fluorescence was measured at excitation wavelengths of 340 nm and 380 nm, and emitted light was monitored at 510 nm. During recording, cells were continuously stirred at 28°C. Although autofluorescence of cells was negligible, autofluorescence of cuvette and solution, among others, was determined before the experiment and subtracted automatically. [Ca2+]i was calculated according the following equation [22 ], [Ca2+]i = (R–Rmin)/(Rmax–R) x Kdx F, with a Kd of Fura-2 for calcium of 220 nmol/l, where R is the ratio of fluorescence of the sample at 340 and 380 nm, and Kd is the dissociation constant. Rmax and Rmin are the ratios for Fura-2 at these wavelengths in the presence of saturating Ca2+ (after application of 10 µM digitonin) and under Ca2+-free conditions (after addition of EGTA, 10 mM final concentration), respectively. F is the ratio of fluorescence intensity at 380 nm under Ca2+-free conditions to the fluorescence intensity at 380 nm under Ca2+-saturating conditions. The increase in intracellular Ca2+ ({Delta}[Ca2+]i) displayed a hyperbolic dependence on the concentration (con.) of tcAP. Data were fitted by nonlinear regression to the following equation (Eq. 1) to obtain estimates for the theoretical maximum increase in intracellular Ca2+ ({Delta}[Ca2+]max) and effective concentration (EC)50 for tcAP: {Delta}[Ca2+]i= {Delta}[Ca2+]maxx [con.tcAP]/(EC50+[con.tcAP]).

ELISA
To study cytokine release by human neutrophils (3–4x106cells/ml), cells were stimulated as described above for indicated time intervals (2 h, 4 h, and 6 h), and supernatants were collected and frozen until used for ELISA assays. For detection of IL-8 and IL-1ß, ELISA kits from BioSource Int. (Solingen, Germany) were used (sensitivity, <2 pg and <0.2 pg per ml, respectively). ELISAs were performed according to the manufacturer’s instructions. To detect IL-6, ELISA kits from Diaclone (via Trinova Biochem, Giessen, Germany) were used (sensitivity, <2 pg per ml). ELISAs were performed according to the manufacturer’s instructions. All data were calculated from triplicate wells and are presented as a mean ± SD. The optical density (OD) of samples was measured using a Microplate Reader 3550 (BioRad, Muenchen, Germany) at 450 nm wavelength.

Soluble L-selectin (sL-selectin) assay
To study changes of the levels of soluble factors such as sL-selectin, collected supernatants (2 h and 6 h) were centrifuged at 1000 g for 10 min before loading onto antibody-coated, 96-well plates. This centrifugation removes cell debris, which could affect the final results of the ELISA. For the measurement of sL-selectin, ELISA kits from Diaclone (via Trinova Biochem) were used (sensitivity, <1 ng/ml). All data were calculated from triplicate wells and are presented as a mean ± SD. The OD of samples was measured using a Microplate Reader 3550 (BioRad) at 450 nm wavelength.

Flow cytometric analysis (FACS)
Neutrophils were treated as described above and subsequently stained and analyzed for surface expression of CD62L, Mac-1, VLA-2, and VLA-4. Briefly, 1 x 106 cells were used for each analysis. Cells were washed in phosphate-buffered saline (PBS; 1% FCS) and subsequently incubated with primary mouse anti-human monoclonal antibodies (1 µg/ml). To detect anti-VLA-2 and -VLA-4 primary antibodies, neutrophils were additionally washed twice in PBS (1% FCS) after 30 min incubation on ice and then incubated with PE-conjugated goat anti-mouse secondary antibody (1 µg/ml) in PBS (1% FCS) for an additional 30 min on ice. After two more washes with PBS (1% FCS), cells were finally resuspended in 500 µl PBS (1% FCS) for analysis. Cells incubated with isotype-control antibodies or with PE-conjugated secondary goat anti-mouse antibody alone served as a negative control. At least 20,000 stained cells were analyzed by FACSCalibur (Becton Dickinson). Results are calculated as percent above negative control.

3-D Collagen matrix assay
3-D Collagen matrices were prepared as described [23 ]. Briefly, 500,000 cells were resuspended in 33 µl minimal essential medium (Flow Laboratories, McLean, VA). The cells were embedded within a total of 100 µl collagen [33 µl cells+66 µl collagen (dermal bovine collagen); Collagen Corp., Palo Alto, CA] at a final concentration of collagen at 1.7 mg/ml. This solution was filled into a small chamber built by a hollowed coverslip on a glass slide and allowed to polymerize for 20–30 min (37°C, 5% CO2). The remaining space in the chamber was filled with PBS (control samples); trypsin at a final concentration of 1 x 10–7 M; PAR2 agonist PAR2-tcAP at a final concentration of 2 x 10–4 M; PAR2-tcAP (2x10–4 M) and KD-IX-73-4 (50 µg/ml) together; and scrambled PAR2-RP (2x10–4 M). Finally, the chamber was then sealed with wax.

Time-lapse video microscopy and computer-assisted cell tracking
Cell migration was documented as described [23 ]. Briefly, cells incorporated within collagen lattices were visualized on a conventional inverted microscope. Time-lapse video microscopy was used to record the movement of 100–200 cells visible within one optical field over a period of up to 4 h. Subsequently, the paths of individual cells were reconstructed from the recorded films by computer-assisted cell tracking as described [24 25 26 ]. Briefly, time-lapse video movie was displayed on a computer screen. From the first frame of the time-lapse sequence, 40 cells were randomly selected, giving a nonbiased, representative sample of the cell population. Subsequently, the movements of each of these cells were individually followed by a trackball. Every 1 min (real time of the time-lapse film), the x-y coordinates of the screen-pointer were registered by computer. Plotting these x-y data on the screen reconstructed the paths of the individual migrating cell. The vector length between two x-y coordinates was used as a direct measure of distance for the calculation of the actual cell velocity. Data representing the differences in neutrophil motility and velocity are presented as graphs. At least three independent experiments have been performed.

Statistical analysis
Results are expressed as mean ± SD or ± SEM (for 3-D collagen assay). At least three independent experiments have been performed (n≥3). Statistical evaluation was performed by an ANOVA and a Student’s t-test. A P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of tcAP on [Ca2+]i in isolated neutrophils
Previously, Howells and colleagues [16 ] demonstrated that PAR2-AP SLIGKV-NH2 induces an increase in [Ca2+]i in human neutrophils. In preliminary studies, we were able to confirm that PAR2-AP SLIGRL-NH2 as well as SLIGKV-NH2 (both peptides sensitive to amino peptidase) cause an increase in intracellular calcium in isolated human neutrophils (data not shown). However, for our studies using neutrophils exposed to a PAR2 agonist over prolonged time-periods, we wished to use the PAR2-AP, tc-LIGRLO-NH2, which is resistant to aminopeptidase action. To confirm that this peptide (tcAP), like SLIGRL-NH2, is able to activate PAR2 expressed by human neutrophils, we performed Ca2+ mobilization studies. tcAP stimulation of human neutrophils in concentrations 1 x 10–4 M and 2 x 10–4 M caused a concentration-dependent increase in intracellular Ca2+(Fig. 1B ). Stimulation by the control reverse-sequence peptide, which cannot activate PAR2, did not induce a Ca2+ response (Fig. 1A) . Thus, PAR2-tcAP stimulates [Ca2+]i increase specifically and effectively in human neutrophils. We analyzed our data according to Eq. 1 (see Materials and Methods) and estimated a theoretical maximum increase in {Delta}[Ca2+]max= 115 ± 9 nM and an EC50 for tcAP of 60 ± 9 µM (Fig. 1B) .



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  		Figure 1. Ca2+mobilization studies revealed a dose-dependent activation of PAR2 in human neutrophils by tcAP. PAR2-tcAP stimulation leads to an increase of intracellular Ca2+ mobilization in buffer, which contains 1.8 mM CaCl2 (A). RP stimulation does not result in any changes of [Ca2+]i (A). In our experiments, formyl-Met-Leu-Phe (fMLP; 10 nM; well-known neutrophil activator) stimulation was used as a positive control. PAR2-tcAP stimulation has a dose-dependent effect at the [Ca2+]i mobilization (B). The data were analyzed according to Eq. 1 (see Materials and Methods). The line shows the fit of the data (the mean±SEM) to Eq. 1.

 
PAR2 agonist stimulation results in enhanced L-selectin (CD62L) shedding from the surface of human neutrophils
L-selectin mediates the initial interaction of circulating leukocytes with endothelial cells, which produces a characteristic "rolling" of leukocytes on the endothelium. L-selectin is shed by proteolytic cleavage from the surface of neutrophils in vitro following activation by a variety of agents [including phorbol esters, lipopolysaccharide (LPS), and fMLP] and in vivo during inflammation [27 28 29 30 31 ]. Enhanced CD62L shedding serves as a well-known marker of neutrophil activation [17 , 32 , 33 ]. Therefore, we wished to determine if PAR2 agonist stimulation affected L-selectin display. In our FACS experiments, stimulation of human neutrophils with tcAP resulted in a time-dependent enhancement of L-selectin shedding (Fig. 2A ). Exposure of neutrophils to the RP did not lead to any significant changes in the process of CD62L shedding (Fig. 2B) . EDTA (complexes ions Ca2+, Mg2+, and Zn2+) is known to reduce, at least partially, L-selectin shedding from the leukocyte surface [34 ]. The addition of 10 mM EDTA to the neutrophils, which were prestimulated by tcAP, resulted in a reduction of PAR2-mediated L-selectin shedding (Fig. 2B) . It was also reported that hydroxamic acid-based metalloprotease inhibitor KD-IX-73-4 inhibits L-selectin shedding from the neutrophil cell surface [31 , 35 ]. In our study, KD-IX-73-4 in concentration 50 µg/ml almost completely (more than 90%) inhibited PAR2-tcAP-induced L-selectin shedding from the surface of isolated human neutrophils (data not shown). Thus, PAR2 agonist stimulation caused an enhanced level of L-selectin shedding from the surface of isolated human neutrophils. This shedding was significantly reduced by metal chelation and almost completely inhibited by a metalloproteinase inhibitor KD-IX-73-4.



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  		Figure 2. PAR2-tcAP stimulation up-regulates L-selectin shedding and expression of Mac-1 and VLA-4 by human neutrophils. PAR2-tcAP (1x10–4 M or 2x10–4 M) stimulation of human neutrophils leads to an enhanced L-selectin shedding. Stimulation with PAR2-tcAP causes time-dependent changes of L-selectin expression by neutrophils (A). PAR2-RP (1x10–4 M) stimulation fails to induce such an effect (B). Addition of 10 mM EDTA to the neutrophils prestimulated with PAR2-tcAP (2x10–4 M) reduces an effect of PAR2 agonist at the L-selectin display (cf., black and gray lines, B, lower panel). Additionally, PAR2-tcAP stimulation leads to increase the expression of Mac-1 and VLA-4 on the surface of neutrophils (C). Isotype control peak is presented as a shaded peak. Three independent experiments were performed.

 
IL-8 stimulation (concentration, 10 ng/ml) is known to enhance L-selectin shedding from the polymorphonuclear neutrophil (PMN) cell surface [36 ]. Therefore, we investigated whether the amount of IL-8 released after PAR2 agonist stimulation of neutrophils is sufficient to up-regulate L-selectin removal from the neutrophil cell surface. However, stimulation of neutrophils with hrIL-8, equal in amount to this cytokine (~300 pg/ml) released after PAR2-tcAP administration, did not lead to any significant changes of neutrophil cell-surface expression of CD62L. Stimulation of neutrophils with IL-8 in two times higher concentration (~600 pg/ml) also did not affect the display of CD62L on neutrophil cell surface (data not shown).

Effects of PAR2 agonist stimulation on Mac-1, VLA-2, and VLA-4 expression by human neutrophils
Integrins are a major group of cell adhesion molecules presented on leukocytes. These molecules are involved in binding of leukocytes to extracellular matrix (ECM) proteins (mostly ß1 integrins) in leukocyte adhesion to endothelium or to other immune cells (mostly ß2 integrins) and also serve as signaling molecules [17 , 32 ]. Therefore, we investigated whether PAR2 agonist stimulation affects the expression of such ß1 and ß2 integrins as VLA-2, VLA-4, and Mac-1. Our results demonstrate that PAR2-tcAP stimulation up-regulates the expression of Mac-1 and VLA-4 on neutrophil cell surface (Fig. 2C) . However, such stimulation failed to induce any changes in VLA-2 display on neutrophils (data not shown). Thus, PAR2 agonist stimulation results in up-regulation of Mac-1 and VLA-4 integrin expression on human neutrophils.

PAR2 agonist stimulation leads to increased levels of sL-selectin
sL-selectin retains bioactivity. High plasma levels of sL-selectin can occur during infection [37 38 39 ]. We investigated whether PAR2 agonists induce an increase of sL-selectin level in neutrophil supernatants. The level of sL-selectin increased at 2 h and 6 h after stimulation of neutrophils with tcAP (Fig. 3 ). This effect was time-dependent and correlated with the increases in L-selectin shedding detected in our FACS experiments (see Fig. 2 ). At 2 h after stimulation of neutrophils with tcAP (1x10–4 M), the level of released sL-selectin increased at ~140 ± 30% above control (Fig. 3) . Trypsin did not have a significant effect at 2 h after stimulation but had almost the same efficiency as tcAP at 6 h after stimulation: ~65 ± 20% (Fig. 3) . PAR2-RP did not have any effect on the sL-selectin level. Thus, PAR2 agonist stimulation of isolated human neutrophils results in an increased level of sL-selectin in the neutrophil environment.



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  		Figure 3. PAR2 agonist stimulation of human neutrophils results in an increased level of sL-selectin in the neutrophil environment. Isolated human neutrophils were cultured during 1 h for recovery and then stimulated with PAR2 agonists: trypsin (10–7 M) or PAR2-tcAP (1x10–4 M). Supernatants were collected and subjected to sL-selectin-specific ELISA. The average values of sL-selectin production by unstimulated neutrophils were at 2 h, 10.12 ± 1.5 ng/ml, and at 6 h, 19.5 ± 0.59 ng/ml. Statistical analysis was performed by Student’s t-test. Results are expressed as an average value of protein concentration (as a percentage to control) ± SD. *, P < 0.05; **, P < 0.01. At least three independent experiments (in triplicates) have been performed.

 
Effect of PAR2 agonists on neutrophil motility in a collagen matrix
After diapedesis, neutrophils migrate along a chemoattractant gradient by the process of chemotaxis until they reach the invading microorganisms. Dynamic 3-D imaging of neutrophils migrating through ECM has revealed that cells crawl along matrix fibers, sometimes pulling elastic portions of these fibers to move through openings but without proteolytic degradation of the matrix [40 ]. Recent studies have identified a role for integrins, especially ß1integrins, in the process of neutrophil ECM migration, but the possible role of other neutrophil surface receptors still remains unclear. Therefore, we were interested to determine if a PAR2 agonist might affect human neutrophil motility in 3-D collagen matrices. We observed that trypsin (10–7 M) as well as PAR2 agonist tcAP stimulated an increase in neutrophil motility ~35% and 40%, respectively (Fig. 4A and 4B ). Neutrophil velocity also significantly increased (approximately three times) after stimulation with both PAR2 agonists (Fig. 4A and 4B) . The neutrophils in control samples and in RP-stimulated samples moved only in a localized region and did not migrate long distances (on average, a distance of 19±5 µm). In contrast, tcAP-stimulated neutrophils migrated over 10 times the distance covered by control cells: 274 ± 40 µm. Furthermore, the tracks of the tcAP or trypsin-stimulated neutrophils were more diverse (Fig. 5A and 5B ). Taken together, these data demonstrate that the PAR2 peptide agonist and trypsin caused an up-regulation of neutrophil motility and velocity in a collagen matrix.



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  		Figure 4. PAR2 agonist stimulation causes increased motility and velocity of human neutrophils in a 3-D collagen assay. PAR2-tcAP (2x10–4 M) stimulation of isolated neutrophils leads to an increased amount of migrated cells and their velocity (A). Trypsin (10–7 M) stimulation of neutrophils also leads to enhanced cell motility and velocity (B). Addition of PBS was used as a control. PAR2-RP (2x10–4 M) stimulation was used as negative control. Three independent experiments have been performed (average value±SEM). Error bar lines presented as fine dotted lines surrounding the average lines of experimental data.

 


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  		Figure 5. PAR2 agonist stimulation leads to more diverse neutrophil paths in 3-D collagen lattices. The path samples of unstimulated (control), tcAP (2x10–4M)-stimulated (A), or trypsin (10–7 M)-stimulated (B) neutrophils are presented. Cells, which did not move, are presented as circles without any paths. The paths of stimulated cells are longer and more diverse as compared with unstimulated control.

 
Effect of the inhibition of L-selectin shedding on PAR2 agonist-induced neutrophil motility in a collagen matrix
We showed that PAR2 agonists up-regulate neutrophil motility in collagen matrix and affect the display of cell adhesion molecules (CAMs) by these cells. Therefore, we investigated whether such an effect on neutrophil motility is associated with changes of CAM expression after PAR2 agonist stimulation. In our experiments, neither VLA-2 nor VLA-4 neutralizing antibodies affected PAR2 agonist-induced neutrophil motility in a collagen matrix (data not shown). However, metalloprotease inhibitor KD-IX-73-4 (50 µg/ml) reduced velocity of PAR2-tcAP-stimulated neutrophils in 3-D collagen matrix (Fig. 6A ). A reduction of neutrophil velocity stimulated with KD-IX-73-4 and tcAP was approximately 2 µm/min as compared with the neutrophils stimulated only with tcAP (Fig. 6A) . Addition of KD-IX-73-4 also caused a down-regulation the number of migrated PAR2-tcAP-stimulated neutrophils. However, this effect was significant only starting from 80 min after KD-IX-73-4 administration (Fig. 6B) . Hydroxamic acid-based metalloprotease inhibitor KD-IX-73-4 inhibits L-selectin shedding from the neutrophil surface [31 , 35 ]. In our experiments, KD-IX-73-4 also inhibited PAR2-tcAP-induced L-selectin shedding from the surface of isolated human neutrophils. Taken together, these data suggest that PAR2-induced neutrophil motility in a 3-D collagen matrix, at least partially, associated with PAR2 agonist-enhanced L-selectin shedding from the neutrophil cell surface.



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  		Figure 6. L-selectin shedding inhibitor KD-IX-73-4 causes a reduction of PAR2 agonist-induced human neutrophil motility in a 3-D collagen assay. PAR2-tcAP (2x10–4 M) and hydroxamic acid-based metalloprotease inhibitor KD-IX-73-4 (50 µg/ml) were used together for stimulation of isolated human neutrophils. Addition of PBS was used as a control. Metalloprotease inhibitor KD-IX-73-4 significantly reduces the velocity of PAR2-tcAP-stimulated neutrophils in 3-D collagen matrix (A). Addition of KD-IX-73-4 also down-regulates the number of migrated neutrophils after PAR2-tcAP stimulation. This effect becomes significant only at 80 min after stimulation (B). Four independent experiments have been performed (average value±SEM). Error bar lines presented as fine dotted lines surrounding the average lines of experimental data.

 
PAR2 agonists up-regulate secretion of IL-6, IL-8, and IL-1ß by human neutrophils
We also examined the ability of PAR2 agonists to affect the secretion of proinflammatory cytokines in human neutrophils. The up-regulatory effects of PAR2-tcAP stimulation were more than 150% for all cytokines investigated. For stimulation of human neutrophils, the agonists were used in the following final concentrations: 5 x 10–5 M and 1 x 10–4 M for tcAP; 5 x 10–8 M and 1 x 10–7 M for trypsin. The effect of 1 x 10–4 M tcAP appeared to be a maximal, as no further increase was observed at twice the concentration (data not shown). Trypsin and PAR2-tcAP up-regulated secretion of IL-6 by human neutrophils. However, the magnitudes of stimulation of IL-6 release by the two agonists differed considerably (Fig. 7A ). Trypsin (1x10–7 M) stimulation resulted in an increase of a maximum 850 ± 80% effect and tcAP (1x10–4 M) 240 ± 50% effect at 6 h after stimulation (Fig. 7A) . Lower concentrations of both agonists also caused a maximal effect on IL-6 release at 6 h after stimulation: trypsin, 550 ± 80%, and tcAP, 340 ± 10% (Fig. 7A) . We also observed an up-regulatory effect of the PAR2 agonists on IL-1ß secretion. Trypsin led to a maximum increase of 450 ± 120% at 6 h after stimulation, whereas the tcAP effect (170±20%) was lower (Fig. 7B) . TcAP stimulation also up-regulated the secretion of IL-8 by human neutrophils. At 2 h after stimulation, tcAP (1x10–4 M) caused an increase in IL-8 of 560 ± 80%. However, later, this effect had a tendency to reduction. At 6 h after, the effect of tcAP caused an increase in IL-8 of 300 ± 80% (Fig. 7C) . The stimulation of neutrophils with a lower concentration of tcAP (5x10–5 M) also induced enhanced IL-8 secretion. However, the effect of a lower tcAP concentration did not change significantly at different time-points after stimulation (Fig. 7C) . An increase in the amount of IL-8 in the medium after trypsin stimulation was not observed, possibly as a result of the degradation of IL-8 by trypsin in the course of the experiment. Thus, all agonists caused an increased secretion of IL-6, IL-8, and IL-1ß in human neutrophils.



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  		Figure 7. Stimulation with PAR2 agonists leads to enhanced secretion of IL-6, IL-8, and IL-1ß by human neutrophils. Supernatants were collected and subjected to IL-6-specific ELISA, IL-8-specific ELISA, and IL-1ß-specific ELISA. Effects of PAR2-tcAP (5x10–5 M and 1x10–4 M) and trypsin (5x10–8 M and 10–7 M) on neutrophil IL-6 levels are presented (A) on IL-1ß levels (B) and on IL-8 levels (C). PAR2-RP (1x10–4 M) stimulation of human neutrophils does not result in any changes of cytokine release. The average values of cytokine production (pg/ml) by unstimulated neutrophils were as follows: for IL-1ß, 1.39 ± 0.3 pg/ml; for IL-6, 9.47 ± 0.26 pg/ml; for IL-8, 55.8 ± 2.7 pg/ml. Statistical analysis was performed by Student’s t-test. Results are expressed as an average value of protein concentration (as a percentage to control) ± SD. *, P < 0.05; **, P < 0.01. At least three independent experiments (in triplicates) have been performed.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the current study, we demonstrated that PAR2 agonists affect various functions in isolated human neutrophils such as motility in 3-D collagen lattices, expression of cell adhesion molecules, and secretion of proinflammatory cytokines. Our findings are in agreement with previous in vivo reports concerning the effects of PAR2 activation at leukocyte functions and inflammatory response [6 , 15 , 41 , 42 ]. In the present study, we focused on the in vitro investigation using PAR2 agonists for human neutrophil stimulation to dissect the underlying mechanism by which PAR2 may regulate neutrophil function.

In very recent studies, Seeliger and colleagues [41 ] investigated the role of PAR2 activation in cutaneous inflammation in vivo. In a model of experimentally induced allergic and toxic contact dermatitis, the authors demonstrated that ear swelling responses, plasma extravasation, and leukocyte adherence were significantly attenuated in PAR2-deficient mice compared with wild-type mice, especially at early stages. Moreover, intradermal injections of PAR2-APs into PAR2+/+ mice induced a marked dermal edema, plasma extravasation, and recruitment of leukocytes in skin by a nitric oxide-dependent mechanism. Such an effect was not observed after the treatment of PAR2–/– mice by PAR2-APs [41 ]. A delayed onset of inflammatory response was also observed in studies performed with PAR2-deficient mice [42 ].

The effects of the administration of PAR2-AP on leukocyte functions in vivo were also recently indicated. Injections of PAR2-tcAP as well as other PAR2-APs into the rat paw led to an acute inflammatory response characterized by edema and granulocyte infiltration [6 , 15 ]. Moreover, peritoneal injection of PAR2-tcAP or other PAR2-APs caused a significant increase in leukocyte migration into peritoneal cavity in a rat model [15 ].

Taken together, these in vivo data strongly support the idea of the important role of PAR2 stimulation on leukocyte functions and during the inflammatory response. Nonetheless, the effects of PAR2 agonist stimulation on human neutrophil functions and their correlation with previously described in vivo findings in animal models remained uncertain. Therefore, it is still necessary to investigate the consequence of PAR2 agonist stimulation on human neutrophil functions in details.

Howells and colleagues [16 ] demonstrated that stimulation of PAR2 expressed by human neutrophils with trypsin or with PAR2-AP (SLIGKV-NH2) results in an increased level of [Ca2+]i and induces shape changes in cells. In preliminary studies, we were able to confirm that SLIGKV-NH2 as well as SLIGRL-NH2 causes an increase of [Ca2+]i in neutrophils. We also found that SLIGRL-NH2 induced a slight (20–40%) increase in cytokine release by human neutrophils (data not shown). However, both PAR2-APs are known to be sensitive to amino peptidase activities and potentially to those derived from neutrophils. This may explain relatively slight effects of PAR2-AP on cytokine production by neutrophils, which we observed in preliminary experiments. Therefore, for further studies using human neutrophils exposed to a PAR2 agonist over prolonged time periods, we applied a PAR2-AP, trans-cinnamoyl-LIGRLO-NH2 (PAR2-tcAP), which is resistant to amino peptidase activity. PAR2-tcAP was documented to be an effective and specific PAR2 agonist in receptor-expressing cells [19 , 43 44 45 ]. However, the ability of this PAR2 agonist on human neutrophils has not been analyzed as of yet.

One of the aims of this study was to investigate the effects of PAR2 agonists on the expression of neutrophil cell adhesion molecules such as L-selectin and some ß1 as well as ß2 integrins. The expression of these molecules is known to be affected upon neutrophil activation by IL-8, fMLP, LPS, and others in vitro and in vivo during inflammation [27 28 29 , 36 , 46 47 48 ]. Down-regulation of L-selectin expression is considered as a marker of neutrophil activation [17 , 32 , 33 ].

Previously, Lindner and colleagues [42 ] reported that SLIGRL-NH2 stimulation did not affect L-selectin or Mac-1 (ß2 integrin) expression on human neutrophils if neutrophils were cultured alone. However, addition of this PAR2-AP to neutrophils coincubated with human endothelial cells resulted in an enhanced L-selectin shedding and up-regulation of Mac-1 expression on the neutrophil surface [42 ].

In the present study, we demonstrated that PAR2-tcAP is able to enhance L-selectin shedding and to up-regulate Mac-1 expression by neutrophils cultured alone without endothelial cells. Moreover, we revealed that PAR2-tcAP stimulation increases the display of {alpha}4ß1 integrin on the surface of isolated human neutrophils.

L-selectin shedding from the PMN surface is known to be a result of autoproteolysis or specific metalloprotease activity (L-selectin sheddase) [17 ]. EDTA (complexes bivalent cations) is known to reduce L-selectin shedding from the surface of human leukocytes [34 ]. In our study, the addition of 10 mM EDTA to human neutrophils, prestimulated by PAR2-tcAP, significantly reduced the enhanced L-selectin shedding. A reduction of this PAR2 agonist-induced effect by addition of EDTA indicates its dependency on the availability of bivalent cations in the neutrophil environment and a possible association with enzymatic activity of the L-selectin sheddase. We also used a hydroxamic acid-based metalloprotease inhibitor KD-IX-73-4 to check its effect on PAR2-tcAP-induced L-selectin shedding from the neutrophil cell surface. The addition of KD-IX-73-4 almost completely inhibited the PAR2-tcAP-induced removal of L-selectin from the PMN cell surface.

One can notice that the maximal effects of PAR2-tcAP stimulation at L-selectin shedding from neutrophil cell surface and at the level of sL-selectin in the neutrophil environment (cell culture medium) appear at different time-points after agonist application (at 4 h and 2 h, respectively). This can be explained by the fact that the level of sL-selectin in the culture medium is relatively low in comparison with the level of L-selectin expressed on the neutrophil cell surface at the beginning of neutrophil culture (2 h after agonist stimulation). Therefore, even a small amount of L-selectin (shed as a result of PAR2-tcAP stimulation) is sufficient to induce strong, relative effects at the level of sL-selectin in the culture medium. However, neutrophils are aging, and during this process, they are known to shed L-selectin even without any activation [33 ]. Indeed, we found that the absolute amount of sL-selectin in control samples increases at 6 h (19.5±0.59 ng/ml) in comparison with 2 h without any stimulation (10.12±1.5 ng/ml). This may (at least partially) explain the decreased magnitude of the PAR2-tcAP-induced effects on sL-selectin in the neutrophil environment at 6 h.

In the present study, we give evidence that PAR2 agonists modulate human neutrophil L-selectin expression. The debate whether up-regulation of L-selectin shedding may be associated with up- or down-regulation of transendothelial migration is still controversial [27 , 28 , 49 ]. Moreover, Allport and colleagues [50 ] demonstrated that treatment of human neutrophils with a shedding inhibitor was not able to affect transendothelial migration of these cells in a complex human endothelial-neutrophil system under flow conditions. Thus, the significance of L-selectin shedding for the regulation of leukocyte transendothelial migration remains unclear. Therefore, in the current study, we were interested in whether inhibition of PAR2-tcAP-induced L-selectin shedding by KD-IX-73-4 affects neutrophil motility in a 3-D collagen environment.

Neutrophil function can be regulated at different steps such as adhesion to endothelial cells or trans- and subendothelial migration. An increased migration of neutrophils within the ECM is associated with a response to chemotactic inflammatory stimuli. However, in contrast to the mechanisms leading to leukocyte adhesion or transendothelial migration, there is still little known about the mechanisms of neutrophil migration through the ECM. Collagen, fibronectin, and laminin are the main components of the ECM, and the 3-D collagen lattices assay is one of the possible and suitable methods to evaluate cell migration in the ECM [24 , 51 ]. In the present study, we revealed that stimulation of human neutrophils with PAR2 agonist leads to an enhanced migration of neutrophils in the 3-D collagen environment. These data allow us to suggest a stimulating role of PAR2 agonists on neutrophil migration through the ECM toward the site of inflammation and/or infection. Moreover, we revealed that addition of a L-selectin shedding inhibitor (KD-IX-73-4) significantly reduced PAR2-induced motility of human neutrophils in 3-D collagen lattices. Therefore, our data suggest that PAR2 agonist-induced neutrophil motility in a 3-D collagen, at least in a part, associated with an up-regulation of L-selectin shedding by PAR2 agonists. These findings are in agreement with the suggestion that leukocytes lose L-selectin from their surface upon leaving the blood vessel to be better adapted to move through ECM. Thus, serine proteases may trigger the migration of human neutrophils to the site of inflammation via PAR2 activation. This may have a high impact in chronic inflammatory diseases such as rheumatoid arthritis (RA) or atopic dermatitis, for example, diseases in which an involvement of serine proteases have been demonstrated [14 , 52 ].

The concept that neutrophils can be a source of cytokines has emerged lately and has been reviewed recently [53 ]. There are some proinflammatory cytokines (IL-1ß, IL-1{alpha}, IL-6) as well as chemokines (IL-8) among those produced by neutrophils. In the present study, we investigated the effects of PAR2 agonists (trypsin and tcAP) on the production and release of IL-1ß, IL-6, and IL-8 by human neutrophils. Stimulation of neutrophils by trypsin as well as tcAP resulted in an increased release of these cytokines. However, the magnitudes of stimulation on cytokine release by trypsin and tcAP varied considerably.

The observed distinctions may be explained, at least in part, by the fact that trypsin and PAR2-tcAP show different affinities to the receptor expressed by neutrophils. Additionally, trypsin, as an enzymatic PAR2 agonist, and PAR2-tcAP, as a synthetic PAR2 agonist, may differ in their sensitivity to neutrophil cell-surface proteases (CD10, for example). Finally, native enzymes have different kinetics and show varieties to endopeptidase inhibition.

Classically, PAR2 is considered as a trypsin receptor and the rest of PARs (PAR1, -3, and-4), as thrombin receptors. Nevertheless, it is well known that trypsin (with less affinity) is capable of activating PAR1, PAR3, and PAR4. [1 , 54 ]. This is especially important, as human neutrophils appear to express not only PAR2 but also PAR3 and under certain circumstances, PAR1 [12 , 55 ]. Several other reports support the idea that trypsin affects cell function not only as a result of PAR2 activation [1 , 2 ]. Additionally, it has been reported previously that neutrophils show a strong variability with regard to their response to enzymatic and synthetic PAR2 agonists [5 , 16 ]. Taken together, these data may explain the observed differences concerning the responses of human neutrophils to trypsin and tcAP after stimulation of human neutrophils.

It is not finally clear whether PAR2 activation of neutrophils occurs before, after, or at both conditions of transendothelial migration in vivo. Although all three possibilities look plausible, activation of neutrophil PAR2 after cell migration into the extracellular compartment seems to be more likely. This scenario may be supported by the following arguments: An essential requirement for PAR2 activation is the presence of sufficient concentrations of the active enzyme (protease) or another natural analog of PAR2-AP [56 ] in the receptor microenvironment. However, it seems contentious that the receptor agonist (especially the active protease) may reach effective concentrations inside the vascular compartment under conditions of blood flow. Additionally, potential, natural PAR2 activators in the extravascular compartment were recently described. For example, certain bacterial and house dust mite-derived proteases are able to activate PAR2 expressed by neutrophils and epithelial cells [5 , 7 , 57 ].

Stimulation of neutrophils with PAR2 agonists after transendothelial migration may be especially important for the resolution of inflammation. In accordance with our data, PAR2 agonists up-regulate the expression of proinflammatory cytokines by human neutrophils. These cytokines are known to affect neutrophil phagocytic activity and apoptosis. For example, IL-6 as well as IL-1ß have been reported to delay neutrophil apoptosis, thereby inhibiting the resolution of inflammation [58 , 59 ]. IL-1ß is known to up-regulate phagocytic activity of neutrophils [60 , 61 ]. Enhanced expression of some integrins (Mac-1, {alpha}4ß1) and the neutrophil interaction via these integrins with ECM proteins are also associated with delayed cell apoptosis [62 , 63 ]. These data suggest an important role of PAR2 agonists for the resolution of inflammation and subsequently for the development of chronic inflammatory diseases.

Indeed, recent studies in a mouse model of RA show that disruption of the PAR2 gene by homologous recombination results in ablation of chronic arthritis and that PAR2 is significantly up-regulated in inflamed tissues [14 ]. In addition, neutrophils are known to be the cells with the highest capacity to inflict damage within joints during RA [64 ], and they appear in numerous amounts within the synovial fluid and joint tissues during early stages of RA [65 ]. Thus, our present findings may have important implications for our understanding of the underlying mechanisms during the development of chronic inflammatory diseases such as RA or psoriasis, respectively.

However, PAR2 activation on neutrophils in the vascular compartment (before transendothelial migration) cannot be completely excluded, as various natural PAR2 agonists are released within the blood vessel. Among such agonists are endothelial-derived trypsin and factor VII/Xa, for example. Thus, further studies should be performed to clarify this question.

In summary, our observations suggest an important effect of PAR2 agonists on human neutrophil function (cytokine release, cell motility, expression of cell adhesion molecules), indicating a significant role of this receptor in acute as well as chronic inflammatory diseases in which neutrophils are involved.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Federal Ministry of Education and Research (IZKF, Fö.01KS9604/0; DFG STE 1014; SFB 293), C.E.R.I.E.S., Paris, Boltzmann-Institute, Münster, Germany, Rosacea Foundation, USA (to M. S.); Schering Foundation (to V. M. S.), and Boehringer-Ingelheim Fonds (to J. B.). Work in the laboratories of M. H. and N. V. is supported by funds from the Canadian Institutes for Health Research, The Kidney Foundation of Canada (M. H.), The Heart and Stroke Foundation of Canada (M. H.), a Johnson and Johnson Focused Giving Grant, and the Ileitis Colitis Foundation (N. V.). We thank Dr. C. Derian for providing PAR2 agonist peptides and A. Lechtermann and A. Grevelhörster for technical assistance.

Received May 14, 2003; revised March 14, 2004; accepted March 15, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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