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Pulmonary Research Group, Department of Medicine, University of Alberta, Edmonton, Alberta, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-hexosaminidase, IL-6, and matrix metalloproteinase-9 release from
BMCMC. Thrombin and the PAR-1-activating peptide
AparafluoroFRCyclohexylACitY-NH2 (cit) induced BMCMC
adhesion to FN in a dose-dependent fashion, while the PAR-1-inactive
peptide FSLLRY-NH2 had no effect. Thrombin and cit induced
also BMCMC adhesion to laminin. Thrombin-mediated adhesion to FN was
inhibited by anti-
5 integrin Ab (51.1 ± 6.7%;
n = 5). The combination of
anti-5 and anti-4 Abs induced
higher inhibition (65.7 ± 7.1%; n = 5).
Unlike what is known for Fc
RI-mediated adhesion, PAR-1-mediated
adhesion to FN did not increase mediator release. We then explored the
signaling pathways involved in PAR-1-mediated mast cell adhesion.
Thrombin and cit induced p44/42 and p38 phosphorylation. Pertussis
toxin inhibited PAR-1-mediated BMCMC adhesion by 57.3 ± 7.3%
(n = 4), indicating that Gi proteins
are involved. Wortmannin and calphostin almost completely inhibited
PAR-1-mediated mast cell adhesion, indicating that PI-3 kinase and
protein kinase C are involved. Adhesion was partially inhibited by the
mitogen-activated protein kinase kinase 1/2 inhibitor U0126 (24.5
± 3.3%; n = 3) and the p38 inhibitor SB203580
(25.1 ± 10.4%; n = 3). The two inhibitors
had additive effects. Therefore, thrombin mediates mast cell adhesion
through the activation of Gi proteins, phosphoinositol
3-kinase, protein kinase C, and mitogen-activated protein kinase
pathways. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Thrombin is a serine protease that is involved in hemostasis as well as in vessel wound healing, revascularization, and tissue remodeling (7). Thrombin exerts many of its actions through protease-activated receptors (PAR) (8, 9). Thrombin activates at least three members of this family of receptors, namely PAR-1, PAR-3, and PAR-4.
Mast cells respond to thrombin with release of granule enzymes, such as
-hexosaminidase (-hex) (10). In some mast cell
populations this response is equipotent with FcRI-mediated
activation. Mast cells express a variety of receptors for thrombin
(11). In the human there is immunohistochemical evidence
that mast cells express PAR-1 (12). PAR-1 has been shown
to be active in the release of IL-6 from murine mast cells
(13) and NO from rat peritoneal mast cells
(14). Thrombin may also affect mast cell biology through
the release of mast cell chemotactic factors from endothelial cells
(15).
In this study we investigated the biologic effects of mast cell
activation through PAR-1. We showed that murine bone marrow cultured
mast cells (BMCMC) adhere to FN following PAR-1-mediated activation,
and this adhesion is dependent primarily on 5
integrin. We also showed that a variety of signaling molecules,
including Gi, protein kinase C (PKC),
phosphoinositol 3-kinase (PI3 kinase), and mitogen-activated
protein (MAP) kinase are involved in PAR-1-mediated BMCMC adhesion.
Finally, we showed that PAR-1-mediated BMCMC activation leads to
release of -hex, IL-6, and matrix metalloproteinase-9 (MMP-9).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RPMI 1640, penicillin/streptomycin, HEPES,
L-glutamine, and nonessential amino acids (BioWhittaker,
Walkersville, MD); TRIzol, Superscript Reverse Transcription kit,
Taq polymerase, plasma FN, human placental laminin (hLN),
and merosin (Life Technologies, Gaithersburg, MD); murine recombinant
stem cell factor (mSCF) and IL-3 (mIL-3; Cedarlane, Hornby, Canada);
anti-4,
anti-5, anti-CD18, anti-CD11b,
anti-CD54, and anti-c-Kit (clone ACK45) mAb (BD PharMingen,
Mississauga, Canada); FITC-labeled anti-murine IgE Ab
(ImmunoKontact, Abingdon, U.K.); flat-bottom, 96-well plates (Limbo,
Flow Laboratories, McLean, VA); calcein-AM (Molecular Probes, Eugene,
OR); pertussis toxin, piceatannol, radicicol, U0126, SB203580,
SB202474, herbimycin A, calphostin, and wortmannin (Calbiochem, San
Diego, CA); FN fragment CS-1, BSA fraction V, murine IgE anti-DNP,
human serum albumin-conjugated DNP (HSA-DNP), Coomassie Brilliant Blue
G-250, and gelatin (Sigma-Aldrich Canada, Oakville, Canada); rabbit
polyclonal anti-p44/42, anti-phospho-p44/42
(Thr202/Tyr204),
anti-p38 and anti-phospho-p38
(Thr180/Tyr182; Cell
Signaling Technology, Beverly, MA); and Hyperfilm (Amersham Pharmacia
Biotech, Little Chalfont, U.K.).
PCR primers were synthesized by the DNA services laboratory, University of Alberta. PAR-1-activating and control peptides were synthesized by the peptide synthesis facility of Faculty of Medicine, University of Calgary. These peptides were >95% pure by HPLC and mass spectrometry criteria. The following peptides were used: a highly specific PAR-1-activating peptide, AparafluoroFRCyclohexylACitY-NH2 (cit), and the inactive peptide, FSLLRY-NH2 (PAR-1 CP).
Cell cultures
BMCMC were obtained as previously described (3) from male BALB/c mice and were cultured in RPMI 1640 medium supplemented with 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 4 mM L-glutamine, 25 mM HEPES, 100 µg/ml penicillin/streptomycin, 50 µM 2-ME, and 10% FBS (cRPMI). The medium was also supplemented with 10 ng/ml mSCF and mIL-3 and was changed every 4 days. BMCMC were used after 3–5 wk in culture when >98% of the cells were mast cells as determined by flow cytometry for c-Kit expression.
Adhesion assay
A fluorescent adhesion assay was used. Cells were loaded with calcein-AM that is fluorescent only after it enters the cells and is hydrolyzed by cell esterases. Briefly, BMCMC were washed in RPMI 1640 and then incubated with 2 µg/ml calcein-AM for 30 min at 37°C to take up the fluorescent dye. The cells were then incubated for another 30 min in cRPMI to metabolize the methyl ester to the fluorescent, non-membrane-permeable form. The cells were then resuspended at 106/ml in cRPMI and used for adhesion assays.
Flat-bottom, 96-well plates were coated at 37°C for 3 h with 100 µl of a 20 µg/ml solution of plasma FN in PBS (pH 7.2). This solution was then discarded, and nonspecific binding blocked with 100 µl 5% BSA in PBS for 1 h at 37°C. The wells were rinsed twice with RPMI 1640 and used in adhesion experiments.
Cells (1 x 105) in 100 µl cRPMI were added in each well alone (for spontaneous adhesion) or together with activating agents for 1 h. The nonadherent cells and medium were then aspirated, and the wells were washed three times with 100 µl cRPMI with vigorous shaking.
Fluorescence was measured using a Millipore Cytofluor 2350 plate reader before washing the plates (total fluorescence) and following the washing procedures described above. The percent adhesion was calculated using the following formula: % cell adhesion = (fluorescence in adherent cells/total fluorescence) x 100. For inhibition experiments, BMCMC were preincubated for 45 min at 37°C with the indicated concentrations of inhibitors or Abs and then employed in the adhesion experiments as described above. For the experiments with pertussis toxin, cells were preincubated for 2 h at 37°C, and for the experiments with calphostin cells were preincubated for 45 min at room temperature under light before being used in adhesion assays.
All assays were performed in duplicate. One value, the mean of the two measurements, was calculated for each condition in each independent experiment. The results in the text are given as the mean ± SEM of n independent experiments.
Flow cytometry
BMCMC (5 x 105) were washed twice with PBS with 0.05% NaN3 and 0.1% BSA (flow buffer) and incubated for 30 min with the appropriate primary Ab at 10 µg/ml or an isotype-matched control in a total volume of 50 µl. The cells were next washed twice with flow buffer and incubated for 30 min with the appropriate secondary PE-labeled Ab. The cells were then washed twice, resuspended in 500 µl flow buffer, and analyzed with a FACScan (BD Biosciences, Mountain View, CA). All procedures were performed at 4°C.
For FcRI analysis the cells were incubated for 30 min with 1 µg/ml
murine IgE in RPMI. They were then washed twice in flow buffer and
incubated for 30 min with FITC-labeled anti-murine IgE Ab. They
were then analyzed as described above.
IL-6 measurement
IL-6 was measured in resting and activated BMCMC supernatants using a commercially available ELISA kit (BioSource, Camarillo, CA).
RT-PCR for PAR-1 mRNA
Total RNA was isolated from BMCMC with TRIzol according to the
manufacturer’s instructions and was used for RT to cDNA with the
Superscript RT kit. Two microliters of the 20 µl RT reaction was used
for PCR amplification with Taq polymerase. The primers used to amplify
murine PAR-1 had the following sequences:
5'-GATCAGCTACTACTTCTCCGGC-3' and 5'-TGGCCGGTGCTGTTGCAACTGT-3'
(732-bp PCR product). Those used to amplify murine -actin had the
following sequences: 5'-CCATGTACGTAGCCATCCA-3' and
5'-GATGGAGCCACCGATCCAC-3' (644-bp PCR product). The PCR products
were separated on a 1.5% agarose-TAE gel and photographed using
Polaroid film (Cambridge, MA).
Western blot analysis
Cells were lysed in borate-buffered saline with 1% Triton X-100 supplemented with 1 mM PMSF, 10 µg/ml aprotinin, 4 µg/ml leupeptin, 10 µg/ml pepstatin, 5 mM EDTA, and 1 mM sodium orthovanadate. Total protein from the lysates of 100,000 cells was separated on a 10% SDS gels and then transferred to a nitrocellulose membrane. Western blot was performed with rabbit polyclonal Abs (1/1000 dilution) in TBS with 0.05% Tween 20 and 5% BSA. The membranes were subsequently incubated with goat anti-rabbit IgG HRP-conjugated secondary Ab (1/5000 dilution) for 1 h at room temperature. Proteins were visualized by ECL on Hyperfilm.
Gelatin zymography
SDS-PAGE gelatin zymography was performed using 7% polyacrylamide gels containing 0.2% gelatin. Resting and activated BMCMC supernatants were assayed to study MMP-9 release. PMA-treated (100 nM, 48 h at 37°C) HT 1080 human fibrosarcoma cell supernatant was used as a positive control for MMP-9 and MMP-2 gelatinolytic activities. Following electrophoresis at 4°C, the gels were washed three times for 20 min each time in 2% Triton X-100 at room temperature. Gels were then incubated in 50 mM Tris-HCl buffer (pH 7.6) supplemented with 0.15 M NaCl, 5 mM CaCl2, and 0.05% NaN3 at 37°C for 24 h. Following incubation the gels were stained for 1 h with 0.05% Coomassie brilliant blue G-250 and then destained overnight in 20% isopropanol/10% acetic acid. Proteolytic activity was identified as clear bands on a blue background.
-Hex release assay
BMCMC were suspended at 5 x 105
cells/ml in color-free RPMI supplemented with 0.2% BSA. One hundred
microliters of cells were prewarmed to 37°C and stimulated for 45 min
with various concentrations of PAR-1-activating peptides or thrombin to
induce -hex release. For FcRI-mediated -hex release, BMCMC
were incubated for 2 h with 1 µg/ml IgE anti-DNP, washed
twice, and then stimulated for 45 min with 20 ng/ml HSA-DNP.
-Hex was measured in the supernatants and cell pellets as described
by Schwartz et al. (16). Briefly, 50 µl sample was
incubated with 50 µl -hex substrate (1 mM
4-methylumbelliferyl-N-acetyl--D-glucosaminide
dissolved in DMSO and 0.2 M sodium citrate) for 2 h at 37°C. One
hundred microliters of 0.2 M Tris base was then added to stop the
reaction. Samples were read using a Cytofluor 2350 fluorescent
spectrophotometer at 450 nm (excitation, 356 nm). Results are shown as
-hex released as a percentage of the total -hex contained in
BMCMC.
Statistics
A paired Student’s t test was used to analyze the results for statistical significance. A value of p < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cells were identified as mast cells by flow cytometry with
anti-c-Kit Ab. On day 14 less than half the cells in culture
expressed c-Kit, while on day 24 >98% of the cells expressed c-Kit
(Fig. 1
A). Furthermore, >95%
of BMCMC expressed FcRI on day 24 (Fig. 1B). We further
characterized adhesion molecule expression by these cells. BMCMC
expressed 4 and 5
integrins, both adhesion receptors for FN. They also expressed ICAM-1
and low levels of CD18, but no CD11b (Fig. 1B).
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C). There was no significant
difference in PAR-1 mRNA levels in cells cultured with IL-3, SCF, or
both growth factors. PAR-1-mediated BMCMC mediator release
Thrombin has been shown to induce histamine and -hex release
(10) from mast cells. We verified that thrombin induced
the release of -hex from BMCMC (Fig. 2). The PAR-1-activating peptide cit
induced similar levels of -hex release from BMCMC with thrombin
(Fig. 2). The highest release through PAR-1 activation was
approximately half the release obtained through FcRI activation. The
inactive control peptide PAR-1 CP (concentrations up to 2 µM) had no
effect on -hex release (Fig. 2). Thrombin and cit also induced the
release of IL-6 (Fig. 3A) and
MMP-9 (Fig. 3B) from BMCMC. Thrombin- and cit-mediated IL-6
release was significantly lower that SCF- or FcRI-mediated release.
PAR-1 CP did not induce IL-6 or MMP-9 release (Fig. 3, A and
B, respectively). Wortmannin (10-7 M)
inhibited resting and thrombin-induced MMP-9 release from BMCMC (Fig. 3B, lower panel).
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Thrombin (0.5 U/ml) and the PAR-1-activating peptide cit (0.5
µM) induced 30.1 ± 2.3 and 24.66 ± 1.9% BMCMC adhesion
to FN-coated wells, respectively (Fig. 4A). PAR-1 CP did not induce
BMCMC adhesion in concentrations up to 50 µM. Also, a
PAR-2-activating peptide (SLIGRL-NH2, PAR-2 AP)
and its inactive control peptide (LRGILS-NH2,
PAR-2 CP) had no effect on BMCMC adhesion to FN in
concentrations up to 50 µM. SCF, used as positive control at 50
ng/ml, induced 49.2 ± 2.7% adhesion to FN (Fig. 4A).
Thrombin and cit-mediated adhesion were dose dependent (Fig. 4, B and C, respectively). Maximal adhesion was
32.4 ± 6.8% with 1 U/ml thrombin and 44.7 ± 0.3% with 50
µM cit.
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Then we examined whether thrombin- and cit-mediated BMCMC
adhesion is restricted to FN. We used two different preparations
of laminin, hLN and merosin, as it has been shown that BMCMC adhesion
varies among different forms of laminin (5). Thrombin (2
U/ml) and cit (2 µM) induced adhesion to both hLN and merosin (Fig. 5), although to significantly lower
levels than adhesion to FN (compare Fig. 5 to Fig. 4A).
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4 and
5 integrins to identify their role in
thrombin- and cit-induced mast cell adhesion to FN.
Anti-5 Abs induced 51.1 ± 6.7 and
50.2 ± 1.8% inhibition of thrombin- and cit-mediated BMCMC
adhesion to FN, respectively (Fig. 6).
Anti-4 Ab, although it had no significant
effect on its own, increased the effect of
anti-5 to 65.7 ± 7.1 and 68.7
± 4.0% for thrombin- and cit-mediated adhesion, respectively
(p < 0.01 compared by paired t test
to anti-5 alone), indicating that
4 integrin may also participate in thrombin-
and PAR-1-mediated mast cell adhesion to FN. The two Abs had a similar
effect when used to inhibit SCF-mediated adhesion to FN (data not
shown). Thrombin and cit had no effect on the level of
5 and 4 expression on
BMCMC, as assessed by flow cytometry (data not shown).
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RI-induced mast cell mediator release (5, 17).
However, in our experiments -hex, IL-6, or MMP-9 release from
thrombin- or cit-activated BMCMC did not change whether the cells were
kept in suspension or allowed to adhere to FN (data not shown).
Microscopic observation of the cells following thrombin- and
cit-induced adhesion to FN showed that in these cases adhesion was not
associated with cell spreading (data not shown). In contrast to
PAR-1-mediated adhesion, SCF- and FcRI-induced adhesions were both
associated with cell spreading (data not shown).
Signal transduction pathways mediating BMCMC adhesion
Because PAR-1 has been shown to activate a variety of signaling
pathways (9), we studied the pathways used by PAR-1 to
induce BMCMC adhesion to FN. PAR-1 has been shown to activate MAP
kinases. We studied the effect of thrombin- and PAR-1-mediated BMCMC
activation on p44/42 and p38 activation status using Western blotting
with Abs against the phosphorylated active form of these proteins.
Thrombin (0.5 U/ml) and cit (0.5 µM) induced a rapid phosphorylation
of p44/42 in BMCMC (Fig. 7A).
Phosphorylation reached maximum at 2 min and had almost returned to
baseline by 10 min. In contrast, SCF induced a more prolonged p44/42
phosphorylation, which was still evident at 30 min (Fig. 7A). Thrombin and cit also induced phosphorylation of p38
(Fig. 7B). In that case the phosphorylation was more
prolonged, but again returned to baseline by 30 min.
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A). Pertussis toxin had no
effect on SCF-mediated BMCMC adhesion to FN. Pertussis induced the
highest inhibition of thrombin- and cit-mediated adhesion at 50 ng/ml
(Fig. 8B). As pertussis does not inhibit adhesion
completely, it is possible that other G proteins activated by PAR-1 in
BMCMC might also be involved in the induction of adhesion.
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PI-3 kinase is also involved in PAR-1-mediated adhesion. The PI-3
kinase inhibitor wortmannin (10-7 M) inhibited
thrombin- and cit-mediated BMCMC adhesion to FN by 77 ± 4.3 and
94.9 ± 5.1%, respectively (n = 4;
p < 0.01; Fig. 8C). The PKC inhibitor
calphostin (10-4 M) also inhibited thrombin- and
cit-mediated BMCMC adhesion to FN by 86.7 ± 6.5 and 91.9 ±
4.1%, respectively (n = 4; p < 0.01;
Fig. 8C).
As we showed above, PAR-1 activates p44/42 and p38 MAP kinases in
BMCMC. U0126 (a MAP kinase kinase 1/2 (MEK1/2) inhibitor) and SB203580
(a p38 inhibitor) inhibited thrombin-mediated BMCMC adhesion to FN by
24.5 ± 3.3 and 25.1 ± 10.4%, respectively (Fig. 8D). These two inhibitors had additive effects (61.9 ±
3.7% inhibition) when used together, indicating that the two MAP
kinase pathways are activated in parallel by PAR-1 in BMCMC. Similar
results were seen for cit-mediated adhesion (Fig. 8D). U0126
had no effect on SCF-mediated BMCMC adhesion (data not shown).
All the data we have presented here indicate that thrombin- and
PAR-1-mediated BMCMC activation have similar characteristics. However,
PAR-1 inhibitors or PAR-1 blocking Abs were not available to us. To
obtain further evidence that thrombin works through PAR-1 activation we
performed cross-desensitization experiments. BMCMC were preactivated
with thrombin (1 U/ml) for 30 min and then activated again with
thrombin (0.5 U/ml), cit (0.5 µM), or SCF (50 ng/ml) for 2 min.
Thrombin preactivation abolished further p44/42 activation by thrombin
and decreased activation by cit by more than half, but had no effect on
SCF-mediated p44/42 activation (Fig. 9).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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51 activation seems
to play a major role in this adhesion, while
41 is also involved.
PAR-1-induced mast cell adhesion to FN is mediated through multiple
signaling molecules. We have shown that Gi
protein, Syk, PI-3 kinase, PKC, and the MAP kinases ERK and p38 are
involved in inducing BMCMC adhesion to FN. Thrombin-mediated adhesion
is not restricted to FN, but is also evident with laminin. Our results offer further evidence of a physiological role for thrombin in mast cell biology. They show that thrombin could mediate the adhesion of recruited mast cells around areas of vascular coagulation, a response independent of up-regulation of adhesion molecule expression, but dependent on increased avidity of adhesion molecules required for adhesion to FN. By inducing mast cell adhesion, thrombin might stabilize the presence of mast cells in the environment for as long as they are needed. Mast cell accumulation in these areas could also have a feedback regulatory role, as enzymes in mast cell granules can inactivate thrombin (18). It is also interesting that the concentration of thrombin is increased in the sputum of asthmatic patients compared with controls (19), implicating thrombin in mast cell-mediated events in asthmatic airway inflammation. Furthermore, other serine proteases, including mast cell products, might also induce PAR-1-mediated mast cell adhesion.
Our BMCMC, developed in the presence of IL-3 and SCF, expressed both
4 and 5 integrins, as
has been shown previously for WEHI-3-conditioned medium-cultured BMCMC
(20). Both 4 and
5 integrin Abs have been shown to inhibit mast
cell adhesion to FN (17, 21). Although
5 integrin seems to be more important for
thrombin-induced BMCMC adhesion to FN, we showed that
4 integrin is also involved. BMCMC used in our
experiments were at late stages of mast cell differentiation (wk 3–5).
It is still possible that 4 might play a more
important role in adhesion at earlier stages of mast cell development
(20). The fact that the two Abs together did not inhibit
mast cells adhesion by 100% indicates that other receptors might also
be involved in thrombin-induced mast cell adhesion to FN.
Thrombin and PAR-1 signaling has been studied extensively in a variety of biological systems and has been reviewed recently (22). However, there is not a lot of information about its signaling in mast cells or in other adhesion systems. In this paper we showed that thrombin- and PAR-1-mediated BMCMC activation induced phosphorylation of p44/42 and p38 MAP kinases.
PAR-1 activates more than one G proteins, including G12/13, Gq, and Gi (22, 23). Our data indicate that PAR-1-mediated Gi activation is involved in BMCMC adhesion. Our results also show that Src kinase family members are not involved in PAR-1-mediated adhesion of BMCMC, since both herbimycin A (24) and radicicol (25), which are known to inhibit c-Src, had no effect on thrombin-induced BMCMC adhesion to FN.
PAR-1 is known to activate Syk kinase in platelets (26).
In our experiments piceatannol, a known inhibitor of Syk, inhibited
thrombin-induced BMCMC adhesion by 41.5 ± 9.5% and also
inhibited SCF-induced adhesion. Piceatannol inhibits FcRI-mediated
Syk activation (27). However, piceatannol is not specific
for Syk, and it inhibits basophil degranulation in the absence of Syk
kinase inhibition (28). Therefore, we cannot be absolutely
certain that Syk is involved in thrombin-induced BMCMC adhesion.
Our data indicate that both PKC and PI-3 kinase are involved in thrombin-induced BMCMC adhesion. Inhibitors of both signaling molecules induced >85% inhibition of adhesion, indicating that the two molecules function in tandem. There is evidence that PI-3 kinase is downstream of PKC, and they are both downstream of Gi in a model of mast cell activation (29). This model would also correlate with our results, as inhibitors of all three signaling molecules induce almost complete inhibition of adhesion. In the same model Syk and MAP kinase activation is downstream of the above signaling molecules. PI-3 kinase and PKC have been also shown to be upstream of MEK/ERK (30) and p38 (31) in thrombin signaling. These observations would explain the reduced inhibitory effect we observed following inhibition of Syk and ERK kinases.
Recent evidence suggests that inflammatory cell adhesion is mediated at
least in part by MEK/ERK pathway activation (32, 33).
Results concerning p38 involvement differ depending on the cell and the
conditions studied (32, 34). The MAP kinase p38 may also
be involved in adhesion through adhesion molecule up-regulation
(35) and cytoskeletal remodeling (36). In our
experiments MEK1/2 and p38 inhibitors each caused 
25% inhibition of
adhesion, and when used together they had additive effects. These
results indicate that MEK/ERK and p38 are involved in pathways that act
synergistically to induce adhesion.
The data we present here indicate that thrombin- and PAR-1-mediated
BMCMC activation have similar characteristics. However, inhibitors of
PAR-1 or blocking Abs were not available to prove that thrombin works
through PAR-1 activation. Cross-desensitization between thrombin and
PAR-1 AP (see Fig. 9) further indicates that thrombin induced BMCMC
adhesion through PAR-1 activation. The pathways involved in
PAR-1-induced mast cell adhesion to FN, based on our results and
information from the literature, are summarized in Fig. 10.
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-hex, IL-6, or MMP-9). This
observation correlates with the lack of spreading of mast cells on FN
following thrombin-mediated adhesion. The physiological significance of
these results is not clear, and we do not have a definitive explanation
for why thrombin did not induce spreading of mast cells. Interestingly,
calpain is important for cell spreading on extracellular matrix
proteins (39, 40), but calpain inhibitors do not affect
cell adhesion (39). Calpain activation is important for
spreading in platelets, but thrombin-induced calpain activation in
platelets is not mediated through a PAR-1 mechanism (41).
It is possible that thrombin fails to activate calpain in BMCMC and
therefore does not induce cell spreading, which might be required for
increased degranulation after adhesion. In conclusion, we have shown that thrombin-induced PAR-1 activation leads to mast cell adhesion to FN. Thrombin-mediated mast cell adhesion could be a link between the coagulation cascade and perivascular inflammation. Therefore, PAR-1-mediated mast cell adhesion could participate in the function of mast cells in inflammation and tissue remodeling.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests Dr. Harissios Vliagoftis, Pulmonary Research Group, Department of Medicine, 550 HMRC, University of Alberta, Edmonton, Alberta, Canada T6G 2S2. E-mail address: harissios.vliagoftis{at}ualberta.ca
3 H.V. is a Canadian Institute of Health Research Scholar and an Alberta Heritage Foundation for Medical Research Clinical Investigator.
4 Abbreviations used in this paper: FN, fibronectin; cit, AparafluoroFRCyclohexylACitY-NH2; BMCMC, bone marrow-cultured mast cells; -hex, -hexosaminidase; hLN, human placental laminin; m, murine; MAP, mitogen-activated protein; MEK, mitogen-activated protein kinase kinase; MMP-9, matrix metalloproteinase-9; PAR-1, protease-activated receptor-1; PAR-2 AP, PAR-2-activating peptide; PAR-1 CP, PAR-1 control peptide (FSLLRY-NH2); PI3 kinase, phosphoinositol 3-kinase; PKC, protein kinase C; SCF, recombinant stem cell factor; HSA-DNP, human serum albumin-conjugated DNP.
Received for publication April 10, 2002. Accepted for publication August 6, 2002.
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) or cit (0.5
µM)-mediated (
) BMCMC adhesion to FN by anti-
