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*
Departments of Medicine (Division of Allergic Diseases) and Immunology, Mayo Clinic, Rochester, MN 55905; and
Department of Microbiology, QEII Medical Centre, University of Western Australia, Nedlands, Western Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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70% of those induced by 100 nM
platelet-activating factor, a positive control. In contrast, thrombin,
an activator for PAR1, PAR3, and PAR4, showed minimal effects. The
stimulatory effect of trypsin was dependent on its serine protease
activity and was blocked 59% by anti-PAR2 Ab. Furthermore, a
specific tethered peptide ligand for PAR2 potently induced superoxide
production and degranulation; the effects of peptide ligands for PAR1,
PAR3, and PAR4 were negligible. These findings suggest that human
eosinophils express functional PAR2, and serine proteases at the
inflammation site may play important roles in regulating effector
functions of human eosinophils. The expression and functional relevance
of other PARs still need to be determined. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There is now substantial evidence that certain proteases can regulate target cells by cleaving and activating a family of G protein-coupled protease-activated receptors (PARs) (17). Four members of this receptor family have been cloned and designated PAR1 (18, 19), PAR2 (20), PAR3 (21), and PAR4 (22, 23). Protease cleavage of these receptors creates a neo-NH2 terminus, which acts as a tethered ligand and activates the seven-transmembrane segment of the PAR. Human PAR1, PAR3, and PAR4 are activated by thrombin (18, 19, 21, 22, 23), whereas PAR2 is activated by trypsin, but not by thrombin (20). The discovery of PAR1 and PAR4 resulted from a successful search for the platelet receptor responsible for the cellular actions of thrombin (i.e., platelet aggregation) (18, 19, 22, 23) and, therefore, it is widely accepted that PAR1 and PAR4 play important roles in hemostasis (24). PAR2 is expressed by endothelial cells, epithelial cells, and smooth muscle cells in a variety of tissues and can regulate the activities of these cell types (reviewed in Ref. 25). For example, recent in vivo studies have demonstrated the ability of PAR2 to regulate blood pressure and vascular tone (reviewed in Ref. 26). PAR2-activating peptide promoted recovery of myocardial function after ischemia-reperfusion injury (27). Activation of PAR2 also elicited the relaxation of murine airway preparations through the release of PGE2 (28). Thus, PAR2 activation appears to play a homeostatic or protective role for the host via the epithelium and vascular endothelium.
Potential roles for PARs in inflammation have also been proposed. For example, because platelets can produce inflammatory mediators, such as serotonin and chemokines, platelet activation by thrombin through PAR1 might amplify inflammatory responses by or recruitment of inflammatory cells (24). Thrombin also triggered endothelial production of IL-6 and IL-8 (29). Trypsin and PAR2-activating peptides stimulated production of neuropeptides, such as calcitonin gene-related peptide and substance P, from spinal afferent neurons and induced edema formation in rat paw in vivo (30). Furthermore, i.p. administration of PAR2-activating peptides induced leukocyte rolling, adhesion, and extravasation through endogenous production of PAF (31). Thus, there is evidence that stimulation of PARs induces production of inflammatory mediators from platelets and interstitial cells and promotes inflammatory responses in the tissues. However, little is known about the direct participation of PAR2 and other PARs in the activation and functioning of immune and inflammatory cells. Indeed, not only interstitial cells, but also inflammatory cells, such as neutrophils and T cells, express PAR2 (32, 33, 34). The potential roles of eosinophils and proteases in allergic inflammation prompted us to investigate whether perturbation of PARs by serine protease might induce cellular activation and release of inflammatory mediators from human eosinophils. In this study, we examined the expression of PARs and the effects of serine protease on eosinophil activation and functions. The involvement of PARs was studied by protease inhibitors, specific PAR-activating peptides, and PAR-blocking Ab.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Trypsin (T8658), thrombin (T7009), and R-PE conjugate goat anti-rabbit IgG (whole molecule, P9795) were purchased from Sigma-Aldrich (St. Louis, MO). Taq DNA polymerase and AMV reverse transcriptase were purchased from Roche Molecular Biochemicals (Indianapolis, IN). TRIzol reagent, oligo(dT)12–18 primer, and dNTP mix were purchased from Life Technologies (Grand Island, NY). CytoStain kit was purchased from PharMingen (San Diego, CA).
Tethered ligand peptides specific for human PAR used in this study are as follows: PAR1 peptide SFLLRN-NH2 (control peptide FSLLRN-NH2) (23, 31), PAR2 peptide SLIGKV-NH2 (control peptide LSIGKV-NH2) (23, 31), PAR3 peptide TFRGAP-NH2 (control peptide FTRGAP-NH2) (31), and PAR4 peptide GYPGQV-NH2 (control peptide GYPGVQ-NH2) (23, 31). All peptides were made in house or purchased from Bachem (Torrence, CA). Z-Ala-Arg-OMe HCl (C-3845), a substrate for the measurement of serine protease activity, was purchased from Bachem.
Polyclonal Abs to human PAR2 and PAR3 were generated by immunizing rabbits with receptor peptides. The synthetic peptides SLIGKVDGTSHVTGKGVC (corresponding to human PAR2 aa 37–53 plus C-terminal cysteine residue) (35) and AKPTLPIKTFRGAPPNSFEEFPFSALEGC (corresponding to human PAR3 aa 31–58 plus carboxyl glycine-cysteine) (33, 36) were conjugated to keyhole limpet hemocyanin and used to generate polyclonal antisera in rabbits. The potency and specificity of Abs were confirmed using an ELISA. Specific Abs were isolated from antisera by affinity chromatography with the immunization peptides used to generate the PAR2 and PAR3 Abs.
Eosinophil isolation
Human eosinophils were purified from normal individuals or patients with mild allergy by Percoll density gradient centrifugation and magnetic cell sorting using MACS anti-CD16 microbeads, as described by Hansel et al. (37). Briefly, after peripheral blood was overlaid on an isotonic Percoll solution (1.084 g/ml; Sigma), the blood was centrifuged at 1000 x g for 30 min at 4°C. Mononuclear cells at the interface were removed, and erythrocytes in the sediment were lysed by two cycles of hypotonic water lysis. Isolated granulocytes were washed twice in PIPES buffer (25 mM PIPES, 50 mM NaCl, 5 mM KCl, 25 mM NaOH, 5.4 mM glucose, pH 7.4) containing 1% bovine calf serum (HyClone Laboratories, Logan, UT). Cells were then incubated with an equal volume of anti-CD16 mAb MACS microbeads for 60 min at 4°C with occasional gentle mixing. After 60 min of incubation on ice, cells were loaded onto the separation column positioned in the MACS magnetic field. Cells were eluted three times with 5 ml PIPES buffer with 1% bovine calf serum. The purity of eosinophils counted by Randolph’s stain was >98%. The contaminating cells were neutrophils, and no mononuclear cells or basophils were present. Purified eosinophils were used immediately for experiments.
Reverse-transcription PCR
Total RNA was prepared from eosinophils using TRIzol reagent. For cDNA synthesis, 1 µg total RNA was denatured and reverse transcribed in a reaction buffer containing 50 µg/ml oligo(dT)12–18 and 50 U AMV reverse transcriptase. PCR amplification (94°C for 1 min, 55°C for 1 min, and 72°C for 1 min; 30 cycles) was performed in a PerkinElmer (Norwalk, CT) thermal cycler. The PCR-amplified samples were run on 1% agarose gel and visualized using ethidium bromide. Primers used in this study were as follows (33): PAR1 (sense, CAG TTT GGG TCT GAA TTG TGT CG and antisense, TGC ACG AGC TTA TGC TGC TGA C), PAR2 (sense, TGG ATG AGT TTT CTG CAT CTG TCC and antisense, CGT GAT GTT CAG GGC AGG AAT G), PAR3 (sense, TCC CCT TTT CTG CCT TGG AAG and antisense, AAA CTG TTG CCC ACA CCA GTC CAC), PAR4 (sense, AAC CTC TAT GGT GCC TAC GTG C and antisense, CCA AGC CCA GCT AAT TTT TG), and G3PDH (sense, GTC AAC GGA TTT GGT CGT ATT and antisense, AGT CTT CTG GGT GGC AGT GAT). All the primers were synthesized at the Mayo Clinic Molecular Biology Core Facility.
Flow cytometry
To detect intracellular PAR, isolated eosinophils were washed once with staining buffer (PBS without Ca2+ or Mg2+, 0.1% calf serum, 0.09% NaN3, pH 7.4), fixed by suspending in Cytofix/Cytoperm solution (BD PharMingen) at 5 x 105 cells/sample, and kept at 4°C for 20 min. After centrifugation, cells were permeabilized and washed with Perm/Wash solution (PharMingen) and incubated in 100 µl Perm/Wash solution with 2 µl each of preimmune rabbit serum, rabbit anti-PAR2 or PAR-3 polyclonal Ab, or rabbit anti-major basic protein polyclonal Ab as a positive control for 30 min at 4°C. After washing in Perm/Wash solution, PE-conjugated goat anti-rabbit IgG was added, and cells were incubated for 30 min at 4°C. The cells were then washed, resuspended in 375 µl staining buffer and 125 µl of 4% paraformaldehyde solution, and kept at 4°C in the dark until analyzed in a FACScan flow cytometer (BD Immunocytometry Systems, Mountain View, CA).
To detect cell surface PAR, eosinophils (5 x 105 cells/sample) were washed once with PAB buffer (PBS containing 0.1% NaN3 and 1% BSA), and incubated in 100 µl PAB with 2 µl rabbit IgG or affinity-purified rabbit anti-PAR polyclonal Ab (1 mg/ml) for 30 min at 4°C. After washing in PAB, PE-conjugated goat anti-rabbit IgG was added, and cells were incubated for 30 min at 4°C. The cells were then washed, resuspended in 375 µl staining buffer and 125 µl 4% paraformaldehyde solution, and kept at 4°C in the dark until analyzed in a FACScan flow cytometer.
Eosinophil superoxide production and degranulation assays
To monitor eosinophil function in response to proteases or synthetic PAR-activating peptides, we used superoxide anion generation by and degranulation of human eosinophils. Superoxide generation was measured by superoxide dismutase-inhibitable reduction of cytochrome c, as previously described (38), with slight modifications. In brief, freshly isolated eosinophils were washed and resuspended in HBSS with 25 mM HEPES and 0.01% gelatin (Sigma) and 100 µM cytochrome c at 5 x 105 cells/ml. Cell suspension (100 µl) was dispensed onto the wells of 96-well tissue culture plates, followed by 100 µl trypsin (1–5 nM), thrombin (100–500 nM), PAR-activating peptides or control peptides (50–1000 µM), PAF (100 nM) as positive control, or medium alone. Immediately after addition of stimuli, the reaction wells were measured for absorbance at 550 nm in a microplate autoreader (Thermomax; Molecular Devices, Menlo Park, CA), followed by repeated readings. Between absorbance measurements, the plate was incubated at 37°C. Each reaction was conducted in duplicate and compared with an identical control reaction that contained 20 µg/ml superoxide dismutase (Sigma). Superoxide anion generation was calculated with an extinction coefficient of 21.1 x 103 cm-1 M-1 for reduced cytochrome c at 550 nm and was expressed as nanomoles of superoxide produced per 106 cells.
After incubation and repeated measurements of superoxide production at 37°C and 5% CO2 for 4 h, cell-free supernatants from 96-well tissue culture plates were collected and stored at -20°C until assayed for eosinophil degranulation. To quantitate eosinophil degranulation, the concentrations of eosinophil-derived neurotoxin (EDN) in the sample supernatants were measured by specific RIA, as previously described (38). The RIA is a double-Ab competition assay in which radioiodinated EDN, rabbit anti-EDN Ab, and burro anti-rabbit IgG are used (5). The sensitivity of EDN RIA was 2 ng/ml. All assays were performed in duplicate.
To examine the role of protease activity, serine proteases were preincubated with or without 2 mM serine protease inhibitor, 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF; Roche Molecular Biochemicals), for 30 min at 37°C before addition to eosinophils. To examine the involvement of PAR2 in the eosinophil response, eosinophils were preincubated with or without 1.5 µg/ml PAR2 Ab or rabbit IgG for 30 min at room temperature. After the preincubation, eosinophils were activated with or without 5 nM trypsin for 4 h at 37°C, and superoxide production by and EDN release from eosinophils were measured.
Quantitation of serine protease activity
To confirm the presence of serine protease activities in trypsin and thrombin and to ensure the effective inhibition of protease activity by AEBSF, we used a microplate protease activity assay with a specific substrate for serine protease (39). In brief, 150 µl substrate mix (6.3 mM Z-Ala-Arg-OMe, 6.3 mM bicine buffer, 0.068% phenol red, pH 9) was mixed with 40 µl enzyme solution (serial dilutions of thrombin or trypsin in distilled water with 2 mM CaCl2 and 1 mg/ml BSA) in the wells of 96-well tissue culture plates. To monitor kinetics, the changes in A580 were read initially at 30 s after the addition of enzyme/substrate mix and subsequently at 1-min intervals. The rate of substrate hydrolysis was determined from OD values within the first 10 min.
Statistical analysis
Data from at least three experiments using different eosinophil preparations from different donors were summarized and presented as mean ± SD. Statistical analyses were performed using Student’s t test, Mann-Whitney U test, or Wilcoxon test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To our knowledge, there were no reports regarding expression of
PARs by human or mouse eosinophils. Therefore, to investigate the roles
of proteases and PARs in human eosinophils, we first investigated the
expression of mRNA for PARs. As shown in Fig. 1
A, RT-PCR analyses of human
eosinophils revealed that they express PAR2 and PAR3 mRNA; in contrast,
PAR1 and PAR4 mRNA were undetectable. Neutrophils also expressed PAR2
and PAR3 mRNA, and PBMC expressed all four classes of PAR mRNA, which
are consistent with previous findings (33). These
observations were reproducible among five different donors that we have
investigated. In Fig. 1B, the results of five experiments
are summarized using densitometric analysis. The results clearly show
that PAR2 mRNA is consistently expressed by human eosinophils, as well
as by neutrophils and PBMC. Furthermore, when we performed the RT-PCR
analysis without reverse transcription, no PCR products were detected,
ruling out the possibility of genomic DNA contamination (data not
shown). Thus, eosinophils as well as neutrophils most likely express
mRNA for PAR2 and PAR3.
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After knowing the expression pattern of PAR mRNA, we sought to
examine the expression of PAR proteins by eosinophils. Antiserum and
affinity-purified Abs to PAR2 and PAR3 were generated in rabbits
immunized with peptides representing the
NH2-terminal exodomains of these receptors
(33, 35, 36). We performed flow cytometry analyses to
detect PAR protein expressed within or on the cell surface of
eosinophils. As shown in Fig. 2A, fixation,
permeabilization, and subsequent intracellular staining with
anti-PAR2 antiserum detected marked intracellular PAR2 protein in
human eosinophils. Affinity-purified anti-PAR2 Ab was used to
detect surface expression of PAR2. As shown in Fig. 2B,
small, but detectable binding of anti-PAR2 Ab to eosinophils was
observed compared with control rabbit IgG. A summary of four different
experiments showed 135 ± 4% mean fluorescence intensity with
anti-PAR2 Ab compared with control Ab (mean ± SEM,
n = 4). Similar experiments were repeated with
anti-PAR3 Ab; however, we were unable to detect any intracellular
and surface expression of PAR3 protein (data not shown). Taken
together, these data suggest the presence and surface expression of the
PAR2 protein in human eosinophils.
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Although human eosinophils seem to express PAR2, the surface
expression level was relatively small. Therefore, we examined the
functional relevance of PAR2 expressed by eosinophils using authentic
agonist proteases for this PAR. It is generally accepted that PAR2 is
activated by a serine protease, trypsin (20, 25), while
another serine protease, thrombin, selectively activates PAR1, PAR3,
and PAR4 (19, 21, 22, 23, 25). As shown in Fig. 3A, isolated human eosinophils
incubated with trypsin produced superoxide anion in a
concentration-dependent manner. Trypsin-induced superoxide production
reached a plateau at 5 nM trypsin, and at this concentration,
superoxide production was approximately 70% of that induced by 100 nM
PAF, which is one of the strongest agonists for human eosinophils
(8, 38).
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B, we also found that trypsin
induced eosinophil degranulation, as measured by EDN release, in a
concentration-dependent manner. At 5 nM, trypsin induced 201.9 ±
49.6 ng EDN release/106 cells (mean ± SD,
n = 5), which corresponds to 46% of the EDN release
induced by 100 nM PAF and approximately 21% of the total cellular EDN
content. In contrast, thrombin induced only 6.7 ± 3.2 and
19.8 ± 15.5 ng EDN release/106 cells at 100
and 500 nM, respectively (mean ± SD, n = 5).
These findings suggest that trypsin, but not thrombin, potently
stimulates human eosinophils and induces superoxide production and
degranulation. Blocking protease activity of trypsin inhibits trypsin-induced eosinophil activation
To address whether the ability of trypsin to stimulate eosinophils is due to its protease activity or due to any unknown contaminants, we examined the effect of a serine protease inhibitor. In a preliminary experiment, we used a microplate protease activity assay to determine the optimal conditions for inhibition of serine protease activity (39). Incubating 5 nM trypsin with a potent serine protease inhibitor, 2 mM AEBSF, for 30 min at 37°C abolished the serine protease activity. Accordingly, this condition was used to test the effect of intact vs inactivated serine protease on eosinophil activation.
Fig. 4A shows the effect of
AEBSF on trypsin- and PAF-induced superoxide anion production by human
eosinophils. Superoxide production from eosinophils stimulated with 5
nM trypsin, which was pretreated with AEBSF, was significantly lower
than that from eosinophils stimulated with untreated trypsin
(p < 0.001, n = 4), suggesting
that serine protease activity of trypsin is required to induce
eosinophil activation. In contrast, superoxide production after
stimulation with 100 nM PAF was not affected significantly by
pretreatment of PAF with AEBSF. Thus, AEBSF most likely does not
adversely affect the ability of eosinophils to respond to other
agonist(s) and to produce superoxide. Furthermore, as shown in Fig. 4B, trypsin-induced eosinophil degranulation was markedly
inhibited when trypsin was pretreated with AEBSF
(p < 0.001, n = 4). There was
little effect after AEBSF pretreatment on PAF-induced eosinophil
degranulation (mean inhibition 7.8%, n = 4). Thus, the
ability of trypsin to stimulate eosinophil effector functions is most
likely dependent on its serine protease activity.
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The results described above suggest that eosinophils express a
functional receptor(s) that responds to the protease activity of
trypsin and is presumably PAR2. Therefore, to examine the role of PAR2
in eosinophil activation induced by trypsin, affinity-purified
anti-PAR2 Ab (see above) was used. As shown in Fig. 5A, preincubation of
eosinophils with anti-PAR2 Ab, but not control rabbit IgG,
significantly inhibited trypsin-induced superoxide anion production by
58% (p < 0.01, n = 5). In
contrast, anti-PAR2 Ab did not affect eosinophil superoxide
production stimulated with PAF. Similarly, as shown in Fig. 5B, pretreatment with anti-PAR2 Ab significantly
inhibited eosinophil degranulation induced by 5 nM trypsin by 59%
(p < 0.01, n = 5). PAF-induced
degranulation was not affected by anti-PAR2 Ab. These findings
suggest PAR2 plays a major role in eosinophil activation and functions
in response to trypsin. In contrast, PAR2 does not seem to be involved
in eosinophil activation stimulated with a nonprotease agonist, such
as PAF.
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One of the ultimate tests for the roles of PAR in cell activation
and functions is the use of peptides that mimic the tethered ligands.
The peptide ligands for PAR1, PAR2, and PAR4 have been used
successfully for studies in vitro and in vivo (23, 25, 31, 33). The potential peptide ligand for PAR3 has also been
proposed (40). As shown in Fig. 6A, incubation of eosinophils
with a PAR2-activating peptide (SLIGKV) induced superoxide production.
A control peptide (LSIGKV) showed no or minimal effects. In contrast,
as shown in Fig. 6B, neither a PAR3 peptide (TFRGAP) nor its
control peptide (FTRGAP) induced eosinophil superoxide production.
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, A and B, EDN
release from eosinophils incubated with the PAR2-activating peptide
(SLIGKV) was most pronounced. At its optimal concentration (500 µM),
PAR2 peptide induced 363.4 ± 73.7 ng EDN
release/106 cells (mean ± SD,
n = 3), corresponding to approximately 40% of total
cellular EDN. In contrast, a control peptide for PAR2 (LSIGKV) showed
no effect. Interestingly, PAR1-activating peptide (SFLLRN) also induced
eosinophil degranulation (57.8 ± 4.1 ng EDN
release/106 cells at 500 µM peptide, mean
± SD, n = 3), but the magnitude was <20% of that
induced by the PAR2-activating peptide (SLIGKV). On the other hand,
PAR3- and PAR4-specifc peptide (TFRGAP and GYPGQV, respectively) showed
no effects. These findings are consistent with the effects of trypsin,
and suggest that PAR2 is a major functional PAR expressed by human
eosinophils.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The differences in the inhibitory effects of the protease inhibitor,
AEBSF, and anti-PAR2 Ab need clarification. As shown in Fig. 4, inhibition of trypsin’s protease activity by AEBSF completely
abolished both trypsin-induced eosinophil superoxide production and
degranulation. On the other hand, as shown in Fig. 5, the inhibitory
effect of anti-PAR2 Ab was partial, resulting in approximately 60%
inhibition of the trypsin-induced cellular responses. This relatively
weaker effect of anti-PAR2 Ab could be explained by a lower
affinity between this Ab and PAR2 compared with the interaction between
trypsin and PAR2. Alternatively, eosinophils may express a low level of
another PAR, such as PAR1 and PAR4, and thus respond to serine protease
in a PAR2-independent manner. This speculation is consistent with Fig. 7, which shows the PAR1-activating peptide inducing small, but
significant, degranulation of human eosinophils. Finally, eosinophils
may also express novel or as yet unidentified subtype(s) of PAR or
PAR-like molecule(s). Indeed, we found recently that human eosinophils
are activated by both serine proteases and by cysteine proteases, such
as papain and a mite allergen, Der p 1 (S. Miike and H.
Kita, manuscript in preparation). Thus, the eosinophil’s expression of
PARs and the consequent responses to proteases may be more common than
anticipated. Therefore, it will be important to define the full
repertoire of PARs and proteases that signal through PARs in
eosinophils.
Eosinophils seem to be exquisitely sensitive to trypsin. PAF is one of
the strongest known agonists for eosinophil degranulation and can
induce eosinophil degranulation at a concentration of 100-1000 nM
(8, 38). Similarly, a complement fragment, C5a, and a
chemokine, eotaxin, induce eosinophil exocytosis at about 100 nM
(9, 41). In this study, trypsin, as low as 3 nM, induced
marked eosinophil degranulation and superoxide production (Fig. 3).
Although the maximal degranulation induced by trypsin was approximately
50% less than that induced by 100 nM PAF, trypsin effectively
stimulated eosinophil responses at <10 nM. A similarly effective
action of trypsin at low concentrations was reported in cells
transfected with PAR2 (42). As a downstream signal
transduction mechanism, PARs as well as receptors for PAF, C5a, and
chemokines use G proteins (24, 43). Therefore, the
question arises as to how low concentrations of trypsin can so
effectively stimulate eosinophil functions. The potential heterogeneity
of G proteins coupled to PARs and other classical G protein-coupled
receptors (e.g., PAF receptor) may explain the differences in their
sensitivities to ligands. Another explanation may be at the level of
the receptor/ligand interaction. Unlike a classical G protein-coupled
receptor (e.g., PAF receptor or C5a receptor), the peptide ligand
unmasked by proteolytic cleavage remains tethered to PARs (24, 43), resulting in longer-lasting activation of the receptors.
Moreover, acting as an enzyme, one trypsin molecule might cleave and
activate several molecules of PAR2. Thus, PAR2 may represent a unique
class of receptors expressed by human eosinophils that enables
eosinophils to respond sensitively to the changes in microenvironment
and to exert their effector functions.
These observations may have important implications in our understanding of the regulation of eosinophil activation at inflammation sites in patients with asthma and allergic diseases. Many allergens, such as fungi (10), mites (11, 12), and pollens (13), are not inert proteins, but most likely have protease activities, such as serine and cysteine protease activities. Furthermore, during an allergic response, mast cells release their granular contents that include serine proteases, such as tryptase (15, 16, 44) and chymase (45). Therefore, once eosinophils are recruited into an allergic inflammation site, these allergens or granule enzymes may directly activate eosinophils through the interaction of their serine protease activities and eosinophil PARs and may induce inflammatory mediator release from eosinophils. Therefore, once allergic responses are initiated, eosinophils interacting with serine proteases may exacerbate the inflammation and disease process by producing more inflammatory mediators. Conversely, removal of protease activity from the sites of inflammation or blockade of eosinophil PARs may benefit patients with allergic diseases by dampening this vicious cycle of allergic inflammation.
It has been known that platelets express PAR1, PAR3, and PAR4 and are activated by a serine protease, thrombin (18, 19, 21, 22, 23). In this study, we found that human eosinophils express PAR2 and are activated by another protease, trypsin, but not by thrombin. Therefore, the specificity of serine proteases to PARs may determine the roles of these proteases in tissue homeostasis and inflammation. Thrombin or thrombin-like serine proteases are most likely critical in coagulation. In contrast, trypsin-like serine proteases may activate inflammatory cells, such as eosinophils, through PAR2 and may be involved in immunity and inflammation. These potential proinflammatory effects of trypsin-like proteases raise several important questions: 1) Among the various serine proteases produced by microorganisms, allergens, and inflammatory cells, which ones actually have trypsin-like activities? 2) Whether and how do these proteases affect functions of inflammatory cells? 3) Are there any diseases that induce cellular expression of other PARs by inflammatory cells (e.g., PAR1), allowing them to respond to different types of serine proteases? The answers to these questions may elucidate the, as yet poorly defined, roles of PARs and serine proteases in the complex inflammatory network of human immunity and diseases.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hirohito Kita, Departments of Medicine and Immunology, Mayo Clinic and Foundation, Rochester, MN 55905. E-mail address: kita.hirohito{at}mayo.edu
3 Abbreviations used in this paper: PAF, platelet-activating factor; AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride; EDN, eosinophil-derived neurotoxin; PAR, protease-activated receptor.
Received for publication June 8, 2001. Accepted for publication September 26, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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