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*
Division of Allergy and Immunology, Department of Pediatrics, and
Division of Infectious Diseases, Department of Medicine, Harbor-UCLA Medical Center, Torrance, CA 90509; and
Tanox, Houston, TX 77025.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The plasminogen activator (PA)3 system has an important role in controlling endogenous fibrosis and regulating extracellular matrix (ECM) proteolysis relevant to tissue remodeling (15). The tissue-type PA (tPA) and urokinase-type PA (uPA) convert plasminogen to plasmin, which enhances proteolytic degradation of the ECM via at least three different mechanisms. First, plasmin removes glycoproteins from the ECM before the matrix metalloprotease (MMP)-dependent degradation of collagen (16). Second, plasmin activates MMPs directly to degrade ECM components (17, 18, 19, 20, 21, 22). Third, plasmin degrades ECM by inhibiting MMP inhibitors. Because of the high concentration of plasminogen in virtually all tissues, the production of small amounts of PA can result in high local concentrations of plasmin. Taken together, plasmin exerts control over MMP activity at the substrate, activation, and inhibitor levels.
An important mechanism in the regulation of PA activity is inhibition of uPA or tPA by three major inhibitors, which are PAI-1, PAI-2, and PAI-3 (23). PAI-3 regulates enzymes involved in fertilization rather than lung tissue fibrosis, whereas altered expression of both PAI-1 and PAI-2 is of potential relevance to the process of lung fibrosis (24, 25). Although PAI-2 exhibits inhibitory activity toward tPA and uPA (26, 27), the efficiency of PAI-2 is 20- to 100-fold less than what is observed for PAI-1 (28). PAI-2 is not generally detectable in human plasma, except during pregnancy (29, 30). Dougherty et al. (31) demonstrated that PAI-2 is not required for normal murine development, survival, or fertility.
Deficiency of PAI-1 in mice (32, 33) and humans (34) is associated with increased fibrinolysis. PAI-1-deficient mice are resistant to pulmonary fibrosis after lung injury, presumably due to accelerated fibrinolysis (35). PAI-1-overexpressing mice suffered a severe lung injury and deposition of ECM after bleomycin challenge (35) or hyperoxia (36), whereas PAI-1-deficient mice were protected against such fibrotic reaction. The finding that the level of PAI-1 gene expression is strongly correlated with the amount of collagen accumulation within lung tissues suggests that the balance of fibrinolytic activity within the lung is an important determinant of the pulmonary response to inflammatory injury. The cellular basis of endogenous fibrinolysis has been the subject of numerous speculations and investigations. Endothelial cells are a major source of tPA and PAIs (37, 38, 39, 40). Activated macrophages and smooth muscle cells are another source of PAs and PAIs (41, 42, 43, 44).
Both murine and human mast cells in various organs (including lung and heart) express tPA as a prestored mast cell granular component (45, 46, 47). Sillaber et al. (46) demonstrated that unstimulated mast cells have fibrinolytic activity due to the presence of tPA. They speculated that mast cell-derived tPA may function as a "repair molecule", preventing fibrin deposition during some pathophysiologic processes. Other investigators demonstrated that the rapid IgE-mediated influx of 125I-labeled fibrinogen into the dermal tissue of mice is mast cell dependent and the fibrin deposition persists for at least 24 h after challenge (48, 49). Taken together, these findings suggest that mast cells may have an important role in modulating the extent of fibrosis in the extracellular environment.
In the present study, we demonstrate that human mast cells secrete an abundant amount of functionally active PAI-1 upon stimulation with a combination of PMA and calcium ionophore (A23187) or by IgE receptor cross-linking. Secretion of PAI-1 is due to increased synthesis of PAI-1 mRNA and protein in mast cells unlike the secretion of tPA. Mast cells are known to have net fibrinolytic activity due to the presence of tPA in unstimulated condition (46). Here we demonstrate that induced PAI-1 from human mast cell line (HMC)-1 completely inhibits tPA activity. Our data provide insight into the relationship between mast cells and endogenous fibrinolysis in various pathophysiologic conditions. Moreover, we demonstrate that mast cells are an active source of PAI-1 in asthmatic airway by immunofluorescence study. These results suggest that mast cells are an important source of PAI-1 that may play a pathophysiologic role in asthmatic airway.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HMC-1 (a kind gift of J. H. Butterfield, Mayo Clinic, Rochester, MN) and primary cultured HMCs (PCHMCs) from cord blood were maintained as described (50, 51). Briefly, HMC-1 cells were cultured in IMDM (Life Technologies, Grand Island, NY) with 10% heat-inactivated FBS (Life Technologies), 4 mM L-glutamine, and antibiotics in a humidified 5% CO2 incubator (50). The cell number was adjusted to 5 x 105 cells/ml twice weekly by adding fresh media. The PCHMCs were derived from human cord blood CD34+ cells grown in the presence of stem cell factor, IL-6, and IL-10, as modified from the procedure of Saito et al. (51). Briefly, heparin-treated umbilical cord blood was purchased from Advanced Biotechnologies (Columbia, Maryland). The cord blood sample was diluted 1:4 in PBS supplemented with 2% BSA and 0.6% citrate, loaded onto Ficoll-Histopaque (Sigma-Aldrich, St. Louis, MO), and centrifuged twice at 200 x g. The interface containing mononuclear cells was mixed with Dynabeads M-450 CD34 according to manufacturer’s protocol (Dynal, Lake Success, NY). The CD34+ cells were magnetically separated and later detached from the beads. The washed mononuclear cells were suspended in cord cell media consisting of RPMI 1640 (Life Technologies) supplemented with 20% FBS (Sigma-Aldrich), 2 mM L-glutamine, 50 µM 2-ME, 100 U/ml penicillin, 100 µg/ml streptomycin, 10 µg/ml gentamicin, 80 ng/ml stem cell factor, 50 ng/ml IL-6, and 5 ng/ml IL-10. The cells were stained with a mAb directed against mast cell tryptase (Chemicon International, Temecula, CA) to determine the percentage of mast cells as we have previously described (52). Over 94% of these cells were mast cells. The cell suspensions were seeded at a density of 1–5 x 105 cells/ml and harvested after 7–8 wk of culture. The cytokine-supplemented medium was replaced on a weekly basis.
Stimulation conditions
Nine-week-old PCHMCs (6 x 105 cells per treatment group) were first incubated with 10 µg/ml mouse IgE (PharMingen, San Diego, CA) for 1 h at 37°C before washing in PBS twice to remove the unbound IgE. Subsequently, the IgE was cross-linked with 10 µg/ml goat anti-mouse F(ab')2 (Jackson ImmunoResearch, West Grove, PA). HMC-1 cells (1 x 106 cells per treatment group) were stimulated with 30 ng/ml PMA (Sigma-Aldrich) and 350 ng/ml A23187 (Sigma-Aldrich). When indicated, 10 µg/ml cycloheximide (CHX; Sigma-Aldrich), 2 µg/ml cyclosporin (CsA; Sigma-Aldrich), and 0.01 or 1 µM dexamethasone (Dex; Sigma-Aldrich) were added. Subsequently, cells were briefly spun down in an Eppendorf Scientific (Westbury, NY) centrifuge (12,000 x g) and the supernatant was separated from the pellet.
DNA microarray
The DNA microarray was constructed and used at Incyte (Fremont, CA) using their proprietary protocol (http://www.rei.edu/researchers/oh.html) (53, 54, 55). Briefly, 200 ng of purified mRNA from resting HMC-1 cells and cells stimulated with PMA and A23187 for 4 h was reverse transcribed with avian myeloblastosis virus reverse transcriptase (Invitrogen, San Diego, CA) at 37°C for 1 h using random hexamers, and labeled with Cy3 (mRNA from resting HMC-1 cells) or Cy5 (mRNA from stimulated HMC-1 cells) fluorescent dyes. The two-color competitive hybridization was then performed on an Incyte human gene chip, UniGEM-V DNA microarray. This gene chip is designed to give a broad view of human gene expression by using genes and expressed sequence tags from the public domain UniGene database. All of the clones containing 7075 elements have been individually sequence verified. Individual cDNA molecules averaging 1000 bp long were isolated into unique pools and amplified. They were then deposited on a glass surface in an array format with each gene occupying a unique location. The cDNAs were subsequently bonded to the glass and the dsDNAs were activated by the removal of its ssDNAs for the hybridization with the fluorescent probes. Following hybridization, the microarray was rinsed and the nonhybridized probes were washed off. Each element on the array was scanned for the two individual fluorescent colors. The ratio of the two fluorescent intensities provided a highly accurate and quantitative measurement of the relative gene expression level in the two treatment conditions.
RT-PCR
Total RNA (5 µg) was reverse transcribed with avian myeloblastosis virus reverse transcriptase (Invitrogen) at 37°C for 1 h using random hexamers in a total volume of 20 µl of reaction buffer. A total of 2 µl cDNA was amplified with 0.2 mM dNTPs, and 2.5 U of Taq DNA polymerase in the buffer recommended by the supplier (Invitrogen). Amplification consisted of denaturation at 94°C for 5 min followed by 30 cycles consisting of 94°C for 1 min, 55°C for 1 min, and 72°C for 3 min, and amplification was ended by a 10-min extension at 72°C. The following primers were used: human tPA (444 bp); sense (5'-CAGGAGAGCAGCGTGGTC-3') and antisense (5'-GTCGGGTGTTCCTGGTCA-3'); and human PAI-1 (348 bp); sense (5'-GGCAACATGACCAGCTG-3') and antisense (5'-GGCCAAGTGAT GGAACCC-3'). Amplification of fragments of the cDNA of GAPDH were performed in the same PCR as the internal control with the following primers: human GAPDH (598 bp); sense (5'-CCACCCATGGCAAATTC CATGGCA-3') and antisense (5'-TCTAGACGGCAGGTCAGGTC CACC-3'). Half of each reaction mixture was analyzed by agarose gel electrophoresis. All experiments were performed three times with similar results.
cDNA probes for Northern blot analysis
Human PAI-1 cDNA was generated by RT-PCR as described above. The 2-kb human ß-actin cDNA was purchased from Clontech Laboratories (Palo Alto, CA). The cDNAs were labeled by random hexamer using protocols suggested by the manufacturer (Stratagene, La Jolla, CA; Ref. 56).
Northern blot analysis
Total cellular RNA was isolated from HMC-1 mast cells by a guanidine thiocyanate-cesium chloride gradient centrifugation as previously described (57). Total cellular RNA (20 µg) was then subjected to electrophoresis in 1.5% agarose-formaldehyde gels and transferred to nylon-reinforced nitrocellulose membranes (Micron Separations, Westboro, MA). Hybridizations and visualization were performed as we have previously described (58). Transcript levels were quantified by densitometry (Model GS-700 Imaging Densitometer; Bio-Rad, Hercules, CA).
Measurement of PAI-1 protein
The HMC-1 cells (1 x 106 cells per
treatment group) or PCHMCs (6 x 105 cells
per treatment group) were stimulated with PMA and A23187 or by Fc
RI
cross-linking, respectively, as described above. The cells were
centrifuged at 800 x g for 10 min at 4°C and the
cell-free supernatant and cell pellet from each group were collected.
Cell pellets were resuspended and lysed with 0.5% Triton X-100 in
Tyrode’s buffer. The concentration of PAI-1 protein in the
supernatants and cell lysates was measured in duplicate by ELISA
(American Bioproducts, Parsippany, NJ). The detection limit was 1.0
ng/ml.
Analysis of tPA activity
The tPA activity of the HMC-1 supernatant was determined by a bioimmunoassay kit using chromogenic substate S-2251 according to the manufacturer’s protocol (Chromogenix, Molndal, Sweden; Ref. 59). Supernatants from unstimulated HMC-1 cells (1 x 106 cells/ml) and cells stimulated with PMA and A23187 for 8 h were assayed for tPA activity. When indicated, 20 µg/ml anti-PAI-1 Ab (Enzyme Research Laboratories, South Bend, IN) was added. The specificity of the Ab was verified by the manufacturer. The bioimmunoassay kit had a detection limit of 0.02 IU/ml.
Clot lysis assay
The clot lysis assay was performed as previously described (60). Briefly, the clot was prepared in 24-well plates (Costar, Cambridge, MA) by mixing 1 mg fibrinogen, 4 mmol CaCl2, 0.1 µmol plasminogen, and 0.5 U thrombin in 300 µl of Tris buffer (pH 7.8). Supernatants from unstimulated cells (1 x 106 cells/ml) and cells stimulated with PMA and A23187 for 8 h were used for the clot lysis assay. Cell supernatants were added to the clot-reaction mixture to a final volume of 1000 µl. When indicated, 20 µg/ml anti-PAI-1 Ab was added. The mixtures were incubated at 37°C for 24 h. Next, the wells were inspected under the microscope for clot lysis, which was defined as a complete dissolution of the fibrin meshwork within 24 h.
Double-immunofluorescence study
R. Barbers (University of Southern California, Los Angeles, CA) provided postmortem lung tissue from a patient who died from an asthmatic attack and normal lung tissue. The tissue was fixed in neutral buffered formalin, embedded in paraffin, and cut into 2-µm sections. Simultaneous double-immunofluorescence staining was performed essentially as previously described (61). Nonspecific binding was blocked with 5% swine or donkey serum (according to the species of the secondary Abs; Jackson ImmunoResearch). After two washes with PBS, primary Abs against PAI-1 (1/100; Santa Cruz Biotechnology, Santa Cruz, CA) or mast cell tryptase (1/500; Chemicon International) were added and the sections were incubated overnight at 4°C. Substitution of each primary Ab with normal rabbit or mouse serum (Jackson ImmunoResearch) at the same dilution was performed to control for nonspecific binding. The sections were washed in PBS twice for 10 min each and then incubated for 1 h at room temperature with the appropriate secondary Abs. The FITC-labeled swine anti-rabbit Abs (Dako, Carpinteria, CA) were diluted 1/20 and the Texas red-labeled donkey anti-mouse Abs (Jackson ImmunoResearch) were diluted 1/100. After four washes in PBS for 10 min each, the coverslips were mounted and then examined using an Eclipse E400 microscope (Nikon, Melville, NY) equipped with epifluorescence optics.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using a DNA microassay, we screened 7075 genes to identify those
that were up-regulated in stimulated mast cells. The HMC-1 human mast
cell line was exposed to a combination of PMA and A23187 to achieve
maximal stimulation. Among the inducible genes that were identified,
PAI-1 mRNA was induced at the highest level followed by uPA receptor
mRNA (Table I
). Other PA genes such as
tPA and uPA were not significantly induced.
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We performed Northern blot analysis to confirm the results from
the DNA microarray (Fig. 1). PAI-1 mRNA
was undetectable in unstimulated HMC-1 cells (Fig. 1, R),
whereas both the 3.2-kb and 2.4-kb PAI-1 mRNAs were expressed at a high
level in HMC-1 cells after treatment with PMA and A23187 for 4 h
(Fig. 1, P/A).
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To determine whether PAI-1 was also induced in PCHMCs upon
physiological stimulation, PCHMCs from human cord blood were stimulated
by IgE receptor cross-linking. Semiquantitative RT-PCR was
performed due to difficulty in obtaining a sufficient number of PCHMCs
for Northern blot analysis. As seen in Fig. 2, PAI-1 mRNA was undetectable in
unstimulated cells (top panel, lane 1) and
was induced in cells stimulated by IgE receptor cross-linking for
3 h (lane 2) and 24 h (lane
3). In contrast, tPA mRNA was detected in unstimulated cells
(middle panel, lane 1) and was decreased in cells
stimulated by IgE receptor cross-linking for 3 h
(lane 2) and 24 h (lane
3).
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The fact that PAI-1 mRNA is induced in primary cultured mast cells
after physiologic stimulation led us to determine the amount of PAI-1
secreted by both HMC-1 cells and PCHMCs. A considerable amount of PAI-1
was secreted in 1 x 106 HMC-1 cells/ml
(171.5 ± 6.6 ng/ml; Fig. 3A) and 6 x
105 PCHMCs/ml (84.5 ± 3.8 ng/ml; Fig. 3B) after overnight stimulation, whereas virtually no PAI-1
was secreted by either group of unstimulated cells (3.9 ± 0.2 and
0 ng/ml, respectively).
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To further characterize the expression of PAI-1 gene in human mast
cells, the kinetics of PAI-1 mRNA expression was examined in HMC-1
cells by Northern blot analysis (Fig. 4).
PAI-1 mRNA was not detected until 3 h, peaked at 6 h, and was
detectable at a lower level for up to 48 h after stimulation.
These results demonstrate that the PAI-1 gene is induced at a high
level with an undetectable level of constitutive expression. We next
determined whether the kinetics of PAI-1 protein secretion correlates
with that of PAI-1 mRNA induction. Minimal amounts of PAI-1 was
secreted by resting HMC-1 cells (1.5 ± 0.8 ng/ml) and cells
stimulated for 30 min (1.7 ± 0.9 ng/ml). PAI-1 was first detected
in the medium 8 h post stimulation (21.1 ± 1.2 ng/ml) and
reached a maximum after 24 h (252.0 ± 13.9 ng/ml; Fig. 5A). When the cells were
pretreated with CHX 10 min before stimulation, the PAI-1 was not
detected in the supernatants or lysates of resting cells (0 ng/ml
and 0 ng/ml, respectively; Fig. 5B). The PAI-1 levels
significantly increased in the supernatants and the cell lysates after
stimulation (265.7 ± 12.3 and 45.3 ± 8.5 ng/ml,
respectively). Pretreatment with CHX almost completely abrogated the
accumulation of PAI-1 in the supernatants and cell lysates (5.3 ±
0.9 and 1.8 ± 0.3 ng/ml, respectively). These data indicate that
the secretion of PAI-1 is due to de novo synthesis of PAI-1 rather than
secretion of preformed PAI-1.
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To determine whether mast cell-derived PAI-1 was active, we
examined its ability to inhibit tPA activity (Fig. 6). The tPA activity in the supernatants
of unstimulated HMC-1 cells was 0.470 ± 0.026 IU/ml. The activity
was almost completely absent in the supernatants of HMC-1 cells that
has been stimulated with PMA and A23187 for 8 h and 24 h
(0.023 ± 0.003 and 0 IU/ml, respectively; Fig. 6A). To
determine whether this reduction in tPA activity was due to inhibition
by PAI-1, the HMC-1 cells were pretreated with a neutralizing Ab
against PAI-1 before stimulation. Neutralizing PAI-1 in the
supernatants restored tPA activity (0.35 ± 0.03 IU/ml; Fig. 6B). Hence, mast cell-derived PAI-1 suppresses tPA activity
upon stimulation.
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To determine the total effect of PAI-1 secretion in the
fibrinolytic system of human mast cells before and after stimulation,
we performed a clot lysis assay (Fig. 7).
In this assay, control medium with or without anti-PAI-1 Ab was
added to a synthetic fibrin meshwork and no clot dissolution was seen
(Fig. 7, A and B). Supernatants from unstimulated
HMC-1 cells induced clot lysis (Fig. 7C). No clot
dissolution was seen with supernatants from stimulated HMC-1 cells
(Fig. 7D). To determine whether the inhibition of clot lysis
upon stimulation was due to PAI-1 protein, the cells were pretreated
with neutralizing Ab against PAI-1 before stimulation and the
supernatants were added to a synthetic fibrin meshwork. The clot lysis
effect was fully recovered in the supernatants from the mast cells
pretreated with the neutralizing Ab (Fig. 7E). These results
demonstrate that mast cell-derived PAI-1 completely suppresses tPA
activity and converts a fibrinolytic environment to a fibrosis-dominant
condition.
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In other cell types such as fibroblasts, glucocorticoids are known
to induce PAI-1 mRNA whereas CsA suppresses PAI-1 gene expression
(62, 63, 64). We determined the effect of CsA or Dex on the
expression of PAI-1 mRNA in HMC-1 cells stimulated with PMA and A23187
by Northern blot analysis. CsA down-regulated PAI-1 gene expression,
whereas high-dose (1 µM) Dex induced PAI-1 mRNA expression (Fig. 8). We also found that CHX abrogated
PAI-1 mRNA expression. This finding indicates that transcription of the
PAI-1 gene requires de novo synthesis of early gene products such as
transcription factors.
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Lung tissue from a patient with asthma was examined using indirect
immunofluorescence to determine whether mast cells express PAI-1 in
vivo (Fig. 9). The lung tissue was
examined through an appropriate red filter (Fig. 9, A,
C, and E) or green filter (Fig. 9, B,
D, and F). Double-immunofluorescence
colocalization of PAI-1 and tryptase in mast cells (white
arrows) is apparent in Fig. 9, C–F. The
yellow-green staining of PAI-1 is seen in airway smooth muscle (SM). A
tryptase-positive but PAI-1-negative mast cell is shown in normal lung
tissue (Fig. 9, A and B, red arrows).
These results suggest that mast cells are an important source of PAI-1
in lungs of an asthmatic patient.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we demonstrate that the stimulated human mast cells are able to secrete functionally active PAI-1. Both 3.2-kb and 2.4-kb PAI-1 mRNAs are induced in HMC-1 cells at a high level after treatment with PMA and A23187 (67). However, Sillaber et al. (46) reported that the PAI-1 message was undetectable in HMC-1 cells by Northern blot analysis even after stimulation with stem cell factor. These discrepancies could be explained by the fact that stimulation for 2 h may be insufficient to induce PAI-1 messages in HMC-1 cells. Also, we used a different stimulus.
PAI-1 is secreted by both HMC-1 cells and PCHMCs after stimulation, whereas PAI-1 is undetectable in either group of unstimulated cells. These results are consistent with the data obtained from Northern blot analysis and RT-PCR. The amount of PAI-1 in HMC-1 cells exceeds the amounts of PAI-1 in the PCHMCs. One reason for this difference might be the usage of different stimuli for the different cell types (i.e., PMA and A23187 for HMC-1 and IgE receptor cross-linking for PCHMCs). Alternatively, PAI-1 production in mast cells is associated with proliferation or differentiation processes, explaining the lower PAI-1 levels in the differentiated primary cells compared with those that are continuously proliferating immature cell line (HMC-1). Sillaber et al. (46) reported that PAI-1 was undetectable in isolated lung mast cells and tissue mast cells in the heart and skin. Because these cells were unstimulated, these results are in agreement with our data showing undetectable level of PAI-1 protein in unstimulated HMC-1 cells or PCHMCs, in vitro.
Unlike tPA, PAI-1 mRNA and protein are newly synthesized in mast cells after stimulation. These results suggest that the constitutive production of tPA in mast cells will lead to the accumulation of tPA over time in the absence of PAI-1, which is not secreted constitutively. Therefore, our data are in agreement with those of Sillaber et al. (46), demonstrating a time-dependent increase in tPA protein and activity in conditioned medium from unstimulated HMC-1.
The immediate response to an allergen in a sensitive individual reaches a maximum 15–30 min after challenge followed by a late phase reaction that peaks by 6–12 h (68). Mekori et al. (48) demonstrated the persistence of fibrin deposition at late intervals (24 h) after elicitation of pure IgE-dependent reactions by either i.v. or dermal challenge with Ag. The amount of fibrin deposition at the 24-h time point was significantly less than those in the same type of reactions assessed 2 or 4 h after challenge. Nevertheless, fibrin persists at sites of IgE-dependent passive cutaneous anaphylaxis in mice for at least 24 h after elicitation of the response. We may speculate that tPA provides active fibrinolytic activity in the resting state and the early phase of activation, after which mast cell-derived PAI-1 may increase enough to overcome tPA-mediated fibrinolysis. Sillaber et al. (46) demonstrated that mast cells have net fibrinolytic activity due to the presence of tPA in the unstimulated condition by tPA activity assay and clot lysis assay. They speculated that mast cell-derived tPA may function as a repair molecule, preventing fibrin deposition during some pathophysiologic processes. Our data in tPA activity and clot lysis assay in unstimulated condition confirm their data. However, in the late phase of stimulated condition, mast cell-derived PAI-1 completely suppresses tPA activity and converts a fibrinolytic environment to a fibrosis-dominant condition. The tPA activity and the fibrinolysis environment are recovered by neutralizing anti-PAI-1 Ab. These data suggest that mast cells produce and accumulate PAI-1 in the target tissues of chronic allergic inflammation and promote tissue remodeling.
Only high-dose (1 µM) Dex further induces PAI-1 mRNA expression in HMC-1 cells stimulated with PMA and A23187. We speculate that it is because mast cells are already strongly (near maximally) stimulated with the combination of PMA and A23187. Our results are consistent with other findings in different cell types. For example, Heaton et al. (62) showed that treating hepatoma tissue culture rat hepatoma cells with Dex caused a rapid decrease in tPA activity, which is secondary to a 5-fold increase in PAI-1 Ag and activity. Bator et al. (63) also showed that PAI-1 mRNA increased 3-fold in cultured murine keratinocytes 2 h after the addition of 1 µM hydrocortisone and remained elevated for at least 8 h. Our data raise a possibility that steroids may enhance tissue fibrosis such as occurs during airway remodeling or recalcitrant atopic dermatitis by up-regulation of PAI-1 expression. CsA down-regulates the expression of the PAI-1 gene. van den Dorpel et al. (64) demonstrated that plasma tPA activity increased with a substantial decrease in PAI-1 activity. CHX abrogates PAI-1 mRNA expression by Northern blot analysis. These results suggest that transcription of the PAI-1 gene requires de novo synthesis of early gene products, including transcription factors. In contrast, CHX has no effect on tPA release because tPA is a prestored cellular constituent that does not require protein synthesis for its immediate release (45). Taken together, the control mechanism of these tPA and PAI-1 gene expression in human mast cells is very different, although these genes have close correlation in controlling microenvironmental balance of PA system.
Airway remodeling in asthma is characterized by subepithelial fibrosis due to extensive deposition of ECM. The MMPs play crucial roles by cleaving the proteins constituting the ECM (69, 70, 71, 72). An imbalance between MMPs and their inhibitors occurs in bronchial asthma and contributes to tissue remodeling (73, 74, 75). PAI-1 is a major inhibitor of the MMPs and it is induced in pulmonary fibrosis. We demonstrated that mast cells are an active source of PAI-1 in asthmatic airway by double-immunofluorescence study of lung tissue from a patient with fatal asthma attack. Severe asthma with airway remodeling is characterized by an increased number of activated mast cells in the airway (76, 77). We thus speculate that these sensitized mast cells release considerable amount of PAI-1, which blocks fibrinolysis and thereby promotes fibrin and collagen deposition. The presence of fibrin and collagen is the main feature of airway remodeling. Whether mast cell-derived PAI-1 indeed provides an essential contribution to the process of airway remodeling in vivo is now under investigation by inducing airway remodeling in PAI-1 knockout and transgenic mice.
In summary, this is the first report demonstrating that stimulated human mast cells are an important source of functionally active PAI-1 upon allergen challenge. These findings suggest the involvement of PAI-1 in the pathogenesis of airway remodeling and the involvement of mast cells in the expression of PAI-1. Our data also suggest that therapeutic interventions designed to enhance fibrinolysis such as administration of plasminogen activators (78) or inhibitors of PAI-1 (79) may inhibit the development of airway remodeling.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Chad K. Oh, Harbor-UCLA Medical Center, Building N25, 1000 West Carson Street, Torrance, CA 90509.
3 Abbreviations used in this paper: PA, plasminogen activator; PAI, PA inhibitor; MMP, matrix metalloprotease; ECM, extracellular matrix; HMC, human mast cell line; PCHMCs, primary cultured HMCs; tPA, tissue-type PA; uPA, urokinase-type PA; A23187, calcium ionophore; CHX, cycloheximide; CsA, cyclosporin; Dex, dexamethasone.
Received for publication February 2, 2000. Accepted for publication June 28, 2000.
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 J. M. Swartz, J. Bystrom, K. D. Dyer, T. Nitto, T. A. Wynn, and H. F. Rosenberg Plasminogen activator inhibitor-2 (PAI-2) in eosinophilic leukocytes J. Leukoc. Biol., October 1, 2004; 76(4): 812 - 819. [Abstract] [Full Text] [PDF]
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 S. H. Cho, C. H. Ryu, and C. K. Oh Plasminogen Activator Inhibitor-1 in the Pathogenesis of Asthma Experimental Biology and Medicine, February 1, 2004; 229(2): 138 - 146. [Abstract] [Full Text] [PDF]
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 B. V. Offersen, B. S. Nielsen, G. Hoyer-Hansen, F. Rank, S. Hamilton-Dutoit, J. Overgaard, and P. A. Andreasen The Myofibroblast Is the Predominant Plasminogen Activator Inhibitor-1-Expressing Cell Type in Human Breast Carcinomas Am. J. Pathol., November 1, 2003; 163(5): 1887 - 1899. [Abstract] [Full Text] [PDF]
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J. D. Savov, D. M. Brass, K. G. Berman, E. McElvania, and D. A. Schwartz Fibrinolysis in LPS-induced chronic airway disease Am J Physiol Lung Cell Mol Physiol, October 1, 2003; 285(4): L940 - L948. [Abstract] [Full Text] [PDF]
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 K. Nielsen, K. Birkenkamp-Demtroder, N. Ehlers, and T. F. Orntoft Identification of Differentially Expressed Genes in Keratoconus Epithelium Analyzed on Microarrays Invest. Ophthalmol. Vis. Sci., June 1, 2003; 44(6): 2466 - 2476. [Abstract] [Full Text] [PDF]
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S. H. Cho, Z. Yao, S.-W. Wang, R. F. Alban, R. G. Barbers, S. W. French, and C. K. Oh Regulation of Activin A Expression in Mast Cells and Asthma: Its Effect on the Proliferation of Human Airway Smooth Muscle Cells J. Immunol., April 15, 2003; 170(8): 4045 - 4052. [Abstract] [Full Text] [PDF]
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 A. M. Vignola, F. Mirabella, G. Costanzo, R. Di Giorgi, M. Gjomarkaj, V. Bellia, and G. Bonsignore Airway Remodeling in Asthma Chest, March 1, 2003; 123(2007): 417S - 422S. [Abstract] [Full Text] [PDF]
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 J. Wojta, C. Kaun, G. Zorn, M. Ghannadan, A. W. Hauswirth, W. R. Sperr, G. Fritsch, D. Printz, B. R. Binder, G. Schatzl, et al. C5a stimulates production of plasminogen activator inhibitor-1 in human mast cells and basophils Blood, June 28, 2002; 100(2): 517 - 523. [Abstract] [Full Text] [PDF]
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S. Page, A. J. Ammit, J. L. Black, and C. L. Armour Human mast cell and airway smooth muscle cell interactions: implications for asthma Am J Physiol Lung Cell Mol Physiol, December 1, 2001; 281(6): L1313 - L1323. [Abstract] [Full Text] [PDF]
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 R. Alam and M. Gorska Genomic Microarrays . Arraying Order in Biological Chaos? Am. J. Respir. Cell Mol. Biol., October 1, 2001; 25(4): 405 - 408. [Full Text] [PDF]
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