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ABSTRACT |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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The lung plasminogen activator (PA) response was examined in four different models of particle- induced pulmonary lesions in NMRI mice (single intratracheal administration, 0.75 to 5 mg/mouse). Sequential changes in cellular (total and differential counts) and biochemical markers of alveolitis (lactate dehydrogenase [LDH], total proteins) were monitored in bronchoalveolar fluid (BALF) and the fibrotic lung response was assessed histologically. An intense but spontaneously resolving alveolitis was produced by manganese dioxide (MnO2) and a fibrosing alveolitis was elicited by crystalline silica (DQ12). Minimal and noninflammatory responses were obtained after instillation of titanium dioxide (TiO2) and tungsten carbide (WC), respectively. The comparison between the resolving and the fibrosing alveolitis model was especially taken into consideration in an attempt to identify fibrinolytic changes associated with the development of fibrosis. At the alveolitis stage, similarly increased BALF PA activities were measured in both the resolving and the fibrosing alveolitis models whereas only slight and no PA modifications were noted after administration of TiO2 and WC, respectively. Persistently (up to 120 d) increased BALF PA activity was selectively associated with the progression to fibrosis (DQ12), suggesting that PA is involved in the fibrotic process. ELISA measurements demonstrated that the changes in BALF PA activity were exclusively related to changes in urokinase (uPA), not tissue-type PA. A rapid and persisting (up to Day 30) upregulation of cell-associated PA activity occurred after DQ12, MnO2, and TiO2 treatment only. Cellular PA activity was however significantly higher in fibrogenic inflammatory cells recovered from DQ12 than from MnO2-treated mice suggesting that the intensity of cellular PA upregulation may represent an early indicator of the progression to fibrosis. The implication of urokinase in the pathogenesis of silica-induced fibrosis was demonstrated by the use of a uPA knockout mice. The acceleration of the fibrotic process in uPA-deficient compared with the wild type animals demonstrated the contribution of uPA to limit the fibrotic process.
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Degradation of connective tissue is an important aspect in the pathogenesis of chronic inflammatory lung disease. Current concepts on the nature of the resolution process taking place in response to lung injury indicate that tissue repair occurs along an insoluble matrix composed of fibrin and both plasma-derived and cell-synthetized products. What happens to this alveolar protein scaffold is an important determinant of whether damaged alveoli can undergo successful repair or are replaced by fibrotic tissue. Persistent matrix-bound fibrin and its soluble degradation products are thought to modulate processes contributing to the formation of the fibrotic tissue including fibroblast cell adhesion/proliferation and subsequent collagen deposition.
The plasminogen activating system may have a potential
role in this repair process. Through the activation of the abundant extracellular plasminogen into the potent protease plasmin, plasminogen activators (PA) promote the clearance of
excessive extravascular fibrin deposition (1). Plasmin can also
degrade most of the glycoproteins and proteoglycans of the
extracellular matrix (ECM) and can indirectly promote degradation of collagen by activating latent forms of collagenase
(2). Urokinase-type PA (uPA) produced by macrophages (3)
and pneumocytes (4) is abundantly found in the alveolar lining fluid and is mainly implicated in extracellular and pericellular proteolysis processes (5). Independently of stromal remodeling processes, plasminogen activation may also activate
cell-associated or EMC-sequestered growth factors such as
transforming growth factor beta (TGF-
) (6). In addition, associated with its specific plasma membrane receptor (uPAR), uPA may also promote in vitro adhesiveness (7) and migration of monocytes (8) as well as fibroblast proliferation (9, 10).
uPA/uPAR interaction might therefore contribute to the cellular proliferation associated with the repair of inflammatory lung injury. Plasminogen activation is counterbalanced by the fast-acting plasminogen activator inhibitors (PAI-1 and PAI-2) produced by macrophages (11), epithelial cells (4), and fibroblasts (12) and plasmin activity is control by 
2-antiplasmin
and 2-macroglobulin (13). The degree of tissue remodeling is
therefore likely to be determined by the net cellular or soluble
fibrinolytic activity expressed locally.
Disruption of normal plasminogen activation control may occur in different pathologic processes including bleeding, thrombotic disorders as well as acute and chronic inflammatory lung diseases (14, 15). Defective or increased fibrinolytic activity has been found in bronchoalveolar fluid (BALF) of patients with adult respiratory distress syndrome (16, 17), sarcoidosis (18), idiopathic pulmonary fibrosis (18), and asbestosis (19), all these diseases being characterized by a certain degree of interstitial fibrosis. Alveolar fibrinolytic activity has also been found enhanced in several experimental models of lung injury including particle- and fiber-induced lung injury (19). Moreover, total deficiency in PAI-1 or local increase of alveolar uPA concentrations were shown to limit the lung fibrotic process induced experimentally by bleomycin (14, 15).
The foregoing human and experimental observations suggest that alterations of the alveolar fibrinolytic activity may influence the course of lung injury. We therefore examined how components of the plasminogen activating system varied in different types of pulmonary lesions. Soluble and cell-associated PA changes were characterized during the course of the pathogenic process occurring in four mouse models of particle-induced lung injury. Crystalline silica (DQ12), manganese dioxide (MnO2), titanium dioxide (TiO2), and tungsten carbide (WC) particles were selected according to their ability or lack of ability to elicit an alveolitis progressing to fibrosis. The implication of uPA in the pathogenesis of silica-induced pulmonary fibrosis was confirmed by the use of a uPA knockout mouse model.
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Reagents
DQ12 was a kind gift of Dr. Armbruster (Essen, Germany), MnO2 was kindly provided by Sedema (Division of Sadacem S.A., Tertre, Belgium), titanium dioxide (anatase form) was from Aldrich (Steinheim, Germany), and tungsten carbide was from Johnson Matthey (Royston, UK). The physicochemical characteristics of these particles are summarized in Table 1. Fibrinogen fragments were obtained from Boehringer (Mannheinn, Germany). Human plasminogen and S2251 (Val-Leu-Lys-para-nitroaniline plasmin substrate) were purchased from Chromogenix (Mölndal, Sweden). Tris-hydroxymethyl-aminomethane (TRIS), glycine, and bovine serum albumin (BSA) were from Sigma (St. Louis, MO), phosphate-buffered saline (PBS) was from GIBCO (Paisley, Scotland), 1,2-phenylenediamine and formaldehyde were from Flucka (Buch, Switzerland), and pentobarbital was from Siegfried Chemie (Brussels, Belgium).
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Animals
Adult female NMRI mice (25 g) were purchased from Iffa Credo
(Brussels, Belgium) and urokinase knockout mice (uPA
/) generated by homologous recombination as well as the wild-type (uPA+/+)
mice were kindly provided by Dr. P. Carmeliet (KUL, Leuven, Belgium) (23). The animals were housed in an air-conditioned room
(22° C, relative humidity 50 ± 10%) with a 12-h light/dark cycle and
fed on a conventional laboratory diet.
In Vivo Treatments
The animals were anesthetized by intraperitoneal injection of pentobarbital (80 mg/kg of body weight). The trachea was exposed using a sterile technique and particulate material suspended in 100 µl sterile saline was slowly instilled into the lung. Control animals were treated with an equivalent volume of saline. Survival was greater than 95%.
In a first series of experiments, the lung response was examined
over a period of 120 d in NMRI mice (5 to 10 mice/group) treated with
at least three (0.75, 2.5, 5 mg/mouse for DQ12) or two doses (0.75, 2.5 mg/mouse for MnO2, TiO2, and WC) including saline controls. In a
second series of experiments (5 to 10 mice/group), the lung response
induced by DQ12 (5 mg/mice) was investigated in urokinase knockout
(uPA/) and wild-type (uPA+/+) mice over a period of 30 d.
Bronchoalveolar Lavage
At selected time intervals after treatment (1, 3, 5, 30, 60, or 120 d), animals were killed by pentobarbital overdose and bronchoalveolar lavage (BAL) was conducted according to the technique previously described (22). The lung was washed 5 times with 1 ml sterile 0.9% NaCl. The lavage fluids (BALF) were centrifuged (1,200 g, 10 min, 4° C). The cell-free supernatants of the first lavage fraction were used for biochemical measurements, and the cell pellets pooled from the five lavages were used for cell-associated PA activity measurements and total/differential cell counts.
Enzymatic Assay for Plasminogen Activators
A chromogenic assay adapted from Leprince and coworkers (24) was used to determine soluble PA activity in unconcentrated BALF samples and associated to the BAL cell fraction. The quantification method used is indirect: the enzymes convert exogenous plasminogen into plasmin and the subsequent plasmin-dependent hydrolysis of a synthetic substrate (S2251) is then monitored. This assay measures PA activity without discrimination between tissue-type PA (tPA) and uPA. Briefly, 50 µl of unconcentrated BALF sample or BAL cell suspension (106 cells/ml) were incubated in 0.1 M Tris/0.1 M glycine/0.5% BSA buffer (pH 8.5) or PBS/BSA 0.5% buffer (pH 7.4), respectively. Incubation was carried out in the presence of S2251 substrate (0.7 mM), fibrinogen fragments (530 pg), and human plasminogen (0.14 mM) in a final volume of 250 µl. The measurements were performed in triplicate in 96-well plates incubated at 37° C. Negative controls in which plasminogen was omitted were assayed simultaneously for each sample. The formation of p-nitroaniline from the hydrolysis of S2251 was followed spectrophotometrically at 405 nm (Twinreader Titertek, Flow Laboratories, Finland) every 15 min over a period of 10 h. The difference in optical density between plasminogen-containing and plasminogen-free wells was plotted against the square of the incubation time and the slope of the regression line calculated. The determination of PA units present in the sample was calculated as described by Schnyder and coworkers (25) and the results were expressed in IU (µmol of substrate hydrolyzed by min).
Measurement of PA-related Antigens
Determinations of murine tPA, uPA, and PAI-1 proteins in unconcentrated BALF samples were performed with a two-site, noncompetitive ELISA as described by Declerck and colleagues (26) using a conventional peroxidase detection method with 1,2-phenylenediamine as substrate. The detection limits for tPA, uPA, and PAI-1 were 0.15, 3, and 1.2 ng/ml, respectively.
Lung Tissue Analysis
Histopathology. Lung tissues (4 mice/group) were fixed in situ via instillation of phosphate-buffered (pH 7.4) 10% formaldehyde, removed and immersed in the same fixative. Paraffin-embedded sections were stained with hematoxylin-eosin and Masson's trichrome for light microscopic examination.
Hydroxyproline measurements. Lung tissues (5 mice/group) were harvested and total hydroxyproline content (OH-proline) was analyzed by high-performance liquid chromatography (HPLC) as previously described by Driscoll and associates (27). All data were expressed as µg OH-proline per lung.
Other Analyses
BALF total protein content was assessed spectrophotometrically using a commercial kit (Systemes Technicon, Doumon, France). BALF lactate dehydrogenase (LDH) activity was assessed spectrophotometrically by monitoring the reduction of NAD+ at 340 nm in the presence of lactate (22). The total number of live cells recovered by lavage was determined by the trypan blue exclusion method and differential cell counts were performed on cytocentrifuge preparations stained with Diff-Quick (Dade NV/SA, Brussels, Belgium).
Statistics
All data are expressed as mean ± SEM. The statistical significance of differences between groups was assessed using ANOVA and the Student-Newman-Keuls test for comparison.
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RESULTS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Particle-induced Lung Injury: A Comparative Study of Four Models
Biochemical and cellular markers of inflammation. The alveolitis response was monitored by measuring LDH activity (Figure 1) and total protein content (Figure 2) in BALF. The inflammatory process induced by DQ12 and MnO2 in the lung was characterized by a rapid and dose-dependent increase of BALF LDH activity and total protein content reaching maximal levels at Days 3 and 5 in the fibrotic and resolving alveolitis model, respectively. BALF protein content and LDH activity progressively returned to control value in the resolving alveolitis model but, up to Day 120, were maintained higher than in controls upon silica treatment. The instillation of TiO2 also induced a dose-dependent increase in LDH activity and total protein content which peaked at Day 3 and remained higher than in controls up to Day 60. Results obtained with WC were in good agreement with the response expected with an "inert particle" because no significant alteration of the biochemical parameters was observed up to Day 60.
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0.05).
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The inflammatory response in the different models was also characterized by an increase in the total cell number and by the recruitment of neutrophils. These changes were monitored until Day 30 and are summarized in Table 2. Cell accumulation resulting from silica and MnO2 treatment were shown dose-dependent with maximal cell number at Days 3 and 1 in the fibrotic and resolving alveolitis models, respectively. This increase in the total cell number was accompanied by a dose-dependent recruitment of neutrophils within the alveoli. Maximal neutrophil recruitment also occurred at Day 3 or 1 representing 50 and 89% of the total cell population in the fibrotic and resolving alveolitis model, respectively. Although the acute phase of inflammation was subsiding after 5 d upon silica and MnO2 treatment, the total cell number in both groups was still higher than in controls after 1 mo. A different pattern occurred after TiO2 treatment which elicited neutrophil recruitment without a concurrent increase in total cell number. Maximal increase in neutrophil population was observed at Day 1 after TiO2 and, as observed after silica treatment, the persistence of neutrophils was noted until Day 30. As for biochemical parameters, WC particles did not induce any significant change in the cellular composition of the BAL cell fraction.
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Lung fibrotic response. The fibrotic response was assessed by microscopic examination 1, 2, and 4 mo after treatment. Figure 3 illustrates lung histologic features observed after 4 mo in particle-treated mice. The morphologic lesions elicited by silica were dose- and time-dependent. One month (5 mg) or 2 mo (2.5 mg) after silica treatment, focal granuloma developed particularly in the bronchiolar regions. With time, these granuloma became progressively fibrous and increased in size (Figure 3A). The presence of collagen within these nodular structures was evidenced by a positive staining with Masson's trichrome (not shown). A dose of 0.5 mg of silica elicited only a weak alveolitis without fibrotic nodules even after 4 mo (not shown). Contrary to the fibrotic model, the instillation of MnO2 elicited a resolving alveolitis only associated with some degree of atelectasia after 4 mo (Figure 3B). The instillation of TiO2 (Figure 3C) or WC (Figure 3D) did not induce any change in the parenchymal lung structure up to 4 mo after exposure.
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Changes in plasminogen activator activity. Changes in
BALF PA activity have been investigated over a period of 120 and 30 d for BALF (Figure 4) and cell-associated activity (Figure 5). The administration of silica, MnO2, and TiO2 induced
dose-dependent increases in BALF PA activity which peaked
at Day 3 except in the low toxicity model which showed only a
slight but constant increase in PA activity up to Day 30. The
intensity of the acute BALF PA response ranked as follows:
fibrotic model 
resolving alveolitis model > low toxicity
model. Contrary to the resolving alveolitis and the low toxicity
models for which soluble PA activity returned to control value
after 60 d, a persistent increase of BALF PA activity (up to
Day 120) was noted in the fibrotic model. The administration
of WC did not induce any significant change in soluble PA activity. In order to obtain a reflection of the total PA cellular
PA activity in the lung, the total proteolytic activity associated
with the BAL cell fraction was calculated as previously described by Brown and Donaldson (20) by multiplying the PA
activity per cell by the total number of leukocytes recovered in
BAL. Concurrently to the increased soluble PA activity, a rapid dose-dependent and persisting upregulation of total cell-associated PA activity was noted in the three models of lung inflammatory reaction. The amplitude of total cell-associated
PA changes was similar in the resolving alveolitis and low toxicity models but higher at a similar dose (2.5 mg) in the fibrotic
model. Similar changes in the expression of PA were observed
in the three inflammatory models when cellular PA activity
was expressed per cell (specific PA activity). For example, PA
activity per cell (IU/cell × 1017) was 4 ± 1 (Day 3) and 2.4 ± 0.32 (Day 30) after 5 mg of silica (p < 0.05) and 1.19 ± 0.23 (Day 3) and 0.51 ± 0.13 (Day 30) after 2.5 mg of MnO2 (p < 0.05). The administration of WC did not induce any significant
change either in total or in specific cell-associated PA activity.
PA activity per cell (IU/cell × 1017) was 0.3 ± 0.006 (Day 3)
and 0.14 ± 0.04 (Day 30) after administration of 2.5 mg of
WC. Measurements of cell-associated PA activity were purposely limited to the first 30 d after treatment in order to avoid possible interferences of the fibrotic process that might affect the recovery of the relevant inflammatory cells which, at
this stage, are mainly entrapped in inflammatory granuloma.
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Changes in PA-related proteins. PA-related proteins (uPA, tPA, PAI-1) have been investigated in unconcentrated BALF over a period of 120 d. Whatever the dose or time point considered, uPA, tPA, and PAI-1 antigens were never detected in BALF either in the control group or in the low toxicity and noninflammatory models. Significant changes in BALF PA- related antigens were only observed during the acute phase of the fibrotic and resolving models (up to Day 5) (Figure 6). The PA protein was of the uPA type and the tPA antigen was never detected. We observed a dose-dependent increase in uPA concentration with a maximum at Days 3 and 5 in the fibrotic and the resolving alveolitis models, respectively. A concomitant dose-dependent increase of PAI-1 antigen was also found. In both models, maximal PAI-1 concentrations were detected at Day 1. No PAI-1 was detected after instillation of 0.75 mg of silica. Overall, while almost similar PA activities were noted in both models (Figure 4), the amplitude of uPA and PAI-1 protein changes was greater in the resolving alveolitis than in the fibrotic model.
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Silica-induced Lung Injury in uPA Knockout Mice
Acute alveolitis response. The aveolitis response to DQ12 was
assessed by measuring biochemical and cellular markers of inflammation (Figure 7). The pulmonary inflammatory process
observed in uPA (
/) and uPA (+/+) mice had a similar
pattern of macrophage accumulation (Figure 7E) and protein
change (Figure 7B). Differences were however observed in
LDH activity (Figure 7A) and cell recruitment pattern after
treatment with DQ12. Total cell number (Figure 7C) and neutrophil accumulation (Figure 7D) were significantly higher at
Days 1 and 3 and significantly lower at Day 30 in uPA knockout than in wild-type mice. BALF LDH activity was significantly lower at Days 3, 5, and 30 in (/) than in (+/+) mice.
No significant biochemical or cellular difference was observed
between the two control groups. One month after silica treatment, significantly (p < 0.01) increased BALF PA activity was
observed in uPA (+/+) (6.5 ± 0.22 versus 0.12 ± 0.05 nIU/ml
in saline controls) whereas, as expected, uPA (/) mice
showed no change in BALF PA activity (0.31 ± 0.08 versus 0.27 ± 0.08 nIU/ml in saline controls).
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Fibrotic response of lung tissue. One month after treatment
with silica, the lung tissue of (/) and (+/+) mice had qualitatively similar histologic features characterized by the presence of granuloma. However, the profusion of these lesions
was apparently greater in uPA/ than in uPA+/+ mice (Figure
8). No prominent histologic changes were observed in both
(/) and (+/+) control groups (not shown).
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At the same time point (Figure 9), we observed a significant increase in OH-proline in both (/) and (+/+) mice in
response to silica treatment. The amplitude of collagen accumulation was however significantly greater in mice carrying
the uPA deficiency than in their wild-type congeners. Similar
OH-proline contents were noted in both (/) and (+/+)
control groups.
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Characterization of the Models
The first objective of this study was to develop experimental
models to differentiate the time course of the PA response occurring during an alveolitis progressing to fibrosis and during
spontaneously resolving alveolitis. Our purpose was therefore
not to compare the intrinsic capacity of the different particles
to induce changes in the PA system but to use them as models
to investigate PA changes associated with varying biologic responses of lung tissue. After a single instillation (up to 5 mg/
mouse), the response to crystalline silica, the reference fibrogenic particle, was consistent with other studies (21). The effect of silica was characterized by a persistent toxic and inflammatory response with increased LDH activity, alveolar
accumulation of proteins, and polymorphonuclear leukocyte
(PMN) recruitment leading to histologic fibrosis after 1 mo.
After administration of 2.5 mg of MnO2, we elicited an acute alveolitis of an almost similar amplitude as after silica treatment (maximum at Days 5 and 3, respectively). Upon MnO2
treatment, however, the alveolitis was spontaneously resolving after about 1 mo. In order to take into account the possible
influence of the aspecific response upon administration of particles, independently of the presence of an alveolitis, we also
examined the PA response after instillation of an inert particle. As previously described in the rat (28), a single dose of
WC (2.5 mg/mouse) was found suitable to develop this type of
noninflammatory model in which no alterations in the markers of inflammation and no long-term adverse effect were observed. In a first attempt to develop this model, we also examined the lung response to TiO2, a particle previously reported
as nonpathogenic up to 50 mg/kg in a rat model (29). In our
mouse model, a single dose of TiO2 (2.5 mg/mouse) produced
a weak but persistent toxic and inflammatory reaction as evidenced by increased LDH activity and alveolar accumulation of proteins, respectively. An inflammatory response associated to TiO2 exposure was previously demonstrated in the rat in an overloading situation (30) and in the presence of a high percentage of ultrafine particles (31). The response induced by
TiO2 in our mouse model could not be explained by the percentage (> 20%) of ultrafine particles present in the sample
used, because a persistent and similar alveolitis was obtained
with another batch of TiO2 (median particle size [d50]: 0.46 µm,
specific surface area: 7.4 m2g1) which contained only 6.5% of
ultrafine material (2.5 mg/mouse; Day 30; LDH: 68 ± 9 IU/L;
total protein: 192 ± 27 µg/ml). As previously observed in a rat
model (30), an overloading effect (32) may therefore remain
as a plausible explanation of the observed effect of TiO2 in our
mouse model.
Is the PA Response Varying with the Type of Alveolitis?
The relationship between the PA system and the long-term fibrotic process is unclear. Various fibrotic diseases are associated with divergent changes in the PA system. Strong reductions of soluble BALF PA activity were found in adult respiratory distress syndrome, idiopathic pulmonary fibrosis, and bleomycin-induced lung fibrosis (16, 17, 33). Early-stage sarcoidosis was associated with increased BALF PA activity (34) whereas reduced activity was observed at later stages of the disease (18). Instillation of crystalline silica was reported to induce an acute elevation of BALF PA activity in mice (21) and in rats (22). Acute inflammation associated with human and experimental sheep asbestosis was associated with high levels of BALF and cell-associated PA activity while chronic and fibrotic disease was characterized by reduced activity (19). Previous studies conducted with inorganic particles have also reported an upregulation of alveolar fibrinolysis (19) whereas reduced BALF PA activity has been found in response to bleomycin and oleic acid (33). This apparent disparity may reflect a selective tissue response to particulate material.
In an attempt to identify PA changes specifically associated with the progression to interstitial fibrosis, we compared the time course of both soluble and cell-associated components of the PA system in the four different models. An early increase of soluble PA activity was noted in the three inflammatory models but persistently increased PA activity (> 30 d) was only observed in the fibrotic model. No significant change in PA activity was detected in the noninflammatory model indicating that PA alterations observed in three other models are associated with alveolitis. The similitude in BALF PA activity observed at the early stage of resolving and fibrotic alveolitis indicates that the persistence rather than the amplitude of the change in soluble PA activity is associated with the development of lung fibrosis. The possible contribution of PA activity in the fibrotic process remained, however, to be demonstrated.
The observed changes in BALF PA activity were found to be exclusively related to changes in uPA. The similar changes in BALF PA activity observed in the resolving and fibrosing alveolitis models were however associated with different levels of uPA antigen, with a higher expression in the resolving than in the fibrotic model. The observed PA activity in the former was therefore lower than would be expected from the antigen levels. This reduction in activity might probably be explained by the formation of PAI-1/UPA complexes. Originating from in situ production or from vascular leakage, soluble PAI-1 was indeed found at a higher concentration in BALF from the resolving alveolitis model. Also, as mentioned previously, the finding of reduced PA activity in experimental models of lung injury produced by other agents than mineral particles (bleomycin, oleic acid) may conceivably be caused by a PAI-1 upregulation that might be of greater amplitude than upon administration of particulate materials.
Concurrently with the increase in soluble PA activity, a rapid and persisting upregulation of total leukocyte-associated PA activity was also noted in the fibrotic model. This upregulation was specifically associated with the inflammatory process since the inert dust was shown without effect. The temporal pattern of the PA upregulation in total inflammatory cells was similar in both the resolving and the fibrosing alveolitis. When comparing the effect of 5 mg of silica and 2.5 mg of MnO2, two situations that produced an inflammatory reaction of an equivalent amplitude (Figures 1, 2, and Table 2), we found that during the first week approximately 10 times more total cellular PA activity was expressed in the fibrotic model. Changes in total leuckocyte-associated PA noted in the fibrotic and resolving models were not simply the consequence of the increased number of cells accumulating in the alveoli, but mainly reflected an increased expression of PA activity per cell, which may be due to upregulation of PA expression or enhancing binding capacity at the membrane level. Changes in cellular PA activity may conceivably be modulated by cell cytotoxicity, negative or positive feedback on the production of uPA and/or uPAR, and interaction with PAI-1 or PAI-2 which promote internalization of membrane-bound uPA. More abundantly expressed in the resolving than in the fibrotic model, the soluble and fast-acting PAI-1 may conceivably also favor a reduction of leukocyte PA activity in the resolving model. Changes in PAI-2, a second inhibitor, could not be investigated in this study because no ELISA was available for the murine protein. However, because PAI-2 is mainly distributed in the intracellular compartment of macrophages its detection in BALF might therefore be compromised and of questionable relevance.
Measurements of cell-associated PA activity were purposely limited to the first 30 d after treatment in order to avoid possible interferences of the fibrotic process that might affect the recovery of the relevant inflammatory cells which, at this stage, are mainly entrapped in inflammatory granuloma. One month after treatment, we observed a persistent and significant upregulation of the total cellular PA activity in both the fibrotic, the resolving, and the low toxicity models which was mainly due to persistent upregulation of PA activity at the surface of inflammatory cells. Once again, the amplitude of the upregulation process was, at this stage, maintained twice greater in the fibrotic than in the resolving alveolitis model. All together, these findings therefore indicate that uPA upregulation is mainly localized in the soluble BAL fraction in the resolving alveolitis model whereas both the cellular and the soluble BAL fraction are increased in the fibrotic alveolitis model. It suggests that the amplitude of PA upregulation in the inflammatory cells could be an indicator of the progression to fibrosis.
Is uPA Involved in the Fibrotic Process?
In view of its multifunctional properties, uPA could exert opposite effects at several stages of the inflammatory and/or fibrotic processes. First, successful repair of damaged alveoli
requires the clearance of plasma exudates, restoration of damaged ECM, and replacement of injured alveolar cells. The implication of the PA system in this clearance process was demonstrated in a combined uPA and tPA knockout mice model
which exhibits spontaneous fibrin deposition in lung tissue
(23). Of possible importance to this repair process is the
BALF PA activity which may limit fibrosis by dismantling fibrin and procollagen scaffolds. The simultaneous presence of
uPA and plasminogen on the macrophage (3, 5) and PMN
(35) cell surface results in the assembly of a highly efficient
system of pericellular proteolysis which may also be operative
in ECM remodeling processes (5). Secondly, at the attempted
repair stage, uPA might exert potential profibrotic effects by
promoting either macrophage adhesiveness, fibroblast proliferation, or growth factors activation. Independently of the proteolytic property of the ligand, uPA/uPAR interaction modulates cellular adhesiveness to several matrix components inclu-
ding vitronectin largely expressed in lung interstitial fibrosis
(36). Recent studies showed that uPAR itself is a high-affinity
adhesion receptor for vitronectin and that uPA is an activator
of this adhesive function (37). In monocytic cells, this adhesion process requires constant occupancy of uPAR and is modulated by uPA/PAI-1 turnover (38). An exacerbation of the
inflammatory cell adhesiveness could therefore promote monocyte/macrophage accumulation and conceivably favor the formation of fibrotic granulomas. The proliferation of lung fibroblasts is also a decisive event in the pathogenic process leading
to pulmonary fibrosis. Persistent increase of soluble uPA might
have considerable relevance to this event because uPA was revealed mitogenic for human skin (9) and lung fibroblasts (10).
Moreover, fibroblasts originating from fibrotic lung showed increased uPAR expression and uPA production and the degree
of uPAR occupancy was shown to correlate with the proliferation rate of these fibrotic fibroblasts (10). Therefore, persistent excess of soluble BALF uPA at the vicinity of interstitial
fibroblasts, as described in the fibrotic model, might contribute to modulate their proliferative activity. Another uPA/
uPAR proteolytic effect potentially relevant for the fibrotic
process relates to the activation of macrophage-associated
growth factors as demonstrated for the latent form of TGF-
(6). Several lines of evidence point to TGF- as a key cytokine
for the control of tissue repair and whose sustained production and/or activation underlies the development of lung fibrosis. With regard to TGF- activation, higher and persisting
cellular PA activity as observed in the fibrotic model might be
related to a higher growth factor activation level.
In order to further clarify the role of uPA expression in the
pathogenic process, the inflammatory and fibrotic responses induced by silica were investigated in uPA knockout mice. Reduced silica-induced cytotoxicity observed in uPA/ mice suggests however that uPA may to some extent mediate cell damage. We found that the amplitude and temporal sequence of
protein and macrophage accumulation induced by silica treatment was not affected by uPA deficiency. Although plasmin
activation has been implicated in the process of inflammatory
cell migration, uPA deficiency did not prevent silica-induced
neutrophil accumulation. Alternative proteolytic mechanisms
may therefore be activated to allow inflammatory cells to infiltrate the lung. Moreover, PMN recruitment was apparently
exacerbated in uPA-deficient mice treated with silica, which
may suggest the involvement of an uncontrolled alternative
pathway in the absence of uPA. Whatever the recruitment pathway that takes place in uPA-deficient mice, the higher
neutrophil accumulation may indicate a possible anti-inflammatory role for uPA during the acute phase of the pathogenic
process. Most importantly, the intensity of the fibrotic reaction was more marked in uPA-deficient mice. This acceleration of the fibrotic process indicates therefore a contribution
of uPA to limit fibrogenesis. In agreement with our result, a
previous study demonstrated that instillation of recombinant
human uPA caused partial reversal of bleomycin-established pulmonary fibrosis (15). The observation that bleomycin produced less fibrosis in PAI-1 knockout mice and more extensive fibrosis in PAI-1 overexpressing mice (14) also supports
this hypothesis. One month after silica treatment, inflammatory cells that accumulated into lung airspace were less numerous in uPA-deficient mice. At this stage, the reduction in
inflammatory cell number within the alveoli of uPA-deficient
mice was consistent with the concurrently higher expansion of
the inflammatory granuloma which may limit BAL cell recovery. All together, these findings indicate that among the possible biologic roles of uPA noted earlier, its antifibrotic activity,
conceivably mediated through the degradation of the fibrin
and procollagen scaffold, is the most relevant in the pathogenic process induced by silica. One month after silica treatment, increased BALF PA activity was observed in uPA+/+
mice whereas uPA/ mice showed no significant change. Together with NMRI mice results, this latter observation in uPA/
mice excludes the possibility that persistent PA activity noted after silica treatment is due to another factor than uPA.
In conclusion, our results indicate that PA can modulate not only to the inflammatory process but also to the tissue repair process. We found differential changes in alveolar PA system during the course of resolving inflammation and fibrosing alveolitis following particle-induced lung injury. A higher level of inflammatory cell PA as well as a sustained BALF PA activity were associated with the progression of fibrosis. However, due to the multifunctional role of uPA, local PA regulation has complex and possibly conflicting effects at several stages of the fibrotic process. Our findings in the uPA-deficient model indicate that uPA contributes to limit fibrogenesis, conceivably through the degradation of fibrin deposits formed during the alveolitis. This result is consistent with previous observations that defective PA activity has been found within the alveolar compartment in various human interstitial lung disorders (16).
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Footnotes |
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Supported by Grant EV5V-CT94-0399 from the European Community.
Correspondence and requests for reprints should be addressed to C. Lardot, Industrial Toxicology and Occupational Medicine Unit, Clos Chapelle-aux-Champs Box 30.54, 1200 Brussels, Belgium. E-mail: Lardot{at}toxi.ucl.ac.be
(Received in original form July 10, 1997 and in revised form September 9, 1997).
Acknowledgments: Mr. P. Rousseau from Sedema-Division of Sadecem S.A. (Belgium) is gratefully acknowledged for providing us with battery-grade manganese dioxide powder.
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References |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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