1 Industrial Toxicology and
Occupational Medicine Unit, Impaired
fibrinolytic activity and persistent fibrin deposits in lung tissue
have been associated with lung fibrotic disorders. The present study
examined the sources of plaminogen activator (PA) changes induced by a
single intratracheal administration of silica particles (5 mg) in the
mouse lung. We found in both control and silica-treated animals that
amiloride almost totally abolished PA activity in bronchoalveolar
lavage (BAL) fluid (BALF), indicating that initial upregulation (from
day 1) as well as sustained PA
activity (up to day 30) observed in
response to silica is related to changes in urokinase-type PA (uPA).
The upregulation of BALF uPA activity was associated with a marked and
persistent increase in uPA mRNA levels in lung tissue. Changes in uPA
expression were also reflected in the BAL cell fraction. A maximal and
constant increase in cell uPA activity was associated with the early
response to silica, whereas significant but lower upregulation was
still noted at the fibrotic stage. From days
3 to 30, a progressive increase in uPA mRNA levels was noted in BAL inflammatory cells elicited by silica. Because the number of BAL neutrophils was strongly
correlated with BALF and BAL cell-associated uPA activity, their
involvement in uPA upregulation was addressed by inducing neutropenia
with cyclophosphamide (200 mg/kg ip) before administration of the
silica. Neutrophilic depletion did not, however, reduce, and even
increased, the BAL cell-associated uPA activity. At the BALF level,
neutropenia did not change PA activity in silica-treated mice, pointing
to alveolar macrophages as the principal source of uPA in response to
silica. Immunohistochemical stainings identified alveolar macrophages
and pneumocytes as uPA-expressing cells in silica-treated animals
(day 30). Intense and heterogenous
staining was observed in silicotic nodules. These findings indicate
that urokinase produced by alveolar macrophages is operative not only at the alveolitis stage but also later in the fibrotic process, produced by silica particles, supporting the role of uPA in
fibrogenesis.
mouse; fibrosis; neutrophil depletion; immunohistochemistry
ALVEOLAR FIBRIN DEPOSITS are often prominent in acute
lung injury, whereas chronic pulmonary immune reactions and granulomas are likewise richly infiltrated by a fibrin stroma. Local fibrin accumulation in the lung, which serves initially to limit lesional hemorrhage, may, however, also amplify inflammation and interfere with
surfactant function (32). At a later stage, the persistence of fibrin
scaffolds may stimulate fibroblast ingrowth, leading subsequently to
the development of interstitial fibrosis.
Fibrin removal is initiated by the potent serine proteinase plasmin,
the activation of which is under the control of two other trypsin-like
proteinases, namely, the tissue-type (tPA) and urokinase-type (uPA)
plasminogen activators (PAs) (36). Plasmin not only solubilizes fibrin
but may also degrade most of the basement membrane components altering
cell-cell and cell-matrix contacts, thereby allowing cell trafficking
through inflammatory foci. Furthermore, plasmin has the capacity to
activate other proteolytic enzymes such as interstitial elastase and
collagenase (27), which contribute to exacerbate tissue injury.
Finally, plasmin plays a role in the generation of proinflammatory and
profibrotic signals by promoting the activation of important cytokines
such as interleukin-1 (23) and transforming growth factor- Silica-induced lung injury is an established murine model of pulmonary
fibrosis. Numerous studies pointed out the crucial role of
proinflammatory mediators (28), anti-inflammatory cytokines (13), and
growth factors (39) in the development of silicosis. Yet, relatively
little is known about the regulation and lung source of uPA during the
course of lung injury induced by silica. In view of its multifunctional
properties, uPA could exert opposite effects at several stages of the
inflammatory and/or fibrotic processes. It may limit fibrosis
by dismantling fibrin and procollagen scaffolds but, associated with
its surface receptor, uPA may also promote in vitro adhesiveness and
migration of monocytes (10) as well as fibroblast proliferation (33).
We have previously shown that on silica administration
(0.75-5 mg/mouse, intratracheal administration), uPA was
upregulated in the lung and that uPA contributed to limit the
fibrogenesis induced by silica particles (17). No change in PA activity
was observed in animals treated with tungsten carbide, an inert
particle (17). In this study, we have investigated possible sources of
uPA changes induced by silica in the mouse lung. The distribution of
the enzyme in the lung was examined by immunohistochemistry, and the
expression of uPA at the mRNA level was quantified in bronchoalveolar
lavage (BAL) cells and lung tissue. The contribution of inflammatory neutrophils in uPA activity was investigated by the induction of
systemic neutropenia after cyclophosphamide pretreatment.
Reagents. Highly toxic crystalline
silica particles (DQ12; median
particle size 2.2 µm) were a kind gift from Dr. L. Armbruster (Essen,
Germany). Polyclonal rabbit IgG against mouse uPA (a-MP48) was a
generous gift from Dr. J. Rømer (Copenhagen,
Denmark). Polyclonal rabbit IgG against mouse glial
fibrillary acidic protein (GFAP) was purchased from Dako (Copenhagen,
Denmark). Anti-rabbit IgG-biotin conjugate
[F(ab')2
fragment], streptavidin-peroxidase conjugate, and fibrinogen
fragments were from Boehringer (Mannheim, Germany). Diaminobenzidine
substrate was from Merck (Belgolabo, Belgium). Human plasminogen and
S2251 (Val-Leu-Lys-p-nitroanilide
plasmin substrate) were purchased from Chromogenix (Mölndal,
Sweden). Tris, glycine, BSA, and lipopolysaccharide (LPS) were from
Sigma (St. Louis, MO). PBS, DMEM, and lactalbumin hydrolysate were from GIBCO BRL (Paisley, UK). TRIzol
reagent and SuperScript I RNAse H Animals. Adult female NMRI mice (8 wk)
purchased from Iffa Credo (Brussels, Belgium) were housed in
positive-pressure air-conditioned units (22°C, relative humidity 50 ± 10%) with a 12:12-h light-dark cycle and fed on a conventional
laboratory diet with free access to tap water.
In vivo treatments. The animals were
anesthetized by an intraperitoneal injection of pentobarbital sodium
(80 mg/kg body weight). The trachea was exposed with sterile technique,
and a single dose of 5 mg of DQ12
suspended in 100 µl of a sterile saline solution was slowly instilled
in the trachea lumen. Control animals were treated with an equivalent
volume of saline.
In a second group of experiments,
DQ12 was administered to mice
depleted in neutrophils. In vivo depletion of neutrophils was obtained,
as previously described (1), by a single intraperitoneal injection of
cyclophosphamide (200 mg/kg suspended in 100 µl of sterile saline) 3 days before the administration of
DQ12. Cyclophosphamide control
groups were treated with an equivalent volume of sterile saline
administered intraperitoneally. Twenty-four hours after the
intratracheal administration of 5 mg of
DQ12 or sterile saline, the extent
of leukocyte depletion was assessed in the BAL cell fraction by
differential cell counting.
In a couple of mice, LPS diluted in sterile saline was injected
intravenously at a dose of 100 µg/mice. The lung tissue from LPS-stimulated mice was recovered 2 h after treatment and used as a
positive control for uPA mRNA expression.
BAL. At selected time intervals after
DQ12 treatment, the animals were
killed by an overdose of pentobarbital sodium, and the lung tissue and
alveolar macrophages were harvested. Alveolar macrophages were
recovered by BAL with sterile saline as previously described (13). The
BAL fluid (BALF) was centrifuged (1,200 g for 10 min at 4°C), and the
cell-free supernatant from the first lavage fraction was used for
biochemical measurements of PA activity. The cell pellet was used for
cell-associated PA activity measurements, mRNA extraction, and
differential cell counts.
In vitro exposure. Resident alveolar
macrophages, recovered by BAL from naive mice, were exposed in vitro (5 × 104 cells/well) to
increasing noncytotoxic concentrations of
DQ12 (0.5, 0.75, 1, and 1.5 µg/well) for a period of 24 h. The culture was performed in 96-well
plates in DMEM supplemented with 0.1% lactalbumin hydrolysate and 1%
antibiotics. At the end of the incubation period, the supernatants were
recovered, and the macrophages were thoroughly rinsed with PBS-0.5%
BSA buffer (pH 7.4). PA activity in the culture supernatants and cell
fractions was assessed as described in Enzymatic assay
for PAs. Each PA determination was done in
triplicate in two separate cultures.
Enzymatic assay for PAs. A chromogenic
assay was used to quantify soluble PA activity in BALF samples and
culture supernatants and to measure cell-associated PA activity in the
BAL cell fractions and macrophage cultures. PA activity was measured
without discrimination between tPA and uPA. Briefly, BALF samples (50 µl) and culture supernatants (50 µl) were incubated in 0.1 M
Tris-0.1 M glycine-0.5% BSA buffer (pH 8.5). The BAL cell suspension
(5 × 104 cells/50 µl) and
macrophage culture were incubated in PBS-0.5% BSA buffer (pH 7.4).
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 at 37°C
in triplicate in 96-well plates. Negative controls in which plasminogen
was omitted were assayed simultaneously for each sample. The formation
of p-nitroaniline from the hydrolysis of the S2251 substrate was followed spectrophotometrically at 405 nm
(Twinreader Titertek, Techgen International) 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
was calculated. The determination of PA units present in the sample was
calculated as described by Schnyder et al. (31), and the results are
expressed in international units (µmol of substrate hydrolyzed/min).
To identify the nature of the BALF PA activity, measurements were also
carried out in the presence of amiloride, a specific inhibitor of the
uPA form (35). In that case, amiloride (1 mM final concentration) was added to the samples 15 min before the addition of plasminogen and the
S2251 substrate.
PCR detection of uPA mRNA. Total RNA
was purified from BAL cells (1.2-2 × 106 cells) obtained from four to
five mice and from lung tissue according to the procedure supplied with
the TRIzol reagent. Single-stranded cDNA was synthesized from 2.5 µg
of the RNA preparation with random hexamers and Moloney murine leukemia
virus reverse transcriptase in a final volume of 50 µl as directed by
the manufacturer. The reverse-transcribed product (2.25 µl) diluted 10-fold was used in the PCR reaction.
Amplification was performed with 10 mM Tris · HCl
buffer (pH 8.3) with 1.5 mM MgCl2,
0.2 mM each deoxynucleoside triphosphate, and 5 pmol of each 5'-
and 3'-specific primer in a final volume of 25 µl. The specific
primers for uPA were sense 5'-GTGGAGAACCAGCCCTGGT-3' and antisense
5'-GGCAGGCAGATGGTCTGTAT-3'; those for Immunohistochemical detection of uPA.
We used the procedure previously described (18) with the following
slight modifications. The lungs were fixed in situ via instillation of
0.01 M phosphate-buffered (pH 7.4) 4% paraformaldehyde, removed, and
immersed for 30 min in the same fixative. After fixation, the lungs
were rinsed twice (10 min) in 0.1 M phosphate buffer (pH 7.4) and
immersed overnight in 0.1 M sodium phosphate buffer, pH 7.3, containing
20% sucrose. The lungs were embedded in Tissue Tek and frozen in
2-methylbutane cooled in liquid nitrogen. Cryostat sections (4 µm)
were mounted on Superfrost Plus slides. Cryostat sections were soaked
in 0.05 M Tris · HCl (pH 7.4) with 0.5 M NaCl
[Tris-buffered saline (TBS)] and 0.5% Triton X-100 (TBS-T)
for 10 min, exposed to 10% NGS in TBS for 30 min at room temperature,
briefly rinsed in TBS-T, and then exposed overnight at 4°C to the
polyclonal rabbit IgG anti-mouse uPA at a concentration of 5 µg/ml in
TBS-1% NGS. Endogenous peroxidase activity was quenched by exposure to
hydrogen peroxide (0.3% in nanopure water) for 30 min followed by
three washes of 5 min each in TBS-T. Adjacent sections not exposed to
the hydrogen peroxide reagent were analyzed in parallel. The site of
antigen-antibody reaction was revealed with the
biotin-streptavidin-peroxidase method with an anti-rabbit IgG-biotin
conjugate and diaminobenzidine as the substrate. The sections were
counterstained with hematoxylin. Control sections included omission of
the primary antibody and substitution of the primary antibody by a
rabbit polyclonal IgG against mouse GFAP (0.5 µg/ml), specific for
glial fibrils. The specificity of the anti-mouse uPA antibody used has
been thoroughly characterized in previous work (18).
Other analyses. Lactate dehydrogenase
(LDH) activity was assessed by monitoring the reduction of
NAD+ at 340 nm in the presence of
lactate. Total protein content was assessed spectrophotometrically with
a commercial kit (Systemes Technicon, Doumon, France). Differential
cell counts were performed on cytocentrifuge preparations stained with
Diff Quick (Dade, Brussels, Belgium).
Statistics. All data are expressed as
means ± SE. The statistical significance of differences between
groups was assessed with ANOVA and the Student-Newman-Keuls or
Student's t-test. Correlations between PA activity and BAL cell data were computed by using the Spearman nonparametric test. Statistical significance was defined as
P Lung PA activity in response to
silica. BALF and cell-associated PA activities were
measured at different time intervals after a single administration of
silica. Soluble PA activity was abundantly detected in BALF from both
control and silica-treated mice. However, in response to silica
treatment, we found a significant increase in BALF PA activity compared
with that of saline control animals (Fig.
1A).
Maximum at day 3, increased BALF PA
activity was maintained higher than in control animals up to
day 30. In the presence of amiloride,
the BALF PA activity in control and silica-treated mice was almost
completely abolished (85-90%) at all time points examined (Fig.
1B). The amiloride-dependent
inhibition of uPA activity was, however, statistically significant in
the silica-treated group only. Although the BAL cell fraction from the
control mice was almost exclusively composed of alveolar macrophages
(>97%), silica treatment induced a strong recruitment of neutrophils
that represented 77 ± 5, 50 ± 5, 26 ± 3, and 13 ± 2%
after 1, 3, 5, and 30 days, respectively. No other cell type was
identified in BAL inflammatory cells elicited by silica treatment.
Silica-induced BALF PA activity was found to correlate with the total
number of BAL cells (r = 0.64;
P < 0.0001) and the number of
polymorphonuclear neutrophils (r = 0.72; P < 0.0001). No significant
association was found with the number of alveolar macrophages
(r = 0.19;
P < 0.08). The PA activity
associated with the BAL cell fraction was investigated up to
day 30. Cell-associated PA activity in silica-elicited leukocytes was significantly greater than that in
control cells (Fig. 2). PA activity
remained maximal and constant during the early phase after silica
treatment (up to day 5). On day 30, PA activity associated with
the BAL cell fraction of silica-treated mice was still eightfold higher
than that in control cells. Cell-associated PA activity in treated
animals was found to correlate with the number of polymorphonuclear
neutrophils (r = 0.81;
P < 0.0001) and to a lesser extent
with the number of macrophages (r = 0.41; P < 0.02). A significant
association was also found with the total number of cells
(r = 0.72;
P < 0.001).
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
(15). Of
the two distinct PAs, uPA is arguably the most important PA that
promotes intra-alveolar fibrin removal. Like most other serine
proteases, uPA is secreted as an inactive single-chain zymogen
(pro-uPA) by several lung cell types including macrophages (37) and
pneumocytes (9). Pro-uPA is hydrolyzed by plasmin and possibly by mast
cell tryptase (34) to form the active two-chain enzyme. uPA is also
spatially confined by the topography of its specific receptor (uPAR)
conferring a proteolytic activity to the epithelium (9) and macrophage (37) cell surface. In addition, uPA binds to extracellular matrix (ECM)
components, with a significant affinity for heparan sulfate proteoglycans (2) and vitronectin (25), suggesting that the ECM may
provide an alternative surface for the assembly and regulation of
plasminogen activation. In turn, the tissue level of active uPA is
regulated locally at multiple levels by a variety of factors including
PA inhibitors (PAIs; PAI-1 and PAI-2) (9, 37) as well as by surface and
soluble uPARs (24). Cell-surface uPA activity is considered to be
transient and cyclic as judged by the ability of uPAR itself to recycle
after turnover of uPA-PAI complexes (38). Local plasmin activity is
controlled by
2-antiplasmin and
2-macroglobulin (20).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Reverse Transciptase were
from GIBCO BRL (Gaithersburg, MD). Taq DNA polymerase and deoxynucleoside
triphosphates were from Perkin-Elmer (Branchburg, NJ). Triton X-100 and
paraformaldehyde were from Fluka (Buch, Switzerland), normal goat serum
(NGS) was from Pansystems (Aidenbach, Germany), and cyclophosphamide
was from Farmitalia (Brussels, Belgium). Tissue Tek was from Sakura Finetek (Zoeterwoude, Netherlands). Sucrose was from Aldrich
(Steinheim, Germany).
-actin were sense
5'-ATGGATGACGATATCGCTGC-3' and antisense 5'-GCTGGAAGGTGGACAGTGAG-3'. The reactions were carried out
in a DNA thermal cycler (Perkin-Elmer) with
Taq DNA polymerase. Each amplification
cycle consisted of denaturing at 94°C for 30 s, annealing primers
to target sequences at 63 (uPA) or 55°C (
-actin) for 30 s, and
primer extension at 72°C for 1.5 min. uPA and
-actin amplification was performed for 36 and 25 cycles, respectively, selected in the exponential phase of PCR. The PCR products [15 (uPA) or 25 (
-actin) µl] were resolved by 2% agarose gel
electrophoresis stained with ethidium bromide. Relative amounts of
specific mRNA were quantitated by densitometric two-dimensional
analysis (peak area) with image-analysis software (MCID Software,
Imaging Research, St. Catharines, Ontario, Canada) and referred to a
calibration curve. Each calibration curve, consisting of serial
dilutions of LPS-stimulated lung cDNA, was generated by plotting the
amount of amplified product as a function of the dilution of the
starting cDNA. To allow a reliable mRNA measurement, three PCR
amplifications per specific gene were performed for each cDNA sample.
Using this procedure, we obtained a variability coefficient of
5-10%. uPA mRNA levels were expressed as a ratio to
-actin
mRNA levels to correct for any variation in RNA content and/or
cDNA synthesis.
0.05.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
View larger version (17K):
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Fig. 1.
Time course of plasminogen activator (PA) activity in bronchoalveolar
lavage (BAL) fluid (BALF) in response to a single intratracheal
administration of crystalline silica particles (5 mg/mouse). PA
activity measurements were performed in absence
(A) and presence
(B) of amiloride (1 mM final
concentration), a specific inhibitor of urokinase-type PA activity.
Values are means ± SE from 4-5 mice. Intergroup comparisons
were assessed with ANOVA and Student-Newman-Keuls test. Significant
difference between saline control and silica groups:
* P 0.05;
** P
0.01. Significant
difference between PA activity with and without amiloride:
§ P
0.05;
§§ P
0.01.
View larger version (12K):
[in a new window]
Fig. 2.
Time course of BAL cell-associated PA activity in response to a single
intratracheal administration of crystalline silica particles (5 mg/mouse). Values are means ± SE from 5-15 mice.
* Significant difference compared with saline control mice,
P 0.01 by ANOVA and
Student-Newman-Keuls test.
Role of BAL neutrophils. The correlation between PA activity and neutrophil recruitment suggested a possible implication of these cells in the lung PA response induced by silica. The relative contribution of neutrophils to the changes in PA activity observed 24 h after silica administration was therefore investigated in mice pretreated with cyclophosphamide, an agent that causes systemic neutropenia. The results are shown in Fig. 3. Pretreatment with cyclophosphamide did not result in lung toxicity, altered vascular permeability, or inflammatory reaction as demonstrated by the comparison of both groups of saline-instilled mice (Fig. 3, A-D). Also, cyclophosphamide did not exacerbate lung toxicity or vascular permeability change induced by silica treatment as evidenced by the measurement of LDH activity (Fig. 3A) and protein content (Fig. 3B) in BALF. After silica treatment, the LDH activity in BALF of cyclophosphamide-pretreated mice was even significantly lower than in the respective control group. Pretreatment with cyclophosphamide significantly reduced the total number of BAL cells (Fig. 3C) and the percentage of neutrophils (Fig. 3D) after administration of silica.
|
Changes in BALF and BAL cell-associated PA activity are shown in Fig. 3, E and F, respectively. Twenty-four hours after saline instillation, similar levels in BALF and cell-associated PA activity were found in control and cyclophosphamide-pretreated mice. No effect of cyclophosphamide pretreatment was found in BALF PA activity measured in silica-treated animals. After silica administration, a significantly increased cell-associated PA activity was found in the cyclophosphamide-treated group compared with silica treatment-alone group.
PA activity after in vitro exposure to silica. Changes in soluble and cell-associated PA activity were investigated in resident alveolar macrophage cultures 24 h after exposure to increasing noncytotoxic concentrations of silica (up to 1.5 µg/well as judged by LDH release). Compared with control macrophages, silica-exposed macrophages did not express enhanced soluble or cell-associated PA activity (data not shown).
Induction of uPA mRNA by silica. To further delineate the regulation of uPA in response to silica, we performed RT-PCR analyses on lung tissue and BAL cells. Sequential measurements in lung tissue (Fig. 4A) demonstrated a rapid and marked upregulation of uPA mRNA levels after silica treatment. The greatest uPA mRNA upregulation was noted on day 3 after silica treatment, with a 17-fold increase compared with the control level. With time, the level of uPA mRNA upregulation after silica treatment decreased progressively but was still fivefold higher than in control levels on day 30. The uPA mRNA level was slightly higher in the control lung tissue on day 1 than at later time points.
|
uPA mRNA levels were also studied in BAL cells (Fig. 4B). After silica instillation, uPA mRNA levels showed a delayed (from day 3 on) and progressive increase, with maximal upregulation on day 30 showing a 12-fold increase compared with the control levels. Similar results were found in two separate determinations performed on days 3, 5, and 30.
Lung cellular source of uPA. In situ immunohistochemical stainings were applied on lung tissue 30 days after treatment. In control mice, the pro-uPA and/or uPA antigen was distributed in alveolar macrophages (Fig. 5F) and virtually all pneumocytes, producing a reticular staining pattern (data not shown). After silica treatment, an intense and heterogenous pro-uPA and/or uPA immunoreactivity was localized in granulomas (Fig. 5, A and D). Some stainings represented extracellular material, whereas others identified intracellular material (Fig. 5, D and E). Negative staining was found in lymphoid nodes of control and silica-treated mice (data not shown). No staining was found when the primary antibody was omitted (Fig. 5B) or after substitution by an unrelated hyperimmune serum (anti-GFAP; Fig. 5C).
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DISCUSSION |
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Inappropriate control of plasmin activity by altered expression of PAs or their inhibitors is of potential relevance in interstitial lung disease. Impaired fibrinolytic activity has been found in BALF of patients with adult respiratory distress syndrome (3), sarcoidosis (5), idiopathic pulmonary fibrosis (5), and asbestosis (4), all these conditions being characterized by a certain degree of pulmonary fibrosis.
Lardot et al. (17) recently demonstrated persistent upregulation of BALF PA activity associated with increased uPA but not tPA antigen levels in BALF in response to silica treatment in the mouse. Using the same experimental model, we found in the present study that amiloride almost totally abolished BALF PA activity, confirming that initial upregulation as well as sustained PA activity observed in response to silica is mainly related to changes in uPA expression. Furthermore, the early upregulation of BALF uPA activity was associated with a marked increase in uPA mRNA in lung tissue, indicating an intense activation of uPA expression rather than a simple secretion and/or activation of preexisting pro-uPA molecules from the intracellular compartment. The slight increase in uPA mRNA observed on day 1 in the control lung tissue is conceivably the reflection of a limited inflammatory reaction induced by the instillation of a saline solution. Although maximal BALF PA activity was measured on day 3, a sustained upregulation of uPA activity and mRNA level was still observed after 30 days, consistent with the possible involvement of uPA at the fibrotic stage. At this stage, immunohistochemistry demonstrated a preferential localization of uPA in the silicotic nodules, nodules that may reflect not only an increase in cell density but also an active uPA production as evidenced by extracellular uPA deposits. The antifibrotic activity of uPA in the pathogenic process induced by silica was demonstrated previously in uPA-deficient mice (17). The observations that instillation of uPA caused a partial reversal of bleomycin-established pulmonary fibrosis (12) and that PAI-1-deficient mice produced less fibrosis (6) also support the antifibrotic role of uPA. It remained, therefore, to identify the source and how silica induces an upregulation of uPA in the lung.
Changes in uPA expression were also reflected in measurements performed in BAL cells. The maximal increase in cell uPA activity was associated with the early response to silica, but significant upregulation was still noted at the fibrotic stage. The relative reduction in uPA activity in BAL cells on day 30 is consistent with immunohistochemical stainings that demonstrated a preferential localization of uPA in silicotic nodules. It is very likely that uPA-positive cells in fibrotic nodules were only poorly recovered by the BAL performed on day 30. One day after silica treatment, the increase in cell-associated PA activity was not associated to an upregulation of mRNA levels, suggesting an early translocation of the intracellular storage pool and/or the binding of extracellular BALF uPA protein, possibly synthesized by pneumocytes. From days 3 to 30, a progressive and marked increase in uPA mRNA levels was noted in BAL inflammatory cells, an observation that may reflect a transcriptional activation in response to silica and/or a progressive enrichment of the BAL cell population in uPA-synthesizing cells. However, this upregulation of uPA mRNA level was not accompanied by a parallel increase in cell uPA activity, which remained almost constant through the first week. One month after silica treatment, we found the highest level of uPA mRNA in BAL cells, whereas a relative reduction in uPA activity was noted. Absence of efficient protein biosynthesis, related to the instability of uPA transcript (26), or, alternatively, absence of suitable translocation to the cell surface are plausible explanations. However, these findings could also indicate that BAL inflammatory cells were maximally triggered as a consequence of cell-surface uPAR saturation. In that case, excess uPA protein could have been released in the extracellular environment, which significantly contributed to the observed increase in BALF uPA activity. In addition, cellular uPA activity may conceivably be modulated by the interaction with PAIs. Enhanced cell recycling of uPA-PAI complexes at the fibrotic stage is supported by the progressive reduction in BALF PAI-1 protein (17), the progressive reduction in BALF PAI activity [from 87 ± 3.1% of urokinase inhibitory activity on day 1 to undetected PAI activity on day 30 in silica-treated mice vs. a constant inhibitory activity (16.7 ± 7.5%) in control mice], and the sustained increase in PAI-2 mRNA levels in silica-elicited inflammatory cells (16). Whatever the mechanism involved, the relative reduction in PA activity at the fibrotic stage may, in limiting cell-surface uPA activity, impair the removal of fibrin and contribute to fibroblast ingrowth.
Because neutrophils have been found to translocate intracellular uPA to the plasma membrane uPAR in response to activation (29) and because the BAL neutrophil number was found to correlate with BALF and BAL cell-associated uPA activity, their involvement in uPA upregulation was examined by inducing systemic neutropenia through cyclophosphamide pretreatment. Although its primary side effect has been marrow suppression, cyclophosphamide was also reported to induce lung damage in mice (14). At the dose (200 mg/kg) and the time point considered in this study (72 h + 24 h post-silica treatment), cyclophosphamide did not, however, induce any pulmonary toxicity or change in vascular permeability in both control and silica-treated mice. Although cyclophosphamide caused a marked neutropenia and attenuated cellular infiltration induced by silica treatment, this intervention did not, however, reduce, and even increased, BAL cell-associated uPA activity. Because no significant change in cell-associated uPA activity could be found in control macrophages after the administration of cyclophosphamide, we can exclude a stimulatory effect of cyclophosphamide on the expression of uPA by these cells. At the BALF level, neutrophilic depletion did not alter PA activity. Therefore, an active and dominant contribution of neutrophils, directly as a uPA producer or indirectly through the production of priming mediators, could be formally excluded. The relationship between neutrophil influx and PA activity could thus represent an epiphenomenon, possibly explained by the chemotactic property of the fibrin degradation products (19) and by the ability of plasmin to activate chemoattractant cytokines (23).
Alveolar macrophage-derived PA is believed to play a central role in
ECM remodeling in some inflammatory disorders such as cryptogenic
fibrosing alveolitis, histiocytosis-X (30), and early asbestosis (4).
In vitro induction of macrophage uPA activity was also observed in
response to asbestos (21) and several inflammatory mediators including
colony-stimulating factor (11) and transforming growth factor- (7),
suggesting that PA activity is a marker of macrophage activation. In
the present silicosis model, the good correlation found between the
number of macrophages and the BAL cell uPA activity would support this view. The simple in vitro interaction of silica particles with alveolar
macrophages was not, however, sufficient to operate a significant
upregulation of uPA activity. The expression of either soluble or
cell-associated PA activity was not affected in resident alveolar
macrophages exposed to silica, indicating that additional cellular or
soluble mediators present at the site of inflammation are required for
efficient uPA activation in alveolar macrophages. Another possibility
would be that uPA expressed in an increased amount at the surface of
alveolar macrophages originates from other cell types such as
pneumocytes. However, this would not be consistent with the higher
degree of uPA mRNA upregulation in BAL inflammatory cells (12-fold on
day 30) than in whole lung tissue
(5-fold on day 30). Alternatively,
it seems more likely that increased uPA expression in the BAL cell
fraction after silica treatment reflects the recruitment of less
differentiated macrophages, which are known to express increased uPA
activity, from the circulation (37).
All together, these findings clearly point to the contribution of inflammatory macrophages as active uPA producers. However, the absence of correlation between BALF uPA activity and the number of macrophages suggests that other lung cell types are also implicated in local uPA production. As judged from the immunoreactivity study, some amount of uPA may originate from pneumocytes. In vitro, alveolar epithelial cells were indeed found to express increased uPA activity in response to asbestos (8) and several inflammatory mediators (22). The preferential localization of uPA within silicotic granulomas is consistent with a focalization of the enzyme at sites where inflammatory macrophages accumulate.
In conclusion, the results discussed above demonstrate the involvement of inflammatory alveolar macrophages in generating and focalizing active uPA and exclude the participation of neutrophils elicited at sites of lung inflammation by silica particles. In addition to alveolar macrophages, it is also evident that pneumocytes express uPA and could therefore contribute to regulation of fibrin metabolism in the bronchoalveolar space in response to silica. The early upregulation of BALF uPA activity observed during silica-induced injury was associated with largely increased uPA mRNA levels in lung tissue, suggesting an active uPA production rather than a simple release of preexisting molecules. Later in the disease process, we found at both the BALF and cell-surface levels a relative reduction in uPA activity compared with that found earlier in the disease process. This finding together with the antifibrotic activity of uPA previously demonstrated (6, 12, 17) is consistent with the implication of this enzyme in the persistence of fibrin and procollagen scaffolds, such as produced by crystalline silica, observed in lung fibrotic disorders.
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ACKNOWLEDGEMENTS |
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P. Thurion (University Hospital of Mont-Godinne, Mont-Godinne, Belgium) is greatly acknowledged for technical assistance in immunohistochemistry.
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FOOTNOTES |
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This work was supported by Grant EV5V-CT94-0399 from the European Community (DGXII-Environment).
Address for reprint requests: C. Lardot, Industrial Toxicology and Occupational Medicine Unit, Clos Chapelle-aux-Champs Box 30.54, 1200 Brussels, Belgium.
Received 19 September 1997; accepted in final form 2 February 1998.
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