Upregulation of urokinase in alveolar macrophages and lung tissue in response to silica particles

Cécile Lardot1, Monique Delos2, and Dominique Lison1

1 Industrial Toxicology and Occupational Medicine Unit, Catholic University of Louvain, 1200 Brussels; and 2 Laboratory of Pathology, University Hospital of Mont Godinne, 5330 Mont Godinne, Belgium

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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-beta (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 alpha 2-antiplasmin and alpha 2-macroglobulin (20).

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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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- 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).

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 beta -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 (beta -actin) for 30 s, and primer extension at 72°C for 1.5 min. uPA and beta -actin amplification was performed for 36 and 25 cycles, respectively, selected in the exponential phase of PCR. The PCR products [15 (uPA) or 25 (beta -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 beta -actin mRNA levels to correct for any variation in RNA content and/or cDNA synthesis.

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 <=  0.05.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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).


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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.


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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.


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Fig. 3.   Effect of neutrophil depletion on BALF changes induced by silica. To achieve neutrophil depletion, mice were pretreated with cyclophosphamide (cyclophosph; 200 mg/kg ip) or saline (control; 100 µl ip) 3 days before administration of crystalline silica particles (5 mg/mouse). A-D: effects of cyclophosphamide on BALF markers of lung injury 24 h after silica or saline administration. LDH, lactate dehydrogenase. E and F: effects of neutrophil depletion in BALF and on BAL cell-associated PA activity, respectively. Values are means ± SE from 8-12 mice from 3 separate experiments. Significant differences from respective control mice were assessed by Student's t-test.

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.


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Fig. 4.   Changes in urokinase-type PA (uPA) mRNA levels in lung tissue (A) and bronchoalveolar cells (B) in response to a single intratracheal administration of crystalline silica particles (5 mg/mouse). Relative changes in uPA mRNA were determined from calibration curves as described in MATERIALS AND METHODS. Values are means of 3-4 PCR amplifications performed on 1:10-diluted cDNA and are expressed as a ratio to beta -actin mRNA level. Nos. above bars, silica-induced multiple of increase in uPA mRNA compared with level found in respective saline control. Gel photographs illustrate representative uPA (405 bp) and beta -actin (1,069 bp) PCR products resolved by 2% agarose gel electrophoresis stained with ethidium bromide.

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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Fig. 5.   Immunohistochemical detection of uPA in cryostat sections of control parenchyma and lung fibrotic tissue 1 mo after a single administration of crystalline silica particles (5 mg/mouse). Antiperoxidase-peroxidase staining with 5 µg/ml of polyclonal rabbit anti-murine uPA IgG was performed. Specific staining occurs in pneumocytes (arrows) and macrophages (arrowheads) of control mice (F) and fibrotic tissue (D). A heterogeneous distribution in staining was observed within silicotic structures (A and D). Some staining apparently represents extracellular material (*), whereas other parts seem to identify intracellular material (E). Fibrotic sections stained in absence of primary antibody (B) or with anti-glial fibrillary acidic protein polyclonal antibody (C) are negative. Magnifications: ×800 in A-C; ×2,000 in D and E; ×4,000 in F.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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-beta (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.

    ACKNOWLEDGEMENTS

P. Thurion (University Hospital of Mont-Godinne, Mont-Godinne, Belgium) is greatly acknowledged for technical assistance in immunohistochemistry.

    FOOTNOTES

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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Lung Cell Mol Physiol 274(6):L1040-L1048
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