1 Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287; 2 Department of Medicine, University of Alabama School of Medicine, Birmingham, Alabama 35294; 3 Department of Internal Medicine, Yale University School of Medicine, New Haven 06520; and 4 Department of Internal Medicine, Veterans Affairs Connecticut Healthcare System, West Haven, Connecticut 06516
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ABSTRACT |
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Pulmonary inflammation and fibrosis are characterized by increased turnover and production of the extracellular matrix as well as an impairment of lung fibrinolytic activity. Although fragments of the extracellular matrix component hyaluronan induce macrophage production of inflammatory mediators, the effect of hyaluronan on the fibrinolytic mediators plasminogen activator inhibitor (PAI)-1 and urokinase-type plasminogen activator (uPA) is unknown. This study demonstrates that hyaluronan fragments augment steady-state mRNA, protein, and inhibitory activity of PAI-1 as well as diminish the baseline levels of uPA mRNA and inhibit uPA activity in an alveolar macrophage cell line. Hyaluronan fragments alter macrophage expression of PAI-1 and uPA at the level of gene transcription. Similarly, hyaluronan fragments augment PAI-1 and diminish uPA mRNA levels in freshly isolated inflammatory alveolar macrophages from bleomycin-treated rats. These data suggest that hyaluronan fragments influence alveolar macrophage expression of PAI-1 and uPA and may be a mechanism for regulating fibrinolytic activity during lung inflammation.
extracellular matrix; lung; chronic fibrosis
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INTRODUCTION |
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CHRONIC INFLAMMATION AND FIBROSIS are characterized by accumulation of inflammatory cells and mediators, increased turnover and production of the extracellular matrix (ECM), and alterations in the fibrinolytic system (2, 8, 21, 26, 39, 61). Activated macrophages play an essential role in inflammation through the release of a variety of mediators including reactive oxygen and nitrogen species, proteases, chemokines, cytokines, and growth factors (10, 21, 32). Although macrophages produce and secrete important mediators of fibrinolysis, plasminogen activator (PA) inhibitors [plasminogen activator inhibitor (PAI)-1] and PAs [urokinase-type plasminogen activator (uPA)], there is a paucity of information regarding the molecular mechanisms regulating macrophage expression of PAI-1 and uPA in inflammatory states (9, 49, 53, 58, 59, 62).
Recent studies (10, 28, 34, 60) have shown that fragments
of ECM components, generated at sites of inflammation, have different
biological activities than their high molecular mass (HMW)
precursor molecules and may, in fact, play a role in the activation of
inflammatory macrophages. For example, fragments of the ECM components
collagen and fibronectin, but not the intact molecules, have been shown
to have proinflammatory properties (52). Hyaluronan (HA)
is a nonsulfated glycosaminoglycan polymer that is a ubiquitously
distributed component of the ECM. In its native form, HA exists as a
HMW polymer of repeating disaccharide units of
(14)-
-D-glucuronic
acid-(1
3)-N-acetyl-
-D-glucosamine, which
functions in water homeostasis, plasma protein distribution, and matrix
structuring in normal tissues (35). At sites of
inflammation, HMW HA is fragmented into lower molecular mass (LMW)
forms that stimulate inflammatory macrophages to produce important
mediators of tissue injury and repair such as cytokines, chemokines,
reactive nitrogen species, and growth factors (1, 4, 20, 22, 23,
25, 42).
Under physiological conditions, the fibrinolytic system is tightly regulated by the balance between PAs and PAIs. uPA, one of the two major plasminogen activators, is a serine protease that is secreted by many different cell types including macrophages (59). Plasminogen is cleaved by uPA to generate plasmin, a protease with broad specificity for fibrin and other matrix proteins. The activity of fluid-phase or receptor-bound uPA is specifically and rapidly inhibited by PAI-1 (37). PAI-1 and PAI-2 are two PAIs, serpins, that have been shown to have potent inhibitory activity against PAs (32, 59). Macrophage PAI-1 is largely secreted; it is a potent inhibitor of both uPA and tissue-type plasminogen activator (tPA), and the congenital absence of PAI-1 produces a phenotype with increased bleeding and fibrinolysis (8, 59). PAI-2 is a less potent inhibitor of uPA and single-chain tPA and is a poor inhibitor of two-chain tPA. PAI-2 is largely intracellular in macrophages, and the congenital absence of PAI-2 produces no distinct murine phenotype (14, 53, 62).
In chronic inflammatory states, there is mounting evidence that the balance between PAs and PAIs may be altered, leading to decreased fibrinolysis, excessive fibrin deposition, and, ultimately, tissue fibrosis and organ damage (2, 9, 26, 49). Indeed, imbalances in PAI-1-uPA regulation have been suggested as playing a role in the pathogenesis of chronic lung inflammation, sepsis, and cancer (3, 5, 12, 15, 47, 56). However, the mechanisms governing fibrinolysis in chronic inflammatory states in vivo are unclear, and, in particular, the role of the ECM in inducing uPA and PAI-1 gene expression in macrophages has not been investigated. Our results show that HA fragments induce PAI-1 and inhibit uPA transcription and activity in murine macrophages and thus suggest a novel mechanism for the regulation of fibrinolysis at sites of inflammation.
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METHODS |
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Macrophage isolation and culture. Mouse bone marrow-derived (BMD) macrophages were isolated from female C3H/HeJ lipopolysaccharide (LPS)-hyporesponsive mice purchased from the Jackson Laboratory (Bar Harbor, ME) as previously described (46). After harvest, the cells (11 × 106 cells/dish) were cultured for 5 days in DMEM supplemented with 10% heat-inactivated low-LPS fetal bovine serum, 15% L-cell medium, and 1% penicillin-streptomycin-1% glutamine (Biofluids, Rockville, MD) at 37°C under 8% CO2. The mouse alveolar macrophage (AM) cell line MH-S (40) was purchased from the American Type Culture Collection (Manassas, VA). The cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated low-LPS fetal bovine serum and 1% penicillin-streptomycin-1% glutamine (Biofluids) at 37°C under 5% CO2. To exclude the effects of contaminating LPS on experimental conditions, cell stimulation was carried out in the presence of 10 µg/ml of polymyxin B (Calbiochem-Novabiochem, La Jolla, CA).
Chemicals and reagents. Purified HA fragments from human umbilical cords were purchased from ICN Biomedical (Costa Mesa, CA). The HA ICN preparation was free of protein (<2%) and other glycosaminoglycans, with a peak molecular mass of 200,000 Da (43). Cycloheximide (10 µg/ml) and actinomycin D (50 µg/ml) were purchased from Sigma (St. Louis, MO). Protein G Sepharose was purchased from Pharmacia Biotech (Piscataway, NJ), and Promix 35S-labeled methionine and cysteine were purchased from Amersham (Arlington Heights, IL). Stock solutions of reagents were tested for LPS contamination with a Limulus amebocyte assay (Sigma).
Northern blot analysis of mRNA production.
RNA was extracted from confluent cell monolayers with 4 M guanidine
isothiocyanate and purified by centrifugation through a 5.7 M cesium
chloride gradient for 12-18 h at 35,000 rpm as previously
described (46). Total RNA (10 µg RNA/lane) was
electrophoresed under denaturing conditions through a 1%
formaldehyde-containing agarose gel and was transferred to Nytran
membranes (Schleicher and Schuell, Keene, NH). RNA was cross-linked to
the filter by ultraviolet cross-linking (Stratagene, La Jolla, CA), and
the blots were hybridized overnight with 106
counts · min1 · ml
1 of
32P-labeled cDNA probes labeled with the random-prime
method (Amersham). The murine PAI-1 probe used was the EcoR
I-Sph 1.1-kb fragment of murine PAI-1, and the murine uPA
probe used was the Hind III 1.1-kb fragment of murine uPA
(13, 47, 51). After hybridization, the blots were washed
in 2× saline-sodium citrate (SSC)-0.1% SDS (once at room temperature
for 30 min) and then 0.1× SSC-0.1% SDS (50°C for 20 min twice).
Autoradiography was performed at
70°C against Kodak XAR diagnostic
film (Eastman Kodak, Rochester, NY). Integrity of RNA was validated by
ethidium bromide staining, and differences in RNA loading were
documented by analysis of blots for mRNA aldolase signal
(36).
Immunoprecipitation of PAI-1. Immunoprecipitation was performed as described by Bonifacino (6). Briefly, the cells were treated with 8 µCi of 35S-Promix with and without HA fragments for 24 h. The samples were cleared of nonspecific antibody binding by treatment with 50 µl/ml of protein G Sepharose (1 h at 4°C). The resultant supernatant was then incubated (overnight at 4°C) with 2 µl of polyclonal antibody specific to PAI-1 (American Diagnostica, Greenwich, CT). The immune complexes were then precipitated with 50 µl of protein G Sepharose (1 h at 4°C). The beads were then washed four times with dilution (10 mM Tris · HCl, pH 8.0, 150 mM NaCl, 0.1% Triton X-100, 0.025% sodium azide, and 0.1% BSA) and wash (50 mM Tris · HCl, pH 6.8) buffers (6). The boiled supernatant was electrophoresed under reducing conditions on a 10% SDS-polyacrylamide gel. The gel was dried (1 h at 80°C), and autoradiography was performed as described in Northern blot analysis of mRNA production.
Direct and reverse fibrin autography.
Direct and reverse fibrin autography (PA and PAI-1 assays,
respectively) were performed on equal volumes of SDS-PAGE-separated conditioned medium as previously described (16, 48).
Briefly, an agarose gel (final concentration 0.83% Sea Plaque; FMC
BioProducts, Rockland, ME) containing purified human plasminogen (final
concentration 8.3 µg/ml), bovine fibrinogen (final concentration 8 mg/ml; Sigma) and -thrombin (final concentration 0.5 U/ml) was cast
and placed over the Triton X-100-washed polyacrylamide gel. The gels
were then incubated for 3-6 h (37°C) in a humidified chamber and
photographed under indirect light. The transparent (dark) zones in the
opaque overlay gel represent PA activity, and the light zones generated in the reverse fibrin gel (uPA 0.05 IU/ml included in overlay gel)
represent PAI activity in locations corresponding to their molecular
mass. As described, amiloride specifically inhibited the action
of a urokinase standard (Ref. 66/46, National Institute for Biological
Standards and Controls, London, UK) but did not affect the activity of
tPA (58). Anti-tPA IgG (final concentration 10 µg/ml), but not preimmune IgG, specifically inhibited the action of
single-chain tPA (Ref. 83/517, National Institute for Biological Standards and Controls) but not that of the urokinase standard (16, 48, 57).
Total PA activity assay. Total PA activity in conditioned medium was measured in triplicate by a plasmin-based chromogenic assay as previously published (48), with minor modifications. The samples were incubated in acetate buffer to destroy anti-plasmin activity followed by incubation (37°C in a humidified chamber for 4-8 h) with plasminogen and the plasmin-sensitive chromogen S-2251 (Chromogenix, Molndal, Sweden) in the presence of CNBr fragments of fibrinogen. In preliminary experiments, amiloride (0.1 mM) was found to inhibit >98% of the activity of a 10 IU/ml urokinase standard (recombinant murine urokinase; American Diagnostica) while reducing the activity of a 10 IU/ml tPA activity standard by only 3%.
Nuclear run-on assay.
Nuclei from confluent monolayers of MH-S cells were harvested by
scraping in ice-cold PBS and subsequently isolated by centrifugation through a sucrose cushion (17, 24). Nuclei were then
incubated (30 min at 30°C) with 1 M dithiothreitol, 20 mM nucleotide
triphosphates, and 100 µCi of [32P]UTP (NEN, Boston,
MA) in transcription buffer (20 mM Tris · HCl, pH 8.3, 100 mM
KCl, 4.5 mM MgCl2, and 20% glycerol). The reaction was
stopped by the addition of termination buffer [50 mM
Tris · HCl, pH 8.3, 500 mM NaCl, 5 mM EDTA, 200 µg/ml of
DNase I (Promega, Madison, WI), and 750 U/ml of RNase inhibitor
(Boehringer Mannheim, Indianapolis, IN)]. The nuclei were then
incubated with tRNA (Sigma) for 15 min before the addition of 10% SDS,
0.2 M EDTA, and 5 U of proteinase K (Sigma). After a 15-min
incubation, RNA was extracted with phenol-chloroform-isoamyl alcohol,
precipitated with 20% trichloroacetic acid, washed with 5%
trichloroacetic acid-5% pyrophosphate, dissolved, and reprecipitated
with 4 M sodium acetate and 100% ethanol (80°C for 30 min). The
purified radiolabeled RNA was washed (once with 70% ethanol), dried
with a Speed Vac concentrator (Savant Instruments, Hicksville, NY), and
resuspended in 100 µl of diethyl pyrocarbonate-treated water. Normalized samples (equal counts per minute) were allowed to bind to
complementary sequences that were prehybridized to Optitran-S membranes
(Schleicher and Schuell). Blots were hybridized for 3-4 days and
then washed in 2× SSC-0.1% SDS (once for 5 min at room temperature)
and in 0.1 × SSC-0.1% SDS (twice for 20 min at 50°C). Band
radioactivity was quantified with a phosphorimager (Molecular Dynamics,
Sunnyvale, CA).
Bleomycin administration and bronchoalveolar lavage. Bleomycin was administrated to male Harlan Sprague-Dawley rats according to published methods (30). The rats were anesthetized with inhaled isofluorane. After a tracheostomy, 500 µl of sterile normal saline with 1 U of bleomycin sulfate were instilled into the lungs through a 25-gauge needle inserted between the cartilaginous rings of the trachea. Control animals received saline alone. The tracheostomy site was sutured, and the animals were allowed to recover until the time of bronchoalveolar lavage (BAL). The rats were killed with a lethal injection of Pentothal Sodium (Ampro Pharmaceutical, Arcadia, CA) at specified time points after intratracheal instrumentation.
BAL was performed by cannulating the trachea and instilling and retrieving ~50 ml of sterile normal saline in 5-ml aliquots. The entire lavage volume was centrifuged, and the cell pellet was resuspended in RPMI 1640 medium supplemented with fetal bovine serum and antibiotics. A cell differential was performed on the lavage fluid by Wright-Giemsa stain before centrifugation, and repooling of the cells consistently showed at least 95% macrophages. Cell viability was determined with trypan blue exclusion. AMs were purified by adherence to plastic tissue culture dishes for 1 h at 37°C in RPMI 1640 medium without serum or antibiotics under 5% CO2; nonadherent cells were removed by aspiration, and adherent macrophages were washed in sterile PBS. All subsequent experiments were performed in RPMI 1640 medium without serum. ![]() |
RESULTS |
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HA fragments augmented PAI-1 and diminished uPA mRNA levels in
mouse macrophages.
To investigate the effect of LMW HA fragments on PAI-1 and uPA mRNA
levels in macrophages, we stimulated both an AM cell line, MH-S, and
primary BMD macrophages from C3H/HeJ mice with HA fragments for varying
time periods and performed Northern blot analysis. Both cell types
expressed detectable levels of uPA and minimal levels of PAI-1 mRNA
under basal conditions (Fig. 1). HA
fragments augmented PAI-1 mRNA yet diminished uPA mRNA levels in a
time-dependent fashion in both macrophage populations, with a peak
induction of PAI-1 mRNA after 3-6 h of HA exposure and a peak
diminution of uPA mRNA after 18-24 h of HA stimulation. However,
in BMD macrophages, HA fragments inhibited uPA expression after an
initial enhancement at 1 h. The effect of HA fragments on PAI-1
and uPA mRNA levels was independent of the presence of serum in the
stimulation medium during HA exposure (data not shown). In summary,
these data demonstrate that HA fragments increased PAI-1 and decreased
uPA steady-state mRNA levels in two different mouse macrophage
populations.
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HA fragments augmented PAI-1 and diminished uPA mRNA levels in a
concentration-dependent and specific fashion in mouse macrophages.
To further delineate the effect of HA fragments on PAI-1 and uPA
expression, we determined the macrophage response to varying concentrations of HA fragments as well as to other glycosaminoglycans. HA fragments (average molecular mass 200 kDa) increased PAI-1 and
diminished uPA mRNA levels in a dose-dependent manner (Fig. 2). PAI-1 expression was induced with as
little as 50 µg/ml of HA fragments and was maximal with 200 µg/ml
of HA fragments. uPA expression was inhibited by 10 µg/ml of HA
fragments, although maximal inhibition required at least 100 µg/ml of
HA. Furthermore, neither PAI-1 nor uPA levels were altered by HMW HA
(molecular mass 1,000 kDa), disaccharide HA (molecular mass 0.4 kDa),
or other glycosaminoglycans such as chondroitin sulfate A or B or heparan sulfate. Thus the effect of HA fragments on PAI-1 and uPA mRNA
levels is concentration dependent and specific.
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HA fragments induced PAI-1 protein production, secretion and
activity but suppressed uPA activity in MH-S cells.
To determine whether HA-induced alterations in PAI-1 and uPA mRNA
levels resulted in physiological changes in fibrinolysis, we
investigated the effect of HA fragments on protein production and
fibrinolytic activity. PAI-1 protein production and secretion were
determined by immunoprecipitation of cell-conditioned medium from
metabolically labeled ([35S]Met or Cys) HA
fragment-stimulated MH-S cells with a polyclonal antibody specific to
PAI-1 followed by SDS-PAGE and autoradiography. No PAI-1 protein was
immunoprecipitated from the conditioned medium from the unstimulated
cells, but a single major band corresponding to a PAI-1 protein (~45
kDa) was immunoprecipitated from the conditioned medium from cells
stimulated with HA fragments (Fig.
3). Thus HA fragments induced
PAI-1 production and pericellular secretion in murine macrophages.
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New protein synthesis is not required for the induction of PAI-1
but is required for the inhibition of uPA by HA fragments.
To examine the mechanism by which HA regulates PAI-1 and uPA, we
pretreated MH-S cells with cycloheximide (CHX) for 30 min before
stimulation with HA fragments for 6 h. We found that HA fragments
in the presence of CHX still augmented PAI-1 steady-state mRNA levels
but no longer diminished uPA mRNA levels (Fig.
5). Thus new protein synthesis appears to
be necessary for HA fragments to inhibit uPA gene expression but not to
induce PAI-1. Of note, CHX alone minimally induced PAI-1 expression as
previously described (38).
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Transcription was required for the induction of PAI-1 and the
inhibition of uPA by HA fragments.
To determine whether HA fragments were regulating PAI-1 and uPA gene
expression at the level of transcription, we performed Northern blot
analysis of HA fragment-stimulated MH-S cells in the presence of the
mRNA synthesis inhibitor actinomycin D. Actinomycin D added before
stimulation of MH-S cells with HA fragments completely blocked the
induction of PAI-1 and the inhibition of uPA gene expression at the
level of steady-state mRNA (Fig. 6).
These results imply that HA fragments do not affect the
stability of PAI-1 and uPA mRNAs.
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HA fragments augmented PAI-1 and diminished uPA mRNA levels in
primary AMs from bleomycin-treated rats.
To extend these observations to primary inflammatory AMs, we employed
an animal model of lung injury and fibrosis. It has been previously
shown in the bleomycin model of lung fibrosis that there was an
increased accumulation of PAI-1 and HA fragments as well as a decreased
activity of uPA (44, 49). Thus bleomycin or saline was
intratracheally administered to rats, and primary AMs were isolated
after 9 days by BAL with differential adherence. These isolated
macrophages were stimulated in vitro with HA (100 µg/ml) for 6 h, and Northern blot analysis was performed. AMs isolated from
saline-treated rats had detectable levels of PAI-1 and uPA mRNA at
baseline that were not altered by stimulation with HA fragments (Fig.
8). In contrast, HA fragments both
induced PAI-1 and diminished uPA mRNA levels in inflammatory AMs from bleomycin-treated rat lungs. Of note, inflammatory AMs from
bleomycin-instilled animals expressed lower basal levels of PAI-1 mRNA
and higher basal levels of uPA mRNA than AMs from saline-treated
animals (Fig. 8). In summary, HA fragments augmented PAI-1 and
diminished uPA mRNA levels selectively in primary AMs isolated from
inflamed lungs but not from saline-treated lungs.
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DISCUSSION |
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The purpose of this study was to examine the effect of LMW ECM fragments on macrophage expression of the modulators of fibrinolysis. Our laboratory (22-25, 41, 42) has previously shown that HA fragments in sizes similar to those generated at sites of inflammation stimulate mouse macrophage expression of numerous cytokines and chemokines. The major finding of this study is that HA fragments, but not native HMW HA, induce transcription and expression of PAI-1 and simultaneously inhibit the transcription and expression of uPA in mouse macrophages. These results identify a novel role for HA fragments as potential regulators of the fibrinolytic system in inflammation.
HA fragments induced PAI-1 and inhibited uPA gene expression in a
time-dependent fashion in the AM cell line MH-S as well as in two types
of primary macrophages, BMD macrophages and AMs. Furthermore, the
decrease in uPA, but not in PAI-1, required new protein synthesis.
Although previous investigators (11, 19, 54) have
suggested a role for tumor necrosis factor (TNF)- in the regulation
of PAI-1 and uPA in macrophages, we found, using macrophages from
TNF-
knockout mice as well as specific TNF-
blocking antibodies,
that HA-induced alterations of PAI-1 and uPA steady-state mRNA levels
were independent of TNF-
(data not shown). These data support a
previously unknown mechanism by which matrix fragments may play a role
in regulating the fibrinolytic process that occurs in lung injury and
repair. Our laboratory (22, 42, 46) has
previously shown that HA fragments induce inflammatory gene expression
in certain macrophage populations. Our laboratory (22, 42,
46) has also provided evidence to support a role for CD44 in
mediating HA fragment-induced gene expression under certain
circumstances. However, Horton et al. (25) and others
(29) have also provided data that there may be
CD44-independent signaling pathways in macrophages as well. Further
studies will be required to characterize the role of other potential HA
receptors in mediating signal transduction pathways.
The regulation of PAI-1 and uPA by HA fragments appears to be dependent
on the state of macrophage activation. Activated and inflammatory
macrophages respond to HA fragment stimulation, whereas inactive or
quiescent macrophages do not respond. SV-40-transformed MH-S cells, BMD
macrophages (which were cultured in colony-stimulating factor-1), and
inflammatory AMs from bleomycin-treated rats are all in a state of
activation, and all respond to HA fragment stimulation by increasing
PAI-1 and decreasing uPA expression. In contrast, noninflammatory AMs
from saline-treated rats display baseline levels of PAI-1 and uPA that
are unresponsive to HA stimulation. These results are consistent with
previously published data from our laboratory (22, 41)
showing that HA fragment-induced macrophage gene expression depends on
both the origin and state of activation of the macrophages studied.
Furthermore, the basal expression of both uPA and PAI-1 and their
response to various agonists (phorbol 12-myristate 13-acetate,
colony-stimulating factor-1, cholera toxin, interferon-, or
LPS) have been shown by other investigators (9, 57) to
also be dependent of the level of macrophage activation.
The effect of HA fragments on the AM cell line expression of PAI-1 and uPA occurred in a dose-dependent fashion, was not a general characteristic of glycosaminoglycans, and was specific for LMW HA fragments (200 kDa). Previous investigators (60, 61) have demonstrated that HA oligosaccharides (12-16 disaccharide units, molecular mass 5 kDa) are angiogenic and stimulate endothelial cell proliferation. Furthermore, these HA oligosaccharides promote in vitro invasion of bovine microvascular endothelial cells into a collagen matrix as well as induce gene expression and activity of both uPA and PAI-1 (43, 60, 61). In this report, only the LMW HA fragments (200 kDa) altered macrophage expression of PAI-1 and uPA. Macrophage expression of PAI-1 and uPA was not affected by the HMW HA (1,000 kDa) or by the HA disaccharides (0.4 kDa). We found that the identical HA oligosaccharide preparation, which was previously shown by West and colleagues (60, 61) to induce both PAI-1 and uPA in endothelial cells, did not alter macrophage mRNA levels of PAI-1 or uPA (data not shown). Furthermore, we demonstrated that LMW HA fragments induced macrophage gene expression of PAI-1 and inhibited macrophage expression of uPA, whereas West and colleagues showed that HA oligosaccharides induced both PAI-1 and uPA in endothelial cells. Thus it appears as if the effect of HA fragments is not only dependent on the size of HA but is also cell-type specific.
A similar coordinated reduction in uPA expression or transcription with
increases in PAI-1 expression or transcription has been observed in
vivo in AMs and in vitro in a murine macrophage cell line (RAW) under
conditions of hypoxia (50). Additionally, a previous work
(31) on the matrix proteins indicated that the basement
membrane protein laminin upregulated uPA expression and fibrinolytic
activity in vitro in two murine macrophage cell lines in an integrin
(6-subunit)-dependent manner, whereas collagen, tenascin, and fibronectin were ineffective. Our findings of coordinated induction of PAI-1 expression and inhibition of uPA expression by
matrix fragments generated at the site of inflammation adds spatial
relevance to the previously described developmental alterations in the
responses of these genes. Together, these data suggest that
monocyte/macrophage lineage cells respond to an inflammatory stimulus
(i.e., cytokines) in a complex manner that is modulated by the
underlying level of activation and matrix environment.
In many chronic inflammatory and fibrotic conditions such as adult respiratory distress syndrome, idiopathic pulmonary fibrosis, and sarcoidosis, there are alterations in fibrinolytic expression (2, 3, 26, 27, 49, 54). Bleomycin-induced lung injury is a well-characterized animal model of pulmonary fibrosis that causes an acute alveolitis followed by a fibroproliferative phase (7). In bleomycin-injured lungs, there is both increased PAI-1 and decreased uPA activity as well as increased production and accumulation of HA fragments (in sizes similar to those of our active HA fragments) (7, 44, 45, 49). In fact, mice deficient in PAI-1 expression are relatively resistant to the fibrosing effects of bleomycin (15). Although the mechanisms responsible for the derangement in fibrinolytic activity found in this model are unclear, primary fibroblast-like cells, macrophages, and epithelial cells appear to play a role (18, 47, 55).
Interestingly, AMs isolated from rats 9 days after bleomycin insult have diminished basal levels of PAI-1 and enhanced mRNA levels of uPA compared with AMs from saline-treated control rats. However, on stimulation with HA fragments, the AMs from bleomycin-treated rats have decreased uPA and increased PAI-1 mRNA levels, whereas the AMs from saline-treated animals are unresponsive to HA. Although one might expect that AMs from bleomycin-injured rats, which are theoretically exposed to HA fragments in vivo, have low levels of uPA and high levels of PAI-1 at baseline, we do not find this to be the case, and, in fact, this phenotype is typical of activated macrophages (33, 62). Additionally, these baseline findings may be a reflection of LMW HA clearance (peak HA levels 2-4 days after bleomycin) or of other microenvironmental stimuli modifying HA signaling. Nevertheless, we have demonstrated that HA fragments modify uPA and PAI-1 levels in primary inflammatory AMs.
In conclusion, we have shown that HA fragments of sizes similar to those found in inflammatory tissues alter macrophage uPA and PAI-1 expression as well as pericellular fibrinolytic activity. Identifying the molecular mechanisms by which HA fragments regulate gene expression may lead to new approaches to treat chronic inflammation and fibrosis.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-09614-01 (to M. R. Horton), R01-HL-53615, P50-HL-52315 (both to C. J. Lowenstein), R01-HL-58655 (to M. A. Olman), K11-HL-02880, R01-HL-60539, and 5F32-HL-09614-02 (all to P. W. Noble); the Cora and John H. Davis Foundation (C. J. Lowenstein); the Bernard Bernard Foundation (C. J. Lowenstein); a Merit Award from the Veterans Administration (to M. A. Olman); and the American Lung Association (P. W. Noble).
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FOOTNOTES |
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Address for reprint requests and other correspondence: P. W. Noble, Yale Univ. School of Medicine, Dept. of Veterans Affairs Connecticut Healthcare System, Pulmonary Section/111A, 950 Campbell Ave., West Haven, CT 06516 (E-mail: paul.noble{at}yale.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 5 January 2000; accepted in final form 27 April 2000.
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