1 Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor 48109; 2 Pulmonary Section of the Department of Veterans Affairs Medical Center, Ann Arbor, Michigan 48108; and 3 College of Natural Sciences, University of Texas, Austin, Texas 78712
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ABSTRACT |
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CC chemokine
receptor 2 (CCR2) /
mice are protected from experimental pulmonary
fibrosis, a disease increasingly recognized as being mediated by
dysfunctional interactions between epithelial cells and fibroblasts. We
have sought to investigate the interactions between alveolar epithelial
cells (AECs) and fibroblasts in these fibrosis-resistant (CCR2
/
)
and fibrosis-sensitive (CCR2 +/+) mice. AECs from CCR2
/
mice
suppress fibroblast proliferation more than AECs from CCR2 +/+ mice (77 vs. 43%). Exogenous administration of the CCR2 ligand monocyte
chemoattractant protein-1 (MCP-1) to the fibroblast-AEC cocultures
reverses the suppression mediated by CCR2 +/+ AECs but has no effect
with CCR2
/
AECs. MCP-1 regulates AEC function but not fibroblast
function. AEC inhibition of fibroblast proliferation was mediated by a
soluble, aspirin-sensitive factor. Accordingly, AECs from CCR2
/
mice produce greater quantities of PGE2 than do AECs from
CCR2 +/+ mice, and MCP-1 inhibits AEC-derived PGE2
synthesis. Diminished PGE2 production by AECs results in enhanced fibroproliferation. Thus an important profibrotic mechanism of
MCP-1/CCR2 interactions is to limit PGE2 production in AECs after injury, thus promoting fibrogenesis.
lipid mediators; lung; chemokines; monocyte chemoattractant protein-1; CC chemokine receptor 2
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INTRODUCTION |
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IDIOPATHIC PULMONARY FIBROSIS (IPF) is an interstitial lung disease of unknown origin in humans. The disease is characterized histologically by areas of inflammation, matrix deposition, and fibroblastic foci (9). An important element of the pathogenesis involves unchecked and excessive fibroblast proliferation and deposition of extracellular matrix proteins (including collagens), leading to alveolar collapse and fibrosis (25). The standard therapy for collective pulmonary fibrosis patients has often included corticosteroids. Unfortunately, few IPF patients respond to this therapy (17). The disease is progressive, generally resulting in respiratory failure within 5 years of diagnosis (16). This disease is difficult to study in humans for at least two reasons. First, patients rarely present at early stages of the disease. Second, the difficulty of obtaining lung tissue and bronchoalveolar lavage fluid (BALF) from patients in a serial fashion has made studying the natural progression of this disease in humans problematic. To circumvent these limitations, investigators have utilized murine model systems that recapitulate cardinal manifestations of the disease process. One commonly used model is administration of bleomycin. We have likewise reported that the intratracheal deposition of fluorescein isothiocyanate (FITC) in mice results in an early acute lung injury (days 1-5) and inflammation (maximal at day 7, but chronic) that progresses to patchy fibrosis by days 14-21 (4).
Historically, it has been suggested that pulmonary fibrosis is the
result of chronic inflammation (10, 30). Therefore, we
have investigated the role that CC chemokines play in the disease process. We found that mice genetically deficient in CC
chemokine receptor 2 (CCR2), the receptor for monocyte chemoattractant
protein-1 (MCP-1) were protected from both FITC- and bleomycin-induced
pulmonary fibrosis (19). The protection was specific for
CCR2 abrogation, as mice deficient in another CC chemokine receptor
(CCR5 /
) were not protected. Unexpectedly, the protection was not
associated with decreases in inflammation, suggesting that MCP-1/CCR2
interactions are not limited to the recruitment of inflammatory cells
within the lung.
More recently, it has been argued that pulmonary fibrosis is the result
of dysfunctional interactions between alveolar epithelial cells (AECs)
and mesenchymal cells within the lung (25). Therefore, we
sought to determine whether we could identify functional differences in
the interactions between AECs and fibroblasts in CCR2 +/+ and CCR2
/
mice. We determined that AECs from CCR2
/
mice are more
suppressive of fibroblast proliferation than are AECs from CCR2 +/+
mice. This enhanced suppressive activity of the CCR2
/
AECs
correlated with increased PGE2 production by the CCR2
/
AECs. Similarly, MCP-1 reduces the synthesis of PGE2 by
CCR2 +/+ AECs. PGE2 has long been known to be a potent
inhibitor of fibroblast proliferation and collagen synthesis (1,
6, 7, 12), and previous work from our laboratory has
demonstrated that PGE2 is an important protective mediator
in pulmonary fibrosis induced by bleomycin administration
(18). Further support for a protective role for
PGE2 comes from the fact that reduced PGE2 levels have been reported in BALF and alveolar macrophage-conditioned medium from IPF patients (2, 21) compared with normal
controls. Our findings demonstrate that an important profibrotic
mechanism of MCP-1/CCR2 interactions is to limit PGE2
production in AECs after injury, thus promoting fibroproliferation and
collagen deposition.
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MATERIALS AND METHODS |
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Mice.
CCR2 +/+ (B6129F2/J; Jackson Laboratory, Bar Harbor, ME) and CCR2 /
mice (B6129F2-Cmkbr2tm1Kuz) (13) bred at the
University of Michigan were housed under specific pathogen-free
conditions in enclosed filter-top cages. Clean water and food were
given ad libitum. The mice were handled and maintained with
microisolator techniques with daily veterinarian monitoring. The
University Committee on the Use and Care of Animals approved these experiments.
AEC purification.
Type II AECs were isolated from CCR2 +/+ and CCR2 /
mice by the
method developed by Corti et al. (5). After anesthesia and
heparinization, the mouse was exsanguinated and the pulmonary vasculature was perfused via the right ventricle with 0.9% NaCl until
the effluent was free of blood. The trachea was cannulated with
20-gauge tubing, and the lungs were filled with Dispase (1-2 ml;
Worthington). Subsequently, 0.45 ml of low-melting-point
agarose was infused via the trachea, and the lungs were placed in iced PBS for 2 min to harden the agarose. The lungs were placed in 2 ml of
Dispase and incubated for 45 min at 24°C. Subsequently, the lung
tissue was teased from the airways and minced in DMEM with 0.01%
DNase. The lung mince was gently swirled for 10 min and passed
successively through 100-, 40-, and 25-µm nylon mesh filters. The
cell suspension was collected by centrifugation and incubated with
biotinylated antibodies (anti-CD32 and anti-CD45) recognizing bone
marrow-derived cells. The cell suspension was incubated with
streptavidin-coated magnetic particles and then was placed in a
magnetic tube separator for removal of the bone marrow-derived cells.
Mesenchymal cells were removed by overnight adherence in a petri dish.
The nonadherent cells after this initial plating were plated at a
density of 50,000 cells/well on 96-well plates coated with fibronectin.
Cells were maintained in DMEM with penicillin-streptomycin and 10%
fetal calf serum at 37°C in 5% CO2. The final adherent
population included only 4% nonepithelial cells at day 2 in
culture by intermediate filament staining.
Enzyme immunoassay. Cell-free AEC supernatants were analyzed by enzyme immunoassay (EIA) for the predominant cyclooxygenase (COX) product PGE2, using a commercially available kit from Cayman Chemicals (Ann Arbor, MI).
Fibroblast purification.
Murine lungs were perfused with 5 ml of normal saline and removed under
aseptic conditions. Lungs were minced with scissors in DMEM complete
medium containing 10% fetal calf serum. One minced lung was placed in
10 ml of medium in 100-cm2 tissue culture plates.
Fibroblasts were allowed to grow out of the minced tissue, and when
cells reached 70% confluence they were passaged by trypsin digestion.
Fibroblasts were grown for 10-14 days (2 passages) before being
used and were always used before passage 6. Murine
fibroblasts isolated in this manner exhibit a myofibroblast phenotype
as evidenced by the expression of -smooth muscle actin.
-Smooth
muscle actin can be detected in these cultures both
immunohistochemically and by Western blot analysis.
AEC-fibroblast proliferation assays. For fibroblast-AEC cocultures, AECs were purified as described and plated onto fibronectin-coated plates on day 2 postisolation. AECs were allowed to adhere to fibronectin-coated plates (50,000 cells/well) for 24 h before being washed three times with 1× PBS. Fresh medium (DMEM, 10% fetal calf-serum, and 1% penicillin-streptomycin) containing fibroblasts was added (5,000 cells/well), and fibroblasts were allowed to grow in the presence or absence of the AECs for 24-48 h. [3H]thymidine was added (10 µCi/well; Amersham) during the final 16 h of culture. As purified AECs grow very poorly in culture and incorporate only low levels of [3H]thymidine, this technique measures fibroblast proliferation in coculture with AECs (15, 32). Control cultures of AECs alone always incorporated <5% of the total counts incorporated in cocultures with fibroblasts. Plates were then harvested, and incorporated radioactivity was determined with a beta scintillation counter. It should be noted that fibroblasts grew as a monolayer on top of the AECs. No detachment of either AECs or fibroblasts were noted in these cultures.
Data analysis. Statistical significance was analyzed using the InStat 2.01 program (Graphpad Software) on a Power Macintosh G3. Student's t-tests were run to determine P values when comparing two groups. When comparing three or more groups, we performed ANOVA analysis with a post hoc Bonferroni test to determine which groups showed significant differences. P < 0.05 was considered significant.
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RESULTS |
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CCR2 /
AECs inhibit
fibroblast proliferation more than CCR2
+/+ AECs do.
It has been hypothesized that one mechanism whereby AECs maintain
alveolar integrity is by limiting the outgrowth of fibroblasts from the
parenchyma (8, 23). If the CCR2 gene deletion increased the functional ability of the AECs in CCR2
/
mice to limit
fibroproliferation, that could help explain the reduced fibrotic
responses that we reported previously in vivo (19). Thus
we sought to test whether there were functional consequences on
fibroblast proliferation mediated by purified AECs from animals of both
genotypes. To perform these experiments, we grew fibroblasts from lung
minces of wild-type, CCR2 +/+ mice and used them at early passages.
AECs were purified from either CCR2 +/+ or CCR2
/
mice and cultured
at 50,000 cells/well in fibronectin-coated 96-well plates. Fibroblasts
(5,000 cells/well) were then seeded alone or on top of the AEC
cultures. After 24 h of culture, [3H]thymidine was
added to each well and the proliferation of the fibroblasts was
assessed by radioactive incorporation. It should be noted that as
previously described, AECs cultured alone proliferate very poorly in
vitro and incorporate almost no [3H]thymidine (15,
32); thus the [3H]thymidine incorporation seen in
fibroblast-AEC cocultures is indicative of fibroblast proliferation.
Figure 1 demonstrates that AECs from both
CCR2 +/+ and CCR2
/
mice inhibit fibroblast proliferation; however,
CCR2
/
AECs are more suppressive. When cocultured with CCR2 +/+
AECs, proliferation of fibroblasts is inhibited by 43% compared with
control fibroblast-only cultures (P < 0.002), whereas
CCR2
/
AECs suppressed fibroblast proliferation by 77%
(P < 0.002). The 77% inhibition mediated by CCR2
/
AECs was also significantly different from the 43% suppression
mediated by CCR2 +/+ AECs (P < 0.0001).
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MCP-1 can reverse CCR2 +/+
AEC-mediated fibroblast suppression.
To determine whether the profibrotic chemokine and CCR2 ligand MCP-1
could alter the suppressive effects of AECs on fibroblasts, cocultures
of either CCR2 +/+ or CCR2 /
AECs and wild-type CCR2 +/+
fibroblasts were incubated in the presence or absence of 10 ng/ml MCP-1
and compared with cultures of fibroblasts alone (Fig. 2). The dashed line in Fig. 2 represents
the proliferation rate of fibroblasts cultured in the absence of AECs.
As seen previously, both CCR2 +/+ and especially CCR2
/
AECs were
able to inhibit fibroblast proliferation in coculture. The addition of
MCP-1 to cocultures containing CCR2 +/+ AECs reversed AEC-mediated
fibroblast suppression. MCP-1 had no effect on proliferation of
fibroblasts cultured with CCR2
/
AECs. Thus in receptor-positive
mice, MCP-1 can reverse AEC-mediated fibroblast inhibition.
|
MCP-1 exerts its effect on AECs, not fibroblasts.
To determine whether the ability of MCP-1 to reverse AEC suppression of
fibroblast proliferation was mediated by effects on AECs or
fibroblasts, we performed experiments using wild-type CCR2 +/+ AECs in
coculture with fibroblasts from either CCR2 +/+ or CCR2 /
mice. As
seen in Fig. 3, wild-type AECs were able to inhibit proliferation of either CCR2 +/+ or CCR2
/
fibroblasts. Proliferation rates of CCR2 +/+ and CCR2
/
fibroblasts alone were
almost identical, and the dashed line in Fig. 3 represents proliferation of each normalized to 100%. MCP-1 could reverse the
wild-type AEC-mediated suppression of fibroblasts from either genotype.
In addition, we examined whether exogenous MCP-1 (ranging in
concentration from 1 pg/ml to 100 ng/ml) had any direct effect on
proliferation of fibroblasts from either genotype in vitro. No effect was seen at any concentration tested (not shown). Thus it
appears that the ability of MCP-1 to reverse AEC-mediated suppression is a direct effect of MCP-1 on AECs, mediated exclusively by CCR2.
|
AEC-mediated suppression of fibroblast proliferation is related to
AEC prostaglandin production.
We hypothesized that the AEC-mediated suppression was related to the
production of prostaglandins by AECs. If this hypothesis were correct,
then the suppressive prostaglandins would be present as secreted
molecules in the conditioned medium from AECs. When AECs were plated
onto the top chambers of transwells, and the fibroblasts were seeded
into the bottom chamber, fibroblast proliferation was inhibited (not
shown). These results suggest that the suppressive factor is secreted
by AECs into the medium and does not require contact between AECs and
fibroblasts. To determine whether AEC-conditioned media could inhibit
fibroblast proliferation, we added conditioned AEC media back to
fibroblasts alone, and proliferation was tested. The addition of either
25 or 50% AEC-conditioned medium to the fibroblasts alone inhibited
their growth in a dose-dependent fashion (Fig.
4). The suppressive effect of the
conditioned medium addition was not due to nutrient deprivation, since
the addition of serum-free medium at the same concentrations had no
effect on basal fibroblast proliferation. To determine whether the
suppressive factor was a prostaglandin, we wished to test the effects
of a COX inhibitor. Prostaglandins are synthesized from free
arachidonic acid via the actions of COX enzymes (26, 27).
We wanted to selectively inhibit COX activity in AECs and not
fibroblasts; therefore, we pretreated the AECs with the irreversible
COX inhibitor aspirin before addition of fibroblasts. AECs were
purified from CCR2 +/+ mice and seeded into 96-well fibronectin-coated
plates. On day 2, AECs were pretreated with 100 µM aspirin
for 1 h to irreversibly inhibit AEC COX activity. AECs were then
washed three times to remove aspirin, and fibroblasts were seeded as
before. Figure 5demonstrates that
aspirin pretreatment of AECs blocks the suppressive action of AECs on
fibroblast proliferation in cocultures. Whereas wild-type untreated
AECs could suppress fibroblast proliferation, and MCP-1 could reverse
this effect, aspirin-treated AECs were unable to suppress fibroblast
proliferation, and MCP-1 had no effect in cocultures with
aspirin-treated AECs. These data strongly indicate that endogenous
prostaglandins produced by the AECs themselves are responsible for
mediating fibroblast suppression. These data are in agreement with
recent studies from our laboratory that demonstrate that AECs derived
from COX-2 /
mice are unable to produce PGE2 and thus
are poor inhibitors of fibroblast proliferation (14).
Furthermore, the addition of MCP-1 to cocultures of COX-2
/
AECs
and fibroblasts had no effect (not shown). These data suggest that
MCP-1, a profibrotic chemokine, can act on the AECs directly to inhibit
prostaglandin production.
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|
CCR2 /
AECs produce more
PGE2 than CCR2 +/+
AECs.
We have previously determined that endogenous prostaglandin production
is beneficial in limiting bleomycin-induced pulmonary fibrosis
(18). AECs have been assigned an important role in limiting pulmonary fibrosis (8, 11, 29, 31), and AECs have
the capacity to synthesize PGE2 (3,
11). Therefore, we investigated whether epithelial
cell-derived prostaglandin production was increased in CCR2
/
mice.
To address this, AECs were isolated from CCR2 +/+ and CCR2
/
mice,
plated on fibronectin, and allowed to adhere for 24 h. The medium
was changed, and 24-h-conditioned medium was collected the next day. In
addition, a maximal stimulus for arachidonic acid release, the calcium
ionophore A-23187 was added to the AECs at this time point, and a
30-min serum-free, ionophore-stimulated supernatant was
collected. These supernatants were then analyzed for
PGE2 by specific EIA. Figure
6 demonstrates that AECs purified from
CCR2
/
mice produce more PGE2 both under basal and
maximally stimulated conditions than do CCR2 +/+ AECs.
|
MCP-1 inhibits PGE2 synthesis in CCR2
+/+ AECs.
To confirm that exogenous MCP-1 could inhibit the production of
PGE2 from CCR2 +/+ AECs, we purified AECs from CCR2 +/+
mice and plated them on fibronectin-coated 96-well plates at 5 × 104/well. The next day, AECs were washed and cultured in
fresh medium in the presence or absence of 10 ng/ml MCP-1 for 24 h. After the culture period, medium was removed and cells were washed
and stimulated in the presence of calcium ionophore for 30 min.
Ionophore-stimulated supernatants were then analyzed for
PGE2 synthesis by specific EIA. Figure
7 demonstrates that exogenous MCP-1
significantly inhibited (P = 0.01) the production of
PGE2 from CCR2 +/+ AECs.
|
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DISCUSSION |
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Our studies demonstrate that MCP-1/CCR2-mediated signals decrease
PGE2 production by AECs. The pathological consequence of diminished PGE2 production by AECs is enhanced
fibroproliferation. Our studies yield several important findings to
support this contention. 1) AECs suppress fibroblast
proliferation, and AECs from CCR2 /
mice are more suppressive than
AECs from CCR2 +/+ mice. 2) Exogenous administration of the
CCR2 ligand MCP-1 to the fibroblast-AEC cocultures reverses the
suppression mediated by CCR2 +/+ AECs but has no effect on the
suppression by CCR2
/
AECs. 3) MCP-1 effects are
dependent on the expression of CCR2 on AECs but not CCR2 expression on
fibroblasts. 4) CCR2
/
AECs produce more PGE2 than do CCR2 +/+ AECs. 5) Exogenous MCP-1
inhibits the synthesis of PGE2 from CCR2 +/+ AECs. Thus
MCP-1/CCR2-mediated signals regulate the production of PGE2
by AECs.
Our studies document that soluble AEC-derived prostanoids (likely
PGE2) play a direct role in suppressing fibroblast
proliferation. Previous studies concluded that AECs suppressed
fibroblast proliferation by inducing changes in fibroblast
PGE2 production (23). Several findings support
our conclusion that PGE2 derived from AECs is a crucial
regulator of fibroblast proliferation. First, selective treatment of
AECs with aspirin, a COX inhibitor, blocked the suppressive action of
AECs on fibroblast proliferation in cocultures. This pharmacological
approach is in complete agreement with recent work from our laboratory
demonstrating that AECs derived from COX-2 /
mice are deficient in
PGE2 synthesis and thus have reduced capacity to inhibit
fibroblast proliferation (14). Second, AEC-mediated suppression of fibroproliferation correlated with PGE2
production. CCR2
/
AECs inhibit fibroblast proliferation more
completely than CCR2 +/+ AECs. CCR2
/
AECs produced significantly
more PGE2 under both basal and maximally stimulated
conditions than did CCR2 +/+ AECs. Exogenous MCP-1 addition to CCR2 +/+
AECs diminished the synthesis of PGE2. The fact that
PGE2 would mediate these effects is not surprising given
that numerous studies have characterized the potent capacity of
PGE2 to limit fibroblast proliferation, limit collagen
synthesis, and promote collagen degradation (1, 6, 7, 12,
14).
Our studies are the first to document that production of
PGE2 by AECs is regulated by MCP-1/CCR2-dependent
mechanisms. Several findings support the contention that
MCP-1/CCR2-dependent regulation resides in AECs. First, CCR2 +/+ AECs
inhibit proliferation of both CCR2 +/+ and CCR2 /
fibroblasts.
Second, MCP-1 reversed CCR2 +/+ AEC-mediated suppression of both CCR2
+/+ and CCR2
/
fibroblasts. Third, exogenous MCP-1 had no direct
effect on proliferation of either CCR2 +/+ or CCR2
/
fibroblasts.
Finally, exogenous MCP-1 diminished the production of PGE2
from CCR2 +/+ AECs.
The biochemical mechanism by which MCP-1 inhibits prostaglandin
production by AECs is currently unknown but could involve regulation at
several biosynthetic steps in the prostaglandin cascade. It is likely,
however, that MCP-1 serves to inhibit an early step in the
prostaglandin pathway as CCR2 /
AECs produce elevated levels of
both PGE2 as well as PGI2 (as measured by the 6-keto-PGF1
derivative, not shown). Both
PGE2 and PGI2 are metabolized from
PGH2 generated by the enzymatic actions of COX-1 and COX-2.
Given our recent report that COX-2 is the predominant COX isoform
responsible for prostaglandin production in AECs (14), we
think it likely that COX-2 or cytosolic phospholipase A2
are the most likely candidates for regulation by MCP-1. A decrease in
phospholipase levels or activity could diminish the amount of free
arachidonic acid released from membrane phospholipids and thus would
decrease substrate for COX. A full understanding of how MCP-1
inhibits AEC production of PGE2 will require a full dissection of both the transcriptional and posttranscriptional regulatory events in this pathway.
Our data do not rule out the possibility that the enhanced ability of
the AECs from CCR2 /
mice to inhibit fibroproliferation may result
from increased secretion of both PGE2 and PGI2.
Both of these prostaglandin molecules can exert inhibitory effects on
fibroblasts via signaling through cAMP-coupled receptors
(20). However, in vitro studies in our laboratory have
demonstrated that PGE2 is more effective at inhibiting lung
fibroblast proliferation than is PGI2, which may reflect
differences in receptor density (Moore and Peters-Golden, unpublished
observations). Furthermore, PGE2 is the predominant
prostaglandin secreted by AECs. For these reasons, we believe that
PGE2 is likely to be the relevant AEC-derived prostanoid
mediating fibroblast suppression.
We are confident that AECs are the predominant cellular source of PGE2 within the AEC cultures. The AEC isolation procedure results in a population of cells that are 96% pure by intermediate filament staining. The contaminating 4% of cells are vimentin positive and may be lung macrophages. Therefore, we performed collagenase digestions of whole lung to isolate lung leukocytes as previously described (19) and adherence-purified a population of lung macrophages to test their ability to secrete PGE2 under basal culture conditions. Whereas AECs at a concentration of 2.5 × 105/ml produced ~5,000 pg/ml PGE2, lung macrophages at 5 × 105/ml produced only ~175 pg/ml PGE2. Additionally, we have previously reported that TxA2, not PGE2, is the major eicosanoid metabolite in alveolar macrophages (24).
The finding that MCP-1/CCR2 regulates PGE2 production
by AECs is of considerable importance and suggests a model for fibrotic progression in vivo. An intact alveolar epithelial barrier is crucial
to the maintenance of normal lung architecture. An intact alveolar
epithelium limits fibroproliferation and collagen synthesis. This is
critical for efficient gas exchange. In the face of insult or injury,
however, resident epithelial cells and recruited chronic inflammatory
cells within the lung rapidly upregulate the secretion of chemotactic
molecules including MCP-1. Additionally, AECs themselves can produce
MCP-1 (22, 28), and its expression can be further induced
under inflammatory stimuli (28). The binding of MCP-1 to
the denuded basement membrane may result in the prolonged
expression of MCP-1 at sites of injury. The persistent expression
of MCP-1 generates a cascade of regulatory events, which results in the expression of profibrotic mediators (TNF-) (19) and the
suppression of antifibrotic mediators [granulocyte-macrophage
colony-stimulating factor (19) and
PGE2]. In the presence of this altered
microenvironment, the ability of the alveolar epithelium to suppress
fibroblast proliferation via elaboration of PGE2 is lost.
Thus at the site of injury, AECs undergo a functional transformation
from cells that suppress fibroblasts to cells that permit fibroblast
proliferation and matrix secretion.
These data provide support for the concept that altered
epithelial/mesenchymal cell interactions play a crucial role in the pathogenesis of fibroproliferative lung disease. Furthermore, these
data highlight the fact that chemokine-mediated signals can perturb the
production of lipid mediators by AECs. These studies also provide proof
of concept that interruption of MCP-1/CCR2-mediated signaling is
beneficial in lung injuries that result in fibroproliferative responses. Finally, our data suggest that PGE2 may be an
effective inhibitor of myofibroblast proliferation, since the
fibroblasts used in this study were -smooth muscle active positive.
Thus these data have important implications for therapeutic
interventions for fibrotic lung disease. Therapeutic strategies aimed
at blocking the effects of MCP-1 via small molecule inhibitors of CCR2
or increasing AEC synthesis of PGE2 may prove clinically
useful in the treatment of fibrotic lung diseases such as IPF.
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ACKNOWLEDGEMENTS |
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The authors thank Ming Du, Carol Wilke, and Deirdra Williams for expert technical assistance.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants R29 CA-79046 (to B. B. Moore) and RO1 HL-51082 (to G. B. Toews); National Heart, Lung, and Blood Institute Specialized Center of Research P50 HL-56402 and P50 HL-60289; Merit Review Awards from the Medical Research Service (to G. B. Toews, P. J. Christensen, and R. Paine III); and Research Enhancement Award Program funds from the Department of Veterans Affairs. R. Paine III is a Career Investigator of the American Lung Association.
Address for reprint requests and other correspondence: B. B. Moore, Univ. of Michigan, 6301 MSRB III, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0642 (E-mail: Bmoore{at}umich.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.
First published October 4, 2002;10.1152/ajplung.00168.2002
Received 30 May 2002; accepted in final form 27 September 2002.
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