Introduction
The concept of lung fibroblasts as effector cells in the
pathogenesis of idiopathic pulmonary fibrosis (IPF) has
recently evolved [ 1 2 ] . Lung fibroblasts respond,
in vitro , to inflammatory cytokines
by producing growth factors and collagen, resulting in
fibroblast proliferation and extracellular matrix
deposition [ 2 3 4 ] . In addition, activated lung
fibroblasts have been shown to produce large amounts of
inflammatory cytokines and chemokines,
in vitro , and hence, these cells may
also have a role as effector-inflammatory cells [ 1 2 ] .
This capacity to produce both inflammatory and fibrotic
factors could mean that phenotypically altered lung
fibroblasts act simultaneously as effector and target
cells, via paracrine and autocrine mechanisms, perpetuating
the fibrotic process [ 2 ] .
Prostanoids are important regulators of fibroblast
function [ 5 6 7 8 9 ] . Prostaglandin (PG)E
2 is thought to have antifibrotic
properties
in vitro , but also can have
proinflammatory effects both
in vivo and
in vitro [ 10 11 12 ] . Thromboxane
(TX)A
2 increases proliferation, and DNA and
RNA synthesis in several cell types, including fibroblasts
and smooth muscle like glomerular mesangial cells [ 13 14
15 16 ] . Conversely, prostacyclin (PGI
2 ) decreases smooth muscle cell
proliferation and collagen synthesis [ 17 18 ] .
Many cell types, including lung fibroblasts, contain
cyclooxygenase (COX), a proximal enzyme in prostanoid
production, and can generate prostanoids [ 19 ] . It has
been previously reported that IPF lung fibroblasts have
decreased COX-2 expression compared to normal lung
fibroblasts and, hence, have decreased PGE
2 production [ 12 20 21 ] . Because of
these findings and the fact that PGs are important
fibroblast regulators, we sought to investigate whether
abnormalities in COX-2 expression could be associated with
an altered balance between profibrotic and antifibrotic
PGs. We hypothesized that fibroblasts from the lungs of
patients with IPF (HF-IPF) have an altered PG balance
compared to normal lung fibroblasts (HF-NL). This
phenotypical abnormality could be an important factor in
the pathogenesis of IPF.
Materials and methods
Primary lung fibroblasts
Fibroblasts from the lungs of seven patients (6 males)
with IPF (HF-IPF) were harvested: a) from excised lung at
the time of lung transplantation; b) during an autopsy
performed within 4 hours from death; or c) during open or
transbronchial lung biopsies at the time of diagnosis. Of
the seven patients, five subjects had advanced lung
fibrosis and were receiving prednisone ±
immunosuppressive agents; 2 patients were at an earlier
stage of their disease and were not receiving
immunosuppressive drugs. The mean age of the patients was
59 (range 43-71)]. HF-NL were cultured from five human
lungs that arrived at our transplant center with the
intention of being used for transplantation, but for
various reasons could not be transplanted; these were
macroscopically and microscopically normal. The cells
were harvested and cultured as per the protocol described
by Kumar
et al. [ 22 ] . Briefly, lung
tissue sections were finely cut with sterile scissors and
incubated with serum free DMEM containing trypsin, DNAse
and collagenase for 30 min. The procedure was repeated
twice, and the supernatants were pooled and cultured in
one 100 mm plate and incubated at 37°C in a 5% CO
2 humidified atmosphere. Culture
medium (DMEM with 5% fetal bovine serum [FBS] and
penicillin/streptomycin) was replaced three times per
week and fibroblasts were passed (1:2 split) at the time
they became confluent. On passage 4 the cells were
resuspended in 1 ml of DMEM with 20% FBS and DMSO and
frozen at -70°C. For each experiment described below the
cells were thawed, cultured and passed at least once. All
the experiments were conducted with cells at passages 6
to 8.
Inducible cyclooxygenase (COX)-2 expression and
eicosanoid production
COX-2 activity was determined by measuring PGE
2 , 6-keto-PGF
1α (stable PGI
2 metabolite), TXB
2 (stable TXA
2 metabolite), and PGF
2α production in stimulated
fibroblasts. HF-IPF (n = 7) and HF-NL (n = 5) were
brought to >90% confluency in 100mm plates and then
placed on serum free DMEM for 24 hours to render them
quiescent. Fibroblasts were then incubated in DMEM with
5% FBS alone or in the same medium with IL-1β (2.5 ng/ml)
for 24 hours. At the end of the incubation period the
supernatant was aspirated and fresh media containing 30
μM of arachidonic acid was added to the plates. After 30
min of incubation the supernatant was collected and saved
at -70°C for later eicosanoid analysis. The cells were
then resuspended and divided into two aliquots, which
were used for RNA and protein extractions, respectively.
The above experiments were repeated in HF-IPF (n = 2) and
HF-NL (n = 2) using serum free media conditions.
Prostanoids were measured by modified stable isotope
dilution assays that used gas chromatography-negative
ion-chemical ionization mass spectrometry as previously
described [ 23 ] . Briefly, deuterium-labeled internal
standards of PGE
2 , PGF
2α , TXB
2 , and 6-keto-PGF
1α were added to the supernatants with
isopropyl alcohol. Isopropyl alcohol was removed by
evaporation under nitrogen. After acidification to pH
3.5, the samples were extracted on preconditioned C-18
PrepSep columns (Fisher Scientific, Fair Lawn, NJ), and
eluted with ethyl acetate. The extract was then converted
to a pentafluorobenzyl ester by treatment with a mixture
of 12.5% pentafluorobenzyl bromide in acetonitrile and
disopropylethylamine at room temperature for 30 min.
After evaporation of reagents, the residue was subjected
to TLC plates, using the solvent system
chloroform/ethanol (93:7, vol/vol) for PGF
2α and TXB
2 , and ethyl acetate/methanol (93:2,
vol/vol) for 6-keto-PGF
1α and PGE
2 . Then PGF
2α was converted to trimethylsilyl
ether derivative by treatment with N,O-bis
(trimethylsilyl) trifluoroacetamide and
dimethylformamide. The methoxime derivative of TXB
2 , PGE
2 and 6-keto-PGF
1α was made by treatment with 2%
methoxamine hydrochloride in pyridine at 70°C for 60 min,
followed by evaporation of the pyridine, addition of
water, and extraction with ethyl acetate. Derivatization
was completed by formation of the trimethylsilyl
derivatives by treatment with N,O-bis (trimethylsilyl)
trifluoroacetamide and pyridine. Eicosanoids were
quantified by measuring the ratio of the intensity of
ions m/z 569/573 for PGF
2α , m/z 614/618 for TXB
2 and 6-keto-PGF
1α , and m/z 524/528 for PGE
2 . An analytical blank for each of
these products was determined by measuring the amount of
nondeuterated material, detected after extracting and
analyzing 2 ml of saline to which the deuterium-labeled
internal standards had been added.
Western analysis
After washing with PBS at pH 7.4, pellets were lyzed
in solubilization buffer containing 50 mM TRIS at pH 8,
1% Tween 20, 10 mM phenylmethylsulphonyl fluoride,
diethyldithiocarbamic acid, leupeptin and pepstatin A
(all from Sigma Chemical), sonicated, boiled with gel
loading buffer (62.5 mM TRIS-HCl, at pH 6.8, 10%
glycerol, 2% SDS, 5% β-mercaptoethanol, and bromophenol
blue), and centrifuged at 15,000 x
g for 10 min. Equal amounts of
protein (70 to 100 μg) were separated by electrophoresis.
SDS-PAGE was performed using a 7.5% separating gel with a
4% stacking gel. The resolved proteins were transferred
electrophoretically to nitrocellulose membranes
(Hybond-ECL, Amersham Corp.). After transfer, the filters
were incubated overnight at 4°C in a blocking solution
(20 mM TRIS base, 137 mM sodium chloride at pH 7.6, 5%
powdered milk, 3% BSA), and incubated with primary
polyclonal rabbit antibodies against COX-2 at a dilution
1:1000 (Cayman Chemical, Ann Arbor, MI), for 1 hour at
room temperature. The filters were washed (TBS-0.1% Tween
20 at pH 7.6) and incubated with horseradish peroxidase
linked secondary antibodies at a dilution 1:4000
(Amersham). After washing, the membranes were incubated
with luminol based chemiluminescence reagent (DuPont NEN
Research Products, Boston, MA).
Northern analysis
Cell pellets were lyzed and RNA extracted using the
RNeasy method ®(Qiagen), following the manufacturer's
instructions. RNA was quantified by determining light
absorbance at 260 nm and then fractioned by
electrophoresis (10 μg per lane) on a 1% agarose
MOPS/formaldehyde gel. The RNA was denatured prior to
loading by incubating the RNA at 65°C for 10 min in a
loading buffer comprising 1X MOPS, 50% formamide, 6.5%
formaldehyde, 5% glycerol, 0.1 mM EDTA, 0.025%
bromophenol blue, 0.025% xylene cyanol. The RNA was
transferred by gravity-assisted capillary method with 6X
SSC to nylon hybridization membrane, and then fixed to
the membrane by UV crosslinking (Stratalinker 1200 μj/cm
2). Prehybridization and hybridization were performed at
42°C and using Quick Hyb ®(Stratagene) as hybridization
solution. The COX-2 probe was random primed following the
directions of the manufacturer (Megaprime ®,
Amersham/Pharmacia). The membrane was then washed at a
final stringency of 0.2X SSC, 0.1% SDS, at 60°C for 30
min. The membrane was wrapped in plastic wrap and exposed
to Kodak XR film at -70°C with intensifier screen
overnight.
Statistical methods
All results are presented as medians with their range.
Comparisons between HF-IPF and HF-NL were done using the
Mann-Whitney test. A
P value of <0.05 was considered
significant.
Results
Baseline and stimulated COX-2 activity in HF-IPF
and HF-NL
Unstimulated eicosanoid production was similar in both
HF-IPF and HF-NL (Fig. 1, a-d). When fibroblasts were
stimulated with IL-1β there was a significant and similar
upregulation of PGE
2 production in both HF-IPF and HF-NL
(28.35 [range: 9.09-89.09] versus 17.12 [8.58-29.33]
ng/10 6cells/30 min, respectively;
P = 0.25; [Fig. 1a]).
IL-1β-stimulated production of TXB
2 (stable metabolite of the active TXA
2 ), PGF
2α , and 6-keto-PGF
1α (stable metabolite of PGI
2 ) increased modestly in every case,
except TXB
2 production by HF-NL, which decreased
(0.75 [0.15-2.58] ng/10 6cells/30 min at baseline versus
0.61 [0.21-1.64] ng/10 6cells/30 min with IL-1β
stimulation) (Fig. 1b). Results of PGE
2 production were similar when
experiments were conducted in serum free media conditions
(results not shown).
IL-1β stimulated TXB
2 production was significantly greater
in HF-IPF (1.92 [1.27-2.57] ng/10 6cells/30 min) than in
HF-NL (0.61 [0.21-1.64] ng/10 6cells/30 min;
P = 0.007) (Fig. 1b); baseline TXB
2 production was not significantly
different between the two cell groups (1.73 [0.77-2.53]
versus 0.75 [0.15-2.58] ng/10 6cells/30 min, in HF-IPF
and HF-NL, respectively;
P = 0.17 [Fig. 1b]). Because PGI
2 and TXA
2 have opposing effects
in vivo , we calculated the ratio
of their metabolites (6-keto-PGF
1α :TXB
2 ) and found a significantly lower
ratio in HF-IPF at baseline (0.08 [0.04-0.52] versus 0.12
[0.11-0.89] in HF-IPF and HF-NL, respectively;
P = 0.028) and a similar trend
under stimulated conditions (0.24 [0.05-1.53] versus 1.08
[0.51-3.79] in HF-IPF and HF-NL, respectively;
P = 0.09 [Fig. 2]).
Baseline and stimulated COX-2 expression
Western blot in unstimulated fibroblasts showed no
detectable COX-2 protein in either group of cells, while
IL-1β significantly induced COX-2 to a similar degree in
IPF and normal lung fibroblasts (Fig. 3). Northern blot
showed minimal COX-2 mRNA in unstimulated cells and
significant upregulation of COX-2 mRNA expression when
stimulated with IL-1β in both HF-IPF and HF-NL (Fig.
4).
Discussion
Several factors modulate fibroblast proliferation and
collagen production, including mitogenic cytokines (e.g.,
transforming growth factor β [TGFβ], platelet-derived
growth factor [PDGF], basic fibroblast growth factor
[bFGF]), eicosanoids (i.e., PGE
2 , TXB
2 , and PGI
2 ), and antifibrogenic cytokines (e.g.
IFN-γ) [ 1 2 3 ] . It is very likely that a complex
interaction among these factors exists in the tissue repair
process, and it is possible that pathologic fibrosis, as in
IPF, results from phenotypical alterations in fibroblasts
that affect the "normal" interaction of these factors.
Our results show that stimulation of primary cultures of
human lung fibroblasts with the proximal cytokine IL-1β
upregulates COX-2 protein and mRNA expression to a similar
degree in normal and IPF fibroblasts. TXA
2 production tended to be greater in IPF
than in normal fibroblasts at baseline; when stimulated
with IL-1β this difference became statistically
significant. The ratio of PGI
2 to TXA
2 metabolites was lower in IPF
fibroblasts at baseline and with IL-1β stimulation. The
above results suggest that a decreased PGI
2 :TXA
2 ratio could be a phenotypic alteration
present in IPF fibroblasts, resulting in a loss of their
capacity to autoregulate proliferation and extracellular
matrix production.
The effects of PGs on cell proliferation and collagen
production have been widely studied in different cell types
[ 13 14 15 16 17 26 ] . TXA
2 has been studied extensively because
of its apparent role in atherosclerosis, due to its
prothrombotic and mitogenic activities on vascular smooth
muscle cells [ 15 16 ] . These mitogenic effects are
potentiated by growth factors [ 15 16 27 28 ] . In vascular
smooth muscle cells TXA
2 stimulates synthesis of bFGF and
increases the expression of the proto-oncogenes
c-fos ,
c-myc , and
egr-1 , which are associated with
entry into the cell growth cycle [ 15 ] . In addition, TXA
2 increases proliferation of fibroblasts
[ 13 ] and smooth muscle-like glomerular mesangial cells [
14 ] .
On the other hand, PGI
2 decreases vascular smooth muscle cell
proliferation and collagen and glycosaminoglycane
synthesis, via activation of adenylyl cyclase and
subsequent production of cAMP [ 17 ] . Betaprost, an analog
of PGI
2 , decreases procollagen I and III mRNA
expression in cardiac fibroblasts [ 18 ] . These effects
may counteract the profibrotic effects seen with TXA
2 and it is possible that an alteration
of a "normal" physiologic balance between PGI
2 and TXA
2 could increase tendency towards
fibrogenesis.
It is important to mention that our experiments were
conducted at similar passage levels (passage 6 to 8) in
both groups, since senescence of fibroblasts is associated
with a shift from the biosynthesis of PGI
2 to TXA
2 [ 24 25 ] . It is possible that the
difference seen in our study between HF-IPF and HF-NL could
result from comparing fibroblasts of different ages. HF-IPF
might have been harvested from fibrotic lesions where
fibroblasts had previously undergone a greater number of
cell divisions than HF-NL, obtained from nonfibrotic lungs.
Although this is a possibility, the age-related shift in PG
production has only been shown at very high cell passages
and has not been documented
in vivo .
We also found that both HF-IPF and HF-NL had similar PGE
2 production at baseline, and a similar
increase when stimulated with IL-1β. PGE
2 can decrease fibroblast proliferation
and collagen synthesis, and increase collagen degradation [
5 6 7 8 ] .
Recent reports suggesting decreased COX-2 expression and
PGE
2 production in IPF fibroblasts have
received significant attention [ 12 20 21 ] . In our study
we found that both COX-2 protein expression and PGE
2 production were upregulated to a
similar degree in IPF and normal lung fibroblasts. We
believe that differences in methodology and patient
selection may explain the discrepancies with other studies.
Vancheri and collaborators [ 20 ] found that
TNF-α-stimulated fibrotic lung fibroblasts had decreased
COX-2 expression and PGE
2 production, but they further showed
that these findings were a result of decreased expression
of TNF-α receptors. The latter finding would argue against
a primary defect in COX-2 expression, since no other
stimulus, other than TNF-α, was tested. In another study,
Keerthisingam
et al. [ 21 ] reported that fibrotic
lung fibroblasts had decreased COX-2 expression and PGE
2 production in response to TGFβ
stimulation. This study differed from ours in that a
different stimulus was used. Of significance is the fact
that the COX-2 gene is known to be NF-κB dependent, and
IL-1β, but not TGFβ, is a potent inducer of NF-κB
activation. Hence, the pathway involved in the induction of
the COX-2 gene by IL-1β and TGFβ may be different.
Furthermore, a significant proportion of the fibroblasts
used in the study by Keerthisingam
et al. [ 21 ] were obtained from
patients with systemic sclerosis, which makes their
fibroblast population more heterogeneous.
Wilborn
et al. [ 12 ] also reported a
decreased production of PGE
2 by IL-1β-stimulated IPF fibroblasts,
due to decreased COX-2 expression [ 12 ] . There is a
possibility that patient selection may have differed
between the two studies. However, we feel certain that the
diagnostic accuracy of our patient population was high, due
to the fact that 5 out of a total of 7 IPF subjects
included in our study underwent lung transplantation with
confirmatory pathology results consistent with IPF. The
other 2 subjects had biopsy-proven IPF. In addition, our
results were similar when comparing lung fibroblasts
obtained from 5 subjects with advanced stage IPF with those
of 2 subjects at an earlier stage of their disease, who had
received no therapy. Although the reasons for our different
results are unclear, the fact that we found similar COX-2
expression and PGE
2 production in normal and IPF lung
fibroblasts suggests that loss of COX-2 expression is not a
universal characteristic of fibroblasts cultured from the
lungs of subjects with IPF.
Conclusion
We have found that fibroblasts cultured from normal and
IPF human lungs have a significant and similar induction of
the COX-2 enzyme when stimulated with IL-1β, but that IPF
fibroblasts produced more thromboxane and had a
significantly lower prostacyclin:thromboxane ratio. We
hypothesize that the lower PGI
2 :TXA
2 ratio seen in HF-IPF may be a
phenotypic alteration that plays a role in the pathogenesis
of IPF.
Abbreviations
COX = cyclooxygenase; HF = human fibroblasts; NL =
normal lungs; IPF = idiopathic pulmonary fibrosis; IFN =
interferon; IL = interleukin; PG = prostaglandin; TX =
thromboxane; PGI
2 = prostacyclin.