
  
    
      
        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.
      
    
  
