Abstract. The ancient drug colchicine has repeatedly been proposed as a noveldrug for therapy of pulmonary fibrosis. The present study was undertaken to add tothe knowledge on colchicine’s antiinflammatory and antifibrotic properties and thushelp determine its actual rank in the treatment of pulmonary fibrosis. In vitro cellculture experiments with stimulated and unstimulated normal donor peripheralblood mononuclear cells (PMNC) and a human lung fibroblast cell line (WI-38)were used to determine the effects of colchicine on PMNC cytokine release (inter-leukin-6 and tumor necrosis factor-a) as well as on fibroblast proliferation andcollagen synthesis rates. Reverse transcriptase polymerase chain amplifications ofa1(III) collagen were done to detect collagen messenger ribonucleic acid (mRNA)expression. Colchicine did not significantly modulate tumor necrosis factor-a(TNF-a) and interleukin-6 (IL-6) release of PMNC. Colchicine inhibited fibroblastproliferation and total collagen synthesis significantly at concentrations obtainablein serum in vivo. Transcription of thea1(III) collagen gene into mRNA continuedunder colchicine. We conclude that colchicine is a potent in vitro inhibitor offibroblast functions in terms of proliferation and collagen synthesis. The mechanismof collagen inhibition is more likely an inhibition of cellular collagen secretion thana switch off of collagen mRNA transcription. On the other hand, although colchicineis known to inhibit many leukocyte functions, it is a poor inhibitor of cytokinesknown to be important for fibrogenesis (e.g. IL-6, TNF-a, IL-1, platelet-derivedgrowth factor, and transforming growth factor-b). This makes colchicine, at leastfrom a theoretical standpoint and as concluded from in vitro studies, a preferablecandidate for a combined therapeutic strategy.
The fibrotic response to lung injury is far from understood. Regardless of the multi-factorial origins of fibrosis, chronic inflammation causes mesenchymal cells to migrateto the site of injury, proliferate, and subsequently synthesize extracellular matrix com-ponents [7]. Neither the factors that contribute to chronic inflammation nor those thatcontribute to excess or sustained matrix production in fibrosis are fully characterized.The effectiveness of current therapeutic strategies is still in doubt. Although 40–70%of patients treated with corticosteroids feel subjective improvement, results of objectiveclinical tests confirm this in only 10–30% [25]. Agents such as azathioprine or cyclo-phosphamide have been reported to be of benefit [3, 19, 29]; the number of controlledclinical trials is, however, limited [24]. The ancient drug colchicine has attractedrenewed interest and has been proposed repeatedly as a novel drug for therapy ofpulmonary fibrosis [37], especially since a survival advantage was reported whentreating patients with cirrhosis of the liver [20]. However, the efficacy of colchicine inpulmonary fibrosis is not established. Only one retrospective study has indicated fa-vorable results of a colchicine treatment in idiopathic pulmonary fibrosis (IPF) [27].There is a lack of published data on in vitro or ex vivo model systems as well, with theresult that many of the colchicine mechanisms at subcellular levels are not yet under-stood. Hypothetically, the drug ought to be effective in fibrosis because of its antipro-liferative and antiinflammatory actions and its suppression of alveolar macrophage-derived growth factor and fibronectin, which might be associated with antifibroticactions [22, 30]. This study was carried out to add to our knowledge of the potentialof colchicine in lung fibrosis. It provides in vitro data on antiinflammatory and anti-fibrotic properties of colchicine in terms of: (1) colchicine effects on peripheral bloodmononuclear cells (PMNC) (generation of tumor necrosis factor-a and interleukin-6 bystimulated PMNC; lymphocyte proliferation) and (2) colchicine effects on prolifera-tion, total collagen synthesis, and typea1(III) collagen messenger ribonucleic acid(mRNA) expression of a human lung fibroblast cell line.
Materials and Methods
PMNC were obtained from buffy coats of five healthy blood donors and were separated by density cen-trifugation over Ficoll-Hypaque. Cells were suspended in RPMI 1640 (Biochrom KG) supplemented with10% heat-inactivated AB serum, 1%L-glutamine, and 1% penicillin-streptomycin (GIBCO). PMNC wereseeded at 1 × 106 cells/ml into 10-ml culture plates (for cytokine measurements) or 96-well plates (forproliferation assays) and stimulated in parallel using phytohemagglutinin (PHA; HA 16/17; Wellcome, UK)at 5 mg/ml. Colchicine (Colchicum Dispertt, Kali-Chemie) was added at 1–25 ng/ml when appropriate. Cellswere cultured at 37°C in a 5% humidified CO2 atmosphere. Supernatants for cytokine measurements wereharvested at 24 h, a time at which preliminary experiments had shown maximum concentrations (results notshown). Cell counts were determined after 72 h and mRNA expression after 4 h of incubation. Trypan blueexclusion was used to assess cell viability.
42 P. Entzian et al.
Assays to Determine Cytokine Concentrations
Tumor necrosis factor-a (TNF-a) activity was determined using a WEHI 164 subclone 13 fibrosarcoma lyticassay [12]. Briefly, serial sample dilutions were incubated with the hybridoma cells for 20 h before stainingwith MTT (dimethylethimazol-diphenyltetrazolium bromide, Sigma, dissolved in phosphate-buffered saline(PBS) at 5 mg/ml). MTT is converted by mitochondria of living cells to the blue colored substance formazan,and the amount of formazan produced is proportional to the number of cells present [8, 16]. After 4 h, theoptical density was determined in a micro-ELISA reader (MR580, Dynatech Laboratories, Inc.) at a wave-length of 550 nm. TNF-a was expressed in pg/ml using an internal standard of human recombinant TNF-a
(Boehringer). An influence of colchicine on this assay system was not detected in the concentrations applied.Interleukin-6 (IL-6) activity was measured using the IL-6-dependent mouse hybridoma cell line B13.29
clone B9 [1]. Serial dilutions of the heat-inactivated samples were incubated with the hybridoma cells for 72h before performing the MTT assay. IL-6 activity was calculated by probit analysis from serial dilutions andexpressed in ng/ml using an internal standard of human recombinant IL-6 (Boehringer). Colchicine did notinfluence the assay in the concentrations used.
Assay of Lymphocyte Proliferation
Cell densities of PHA- or IL-2-stimulated PMNC were determined in the presence or absence of colchicine(1–25 ng/ml) using the [3H]thymidine incorporation method (185 GBq/mmol; Amersham Buchler). Radio-labeling was allowed during the last 4 h of theincubation period by adding 20 ml of medium containing[3H]thymidine to each well (final activity, 18.5 kBq/200 ml of medium). Cells were lysed and harvested ontofilter discs. Radioactivity was measured by liquid scintillation (Minaxi 3200, Packard). Lymphoproliferationwas expressed in counts per minute (cpm).
Assay of Fibroblast Proliferation
A human lung fibroblast cell line was obtained from the American Type Culture Collection (WI-38,derived from normal embryonic lung tissue of a Caucasian female) and grown in basal medium (Eagle)(BME) according to standard protocols [38]. Only early passage cell cultures (days 15–35, split ratio 1:2 onceweekly) were used in these experiments. Fibroblasts were seeded at subconfluent density of 0.1 × 106
cells/ml (BME, 10% fetal calf serum (FCS) added) into 24-well flat bottom microtiter plates (0.5 ml/well,Greiner) and cultivated for 24 h. After a washout period, medium was changed into BME supplemented withFCS 10%, and colchicine (10 or 25 ng/ml) was added. Medium was removed completely after 72 h, and cellswere lysed with Triton X-100 (1.5 ml, Serva). After 5 min, cell nuclei were fixed using 10% formaline, andthe number of cell nuclei was determined in an electronic particle counter (Coulter counter).
Assay of Collagen Synthesis by In Vitro Cultured Fibroblasts
The principle of the assay is the incorporation of [3H]proline into proteins and its hydroxylation to [3H]hy-droxyproline in collagenous proteins as outlined in Ref. 38. Since quantitative amounts of hydroxylation inproteins other than collagen are insignificant, the content of hydroxyproline reflects concentration of collagen[21]. The method was adapted such that nonincorporated radioactivity was removed by ultrafiltration insteadof dialysis. Briefly, fibroblasts (WI-38) were grown in 24-well flat bottom microtiter plates to visualconfluence and incubated in presence of 50 mg/ml ascorbate, 10% FCS, or 0.4% FCS + 50 ng/ml basicfibroblast growth factor (bFGF), and 10 mCi [3H]proline/ml medium (L-[2,3-3H]proline, Dupont) and 1–10ng/ml colchicine were added. After 24 h, cell pellets as well as cell-free supernatants were harvested,freeze-thawed three times, pelleted by centrifugation, and the supernatant was washed four times andultrafiltrated (centrifugal concentrators Microsep Filtron, Karlstein; molecular weight cutoff 10,000; aquadest. supplemented with 0.3 ml/ml Phenylmethylsulfonyl fluoride (PMSF), proteinase inhibitor, Roth).Subsequently, samples were resuspended in 6N HCl (Merck), hydrolyzed (110°C, 24 h), dried in a vacuum-
Colchicine and Pulmonary Fibrosis 43
desiccator, and the amounts of [3H]proline and [3H]hydroxyproline were determined by automated aminoacid analysis. Collagen concentrations were calculated according to the method of Wiestner et al. [38].Collagen synthesis was expressed as percent collagen of generated total protein and as radiolabeled hy-droxyproline/cell.
Detection of In Vitroa1(III) Collagen Messenger Ribonucleic Acid (mRNA)Generation by Cultured Fibroblasts
Extraction of mRNA and Reverse Transcriptase Reaction.WI-38 cells were grown to visual confluencein 24-well flat bottom microtiter plates and incubated in the presence or absence of 10% FCS, and 10 or 25ng/ml of colchicine. After 4 h, poly(A)+ mRNA was isolated by magnetic isolation with Dynabeadsoligo(dT)25 (Dynal) according to the manufacturer’s protocol. The quantity and quality of the RNA wereassessed spectrophotometrically. First-strand complementary DNA (cDNA) was synthesized using Super-script reverse transcriptase (Life Technologies, Inc.).
Polymerase Chain Reaction.Oligonucleotide primers were designed by computer-assisted search onPC GENE software (Intelligenetics) and synthesized on an oligonucleotide synthesizer (MilliGen Biosearch,Burlington). Sense primers: 58-GGT CAG CAG GGT GCA ATC GG-38 and antisense 58-CTC ACA GCCTTG CGT GTT CG-38 generate a 960-base pair (bp) product of the procollagena1(III) transcript; senseprimers: 58-AGC GGG AAA TCG TGC GTG-38 and antisense 58-CAG GGT ACA TGG TGG TGCC-38 yield a 309-bp product of theb-actin transcript which was used as a control. The PCR was performedwith 5-ml template in a total volume of 50 ml, containing 30 mM Tricine, pH 8.4, 2 mM MgCl2, 5 mMb-mercaptoethanol, 0.1% thesit, 0.01% gelatin, 1 mM sense and antisense primers, 200 mM of each dNTPs,and 2.5 units Taq polymerase. After 35 PCR cycles (Biometra personal cycler 20, thermal profile: denatur-ation 94°C 2 min, primer annealing 59°C 2 min, extension 72°C 3 min), 10 ml of each PCR mixture waselectrophoresed on 1% agarose gel, and DNA was visualized by ethidium bromide staining. A 1,114-bpladder (Boehringer Mannheim) was used as a DNA molecular weight marker.
Statistics
Data are expressed as means ± S.E., and comparisons between values were made using the rank sum test ofWilcoxon, Mann, and Whitney, or the rank correlation test of Spearman.
Results
Colchicine Does Not Significantly Influence TNF-a And IL-6 Release
The rationale to determine TNF-a and IL-6 was their importance in early events ofinterstitial lung diseases, e.g. in sarcoidosis [18]. Control experiments demonstratedthat unstimulated PMNC produced neither TNF-a nor IL-6 in significant amounts. Cellviability remained acceptable throughout the experiments (>93%). Colchicine effectson TNF-a and IL-6 synthesis remained insignificant (Fig. 1), although there was aslight trend toward lower TNF-a concentrations when augmenting colchicine supple-ment.
Lymphocyte Proliferation Is Inhibited by Colchicine Independently of IL-2
Colchicine-induced inhibition of lymphocyte proliferation is depicted in Figure 2. Thisoccurred independently of IL-2 since addition of this lymphocyte proliferation inducing
44 P. Entzian et al.
cytokine at 8.75 units/ml did not prevent the inhibition by colchicine. Control experi-ments revealed that unstimulated PMNC incorporated insignificant amounts of radio-activity, whereas PHA or IL-2 stimulation resulted in distinctly enhanced incorpora-tion. Decreased viability of cells did not influence results significantly since trypanblue staining increased slightly but remained below 7%. Levels of significance were *p< 0.005; **p < 0.05.
Colchicine Inhibits Both Fibroblast Proliferation and Collagen Synthesis Rates
Colchicine influenced both fibroblast proliferation and collagen synthesis rates calcu-lated as percent of total protein generated (Figs. 3 and 4). Analogous results have beenobtained when calculating the radiolabeled hydroxyproline/cell (not shown). Colchi-cine inhibited fibroblast functions dose dependently (p < 0.005 in proliferation,p <0.001 in collagen synthesis assays). Control experiments revealed no cell proliferationand insignificant collagen synthesis using culture medium supplemented with only0.4% FCS.
Colchicine Does Not Switch Off Collagena1(III) mRNA Expression
Maximum a1(III) gene transcription was observed at 4 h after cell stimulation (notshown). Addition of 10 or 25 ng/ml colchicine did not prevent fibroblasts from tran-scribing the collagena1(III) gene as demonstrated in Figure 5. The figure shows a
Fig. 1. Colchicine failed to exert significant influence on IL-6 and TNF-a synthesis by cultured PMNCstimulated by PHA (5 mg/ml). Cytokines were determined by bioassays that were not influenced by col-chicine in the applied concentrations (not shown). Data are given in means ± S.E. of the respective cytokine.
Colchicine and Pulmonary Fibrosis 45
representative gel electrophoresis of experiments done threefold. Amplification ofb-actin was used as a control experiment (‘‘housekeeping gene’’).
Discussion
The management of pulmonary fibrosis remains controversial. An ideal drug or a drugcombination has not yet been found [6]. The ancient substance colchicine has attractedconsiderable interest and has been proposed repeatedly for use in the therapy ofpulmonary fibrosis [37]. Since chronic inflammation and subsequent fibrogenesis arethe main parameters in fibrosis, we were interested in the antiinflammatory and anti-fibrotic properties of colchicine. This study presents in vitro data questioning themonotherapeutic use of colchicine in lung fibrosis. We found a different effect ofcolchicine on inflammation and fibrogenesis. In concentrations obtainable in vivo(serum; approximately 4 ng/ml; tissue, probably even higher since the volume ofdistribution is nearly 700 liters [31]), fibrogenesis is inhibited more effectively thanrelease of proinflammatory mediators.
Specific antiinflammatory properties of colchicine are demonstrated by its well-known ability to inhibit in vitro lymphocyte proliferation (see Fig. 2). In acute goutyarthritis, it interferes with polymorphonuclear leukocyte migration and phagocytosis[22]. Furthermore, colchicine has been shown to protect against TNF-induced toxicityin mice [34]. On the other hand, colchicine failed to inhibit, or had a very slight effectonly, on two cytokines that are thought to orchestrate nonspecific inflammatory re-sponse; that is, colchicine did not significantly inhibit TNF-a or IL-6 synthesis byPMNC in our hands (Fig. 1). Recently, Allen et al. [2] found opposite results and
Fig. 2. Colchicine inhibits PMNC proliferation independently of IL-2. Addition of the proliferation-inducingcytokine IL-2 (8.75 units/ml) did not prevent the colchicine effects. Levels of significance: *p < 0.01; **p< 0.05. Data are given in means ± S.E. of incorporated [3H]thymidine in cpm.
46 P. Entzian et al.
reported a significant reduction in TNF-a generation. The data are, however, notcomparable because (1) Allen used lipopolysaccharide (LPS)-stimulated isolatedmonocytes and thus detected not PMNC, but only monocyte-derived TNF; and (2)colchicine was added at 50 ng/ml, which exceeds the in vivo obtainable concentrationsby far.
Additional evidence that colchicine might not be an ideal drug to inhibit inflam-matory mediators can be seen in its in vitro induction of the acute phase cytokine IL-1[14, 23, 32]. Furthermore, colchicine failed to inhibit TGF-b and induced platelet-derived growth factor (B) [36]. Both TGF-b and PDGF have been considered keycytokines in the regulation of not only the inflammatory response (especially TGF-b)but of the fibrogenic effects as well. PDGF is an important stimulator of fibroblastproliferation, and TGF-b of fibroblast extracellular matrix production [4, 5, 13]. Thismight provide one explanation for the failing therapeutic efficacy of colchicine in 9 outof 23 patients with IPF studied by Peters et al. [27]. Although only few clinical data aregiven in their paper, we assume that differences in mediator profiles and activities ofinflammation cause the therapeutic failures rather than differences in severity of dis-ease, since pulmonary function tests before colchicine treatment were similar in theresponding and the nonresponding study groups [27].
We found in vitro fibroblast proliferation and collagen synthesis inhibited bycolchicine (Figs. 3 and 4); these results are confirmatory of reports published long ago,e.g. [9, 28]. Results are nevertheless relevant and worth bringing to the attention ofpulmonary clinicians since the suggestion of colchicine as an antifibrotic drug is basedmainly on these findings.
Fig. 3. Colchicine at 10 and 25 ng/ml inhibited in vitro proliferation of a human lung fibroblast cell line(WI-38) significantly. Cells were stimulated for 72 h by 10% FCS; the number of cells was determinedelectronically (hemocytometer, *p < 0.05; **p < 0.005). Data are given in means ± S.E. of quadruplicateexperiments.
Colchicine and Pulmonary Fibrosis 47
Changes in the production of collagen do not necessarily reflect changes in mRNAgeneration since collagens are known to undergo extensive posttranscriptional modi-fications [7], and colchicine is known to inhibit procollagen secretion [11]. This isprobably predominant in type III collagen synthesis where mRNA expression of thea1chain of the collagen helix continued to occur under colchicine (Fig. 5). However,although a switchoff on the transcriptional level is not likely to exist, this cannot beexcluded as a possibility since analysis of collagen transcripts was restricted to thea1chain of type III collagen.
Many cell culture studies on fibroblast functions, including our data, give rise tospeculation that fibrogenesis might be inhibited by colchicine in vivo as well [24, 25].Beneficial colchicine effects have been confirmed in rat models with bleomycin andirradiation-induced pulmonary fibrosis [10, 26, 39]; encouraging results also emergedfrom the retrospective therapeutic IPF study mentioned above [27]. Since prospectivestudies on IPF are not available, the available experience with colchicine treatment infibrosis of the liver and primary biliary cirrhosis might help us arrive at a preliminaryevaluation of this therapeutic strategy. The results cannot, however, be considereduncontradicted since no clear-cut advantages have emerged, although some papers arerather optimistic, and a trend toward less clinical deterioration and improved survivalhad appeared [15, 22].
It may be that the point of time at which therapy is initiated largely determinestherapeutic outcome. Prognosis of IPF is associated positively with early stages ofdisease [33]. Early disease is also associated with a better response to immunosup-
Fig. 4. Colchicine inhibited both 10% FCS- or bFGF (50 ng/ml)-stimulated collagen synthesis by a humanlung fibroblast cell line (WI-38). Data are given in means ± S.E. of percent collagen of total protein generatedby the fibroblasts; at 10 ng/ml colchicine, results were highly significant (p < 0.001) regarding both stimuli.Analogous results have been obtained when calculating the amount of radiolabeled hydroxyproline/cell (notshown).
48 P. Entzian et al.
pressive therapy [35]. This accounts, at least in part, for the colchicine benefit in theanimal model with bleomycin-induced pulmonary fibrosis and the failure of therapy inIPF patients. Whereas colchicine treatment had been started simultaneously with theharmful event in animal experiments [26, 39], this is not the case in IPF patients forobvious reasons.
In summary, colchicine has antiinflammatory properties because of its interferencewith PMNC functions in terms of cell motility and secretion of chemotactic factors[22]. It arrests mitosis independently of IL-2 (Fig. 2) and reduces DNA synthesis [22].It is a good inhibitor of fibroblast proliferation and of collagen synthesis. The mecha-nism of type III collagen inhibition is most likely a posttranscriptional one. Colchicinefurthermore has been shown to inhibit alveolar macrophage-derived growth factor andfibronectin [30] and to augment collagenase generation [17]. On the other hand, we andothers found colchicine a poor inhibitor of inflammatory and fibrosis-associated cyto-kines (IL-1, IL-6, TNF-a, PDGF, TGF-b). This makes colchicine, at least from atheoretical standpoint and concluded from in vitro studies, a preferable candidate for acombined therapeutic strategy. Besides taking advantage of the described colchicinebenefits on fibroblast functions, we should look for one or more other drugs that effectinflammatory and fibrosis-associated cytokines as well as collagen gene transcriptionmore potently than colchicine.
Fig. 5. Colchicine does not switch off fibroblasta1(III) collagen mRNA generation. mRNA synthesis wasdetermined by PCR amplification of the cDNA of collagen typea1(III). Panel A,control experiment usingtheb-actin gene (housekeeping gene).Panel B,amplification ofa1(III) collagen.Lanes 1and7,molecularweight marker;lane 2,PCR-positive control experiment;lane 3,PCR-negative control;lane 4,experimentwithout colchicine supplement;lanes 5and6,addition of colchicine at 10 and 25 ng/ml. Colchicine does notswitch off collagen typea1(III) mRNA expression. Data show a representative gel electrophoresis; resultshave been reproduced three times.
Colchicine and Pulmonary Fibrosis 49
Acknowledgments.We acknowledge gratefully the methodological help by Prof. J. Gerdes, Forschungsin-stitut Borstel, and by Prof. P. K. Mu¨ller, University of Lubeck. We are deeply indebted to S. Ross and S.Kutsch for expert technical assistance.
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