Clinical observations indicate that in chronic obstructive pulmonary disease patients, the long-acting muscarinic antagonist tiotropium delays decline in airway function, suggesting that cholinergic mechanisms contribute to long-term structural changes. Human lung fibroblasts express muscarinic receptors and the present study aimed to explore their role in controlling collagen synthesis.
MRC-5, HEL-299 and primary human lung fibroblasts (phLFb) were cultured. Incorporation of [3H]-proline into cellular proteins was determined as measure of collagen synthesis.
In MRC-5 cells, the muscarinic agonist carbachol enhanced [3H]-proline incorporation in a concentration-dependent manner (effective concentration of 50%: 220 nM, increase at 10 µM by 40–55%, in a different series of experiments). Likewise, 10 µM oxotremorine caused an increase of ∼65%. For comparison, transforming growth factor-β1 (5 ng·mL−1) caused an increase of ∼80%. Effects of carbachol on total [3H]-proline incorporation and collagenase-sensitive [3H]-proline fraction were similar. The effect of 10 µM carbachol was inhibited by tiotropium (inhibitory concentration of 50%: 110 pM), prevented by pertussis toxin and the mitogen-activated protein kinase inhibitor, PD 98059. Muscarinic agonists also enhanced [3H]-proline incorporation in a tiotropium-sensitive manner in HEL-299 cells and phLFb.
In human lung fibroblasts, muscarinic receptors exert stimulatory effects on collagen synthesis. Prolonged blockade of muscarinic-induced collagen synthesis may contribute to reported beneficial long-term effects of anticholinergics in chronic obstructive pulmonary disease.
- Airway remodelling
- collagen synthesis
- lung fibroblasts
- mitogen-activated protein kinase
- muscarinic receptors
Airway remodelling is a pathological feature observed in chronic inflammatory and obstructive airway diseases. Although airway remodelling processes in asthma and chronic obstructive pulmonary disease (COPD) are somewhat different, fibrotic alterations are observed in both diseases 1–3. Based on their bronchodilatatory effects, anticholinergic drugs constitute an essential element in the therapy of obstructive airway diseases, particularly in COPD 4. Moreover, preliminary clinical data indicate that, in patients with COPD, treatment with the long-acting muscarinic antagonist tiotropium may delay the decline in airway function 5, 6, suggesting that cholinergic mechanisms may contribute to long-term structural changes, in addition to the well-known acute bronchoconstrictory and secretory effects 7, 8. Indeed, muscarinic agonists were found to enhance the proliferative response of human and bovine airway smooth muscle to growth factors 9, 10. Moreover, in sensitised guinea pigs, tiotropium attenuated airway smooth-muscle mass thickening and prevented mucus-gland hypertrophy induced by repeated allergen challenges 11, 12.
Since fibrotic alterations are crucial in airway remodelling 1–3, investigatons have recently begun into the expression and function of muscarinic receptors in pulmonary fibroblasts. The expression of different muscarinic receptors in primary human lung fibroblasts (phLFbs) and MRC-5 cells, a human fibroblast cell line derived from normal foetal lung tissue 13, has been revealed. In both cell types, M2 appeared to be the predominant receptor sub-type 14. Moreover, in both cell types, muscarinic agonists induced a proliferative response 14, 15.
The present study aimed to further characterise the functional significance of muscarinic receptors expressed in human pulmonary fibroblasts and investigated whether collagen synthesis is controlled by muscarinic mechanisms.
MATERIALS AND METHODS
Culture of lung fibroblasts
MCR-5 human lung fibroblasts (CCL-171; American Type Culture Collection, Manassas, VA, USA) and HEL-299 fibroblasts (CCL-137; American Type Culture Collection) were grown in Eagle's minimal essential medium (MEM) supplemented with 10% foetal calf serum (FCS), 2 mM l-glutamine, Earle's balanced buffered solution adjusted to contain 2.2 g·L−1 sodium bicarbonate, 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate, 100 U·mL−1 penicillin and 100 µg·mL−1 streptomycin. Cells were grown in a humidified incubator at 37°C and 5% CO2 and passaged by trypsinisation at near confluence.
Normal areas of surgically resected lung tissue obtained from lung cancer patients after thoracotomy were used to establish phLFbs. Anonymous lung tissue of male Caucasians was obtained from patients who gave informed consent. The protocol for obtaining human tissue was approved by the local ethical review board for human studies (Medical Faculty, University of Bonn, Bonn, Germany). Tissue was cut into small pieces, treated with pronase (1 mg·mL−1; Calbiochem Novabiochem, San Diego, CA, USA) at 37°C for 30 min, placed in cell culture plates and incubated in Eagle's MEM, supplemented as described previously 14 with FCS increased to 15%. After 2 weeks, fibroblasts had grown from the tissue and were passaged by standard trypsinisation. In all experiments described in the present study, cells from passages 3–11 were used.
Extraction of RNA and RT-PCR
Total RNA was isolated using silica-gel-based membranes according to manufacturers’ instructions but including an additional DNase digestion protocol in order to eliminate contamination by genomic DNA (Qiagen, Hilden, Germany). First strand cDNA was synthesised using Omniscript reverse transcriptase (Qiagen). Specific oligonucleotide primers were constructed based on human EMBL sequences (The European Molecular Laboratory, Heidelberg, Germany): β-actin, 5′-CACTCTTCCAGCCTTCCTTC-3′ and 5′-CTCGTCATACTCCTGCTTGC-3′; M1 receptor, 5′-CAGGCAACCTGCTGGTACTC-3′ and 5′-CGTGCTCGGTTCTCTGTCTC-3′; M2 receptor, 5′-CTCCTCTAACAATAGCCTGG-3′ and 5′-GGCTCCTTCTTGTCCTTCTT-3′; M3 receptor, 5′-GGACAGAGGCAGAGACAGAA-3′ and 5′-GAAGGACAGAGGTAGAGTGG-3′; M4 receptor, 5′-ATCGCTATGAGACGGTGGAA-3′ and 5′-GTTGGACAGGAACTGGATGA-3′; and M5 receptor, 5′-ACCACAATGCAACCACCGTC-3′ and 5′-ACAGCGCAAGCAGGATCTGA-3′. PCR amplification was performed using Taq DNA polymerase in a programmable thermal cycler with initial heating for 3 min at 94°C, followed by 23 and 35 cycles (for β-actin and M1–M5, respectively) of 45 s denaturation at 94°C, annealing at 53–60°C (30 s), extension at 72°C (1 min) and final extension for 10 min at 72°C. PCR products were separated using 1.2% agarose gel electrophoresis. Optical density of bands was quantified by RFLPscan version 2.01 software (MWG-biotech AG, Ebersberg, Germany), corrected over β-actin and referred to respective amplification of genomic DNA to normalise for variations in PCR effectiveness.
Collagen synthesis and deposition into the extracellular matrix was assessed by [3H]-proline incorporation assays originally developed by Peterkofsky and Diegelmann 16 and subsequently used in many studies 17–20. Cells were trypsinised, harvested and seeded into 12-well dishes at a density of 105 cells per well. Cells were first cultured for 24 h in the presence of 10% FCS, followed by an additional 24 h under FCS-free conditions. Following this, [3H]-proline (37 kBq) was added alone or in combination with the test drugs and the cells were cultured for a further 24 h. After incubation, culture medium was removed and cells were washed twice with PBS at 4°C followed by 1 h incubation in 1 mL 20% trichloric acid (TCA) at 4°C. Denaturated cells were scraped off, transferred into a reaction tube and centrifuged. The pellet was washed with 1 mL 10% TCA and dissolved in 300 µL 0.2 M NaOH followed by neutralisation with 300 µL 0.2 M HCl. A scintillation cocktail (PerkinElmer, Jügesheim, Germany) was combined with 300-μL portions of the pellet and radioactivity was determined by liquid scintillation spectrometry using a Packard 2100 TR liquid scintillation analyser (PerkinElmer). External standardisation was used to correct for counting efficiency. Three wells of each 12-well culture dish served as controls and data were expressed as percentage of the mean of these individual controls of each cell preparation. In addition, [3H]-proline incorporation was also expressed in absolute terms (dpm).
In selected experiments, 100 µL of collagenase II solution (2 mg·mL−1 in buffer containing 50 mM Tris-Base, 5 mM EDTA, 20 mM NaCl and 5 mM CaCl2) was added to 300 µL of neutralised protein extract followed by 1 h incubation at 37°C. The digestion was terminated by addition of 100 µL 50% TCA. Samples were centrifuged at 18,000×g for 10 min and radioactivity in the supernatant (as a measure of collagenase-sensitive [3H]-proline) was determined by liquid scintillation counting. Releasable radioactivity was expressed as percentage of the total incorporated radioactivity in each individual experiment.
Data are presented as scatter plots of the individual observations. In addition, mean±sem values were calculated; these values are referred to when drug effects are described quantitatively in the text. Normal distribution was confirmed by performing the D’Agostino–Pearson omnibus normality test. Statistical significance of differences was evaluated by ANOVA followed by Dunnett or Bonferroni test. When normal distribution could not be confirmed (i.e. in all experimental series in which one treatment group had less than eight observations) significance of differences was evaluated by the Kruskal–Wallis test followed by Dunn’s test. A p-value of <0.05 was considered as being significant. The inhibitory concentration of 50% (IC50) value was calculated.
Drugs and materials
Butaprost, carbachol (carbamylcholine chloride), nicotine bitatrate, oxotremorine sesquifumerate, PD-98059 (2-(2′-amino-3′-methoxyphenyl)-oxanaphthalen-4-1), penicillin–streptomycin solution, pertussis toxin, transforming growth factor (TGF)-β1 and trypsin were all purchased from Sigma (Deisenhofen, Germany); collagenase II was obtained from Cell Systems (St. Katharinen, Germany). Tiotropium bromide was a gift from Boehringer Ingelheim (Biberach, Germany). Eagle’s MEM with Earl’s salts and l-glutamine, and nonessential amino acids were obtained from PAA (Cölbe, Germany), FCS from Biochrom (Berlin, Germany), Taq DNA-polymerase from Invitrogen (Karlsruhe, Germany) and Omniscript reverse transcriptase, RNeasy Mini kit and RNase-free DNase set were all obtained from Qiagen. Oligodesoxynucleotides for RT-PCR were obtained from MWG-biotech.
Muscarinic receptor expression in human lung fibroblasts
In addition to the previously mentioned observations, abundant expression of M2 receptor mRNA was confirmed in a large number of observations in phLFbs and MRC-5 cells, as well as in HEL-299 cells, a further human lung fibroblast cell line 21 (figs 1⇓ and 2⇓). In all cells, mRNA encoding M2 receptor was by far the most prominent muscarinic receptor transcript. The levels of M2 receptor mRNA appeared to be quite similar in phLFbs and the two other cell lines studied (fig. 2a⇓). There were some differences with regard to the expression of other muscarinic receptor subtypes. M1 receptor transcript was only detected in primary cells whereas the levels of M3 transcripts were highest in MRC-5 cells followed by HEL-299 cells and primary cells (fig. 2b–d⇓).
Muscarinic stimulation of collagen synthesis in MRC-5 human lung fibroblasts
Under control conditions, i.e. absence of FCS and any test drug, [3H]-proline incorporation into cellular proteins amounted to 18,303±981 dpm (84 individual observations under control conditions), since there was no significant difference between the different series of experiments, a pooled value is given. The muscarinic receptor agonist carbachol enhanced [3H]-proline incorporation in a concentration-dependent manner, maximally by ∼55% at 10 µM and with a half maximally effective concentration of 220 nM (fig. 3a⇓). Likewise, 10 µM oxotremorine caused an increase of ∼65% (fig. 3a⇓). In comparison, TGF-β1, a well known activator of fibroblast collagen synthesis, caused an increase of ∼85%, whereas the prostanoid EP2 receptor agonist butaprost (100 nM) and forskolin (10 µM), a direct activator of adenylyl cyclase, inhibited [3H]-proline incorporation by ∼50% (fig. 3b⇓). Following 1 h digestion by collagenase II, ∼80% of the [3H]-proline was released from the protein fraction (fig. 4a⇓), confirming that total [3H]-proline incorporation largely reflects specific collagen synthesis. Furthermore, the effects of carbachol and butaprost on total [3H]-proline incorporation were comparable to the effects on collagen-sensitive [3H]-proline (fig. 4b⇓ and c).
The stimulatory effect of 10 µM carbachol on [3H]-proline incorporation was inhibited in a concentration-dependent manner by the muscarinic receptor antagonist tiotropium with an IC50 of 110 pM (fig. 5⇓). Pre-treatment of the cells with pertussis toxin tended to decrease (by ∼20%) basal [3H]-proline incorporation and prevented the stimulatory effect of 10 µM carbachol (fig. 6a⇓). Furthermore, PD 98059 (30 µM), a specific inhibitor of the mitogen-activated protein kinase-activating enzymes 22 did not affect basal [3H]-proline incorporation, but prevented the stimulatory effect of 10 µM carbachol on [3H]-proline incorporation (fig. 6b⇓).
Nicotine failed to significantly affect [3H]-proline incorporation. In the presence of 1, 10 and 100 µM nicotine, [3H]-proline incorporation amounted to 103±9, 119±6 and 117±10%, respectively, of controls (six observations from each).
Muscarinic stimulation of collagen synthesis in HEL-299 and phLFbs
In HEL-299 cells, basal [3H]-proline incorporation (14,915±557 dpm, 18 observations under control conditions) was not significantly different from that observed in MRC-5 cells, whereas in phLFbs, it was significantly lower compared with either cell line (10,122±745 dpm; 42 observations under control conditions; p<0.01). Nonetheless, in both cell types, muscarinic agonists stimulated [3H]-proline incorporation (fig. 7⇓) and the half maximally effective concentration for carbachol obtained in primary cells (165 nM) was comparable with that in MRC-5 cells. Finally, in both HEL-299 and the primary cells, the stimulatory effect of the muscarinic agonists (10 µM oxotremorine or 10 µM carbachol) was blocked by 10 nM tiotropium (fig. 7⇓).
In human lung fibroblasts (primary cell and different cell lines), RNA encoding multiple muscarinic receptors was found to be transcribed. However, in all cells studied to date, M2 receptor mRNA was by far the most prominent transcript. In this regard, previous observations on phLFbs and MRC-5 cells 14 were confirmed in the present study in a larger series of experiments and extended to HEL-299 cells (figs 1⇑ and 2⇑), another human lung fibroblast cell line 21. In agreement with the current observations, two other recent studies demonstrated muscarinic receptor mRNA expression in phLFbs and also observed a predominant expression of mRNA encoding the M2 subtype 23, 24.
In previous functional studies, it has been observed that muscarinic receptor activation can exert proliferative effects in both primary lung fibroblasts and MRC-5 cells 14, 15, 23. The present study aimed to further explore the functional significance of muscarinic receptors in pulmonary fibroblasts and studied possible effects on collagen synthesis in MRC-5 and HEL-299 cells as well as in primary lung fibroblasts.
Collagens are proteins of particular high proline content and [3H]-proline incorporation into newly synthesised proteins has often been used as a measure of collagen synthesis 16–20. In the present study, digestion with collagenase confirmed that [3H]-proline incorporation largely reflects collagen synthesis (fig. 4a⇑) and that drug effects on total [3H]-proline incorporation paralleled those on the collagenase-sensitive [3H]-proline fraction (fig. 4b⇑ and c). Moreover, stimuli known to inhibit (butaprost and forskolin) or to stimulate (TGF-β1) collagen synthesis 20, 25 also showed corresponding effects in the present study.
In MRC-5 cells, carbachol as well as oxotremorine, two well known muscarinic receptor agonists, caused a clear increase in collagen synthesis. In contrast to oxotremorine, which is a pure muscarinic agonist, carbachol also has some nicotine receptor agonistic activity. However, ≤100 µM nicotine did not significantly affect [3H]-proline incorporation, which argues against a role of nicotine receptors in the effect of carbachol. Finally, interaction experiments, in which the effect of carbachol was antagonised by the long-acting muscarinic antagonist tiotropium (fig. 5⇑), proved mediation via specific muscarinic receptors.
In previous experiments, it has been observed that the muscarinic receptors mediating proliferative effects in lung fibroblasts had pharmacological characteristics of M2 receptors and coupled to pertussis toxin sensitive G proteins 14. Likewise, the muscarinic stimulation of collagen synthesis was also prevented by pertussis toxin (fig. 6⇑) indicating the involvement of Gi/o proteins in this effect. In the present study, a detailed pharmacological characterisation of the muscarinic receptors mediating stimulatory effects on collagen synthesis was not performed; however, of the five muscarinic receptor subtypes, only M2 and M4 receptors couple to pertussis-toxin sensitive proteins 26. Moreover, MRC-5 cells express substantial amounts of transcript for M2 receptors, but only traces of mRNA encoding M4 receptors (figs 1⇑ and 2⇑). Thus, M2 receptors are also likely to mediate the muscarinic stimulation of collagen synthesis. Is this conclusion compatible with the high potency of tiotropium in antagonising the stimulatory effect of carbachol on collagen synthesis? Affinity constants of tiotropium are known for M1, M2 and M3 receptors and are similar for all three receptor subtypes (0.1–0.3 nM) 27, 28. However, the dissociation of the respective receptor complexes shows different kinetics, with the longest half-life for the tiotropium–M3 receptor complex 27, 28. This kinetic subtype selectivity of tiotropium may not be of significance in the present study because those experiments were performed under equilibrium conditions. However, whether the kinetic subtype selectivity of tiotropium is of clinical significance is still a matter of debate 29. In any case, it is very likely that, under clinical conditions, tiotropium causes a significant blockade of M2 receptors in the lung, in addition to the blockade of M3 and M1 receptors, because the half-life of the tiotropium–M2 receptor complex, although shorter than that of the M3 and M1 receptors, is still quite long at 3.6 h compared with ipatropium, for which the dissociation half-life is in the range of 2–6 min for all three muscarinic receptors 27, 28.
Furthermore, in both MRC-5 cells and phLFbs, the muscarinic proliferative effects were mediated via activation of the extracellular signal-regulated kinase–mitogen-activated protein kinase pathway 15. The present observations showed that PD 98059 prevented the carbachol-induced stimulation of [3H]-proline incorporation indicating that this pathway is also essential for the muscarinic stimulation of collagen synthesis.
As mentioned previously, in HEL-299 and phLFbs, mRNA encoding M2 receptors is also the predominant muscarinic receptor transcript. In the present study, muscarinic agonists also stimulated collagen synthesis in these cells. Thus, as in previous studies investigating proliferative effects 14, 15, muscarinic receptor activation elicited the same response in phLFbs and human lung fibroblast cell lines.
In conclusion, the present results demonstrate muscarinic stimulation of collagen synthesis in cultured human lung fibroblasts. Together with previous observations showing muscarinic receptor-mediated proliferative effects 14, 15, 23, the current findings support the notion that cholinergic mechanisms could play a role in pro-fibrotic airway remodelling processes, which occur in chronic inflammatory and obstructive airway diseases. Inhibition of these processes, by prolonged blockade of airway muscarinic receptors, may contribute to the long-term beneficial effects of anticholinergics, as observed for the long-acting muscarinic antagonist tiotropium in chronic obstructive pulmonary disease.
Statement of interest
Statements of interest for K. Racké and the study itself can be found at www.erj.ersjournals.com/misc/statements.shtml
- Received October 2, 2007.
- Accepted April 29, 2008.
- © ERS Journals Ltd