Abstract
Macrophages increase in number and are highly activated in chronic obstructive pulmonary disease (COPD). Muscarinic receptor antagonists inhibit acetylcholine-stimulated release of neutrophilic chemoattractants, suggesting that acetylcholine may regulate macrophage responses. Therefore, expression and function of components of the non-neuronal cholinergic system in monocyte-macrophage cells was investigated.
RNA was isolated from monocytes, monocyte-derived macrophages (MDMs), lung and alveolar macrophages from nonsmokers, smokers and COPD patients, and expression of the high-affinity choline transporter, choline acetyltransferase, vesicular acetylcholine transporter and muscarinic receptors (M1–M5) ascertained using real-time PCR. M2 and M3 receptor expression was confirmed using immunocytochemistry. Release of interleukin (IL)-8, IL-6 and leukotriene (LT)B4 were measured by ELISA or EIA.
All monocyte-macrophage cells expressed mRNA for components of the non-neuronal cholinergic system. Lung macrophages expressed significantly more M1 mRNA compared with monocytes, and both lung macrophages and alveolar macrophages expressed the highest levels of M3 mRNA. Expression of M2 and M3 protein was confirmed in MDMs and lung macrophages. Carbachol stimulated release of LTB4 from lung macrophages (buffer 222.3±75.1 versus carbachol 1,118±622.4 pg·mL−1; n=15, p<0.05) but not IL-6 or IL-8. LTB4 release was attenuated by the M3 antagonist, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP; half maximal effective concentration 5.2±2.2 nM; n=9).
Stimulation of macrophage M3 receptors promotes release of LTB4, suggesting that anti-muscarinic agents may be anti-inflammatory.
Macrophages are the predominant inflammatory cell found in the lung. Their role is primarily to remove any inhaled particles and pathogens and maintain sterility of the respiratory tract. However, in lung diseases such as chronic obstructive pulmonary disease (COPD), macrophage numbers increase by more than 10-fold and are highly activated, producing increased levels of inflammatory mediators [1]. At present, pharmacotherapy for COPD is largely symptomatic with no treatments capable of decreasing the underlying inflammatory response and improving lung function [2]. Long-acting muscarinic antagonists, such as tiotropium bromide, have been shown to have efficacy in patients with COPD [3], although whether this drug could also act to control the inflammatory components of the disease or act upon the small airways where COPD is manifest is unclear. Recently, tiotropium bromide has been shown to suppress chemotactic activity released by macrophages following stimulation with acetylcholine (ACh) [4]. This suggests that macrophage-mediated inflammation may, in part, be regulated by components of the non-neuronal cholinergic system.
Classically, ACh is synthesised in nerve terminals and is released to regulate many activities, including regulation of airway contraction and dilation of vessels [5, 6]. More recently, essential components of the non-neuronal cholinergic system, including the high-affinity choline transporter (CHT1), ACh, choline acetyltransferase (ChAT), and muscarinic and nicotinic ACh-receptors, are expressed by a number of non-neuronal cells, including peripheral blood mononuclear cells and lymphocytes [7, 8]. Many of these cells not only release ACh upon stimulation but also can be activated by ACh [9–11]. Bovine alveolar macrophages release neutrophil, monocyte and eosinophil chemotactic factors after stimulation with ACh [12] and it has been suggested that human alveolar macrophages release leukotriene (LT)B4 in response to ACh stimulation [4]. Cells obtained from induced sputum have also been shown to express muscarinic M2 and M3 receptors, and following stimulation with ACh the release of LTB4 increased in cells from COPD patients but not in cells from nonsmokers or smokers [13]. Taken together, these data suggest that non-neuronal ACh might be involved in the pathophysiology of COPD by stimulating the release of inflammatory mediators from macrophages.
This study examined the expression of components of the non-neuronal cholinergic system including CHT1, ChAT and vesicular acetylcholine transporter (VAChT) in cells of the monocyte-macrophage lineage together with expression of muscarinic receptors (M1–M5). The putative roles of these receptors on macrophages were then evaluated using functional assays.
METHODS
Subject selection
Subjects were recruited from clinics at the Royal Brompton Hospital NHS Trust, from staff of the Royal Brompton Hospital and National Heart and Lung Institute, or volunteers known to the clinical research group of the Asthma Laboratory, National Heart and Lung Institute (all London, UK). Alveolar macrophages from bronchoalveolar lavage (BAL) fluid of nonsmokers, smokers and patients with COPD were obtained from consenting patients at Heatherwood Hospital, Ascot, and Wexham Park Hospital, Slough, UK. Smokers had a smoking history of at least 10 pack-yrs and COPD patients were stable and fulfilled the American Thoracic Society criteria [14]. All studies were approved by the ethics committee of the Royal Brompton and Harefield NHS Trust, the National Heart and Lung Institute ethics committee, the East Berkshire local research ethics committee or St Mary's NHS Trust ethics committee. All subjects gave informed written consent.
Preparation of monocytes
Monocytes were isolated from peripheral blood, centrifuged on discontinuous Percoll gradients, and purified either by adherence to tissue culture plastic [15] or by negative immunoselection using a MACS monocyte isolation kit (Miltenyi Biotec, Bisley, UK) and magnetic depletion columns according to the manufacturer's instructions. Cells were cultured in complete media (RPMI-1640 media supplemented with 10% (v/v) fetal calf serum, 100 U·mL−1 penicillin, 100 μg·mL−1 streptomycin and 2 mM l-glutamine) in a six-well plate and were then resuspended in lysis buffer and stored at -70°C.
Preparation of monocyte-derived macrophages
After separation of peripheral blood mononuclear cells, cells were resuspended at 2×106 cells per mL in complete media. The cells were seeded onto 48-well plates or chamber slides, and incubated for 2 h at 37°C in a humidified incubator with 5% (v/v) CO2. After incubation, the supernatant was removed and replaced with complete media supplemented with 2 ng·mL−1 granulocyte-macrophage colony-stimulating factor. The cells were differentiated in culture for 12 days towards a macrophage phenotype.
Preparation of lung-derived macrophages
Lung-derived macrophages were isolated from lung tissue as previously described [16]. Briefly, lung tissue from patients undergoing surgical resection for carcinoma was lavaged by injection of RPMI-1640 containing 100 U·mL−1 penicillin, 100 μg·mL−1 streptomycin, 2.5 μg·mL−1 amphotericin and 2 mM l-glutamine. The cells were washed and resuspended in 2 mL PBS and separated by centrifugation (25 min; 18°C; 1,100×g) using Percoll density gradient (65%/35%/25% (v/v)). The macrophage-enriched fraction was collected at the 25% and 35% Percoll interface. The cells were washed in Hank’s balanced salt solution (HBSS) and resuspended in RPMI-1640 medium supplemented with 10% (v/v) fetal calf serum, 100 U·mL−1 penicillin, 100 μg·mL−1 streptomycin, 2.5 μg·mL−1 amphotericin and 2 mM l-glutamine resuspended at 1×106 cells per mL and seeded into 24-well plates at 5×105 cells per well, 48-well plates at 3.2×105 cells per well or chamber slides at 4×105 cells per well. After 2 h of incubation at 37°C in a humidified incubator with 5% (v/v) CO2, the nonadherent cells were removed and fresh medium was added. The adherent purified macrophages were incubated overnight and the medium was changed the next day before the experiment was started. Macrophage purity was confirmed by anti-CD68 staining as described previously [17].
Preparation of alveolar macrophages
Bronchoscopy and processing were performed according to the guidelines of the European Respiratory Society task force [18] and alveolar macrophages isolated as described previously [19].
Preparation of sputum cells
Sputum was induced by inhalation of hypertonic saline and processed with 0.05% (w/v) dithiothreitol [20]. After centrifugation, the cell pellet was resuspended with HBSS and cytospins prepared.
Real-time PCR
Total RNA was extracted from cells using an RNeasy RNA extraction kits (Qiagen, Crawley, UK) and isolated RNA was quantified using the Ribogreen quantification Assay (Molecular Probes, Leiden, the Netherlands). RNA was reverse transcribed using a Taqman reverse transcriptase mastermix (Taqman RT buffer, MgCl2 5.5 mM, deoxynucleotide triphosphates 500 μM, random hexamers 2.5 μM, RNase inhibitor 0.4 U·μL−1 and reverse transcriptase enzyme 1.25 U·μL−1) (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. Reverse transcription products were amplified by PCR. cDNA (5 μL) was added to 20 μL of a solution containing universal master mix, water, sense and antisense primers, and 6-carboxy-tetramethyl-rhodamine carboxyfluorescein-labelled probe. Primers and probes were designed by Applied Biosystems. An ABI Prism 7500 Sequence Detection System (Applied Biosystems) was used for thermal cycling, which consisted of an initial activation step of 50°C for 2 min and 95°C for 10 min, followed by 45 cycles of 95°C for 15 s and 60°C for 1 min. Each analysis included a standard curve (1.25–20 ng) consisting of cDNA synthesised from a panel of five control human RNAs (human RNA control panel; Becton Dickinson, Oxford, UK). Samples were analysed in duplicate and levels of expression for each specific gene calculated by extrapolating from the standard curve. For each test gene, endogenous control (HPRT) levels were also analysed on the same plate, calculated using extrapolation of standard curve values.
Immunocytochemistry of M2 and M3 receptors
Slides were immersed for 10 min with 4% (w/v) paraformaldehyde in PBS (pH 7.4). After washing with PBS, the slides were incubated with either rabbit anti-human M2 polyclonal antibody (diluted 1:500) or rabbit anti-human M3 polyclonal antibody (diluted 1:200) (Life Span Biosciences, Seattle, WA, USA) or rabbit immunoglobulin (Ig)G antibody control (Dako, Ely, UK) in PBS containing 10% (v/v) normal human serum for 1 h at room temperature. After washing with PBS, the slides were incubated with Alexa Flour 488-conjugated goat anti-rabbit IgG antibody (diluted 1:1,000) (Molecular Probes) in PBS (pH 7.4) containing 10% (v/v) normal human serum for 1 h at room temperature. The slides were washed with PBS and then incubated with 4,6-diamino-2-phenylindole (DAPI) at 5 μM in HBSS for 3 min. After washing, the slides were mounted with 50% (v/v) PBS/50% (v/v) glycerol. The slides were examined using a Leica TCS 4D confocal microscope (Leica Microsystems, Milton Keynes, UK) equipped with argon, krypton and ultraviolet lasers.
Fluorescence-activated cell sorting analysis of M2 and M3 receptors
Lung macrophages (106 cells per mL) were permeabilised by the addition of ice-cold methanol. Cells were then incubated in the absence or presence of either anti-rabbit IgG, anti-M2 receptor or anti-M3 receptor antibodies for 1 h at 4°C. The cells were washed with PBS and then resuspended in PBS containing 1% (v/v) bovine serum albumin. All tubes were then incubated with the secondary antibody (goat anti-rabbit IgG) conjugated with phycoerythrin–Cy5.5 for 30 min. Cells were washed with PBS and resuspended in fluorescence-activated cell sorting (FACS) fix solution and samples analysed using a BD FACS Canto II flow cytometer and analysed using FACS Diva software (BD Biosciences, Oxford, UK). Data are expressed as the percentage of macrophages expressing the receptor of interest and as the ratio of the median fluorescence intensity (MFI) relative to the isotype control.
Measurement of IL-8 and IL-6 using ELISA
Interleukin (IL)-8 and IL-6 were measured in the supernatants from monocyte-derived macrophages (MDMs) and tissue-derived macrophages incubated with ACh or carbachol using ELISA (R&D Systems, Abingdon, UK). The lower limit of detection of both these assays was 16 pg·mL−1.
Measurement of LTB4
Release of LTB4 into cell culture media was measured using a commercially available kit from GE Healthcare (Little Chalfont, UK) according to the manufacturer's instructions.
Measurement of ACh release
Release of ACh into the cell culture media was measured using a commercially available kit from Invitrogen Ltd (Paisley, UK), according to the manufacturer's instructions. The lower limit of detection of this assay is 1.5 μM.
Cell viability assays
Cell viability was determined colorimetrically by measuring the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan by mitochrondial dehydrogenases, as described previously [21]. None of the cell treatments altered cell viability.
Statistical analysis
GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA) was used to perform all statistical tests. When the data were analysed nonparametric distribution was assumed, therefore the Wilcoxon matched paired test or Kruskal–Wallis test was used, initially with Dunn's post-test for ANOVA analysis. Results were considered significant when p<0.05.
RESULTS
Expression of components of the ACh synthesis pathway
In order to examine whether cells of the monocyte-macrophage lineage have the capacity to synthesise ACh, the expression of components of the ACh synthesis pathway, CHT1, ChAT and VAChT were examined by real-time PCR. All three components were expressed by monocytes, MDMs, lung macrophages and alveolar macrophages (table 1). There was no difference in the level of expression of any of the components of the ACh synthesis pathway in any of the cells examined, with the exception of significantly reduced expression of ChAT in lung macrophages (table 1).
Low levels of ACh were released and could be measured from lung macrophages (6±2 μM, n=4); however, these levels were near the limit of detection of the assay.
Muscarinic receptor expression analysed by real-time PCR
Having ascertained that cells of the monocyte-macrophage lineage express mRNA for proteins to drive synthesis of ACh, we next determined whether these cells could respond to this mediator. To this end, we examined the level of mRNA expression of M1–M5 muscarinic receptors in cells of the monocyte-macrophage lineage. Expression of the muscarinic receptors M1–M5 was detected on all cell types (fig. 1). The expression of M1 receptor mRNA in lung-derived macrophages was significantly greater than that on monocytes and MDMs (monocytes: median (range) 0.14 (0.06–1.6)%, n=24; MDMs: 0.16 (0.03–0.8)%, n=52; lung-derived macrophages: 1.22 (0.5–4.8)%, n=27; alveolar macrophages: 0.65 (0.2–3.5)%, n=16) (fig. 1a). Expression of M3 receptor mRNA in lung-derived macrophages and alveolar macrophages was significantly greater than that in monocytes and MDMs (monocytes: 2.1 (0.9–4.7)%, n=15; MDMs: 0.06 (0–0.3)%, n=46; lung-derived macrophages: 51.4 (17–179.4)%, n=22; alveolar macrophages: 42.1 (32–90)%, n=13) (fig. 1c).
M2 and M3 receptor expression estimated by immunohistochemistry
In order to confirm the expression data obtained using Taqman analysis, regarding the expression of muscarinic receptors in cells of the monocyte-macrophage lineage, we performed immunocytochemistry. Due to the poor quality and availability of antibodies against human muscarinic receptors, this study was limited to expression of M2 and M3 receptors. M2 and M3 receptor expression was evaluated on MDMs and lung-derived macrophages (fig. 2) with the M2 receptor predominant on the cell membrane. Expression of the M3 receptor on MDMs appeared to be both membrane-associated and cytosolic (fig. 2). In lung-derived macrophages, expression of M2 and M3 receptors was also detected on the cell membrane (fig. 2a). The immunocytochemistry was validated using FACS (fig. 2b), by which M2 expression was observed on mean±sem 50.8±17.3% of lung macrophages with an MFI of 5.4±1.2 (n=5) and M3 expression was observed on 66.5±17.3% of lung macrophages with an MFI of 6.9±1.3 (n=5). Expression was also determined in cells obtained from BAL and sputum samples from nonsmokers, smokers and patients with COPD. Cells obtained from BAL from all three groups expressed similar levels of both M2 and M3 receptor (fig. 3), with expression associated with the macrophages. Similarly, macrophages obtained from induced sputum expressed similar levels of the M2 receptor (fig. 4) with less expression of the M3 receptor (fig. 4).
Function of muscarinic receptors on MDMs and lung-derived macrophages
Having determined the expression of muscarinic M2 and M3 receptors on the surface of cells of the macrophage lineage, the function of these receptors was then investigated. Neither MDMs nor lung macrophages stimulated with the stable analogue of ACh, carbachol (100 μM), for up to 24 h released measurable levels of either IL-8 or IL-6 (data not shown). Similarly, MDMs exposed to 100 μM carbachol did not lead to the release of LTB4 (mean±sem, buffer 278.9±84.9 versus carbachol 230.4±84.8pg·mL−1; n=6) (fig. 5a). In contrast, carbachol stimulated LTB4 release from lung-derived macrophages (buffer 222.3±75.0 versus carbachol 1,118±622.4 pg·mL−1; n=15) (fig. 5b). In order to investigate the mechanism of carbachol-stimulated LTB4 release from lung macrophages, cells were pre-treated with the muscarinic receptor antagonists 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP) or AF-DX116 prior to stimulation with carbachol. The release of LTB4 from carbachol-stimulated lung macrophages was inhibited in a concentration-dependent manner by 4-DAMP (fig. 6a). Maximal inhibition (58.9±6.6%, n=9) occurred at 30 nM with a half maximal effective concentration of 5.2±2.2 nM. The effect of pre-treating these cells with AF-DX116 and gallamine were less effective with maximal inhibition of 42±15.1% and 36.4±15.6%, respectively (n=5).
DISCUSSION
This study demonstrated that mRNA for components of the ACh synthesis pathway was expressed by both monocytes and macrophages. Human mononuclear cells have been reported to contain ACh [22] and ChAT is expressed by rat monocytes [23] and human alveolar macrophages [24]. However, we have now demonstrated expression of CHT1 and VAChT in cells of the monocyte-macrophage lineage, suggesting that these cells are capable of ACh synthesis and release of ACh from lung macrophages could be measured, but was very low and near to the limit of detection of the assay. Nevertheless, ACh may reach sufficient concentrations to act locally and thus contribute to the inflammatory response. This is further substantiated by expression of muscarinic receptor mRNA in cells of the monocyte-macrophage lineage.
The present study demonstrated expression of mRNA for muscarinic M1–M5 receptors in human monocytes in contrast to a previous report [25]. This may reflect increased sensitivity of real-time PCR methodology. However, the present study showed increased expression of M1 and M3 mRNA in lung-derived macrophages compared with monocytes. This may suggest that as monocytes differentiate towards a macrophage phenotype there is a concomitant change in expression of muscarinic receptors. However, lung tissue macrophages were obtained from the tissue of patients undergoing surgery for lung cancer. Although the tissue was macroscopically normal, it is not known whether the tumour-promoting environment may alter the expression of muscarinic receptors locally. Nevertheless, these data are consistent with reports of M3 receptor expression in alveolar macrophages [12, 13] and were further substantiated with immunocytochemistry. Using this technique, it was observed that, despite little difference between MDMs and lung macrophages with respect to M2 mRNA expression, there appeared to be increased protein expression in the MDMs. Similarly, despite lung-derived macrophages expressing significantly greater quantities of M3 mRNA compared with MDMs, protein expression by immunocytochemistry appeared reduced. This suggests that mRNA levels of muscarinic receptors may not reflect protein expression in these cell types. To address this further, we used FACS analysis of lung macrophage expression of M2 and M3 receptors and demonstrated expression of both receptors. Of note in induced sputum samples, muscarinic receptor M2 and M3 expression appeared to be restricted to the macrophage population. However, in contrast to a previous study [13], we did not see an increase in macrophage M3 receptor expression in cells from COPD patients and this was corroborated in BAL macrophages.
Despite a lack of alteration of either M2 or M3 expression in macrophages from COPD patients, there is no doubt that these receptors are expressed by macrophages and MDMs. These data led to a subsequent investigation into the role of these receptors on the macrophage surface. Stimulation of macrophages with carbachol did not stimulate the release of either IL-8 or IL-6, confirming a previous study whereby stimulation with ACh did not release IL-8 or monocyte chemotactic protein 1 (CCL2) from alveolar macrophages [4]. Bovine alveolar macrophages produce LTB4 following stimulation with ACh [12] and we demonstrated a similar effect of carbachol on lung-derived macrophages. This appeared to be mediated via the M3 receptor, since this effect could be abrogated by 4-DAMP but not AF-DX116 or gallamine. It is possible that ACh may mediate inhibitory effects via nicotinic receptor activation, but Birrell et al. [26] demonstrated that nicotine is not inhibitory in human lung macrophages. However, ∼40% residual LTB4 release was not affected by blockade of the M3 receptor, indicating that LTB4 release from lung macrophages stimulated with carbachol may invoke other pathways. For example, activation of the extracellular signal-regulated kinase pathway has been shown to be involved in LTB4 release from ACh activated sputum cells from patients with COPD and in isolated monocytes from healthy volunteers [13]. It is unlikely that this could be attributed to a feedback of LTB4 stimulation on the macrophage as we have shown previously that this does not occur in lung macrophages [27]. The release of LTB4 by carbachol stimulation was not observed in MDMs, despite expression of both M2 and M3 receptors on the surface of these cells. This may reflect uncoupling of these receptors from subsequent downstream signalling events. However, we have recently demonstrated that lung-derived macrophages exhibit a greater capacity to synthesise and release LTB4 when compared to MDMs and, therefore, may not be the best cell type to use for study of these responses [27].
In summary, cells of the monocyte-macrophage lineage express components of the non-neuronal cholinergic system with the capacity to both synthesise and respond to ACh. The role of this system in regulating macrophage function is less clear but appears to regulate the release of LTB4, in part, via the muscarinic M3 receptor. Therefore, antagonists of the M3 receptor might contribute to the control of inflammatory status, such as through the release of LTB4 from macrophages, in addition to the inhibitory effect of smooth airway contraction, suggesting an additional role for these drugs in COPD and other inflammatory lung diseases [28].
Footnotes
Support Statement
This study was supported by Boehringer Ingelheim GmbH. It was supported by the NIHR Respiratory Disease Biomedical Research Unit at the Royal Brompton and Harefield NHS Foundation Trust and Imperial College London, UK. A. Koaraiwas supported by fellowships from Pfizer and Kanae Foundation for Life and Socio-medical Science, Japan.
Statement of Interest
Statements of interest for A. Koarai, P.J. Barnes and L.E. Donnelly, and for the study itself, can be found at www.erj.ersjournals.com/site/misc/statements.xhtml
- Received August 25, 2010.
- Accepted August 16, 2011.
- ©ERS 2012