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Role of M3 mAChR in in vivo and in vitro models of LPS-induced inflammatory response
International Immunopharmacology 14 (2012) 320–327
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Role of M3 mAChR in in vivo and in vitro models of LPS-induced inflammatory response Zu-Peng Xu a, 1, Kai Yang a, 1, Guang-Ni Xu a, Liang Zhu a, Li-Na Hou a, Wen-Hui Zhang b, Hong-Zhuan Chen a,⁎, Yong-Yao Cui a,⁎ a b
Department of Pharmacology, Shanghai Jiao Tong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, China Department of Physiology, Shanghai Jiao Tong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, China
a r t i c l e
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Article history: Received 20 January 2012 Received in revised form 23 April 2012 Accepted 27 July 2012 Available online 19 August 2012 Keywords: nAChR mAChR Mice Alveolar macrophage Inflammation
a b s t r a c t Objective: We tested the potential role of the mAChR in lipopolysaccharide (LPS)‐induced inflammatory response in in vivo and in vitro models and a possible signaling pathway involved in the inflammatory process. Methods: Anesthetized mice were challenged with intratracheal LPS to induce acute lung injury. The cytology and histopathology changes, expression of cytokines and pulmonary vascular permeability were used to evaluate the effects of the cholinergic agent. Alveolar macrophage cell line NR8383 was also used to confirm the role of mAChRs and the molecular mechanisms underlying the LPS-induced events. Results: LPS-induced acute lung injury (ALI) was significantly improved by atropine (a non-selective mAChR antagonist) and 4-DAMP (a M3 mAChR antagonist), as indicated by the diminution of neutrophil infiltration, pulmonary vascular permeability and IL-6 and TNF-α production. LPS-induced TNF-α production from the alveolar macrophage was significantly inhibited by atropine and 4-DAMP, but not pirenzepine (a M1 mAChR antagonist) and methoctramine (a M2 mAChR antagonist). Interestingly, LPS-induced TNF-α production was enhanced by the muscarinic receptor agonist pilocarpine, and treatment with pilocarpine alone was able to trigger TNF-α production from the alveolar macrophage, which was effectively attenuated by 4-DAMP. Western blot analysis showed that LPS-induced degradation of IκBα was strongly blocked by atropine/4-DAMP both in vivo and in vitro, indicating that M3 mAChR was involved in LPS-induced lung inflammation by mediating the NF-κB signaling pathway. Conclusion: Our findings bring the evidence that the blockage of mAChR exerts anti-inflammatory properties, in which the M3 mAChR plays an important role in the LPS-induced lung inflammation. © 2012 Elsevier B.V. All rights reserved.
1. Introduction An increasing body of knowledge indicates that acetylcholine can suppress the production of multiple cytokines directly via the α7 nicotinic acetylcholine receptor (α7 nAChR) expressed on macrophages [1], which is called the cholinergic antiinflammatory pathway. Pharmacological stimulation of the peripheral α7 nAChR by nicotinic receptor agonists prevents NF-κB activation and inhibits cytokine release in wild-type but not in α7 nAChR-deficient macrophages [1,2]. In vivo, nicotinic agonists decrease inflammation and increase survival in severe sepsis [3]. Although nAChR and mAChR respond to the same neuromediator—acetylcholine and activation of α7 nAChR could
⁎ Corresponding authors. Tel.: +86 21 6384 6590x776451; fax: +86 21 6467 4721. E-mail addresses: [email protected] (H.-Z. Chen), [email protected] (Y.-Y. Cui). 1 Both authors contributed equally to this article. 1567-5769/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.intimp.2012.07.020
attenuate sepsis-induced ALI [4,5], the role of the mAChR remains relatively unexplored. The mAChR belongs to the superfamily of G-protein coupled receptors (GPCRs) that are commonly expressed in a variety of tissues and are classified into five known subtypes (M1–M5 mAChRs). M1, M3, and M5 mAChRs are selectively coupled to Gq proteins while M2 and M4 mAChRs are linked to Gi/G0 proteins [6,7]. Several studies have demonstrated that mAChR activation has pro-inflammatory effects in a wide variety of cells implicated in airway inflammation. Acetylcholine, acting at the M3 mAChR, can stimulate bovine alveolar macrophages and human sputum macrophages to release lipoxygenase-derived mediators, predominantly LTB4. The anticholinergic drug has been shown to regulate the release of chemotactic factors from human epithelial cells and macrophages in vitro [8]. Studies in animals have shown that mAChR antagonists inhibit vagus nerve stimulation or ACh induction of vascular edema and eosinophil infiltration into the guinea pig lung, and lung neutrophilia in the rat in response to diesel soot inhalation [9]. In previous studies, we have demonstrated the critical importance of mAChR in lung inflammation induced by repeated HCl intra-esophageal instillation
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in mice [10,11]. The modulatory effect of mAChRs for inflammatory responses was also confirmed in a mouse model of asthma [12]. In addition, the change of expression of M2 and M3 mAChRs on inflammatory cells recovered from COPD subjects is also related to the inflammation response. Overall, these observations suggest that inflammatory responses might be induced by the stimulation of mAChR and release of inflammatory cytokines, which can be diminished by mAChR antagonists. Inflammation of the lung marked by excessive recruitment of neutrophils from circulation to the airway is a common feature among several pathological lung disorders, particularly those involving infection [13–15]. ALI is characterized by extensive neutrophil influx into the lungs, the production of pro-inflammatory mediators from inflammatory cells, and pulmonary microvascular leakage, followed by severe lung damage [16–18]. The manifestations of ALI are related to the activation of antigen presenting cells like alveolar macrophages, up-regulation of cell surface adhesion molecules and subsequent production of cytokines and chemokines. Activated alveolar macrophages are the main sources of TNF-α, which is suggested as an important early mediator of ALI. In the inflammatory process, over-production of TNF-α is pivotal in the induction of inflammatory genes and in the recruitment and activation of immune cells. A lipopolysaccharide (LPS) is an outer membrane component of Gram negative bacteria. A model of LPS-induced ALI was applied to mimic the morphological and functional changes observed in clinical situations resulting from circulating LPS. It is well documented that LPS administration triggers a network of inflammatory responses mediated by a number of immune cells, such as neutrophil and macrophage, which is followed by the release of various inflammatory molecules including TNF-α and IL-6, which play a very important role in the pathogenesis of acute lung injury [19–24]. LPS-treated animals could induce ALI characterized by increased infiltration of inflammatory cells and production of inflammatory mediators and tissue edema [25]. The current study was undertaken to test the efficacy of mAChR antagonists compared to α7 nAChR agonists, administered right before exposure, and attenuated the extent of lung inflammation. In vivo, exposure of mice to LPS by intratracheal instillation increased proinflammatory cytokine production, neutrophil infiltration into the lung and pulmonary vascular permeability, and in vitro, exposure of alveolar macrophage cell line NR8383 to LPS increased cytokine release. Furthermore, we also evaluated whether inhibition of the NF-κB signaling pathway was involved in the protective effects of mAChR antagonists on LPS-induced lung inflammatory injury.
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dose of 4 mg/kg [26,27]. After intratracheal instillation, the mice were placed in a vertical position and rotated for 0.5–1 min to distribute the instillation evenly within the lungs. Sham-operated mice underwent the same procedure with intratracheal (i.t.) injection of 50 μl sterile saline without LPS. Atropine (3 mg/kg, i.p.) and PNU-282987 (3 mg/kg, i.p.) were given 30 min before LPS instillation. 2.3. Collection of bronchoalveolar lavage fluid (BALF) and cell counting Four hours after LPS challenge, mice were sacrificed by receiving euthanasia. The bronchoalveolar lavage was performed by infusion and extraction of 1 ml of phosphate buffered saline (PBS) via a tracheal cannula. This was repeated twice, and the recovered BALF was pooled. The total number of cells was counted with a hemocytometer. The percentage of neutrophils was determined on cytospin smears of BAL samples from individual mice, and stained with Wright–Giemsa after counting 300 cells. Results are expressed as cell number × 10 4/ml of BALF. 2.4. Histopathological examination of the lung The right lung was harvested and fixed in 10% phosphatebuffered formalin. After 24 h of fixation, lungs were embedded in paraffin, and 8 μm sections were cut and stained with hematoxylin and eosin (H&E), as in our prior studies [11]. All samples were analyzed based on a scaled grading system by a pathologist who was blinded to the experimental protocol and the region of sampling. A total of three slides from each lung sample were randomly screened and the mean was taken as the representative value of the sample. Each section was assigned a numerical histological injury score ranging from 0 to 3, based on the degree of inflammatory cell infiltration, hemorrhage and edema in the interstitial and alveolar spaces as follows: 0 (normal), normal appearing lung; 1 (mild injury), mild congestion, interstitial edema, and interstitial inflammatory cell infiltrate with occasional red blood cells and neutrophils in the alveolar spaces; 2 (moderate injury), moderate congestion and interstitial edema with inflammatory cell partially filling the alveolar spaces but without consolidation; 3 (severe injury), marked congestion and interstitial edema, with inflammatory cell infiltrate nearly filling the alveolar spaces, or with frank lung consolidation [4–6]. Results were graded from 0 to 3 for each item, as described above, where 0 = minimal damage, 1 = mild damage, 2 = moderate damage and 3 = severe damage.
2. Materials and methods 2.5. Measurement of lung vascular permeability 2.1. Animals and agents Male Kunming mice from Shanghai SLAC Laboratory Animal Co. Ltd, which were 8 weeks old and weighed 18–24 g, were used. All experiments were conducted in accordance with the guidelines for the care and use of laboratory animals from Shanghai Jiao Tong University. Animals were housed in the animal unit for at least 24 h before experimentation and given free access to food and water. Lipopolysaccharide (LPS, Escherichia coli serotype O55:B5), Evan blue, PNU-282987, pirenzepine, methoctramine, 4-DAMP, atropine and pilocarpine were purchased from Sigma Chemical (St Louis, Mo, USA).
The vascular permeability was expressed by the leakage of Evans blue in the lung as an index of pulmonary inflammation by the method described previously [10]. Mice were anesthetized with urethane (1.25 g/kg, i.p.) placed in the supine position. Evans blue (20 mg/kg) was administered via a tail vein, 30 min prior to termination of the experiment. 4 h later by i.t. instillation of LPS (4 mg/kg) or saline, lungs were removed en bloc, blotted dry and weighed, and their dye content was extracted in formamide at 37 °C for 24 h. Dye concentration was quantified by light absorbance at 620 nm, and tissue content (μg dye/mg wet weight tissue) was calculated from a standard curve of dye concentrations.
2.2. Murine model of LPS-induced acute lung inflammatory response
2.6. Culture and stimulation of alveolar macrophage cells
For induction of acute lung inflammation, mice were anesthetized with urethane (1.25 g/kg, i.p.) and placed in a supine position head up on a board tilted at 50°. A midline incision was performed in the neck and the trachea was exposed. LPS was instilled intratracheally (i.t.) in a total volume of 50 μl in saline to a final
The alveolar macrophage cell line NR8383 was cultured in Ham's F-12 medium containing 15% fetal bovine serum, 100 μg/ml streptomycin and 100 U/ml penicillin in a humidified incubator at 37 °C with 5% CO2. The mAChR antagonists such as pirenzepine, methoctramine, 4-DAMP and atropine, or the nicotinic receptor
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Fig. 1. Histological changes of LPS-induced acute lung injury. A: Control, B: LPS, C: LPS+PNU-282987, D: LPS+atropine, E: LPS+4-DAMP. F: Histology score of acute lung injury. PNU-282987 (3 mg/kg) or atropine (3 mg/kg) and 4-DAMP (1 mg/kg) were administrated 30 min before LPS (4 mg/kg). All animals were killed 4 h after LPS challenge. Data are expressed as mean±S.E.M. ##pb 0.01 vs the control group; *pb 0.05, **pb 0.01 vs the LPS challenged group. N=7–10 in each group.
agonist PNU-282987 was, where appropriate, added to the cultures 30 min before LPS. The supernatants were collected after 12 h simulated with LPS (1 μg/ml) for the detection of TNF-α. 2.7. Measurement of cytokines The amount of cytokines in the supernatants of cultured cells and in lung tissue homogenates was determined by ELISA kits (Rapidbio Lab, California, USA) according to the manufacturer's instruction. 2.8. Western blot analysis
that i.t. instillation of LPS induced significant ALI in mice. As shown in Fig. 1B, compared to lung tissue from mice exposed to saline control, the recruitment of inflammatory cells to airways, perivascular and parenchymal compartments and occasional alveolar hemorrhages were observed in some portions of the LPS-exposed lungs. Fig. 1F was the result of blinded pathologic examination and semi-quantitative analysis of the measurement of the severity and extent of inflammatory change. The pathological score in the LPS group was significantly heightened than that in the saline control group (pb 0.01). After challenged with LPS, the pulmonary vascular permeability is dramatically increased compared to the saline control mice (pb 0.01) (Fig. 2). The
Equal amounts of protein were loaded on 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes. To avoid non-specific binding, membranes were blocked with 5% non-fat milk in TBS-T (Tris–HCl 50 mM, NaCl 150 mM, Tween-20 0.1%) for 1 h at room temperature. The membranes were then incubated with primary antibodies specific for IκBα or GAPDH overnight at 4 °C. After washing the membranes three times with TBS-T for 10 min, incubation with the secondary antibody conjugated to HRP was performed within 1 h at room temperature, followed by additional washing for three times with TBS-T. Bands were subsequently visualized on film using enhanced chemiluminescence reagents. 2.9. Data analysis Data are presented as mean ± SEM. The statistical significance of differences between groups was assessed with one-way analysis of variance (ANOVA) followed by a Dunnett's test for selected pairs if appropriate. For individual comparison, statistical analysis was performed using a Student's t test. All tests were performed using Prism version 5.0 (Graph-Pad Software, San Diego, CA, USA). Values of p b 0.05 were considered significant. 3. Results 3.1. Intratracheal instillation of LPS-induced ALI Histological examination, pulmonary vascular permeability examination, measurement of BAL cells and detection of cytokines suggested
Fig. 2. Effects of PNU-282987, atropine and 4-DAMP on lung microvascular leakage induced by i.t. instillation of 4 mg/kg LPS. Mice were pretreated with PNU-282987 (3 mg/kg) or atropine (3 mg/kg) or 4-DAMP (1 mg/kg) 30 min before LPS. Evans blue (20 mg/kg) was administered via a tail vein, 10 min prior to termination of the experiment. All animals were killed 4 h after LPS administration. Data are expressed as mean ± S.E.M. ##p b 0.01 vs the saline group; **p b 0.01 vs the LPS challenged group. N = 7–10 in each group.
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Fig. 3. Effects of PUN-282987 (3 mg/kg), atropine (3 mg/kg) and 4-DAMP (1 mg/kg) on the total cell counts and percentage of neutrophils in BAL fluid recovered from mice with LPS-induced ALI. Data are expressed as mean ± S.E.M. ##pb 0.01 vs the saline group; **p b 0.01 vs the LPS challenged group. N = 7–10 in each group.
total cell number and percentage of neutrophils in the BAL fluid of the LPS group were significantly increased compared to the control group (pb 0.01) (Fig. 3A, B). The level of TNF-α or IL-6 in the lung of the LPS group was significantly higher than that of the control group (pb 0.01) (Fig. 4). Taken together, these results suggested that the model of ALI induced by i.t. instillation of LPS is successful.
3.2. Effects of PNU-282987 on LPS-induced ALI in mice PNU-282987 (a highly specific α7 nAChR agonist) (3 mg/kg) significantly reduced the inflammatory histological changes (Fig. 1C, F) (pb 0.05) and attenuated the pulmonary vascular permeability induced by LPS (Fig. 2) (pb 0.01). PNU-282987 also decreased the
Fig. 4. Effects of PUN-282987, atropine and 4-DAMP on the concentration of TNF-α or IL-6 in lung tissue homogenates in a mouse model of LPS-induced ALI. PNU-282987, atropine and 4-DAMP were administrated 30 min before LPS (4 mg/kg). All animals were killed 4 h after LPS administration. The TNF-α or IL-6 secretion was measured using specific ELISA. Data are expressed as mean ± S.E.M. ##p b 0.01 vs the saline group; *p b 0.05, **p b 0.01 vs the LPS challenged group. N = 7–10 in each group.
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Fig. 5. Effects of PUN-282987 (A), atropine (B), different muscarinic receptor antagonists (C) and 4-DAMP (D) on the concentration of TNF-α in the supernatant of alveolar macrophage after stimulation with 1 μg/ml of LPS for 12 h. PNU-282987, atropine, pirenzepine (PZ), methoctramine (Methoc) or 4-DAMP was administrated 30 min before LPS. The levels of TNF-α were evaluated by specific ELISA. Data are expressed as mean ± S.E.M. from at least three experiments. ##p b 0.01 vs control; *p b 0.05 vs LPS; **p b 0.01 vs LPS.
total cell number and percentage of neutrophils in the BAL (Fig. 3A, B) (pb 0.01). PNU-282987 (3 mg/kg) prevented the LPS-induced increase in the levels of TNF-α (pb 0.01) and IL-6 (pb 0.01) in the lung tissue homogenates (Fig. 4A, C). 3.3. Effects of atropine and 4-DAMP on LPS-induced ALI in mice To evaluate the role of mAChR in LPS-induced ALI, atropine, a non-selective mAChR antagonist or 4-DAMP, a M3 mAChR antagonist was given to mice 30 min before LPS. Atropine (3 mg/kg) significantly reduced the histological inflammatory changes (Fig. 1D, F) (p b 0.05) and the pulmonary microvascular leakage induced by LPS (Fig. 2) (p b 0.01). The total cell number and percentage of neutrophils in the BAL of the atropine group (3 mg/kg) were also decreased compared to the LPS group (Fig. 3A, B) (p b 0.01). Atropine also significantly reduced the levels of TNF-α in the lung induced by LPS at doses of 0.1, 1 and 3 mg/kg (Fig. 4B) and the level of IL-6 at the dose of 3 mg/kg (Fig. 4C) compared to the LPS-exposed group, while IL-8 and MIP-2 only showed a declined trend without reaching statistical significance (data not shown). Similar results were also obtained for the prevention of LPS-induced events in mice by 4-DAMP at a dose of 1 mg/kg in comparison with atropine at a dose of 3 mg/kg (Figs. 1–4). These results suggested that blockage of mAChR by atropine or 4-DAMP exerted similar anti-inflammatory effects as activating α7 nAChR by PNU-282987 in our mouse model of LPS-induced ALI.
3.4. Effect of cholinergic agents on LPS-induced TNF-α production in alveolar macrophage The levels of TNF-α in the supernatants of cultured cells under LPS stimulation were significantly (pb 0.01) increased, and this increased effect was significantly attenuated by PNU-282987 at the concentrations of 10 μM and 100 μM (Fig. 5A). Similarly, atropine also showed a significant effect on decreasing the TNF-α production at concentrations of 10 μM and 100 μM compared to the supernatant from the alveolar macrophage cultured with LPS alone (Fig. 5B). Reduction in the increase of TNF-α production reached 10.21% and 17.64% at the concentration of 10 μM; 27.96% and 47.13% at the concentration of 100 μM, respectively for PNU-282987 and atropine. In order to better understand which mAChR subtypes are involved in the release of TNF-α by alveolar macrophage, we used the following anticholinergic drugs: pirenzepine (10 μM), methoctramine (10 μM) and 4-DAMP (10 μM), respectively for M1, M2 and M3 mAChR antagonists 30 min before the LPS. Pre-incubation of cells with pirenzepine or methoctramine showed no significant inhibitory effect on LPS-induced TNF-α release (Fig. 5C), however, pre-incubation with 4-DAMP from 1 μM to 100 μM resulted in a concentration-dependent significant decrease in TNF-α release (Fig. 5D). Taken together, these results demonstrated that activation of α7 nAChR by PNU-282987 or blockage of mAChR by atropine or 4-DAMP exerted similar anti-inflammatory properties and M3 mAChR is the mainly responsible mAChR subtype in the progress of TNF-α release in alveolar macrophage.
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3.5. Influence of pilocarpine on TNF-α production from alveolar macrophage In order to further investigate the involvement of mAChRs, alveolar macrophage cells were pre-incubated with 10 μM pilocarpine alone or in combination with 1 μg/ml LPS, which was added 15 min before pilocarpine, and the supernatants were collected after 12 h. As shown in Fig. 6A, ELISA assay data for pilocarpine plus LPS-treated cells presented in a strongly synergistic manner (pb 0.01) an increase of TNF-α release when compared to the LPS treated alone. The synergistic effect was 18% greater than the LPS treated alone (pb 0.01). In other experiments, the effects of atropine to cause a pronounced decrease in LPS-induced TNF-α release were almost completely reversed by pilocarpine in a concentration-dependent manner (Fig. 6B). Interestingly, treatment with pilocarpine alone was able to significantly increase the level of TNF-α in the supernatants (pb 0.01), and this effect was also inhibited by 4-DAMP (1 μM) (pb 0.01), whereas pirenzepine (10 μM) or methoctramine (10 μM) was without inhibitory effect (Fig. 6A, C), suggesting that LPS activates a non-neuronal cholinergic system and endogenous acetylcholine exerts its activity through the activation of M3 mAChRs, which are also the most expressed in alveolar macrophage cells. 3.6. NF-κB pathway is involved in the anti-inflammation effect of muscarinic receptors both in mice and in alveolar macrophage Activation of the NF-κB pathway promotes the production of proinflammatory cytokine in multiple cell type. To understand whether the anti-inflammatory properties exerted by mAChR antagonists are related to the NF-κB pathway, LPS-induced IκBα degradation, which eventually leads to suppression of NF-κB activation, was assessed by western blot analysis in our study. As shown in Fig. 7, LPS-induced degradation of IκBα was effectively prevented in atropine or 4-DAMP-pretreated group both in vivo and in vitro. A similarity effect was found in the pretreatment with PNU-282987, demonstrating that M3 mAChR is involved in LPS-induced lung inflammatory processes. 4. Discussion ACh receptors, like many other ligand-activated neurotransmitter receptors, consist of two major subtypes, mAChRs and nAChRs. Each group is further subdivided based on pharmacology, location, mode of action, and molecular biology. Both share the property of being activated by the endogenous ACh. The cholinergic anti-inflammatory pathway is important in modulating the inflammatory response in local and systemic diseases, including ALI [28]. Activation of α7 nAChR by its exogenous agonist could limit lung inflammation. We extended, in the present study, this notion and provided empirical evidence that blockage of mAChRs, in particular, M3 mAChR could influence the lung inflammatory process elicited by LPS, and as indicated by the diminution of neutrophil accumulation in the lungs, pulmonary vascular permeability and TNF-α and IL-6 production exerted similar anti-inflammatory effects as the activation of α7 nAChR by PNU-282987. It is well known that lung immune cells (monocytes, macrophages and neutrophils) play an important role in the pathogenesis of acute lung inflammatory responses. LPS stimulates macrophages/monocytes to sequentially release various inflammatory molecules like proFig. 6. A) Effects of LPS, pilocarpine or LPS+pilocarpine; B) influence of pilocarpine on the inhibitory effect of atropine in LPS-induced TNF-α release; C) effect of the selective muscarinic receptor antagonists pirenzepine (10 μM PZ), methoctramine (10 μM Methoc) and 4-DAMP (1 μM) on pilocarpine-induced TNF-α release. Alveolar macrophages were pretreated with pilocarpine (10 μM Pilo) 15 min before LPS. TNF-α concentrations in the supernatant were detected after stimulation with LPS for 12 h. The secretion of TNF-α was evaluated by specific ELISA. Data are expressed as mean±S.E.M. from at least three experiments. ##pb 0.01 vs control; **pb 0.01 vs LPS; ++pb 0.01 vs LPS; &&pb 0.01 vs atropine.
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Fig. 7. Effects of PUN-282987, atropine and 4-DAMP on the degradation of IKBα in lung tissues of ALI mice or in LPS-stimulated alveolar macrophage. In animals, atropine (3.0 mg/kg, i.p.), 4-DAMP (1.0 mg/kg, i.p.) and PUN-282987 (3.0 mg/kg, i.p.) were administrated 30 min before LPS (4 mg/kg). All animals were killed 4 h after LPS administration. In alveolar macrophage, atropine (10 μM), 4-DAMP (10 μM) and PUN-282987 (10 μM) were administrated 30 min before LPS (1 μg/ml) and the cells were stimulated with LPS for 30 min. The gel images shown are representative of 5–6 animals or 3 separate in vivo experiments.
inflammatory cytokines including TNF-α, which is an important mediator of immuno-inflammatory responses and inducer of other proinflammatory cytokines, associated with the development and outcome of ALI [22]. The correlation between the level of TNF-α and development of ALI is highest when this cytokine is measured in the lung or BAL fluid [15]. Interestingly, LPS-induced TNF-α production was enhanced by the mAChR agonist pilocarpine, and pilocarpine treatment alone was able to trigger TNF-α production from alveolar macrophage, which was effectively attenuated by the M3 mAChR antagonist. This finding could be explained by augmented endogenous ACh release after LPS stimulated, and demonstrated that inflammatory cytokines are likely to play a crucial part in the observed effects of endogenous ACh. Accumulating evidence has demonstrated that ACh and its synthesizing enzyme, choline acetyltransferase, are present not only in airway nerves, but also are localized to lung immune cells [29,30]. ACh is known to have different immunological effects on these cells. It is predominantly pro-inflammatory for lymphocytes, while anti-inflammatory for macrophages. It can be either pro- or anti-inflammatory for monocytes, and may have variable effects on neutrophils [29]. Furthermore, recent studies reported that lymphocytes and macrophage expressed mRNA for components of the non-neuronal cholinergic system, which possessed acting sites for endogenous and exogenous mAChR agonists [30–33]. Therefore, it is likely that mAChRs on immune cells play a role in regulating the inflammatory responses. Our research findings lend further support to this notion and demonstrate that mAChRs on macrophage mediated the LPS-induced release of TNF-α, evidenced by the fact that both non-specific mAChR atropine and M3-preferring antagonist 4-DAMP, but not M1 and M2 mAChR antagonists, could attenuate LPS stimulated TNF-α production in lung macrophage. LPS-induced macrophage TNF-α production that is either attenuated by a mAChR antagonist or enhanced by a mAChR agonist leads us to presume that endogenous ACh might be involved in the modulation of the function of macrophage through paracrine and/or autocrine mechanisms. If the presumption is
true, then it was reasonable to believe that the effect of endogenous ACh could be reproduced by exogenous mAChR agonists. Our study was further confirmed by our present findings that production of TNF-α from macrophage was triggered by the mAChR agonist pilocarpine alone and was blocked by a mAChR antagonist, in particular, blockage of M3 mAChR. The present findings are consistent with other research which found that activation of lung macrophages with carbachol could induce LTB4 release and is attenuated by the M3 mAChR antagonist [33] and indicate the ability of endogenous ACh, which might serve as a stimulator in inducing inflammatory cytokine release through direct interaction with M3 mAChRs to regulate the functions of macrophage, and provide proof to the concept of an anti-inflammatory activity of anti-muscarinic agents in inflammatory disease. GPCRs and their signaling molecules have diverse and central roles in regulating macrophage function [34–36]. A recent study shows that the GPCRs are also important regulators of innate and acquired immunity [36]. The mAChRs belong to the superfamily of GPCR genes and mediate the effects of ACh [37] and also contribute to control inflammatory processes via interactions with inflammatory signaling molecules [38]. Recently, Razani-Boroujerdi S. et al. [32] reported that oxotremorine stimulated and atropine inhibited the antibody and T-cell proliferative responses. Moreover, atropine also suppressed the turpentine-induced leukocytic infiltration and tissue injury, and inhibited chemotaxis of leukocytes toward neutrophil and monocyte/lymphocyte chemoattractants. The underlying mechanisms responsible for the inflammatory process remain unclear. We now have the evidence that LPS stimulation leads to increase in TNF-α production by macrophage which is dependent on downstream signaling to the IκBα/NF-κB pathways. In addition, LPS-induced IκBα degradation to regulate TNF-α secretion could be blocked by atropine or 4-DAMP, suggesting that blockage of the M3 mAChR, produced a similar effect of PNU-282987 to influence LPS-induced degradation of IκBα seen during intratracheal instillation of LPS to mice [39].
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Thus, the present study suggests that blockage of mAChRs may lead to modulation of the inflammatory response elicited by LPS both in mice and in alveolar macrophage, and endogenous ACh, acting on mAChRs, in particular, M3 mAChR, plays a broad role in the inflammatory responses. Therefore, understanding the role of M3 mAChR may offer novel therapeutic strategies to approach inflammatory lung diseases. Funding This work was supported by the National Key Basic Research Program (2010CB529800), the National Natural Science Foundation of China (Nos. 81070010, 30873109), the Key Project of Science and Technology Commission of Shanghai Municipality (No. 09JC1408800) and the Research Fund for the Doctoral Program of Shanghai Jiao Tong University School of Medicine (BXJ201007). References [1] Wang H, Yu M, Ochani M, Amella CA, Tanovic M, Susarla S. Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation. Nature 2003;421:384-8. [2] van Westerloo DJ, Giebelen IA, Florquin S, Daalhuisen J, Bruno MJ, de Vos AF, et al. The cholinergic anti-inflammatory pathway regulates the host response during septic peritonitis. J Infect Dis 2005;191:2138-48. [3] Wang H, Liao H, Ochani M, Justiniani M, Lin X, Yang L, et al. Cholinergic agonists inhibit HMGB1 release and improve survival in experimental sepsis. Nat Med 2004;10:1216-21. [4] Giebelen IA, van Westerloo DJ, LaRosa GJ, de Vos AF, van der Poll T. Local stimulation of alpha7 cholinergic receptors inhibits LPS-induced TNF-alpha release in the mouse lung. Shock 2007;28:700-3. [5] Su X, Matthay MA, Malik AB. Requisite role of the cholinergic alpha7 nicotinic acetylcholine receptor pathway in suppressing Gram-negative sepsis-induced acute lung inflammatory injury. J Immunol 2010;184:401-10. [6] Nathanson NM. Molecular properties of the muscarinic acetylcholine receptor. Annu Rev Neurosci 1987;10:195-236. [7] Wess J. Molecular biology of muscarinic acetylcholine receptors. Crit Rev Neurobiol 1996;10:69-99. [8] Bühling F, Lieder N, Kühlmann UC, Waldburg N, Welte T. Tiotropium suppresses acetylcholine-induced release of chemotactic mediators in vitro. Respir Med 2007;101:2386-94. [9] McQueen DS, Donaldson K, Bond SM, McNeilly JD, Newman S, Barton NJ, et al. Bilateral vagotomy or atropine pre-treatment reduces experimental diesel-soot induced lung inflammation. Toxicol Appl Pharmacol 2007;219:62-71. [10] Cui Yong-Yao, Zhu Liang, Wang Hao, Advenier Charles, Devillier Philippe, Chen Hong-Zhuan. Muscarinic receptors involved in airway vascular leakage induced by experimental gastro-oesophageal reflux. Life Sci 2008;82:949-55. [11] Cui Y, Devillier P, Kuang X, Wang H, Zhu L, Xu Z, et al. Tiotropium reduction of lung inflammation in a model of chronic gastro-oesophageal reflux. Eur Respir J 2010;35:1370-6. [12] Ohta S, Oda N, Yokoe T, Tanaka A, Yamamoto Y, Watanabe Y, et al. Effect of tiotropium bromide on airway inflammation and remodelling in a mouse model of asthma. Clin Exp Allergy 2010;40:1266-75. [13] Beeh KM, Beier J. Handle with care: targeting neutrophils in chronic obstructive pulmonary disease and severe asthma? Clin Exp Allergy 2006;36:142-57. [14] Lyczak JB, Cannon CL, Pier GB. Lung infections associated with cystic fibrosis. Clin Microbiol Rev 2002;15:194-222. [15] Meduri GU. Clinical review: a paradigm shift: the bidirectional effect of inflammation on bacterial growth. Clinical implications for patients with acute respiratory distress syndrome. Crit Care 2002;6:24-9.
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Role of M3 mAChR in in vivo and in vitro models of LPS-induced inflammatory response Zu-Peng Xu a, 1, Kai Yang a, 1, Guang-Ni Xu a, Liang Zhu a, Li-Na Hou a, Wen-Hui Zhang b, Hong-Zhuan Chen a,⁎, Yong-Yao Cui a,⁎ a b
Department of Pharmacology, Shanghai Jiao Tong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, China Department of Physiology, Shanghai Jiao Tong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, China
a r t i c l e
i n f o
Article history: Received 20 January 2012 Received in revised form 23 April 2012 Accepted 27 July 2012 Available online 19 August 2012 Keywords: nAChR mAChR Mice Alveolar macrophage Inflammation
a b s t r a c t Objective: We tested the potential role of the mAChR in lipopolysaccharide (LPS)‐induced inflammatory response in in vivo and in vitro models and a possible signaling pathway involved in the inflammatory process. Methods: Anesthetized mice were challenged with intratracheal LPS to induce acute lung injury. The cytology and histopathology changes, expression of cytokines and pulmonary vascular permeability were used to evaluate the effects of the cholinergic agent. Alveolar macrophage cell line NR8383 was also used to confirm the role of mAChRs and the molecular mechanisms underlying the LPS-induced events. Results: LPS-induced acute lung injury (ALI) was significantly improved by atropine (a non-selective mAChR antagonist) and 4-DAMP (a M3 mAChR antagonist), as indicated by the diminution of neutrophil infiltration, pulmonary vascular permeability and IL-6 and TNF-α production. LPS-induced TNF-α production from the alveolar macrophage was significantly inhibited by atropine and 4-DAMP, but not pirenzepine (a M1 mAChR antagonist) and methoctramine (a M2 mAChR antagonist). Interestingly, LPS-induced TNF-α production was enhanced by the muscarinic receptor agonist pilocarpine, and treatment with pilocarpine alone was able to trigger TNF-α production from the alveolar macrophage, which was effectively attenuated by 4-DAMP. Western blot analysis showed that LPS-induced degradation of IκBα was strongly blocked by atropine/4-DAMP both in vivo and in vitro, indicating that M3 mAChR was involved in LPS-induced lung inflammation by mediating the NF-κB signaling pathway. Conclusion: Our findings bring the evidence that the blockage of mAChR exerts anti-inflammatory properties, in which the M3 mAChR plays an important role in the LPS-induced lung inflammation. © 2012 Elsevier B.V. All rights reserved.
1. Introduction An increasing body of knowledge indicates that acetylcholine can suppress the production of multiple cytokines directly via the α7 nicotinic acetylcholine receptor (α7 nAChR) expressed on macrophages [1], which is called the cholinergic antiinflammatory pathway. Pharmacological stimulation of the peripheral α7 nAChR by nicotinic receptor agonists prevents NF-κB activation and inhibits cytokine release in wild-type but not in α7 nAChR-deficient macrophages [1,2]. In vivo, nicotinic agonists decrease inflammation and increase survival in severe sepsis [3]. Although nAChR and mAChR respond to the same neuromediator—acetylcholine and activation of α7 nAChR could
⁎ Corresponding authors. Tel.: +86 21 6384 6590x776451; fax: +86 21 6467 4721. E-mail addresses: [email protected] (H.-Z. Chen), [email protected] (Y.-Y. Cui). 1 Both authors contributed equally to this article. 1567-5769/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.intimp.2012.07.020
attenuate sepsis-induced ALI [4,5], the role of the mAChR remains relatively unexplored. The mAChR belongs to the superfamily of G-protein coupled receptors (GPCRs) that are commonly expressed in a variety of tissues and are classified into five known subtypes (M1–M5 mAChRs). M1, M3, and M5 mAChRs are selectively coupled to Gq proteins while M2 and M4 mAChRs are linked to Gi/G0 proteins [6,7]. Several studies have demonstrated that mAChR activation has pro-inflammatory effects in a wide variety of cells implicated in airway inflammation. Acetylcholine, acting at the M3 mAChR, can stimulate bovine alveolar macrophages and human sputum macrophages to release lipoxygenase-derived mediators, predominantly LTB4. The anticholinergic drug has been shown to regulate the release of chemotactic factors from human epithelial cells and macrophages in vitro [8]. Studies in animals have shown that mAChR antagonists inhibit vagus nerve stimulation or ACh induction of vascular edema and eosinophil infiltration into the guinea pig lung, and lung neutrophilia in the rat in response to diesel soot inhalation [9]. In previous studies, we have demonstrated the critical importance of mAChR in lung inflammation induced by repeated HCl intra-esophageal instillation
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in mice [10,11]. The modulatory effect of mAChRs for inflammatory responses was also confirmed in a mouse model of asthma [12]. In addition, the change of expression of M2 and M3 mAChRs on inflammatory cells recovered from COPD subjects is also related to the inflammation response. Overall, these observations suggest that inflammatory responses might be induced by the stimulation of mAChR and release of inflammatory cytokines, which can be diminished by mAChR antagonists. Inflammation of the lung marked by excessive recruitment of neutrophils from circulation to the airway is a common feature among several pathological lung disorders, particularly those involving infection [13–15]. ALI is characterized by extensive neutrophil influx into the lungs, the production of pro-inflammatory mediators from inflammatory cells, and pulmonary microvascular leakage, followed by severe lung damage [16–18]. The manifestations of ALI are related to the activation of antigen presenting cells like alveolar macrophages, up-regulation of cell surface adhesion molecules and subsequent production of cytokines and chemokines. Activated alveolar macrophages are the main sources of TNF-α, which is suggested as an important early mediator of ALI. In the inflammatory process, over-production of TNF-α is pivotal in the induction of inflammatory genes and in the recruitment and activation of immune cells. A lipopolysaccharide (LPS) is an outer membrane component of Gram negative bacteria. A model of LPS-induced ALI was applied to mimic the morphological and functional changes observed in clinical situations resulting from circulating LPS. It is well documented that LPS administration triggers a network of inflammatory responses mediated by a number of immune cells, such as neutrophil and macrophage, which is followed by the release of various inflammatory molecules including TNF-α and IL-6, which play a very important role in the pathogenesis of acute lung injury [19–24]. LPS-treated animals could induce ALI characterized by increased infiltration of inflammatory cells and production of inflammatory mediators and tissue edema [25]. The current study was undertaken to test the efficacy of mAChR antagonists compared to α7 nAChR agonists, administered right before exposure, and attenuated the extent of lung inflammation. In vivo, exposure of mice to LPS by intratracheal instillation increased proinflammatory cytokine production, neutrophil infiltration into the lung and pulmonary vascular permeability, and in vitro, exposure of alveolar macrophage cell line NR8383 to LPS increased cytokine release. Furthermore, we also evaluated whether inhibition of the NF-κB signaling pathway was involved in the protective effects of mAChR antagonists on LPS-induced lung inflammatory injury.
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dose of 4 mg/kg [26,27]. After intratracheal instillation, the mice were placed in a vertical position and rotated for 0.5–1 min to distribute the instillation evenly within the lungs. Sham-operated mice underwent the same procedure with intratracheal (i.t.) injection of 50 μl sterile saline without LPS. Atropine (3 mg/kg, i.p.) and PNU-282987 (3 mg/kg, i.p.) were given 30 min before LPS instillation. 2.3. Collection of bronchoalveolar lavage fluid (BALF) and cell counting Four hours after LPS challenge, mice were sacrificed by receiving euthanasia. The bronchoalveolar lavage was performed by infusion and extraction of 1 ml of phosphate buffered saline (PBS) via a tracheal cannula. This was repeated twice, and the recovered BALF was pooled. The total number of cells was counted with a hemocytometer. The percentage of neutrophils was determined on cytospin smears of BAL samples from individual mice, and stained with Wright–Giemsa after counting 300 cells. Results are expressed as cell number × 10 4/ml of BALF. 2.4. Histopathological examination of the lung The right lung was harvested and fixed in 10% phosphatebuffered formalin. After 24 h of fixation, lungs were embedded in paraffin, and 8 μm sections were cut and stained with hematoxylin and eosin (H&E), as in our prior studies [11]. All samples were analyzed based on a scaled grading system by a pathologist who was blinded to the experimental protocol and the region of sampling. A total of three slides from each lung sample were randomly screened and the mean was taken as the representative value of the sample. Each section was assigned a numerical histological injury score ranging from 0 to 3, based on the degree of inflammatory cell infiltration, hemorrhage and edema in the interstitial and alveolar spaces as follows: 0 (normal), normal appearing lung; 1 (mild injury), mild congestion, interstitial edema, and interstitial inflammatory cell infiltrate with occasional red blood cells and neutrophils in the alveolar spaces; 2 (moderate injury), moderate congestion and interstitial edema with inflammatory cell partially filling the alveolar spaces but without consolidation; 3 (severe injury), marked congestion and interstitial edema, with inflammatory cell infiltrate nearly filling the alveolar spaces, or with frank lung consolidation [4–6]. Results were graded from 0 to 3 for each item, as described above, where 0 = minimal damage, 1 = mild damage, 2 = moderate damage and 3 = severe damage.
2. Materials and methods 2.5. Measurement of lung vascular permeability 2.1. Animals and agents Male Kunming mice from Shanghai SLAC Laboratory Animal Co. Ltd, which were 8 weeks old and weighed 18–24 g, were used. All experiments were conducted in accordance with the guidelines for the care and use of laboratory animals from Shanghai Jiao Tong University. Animals were housed in the animal unit for at least 24 h before experimentation and given free access to food and water. Lipopolysaccharide (LPS, Escherichia coli serotype O55:B5), Evan blue, PNU-282987, pirenzepine, methoctramine, 4-DAMP, atropine and pilocarpine were purchased from Sigma Chemical (St Louis, Mo, USA).
The vascular permeability was expressed by the leakage of Evans blue in the lung as an index of pulmonary inflammation by the method described previously [10]. Mice were anesthetized with urethane (1.25 g/kg, i.p.) placed in the supine position. Evans blue (20 mg/kg) was administered via a tail vein, 30 min prior to termination of the experiment. 4 h later by i.t. instillation of LPS (4 mg/kg) or saline, lungs were removed en bloc, blotted dry and weighed, and their dye content was extracted in formamide at 37 °C for 24 h. Dye concentration was quantified by light absorbance at 620 nm, and tissue content (μg dye/mg wet weight tissue) was calculated from a standard curve of dye concentrations.
2.2. Murine model of LPS-induced acute lung inflammatory response
2.6. Culture and stimulation of alveolar macrophage cells
For induction of acute lung inflammation, mice were anesthetized with urethane (1.25 g/kg, i.p.) and placed in a supine position head up on a board tilted at 50°. A midline incision was performed in the neck and the trachea was exposed. LPS was instilled intratracheally (i.t.) in a total volume of 50 μl in saline to a final
The alveolar macrophage cell line NR8383 was cultured in Ham's F-12 medium containing 15% fetal bovine serum, 100 μg/ml streptomycin and 100 U/ml penicillin in a humidified incubator at 37 °C with 5% CO2. The mAChR antagonists such as pirenzepine, methoctramine, 4-DAMP and atropine, or the nicotinic receptor
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Fig. 1. Histological changes of LPS-induced acute lung injury. A: Control, B: LPS, C: LPS+PNU-282987, D: LPS+atropine, E: LPS+4-DAMP. F: Histology score of acute lung injury. PNU-282987 (3 mg/kg) or atropine (3 mg/kg) and 4-DAMP (1 mg/kg) were administrated 30 min before LPS (4 mg/kg). All animals were killed 4 h after LPS challenge. Data are expressed as mean±S.E.M. ##pb 0.01 vs the control group; *pb 0.05, **pb 0.01 vs the LPS challenged group. N=7–10 in each group.
agonist PNU-282987 was, where appropriate, added to the cultures 30 min before LPS. The supernatants were collected after 12 h simulated with LPS (1 μg/ml) for the detection of TNF-α. 2.7. Measurement of cytokines The amount of cytokines in the supernatants of cultured cells and in lung tissue homogenates was determined by ELISA kits (Rapidbio Lab, California, USA) according to the manufacturer's instruction. 2.8. Western blot analysis
that i.t. instillation of LPS induced significant ALI in mice. As shown in Fig. 1B, compared to lung tissue from mice exposed to saline control, the recruitment of inflammatory cells to airways, perivascular and parenchymal compartments and occasional alveolar hemorrhages were observed in some portions of the LPS-exposed lungs. Fig. 1F was the result of blinded pathologic examination and semi-quantitative analysis of the measurement of the severity and extent of inflammatory change. The pathological score in the LPS group was significantly heightened than that in the saline control group (pb 0.01). After challenged with LPS, the pulmonary vascular permeability is dramatically increased compared to the saline control mice (pb 0.01) (Fig. 2). The
Equal amounts of protein were loaded on 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes. To avoid non-specific binding, membranes were blocked with 5% non-fat milk in TBS-T (Tris–HCl 50 mM, NaCl 150 mM, Tween-20 0.1%) for 1 h at room temperature. The membranes were then incubated with primary antibodies specific for IκBα or GAPDH overnight at 4 °C. After washing the membranes three times with TBS-T for 10 min, incubation with the secondary antibody conjugated to HRP was performed within 1 h at room temperature, followed by additional washing for three times with TBS-T. Bands were subsequently visualized on film using enhanced chemiluminescence reagents. 2.9. Data analysis Data are presented as mean ± SEM. The statistical significance of differences between groups was assessed with one-way analysis of variance (ANOVA) followed by a Dunnett's test for selected pairs if appropriate. For individual comparison, statistical analysis was performed using a Student's t test. All tests were performed using Prism version 5.0 (Graph-Pad Software, San Diego, CA, USA). Values of p b 0.05 were considered significant. 3. Results 3.1. Intratracheal instillation of LPS-induced ALI Histological examination, pulmonary vascular permeability examination, measurement of BAL cells and detection of cytokines suggested
Fig. 2. Effects of PNU-282987, atropine and 4-DAMP on lung microvascular leakage induced by i.t. instillation of 4 mg/kg LPS. Mice were pretreated with PNU-282987 (3 mg/kg) or atropine (3 mg/kg) or 4-DAMP (1 mg/kg) 30 min before LPS. Evans blue (20 mg/kg) was administered via a tail vein, 10 min prior to termination of the experiment. All animals were killed 4 h after LPS administration. Data are expressed as mean ± S.E.M. ##p b 0.01 vs the saline group; **p b 0.01 vs the LPS challenged group. N = 7–10 in each group.
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Fig. 3. Effects of PUN-282987 (3 mg/kg), atropine (3 mg/kg) and 4-DAMP (1 mg/kg) on the total cell counts and percentage of neutrophils in BAL fluid recovered from mice with LPS-induced ALI. Data are expressed as mean ± S.E.M. ##pb 0.01 vs the saline group; **p b 0.01 vs the LPS challenged group. N = 7–10 in each group.
total cell number and percentage of neutrophils in the BAL fluid of the LPS group were significantly increased compared to the control group (pb 0.01) (Fig. 3A, B). The level of TNF-α or IL-6 in the lung of the LPS group was significantly higher than that of the control group (pb 0.01) (Fig. 4). Taken together, these results suggested that the model of ALI induced by i.t. instillation of LPS is successful.
3.2. Effects of PNU-282987 on LPS-induced ALI in mice PNU-282987 (a highly specific α7 nAChR agonist) (3 mg/kg) significantly reduced the inflammatory histological changes (Fig. 1C, F) (pb 0.05) and attenuated the pulmonary vascular permeability induced by LPS (Fig. 2) (pb 0.01). PNU-282987 also decreased the
Fig. 4. Effects of PUN-282987, atropine and 4-DAMP on the concentration of TNF-α or IL-6 in lung tissue homogenates in a mouse model of LPS-induced ALI. PNU-282987, atropine and 4-DAMP were administrated 30 min before LPS (4 mg/kg). All animals were killed 4 h after LPS administration. The TNF-α or IL-6 secretion was measured using specific ELISA. Data are expressed as mean ± S.E.M. ##p b 0.01 vs the saline group; *p b 0.05, **p b 0.01 vs the LPS challenged group. N = 7–10 in each group.
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Fig. 5. Effects of PUN-282987 (A), atropine (B), different muscarinic receptor antagonists (C) and 4-DAMP (D) on the concentration of TNF-α in the supernatant of alveolar macrophage after stimulation with 1 μg/ml of LPS for 12 h. PNU-282987, atropine, pirenzepine (PZ), methoctramine (Methoc) or 4-DAMP was administrated 30 min before LPS. The levels of TNF-α were evaluated by specific ELISA. Data are expressed as mean ± S.E.M. from at least three experiments. ##p b 0.01 vs control; *p b 0.05 vs LPS; **p b 0.01 vs LPS.
total cell number and percentage of neutrophils in the BAL (Fig. 3A, B) (pb 0.01). PNU-282987 (3 mg/kg) prevented the LPS-induced increase in the levels of TNF-α (pb 0.01) and IL-6 (pb 0.01) in the lung tissue homogenates (Fig. 4A, C). 3.3. Effects of atropine and 4-DAMP on LPS-induced ALI in mice To evaluate the role of mAChR in LPS-induced ALI, atropine, a non-selective mAChR antagonist or 4-DAMP, a M3 mAChR antagonist was given to mice 30 min before LPS. Atropine (3 mg/kg) significantly reduced the histological inflammatory changes (Fig. 1D, F) (p b 0.05) and the pulmonary microvascular leakage induced by LPS (Fig. 2) (p b 0.01). The total cell number and percentage of neutrophils in the BAL of the atropine group (3 mg/kg) were also decreased compared to the LPS group (Fig. 3A, B) (p b 0.01). Atropine also significantly reduced the levels of TNF-α in the lung induced by LPS at doses of 0.1, 1 and 3 mg/kg (Fig. 4B) and the level of IL-6 at the dose of 3 mg/kg (Fig. 4C) compared to the LPS-exposed group, while IL-8 and MIP-2 only showed a declined trend without reaching statistical significance (data not shown). Similar results were also obtained for the prevention of LPS-induced events in mice by 4-DAMP at a dose of 1 mg/kg in comparison with atropine at a dose of 3 mg/kg (Figs. 1–4). These results suggested that blockage of mAChR by atropine or 4-DAMP exerted similar anti-inflammatory effects as activating α7 nAChR by PNU-282987 in our mouse model of LPS-induced ALI.
3.4. Effect of cholinergic agents on LPS-induced TNF-α production in alveolar macrophage The levels of TNF-α in the supernatants of cultured cells under LPS stimulation were significantly (pb 0.01) increased, and this increased effect was significantly attenuated by PNU-282987 at the concentrations of 10 μM and 100 μM (Fig. 5A). Similarly, atropine also showed a significant effect on decreasing the TNF-α production at concentrations of 10 μM and 100 μM compared to the supernatant from the alveolar macrophage cultured with LPS alone (Fig. 5B). Reduction in the increase of TNF-α production reached 10.21% and 17.64% at the concentration of 10 μM; 27.96% and 47.13% at the concentration of 100 μM, respectively for PNU-282987 and atropine. In order to better understand which mAChR subtypes are involved in the release of TNF-α by alveolar macrophage, we used the following anticholinergic drugs: pirenzepine (10 μM), methoctramine (10 μM) and 4-DAMP (10 μM), respectively for M1, M2 and M3 mAChR antagonists 30 min before the LPS. Pre-incubation of cells with pirenzepine or methoctramine showed no significant inhibitory effect on LPS-induced TNF-α release (Fig. 5C), however, pre-incubation with 4-DAMP from 1 μM to 100 μM resulted in a concentration-dependent significant decrease in TNF-α release (Fig. 5D). Taken together, these results demonstrated that activation of α7 nAChR by PNU-282987 or blockage of mAChR by atropine or 4-DAMP exerted similar anti-inflammatory properties and M3 mAChR is the mainly responsible mAChR subtype in the progress of TNF-α release in alveolar macrophage.
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3.5. Influence of pilocarpine on TNF-α production from alveolar macrophage In order to further investigate the involvement of mAChRs, alveolar macrophage cells were pre-incubated with 10 μM pilocarpine alone or in combination with 1 μg/ml LPS, which was added 15 min before pilocarpine, and the supernatants were collected after 12 h. As shown in Fig. 6A, ELISA assay data for pilocarpine plus LPS-treated cells presented in a strongly synergistic manner (pb 0.01) an increase of TNF-α release when compared to the LPS treated alone. The synergistic effect was 18% greater than the LPS treated alone (pb 0.01). In other experiments, the effects of atropine to cause a pronounced decrease in LPS-induced TNF-α release were almost completely reversed by pilocarpine in a concentration-dependent manner (Fig. 6B). Interestingly, treatment with pilocarpine alone was able to significantly increase the level of TNF-α in the supernatants (pb 0.01), and this effect was also inhibited by 4-DAMP (1 μM) (pb 0.01), whereas pirenzepine (10 μM) or methoctramine (10 μM) was without inhibitory effect (Fig. 6A, C), suggesting that LPS activates a non-neuronal cholinergic system and endogenous acetylcholine exerts its activity through the activation of M3 mAChRs, which are also the most expressed in alveolar macrophage cells. 3.6. NF-κB pathway is involved in the anti-inflammation effect of muscarinic receptors both in mice and in alveolar macrophage Activation of the NF-κB pathway promotes the production of proinflammatory cytokine in multiple cell type. To understand whether the anti-inflammatory properties exerted by mAChR antagonists are related to the NF-κB pathway, LPS-induced IκBα degradation, which eventually leads to suppression of NF-κB activation, was assessed by western blot analysis in our study. As shown in Fig. 7, LPS-induced degradation of IκBα was effectively prevented in atropine or 4-DAMP-pretreated group both in vivo and in vitro. A similarity effect was found in the pretreatment with PNU-282987, demonstrating that M3 mAChR is involved in LPS-induced lung inflammatory processes. 4. Discussion ACh receptors, like many other ligand-activated neurotransmitter receptors, consist of two major subtypes, mAChRs and nAChRs. Each group is further subdivided based on pharmacology, location, mode of action, and molecular biology. Both share the property of being activated by the endogenous ACh. The cholinergic anti-inflammatory pathway is important in modulating the inflammatory response in local and systemic diseases, including ALI [28]. Activation of α7 nAChR by its exogenous agonist could limit lung inflammation. We extended, in the present study, this notion and provided empirical evidence that blockage of mAChRs, in particular, M3 mAChR could influence the lung inflammatory process elicited by LPS, and as indicated by the diminution of neutrophil accumulation in the lungs, pulmonary vascular permeability and TNF-α and IL-6 production exerted similar anti-inflammatory effects as the activation of α7 nAChR by PNU-282987. It is well known that lung immune cells (monocytes, macrophages and neutrophils) play an important role in the pathogenesis of acute lung inflammatory responses. LPS stimulates macrophages/monocytes to sequentially release various inflammatory molecules like proFig. 6. A) Effects of LPS, pilocarpine or LPS+pilocarpine; B) influence of pilocarpine on the inhibitory effect of atropine in LPS-induced TNF-α release; C) effect of the selective muscarinic receptor antagonists pirenzepine (10 μM PZ), methoctramine (10 μM Methoc) and 4-DAMP (1 μM) on pilocarpine-induced TNF-α release. Alveolar macrophages were pretreated with pilocarpine (10 μM Pilo) 15 min before LPS. TNF-α concentrations in the supernatant were detected after stimulation with LPS for 12 h. The secretion of TNF-α was evaluated by specific ELISA. Data are expressed as mean±S.E.M. from at least three experiments. ##pb 0.01 vs control; **pb 0.01 vs LPS; ++pb 0.01 vs LPS; &&pb 0.01 vs atropine.
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Fig. 7. Effects of PUN-282987, atropine and 4-DAMP on the degradation of IKBα in lung tissues of ALI mice or in LPS-stimulated alveolar macrophage. In animals, atropine (3.0 mg/kg, i.p.), 4-DAMP (1.0 mg/kg, i.p.) and PUN-282987 (3.0 mg/kg, i.p.) were administrated 30 min before LPS (4 mg/kg). All animals were killed 4 h after LPS administration. In alveolar macrophage, atropine (10 μM), 4-DAMP (10 μM) and PUN-282987 (10 μM) were administrated 30 min before LPS (1 μg/ml) and the cells were stimulated with LPS for 30 min. The gel images shown are representative of 5–6 animals or 3 separate in vivo experiments.
inflammatory cytokines including TNF-α, which is an important mediator of immuno-inflammatory responses and inducer of other proinflammatory cytokines, associated with the development and outcome of ALI [22]. The correlation between the level of TNF-α and development of ALI is highest when this cytokine is measured in the lung or BAL fluid [15]. Interestingly, LPS-induced TNF-α production was enhanced by the mAChR agonist pilocarpine, and pilocarpine treatment alone was able to trigger TNF-α production from alveolar macrophage, which was effectively attenuated by the M3 mAChR antagonist. This finding could be explained by augmented endogenous ACh release after LPS stimulated, and demonstrated that inflammatory cytokines are likely to play a crucial part in the observed effects of endogenous ACh. Accumulating evidence has demonstrated that ACh and its synthesizing enzyme, choline acetyltransferase, are present not only in airway nerves, but also are localized to lung immune cells [29,30]. ACh is known to have different immunological effects on these cells. It is predominantly pro-inflammatory for lymphocytes, while anti-inflammatory for macrophages. It can be either pro- or anti-inflammatory for monocytes, and may have variable effects on neutrophils [29]. Furthermore, recent studies reported that lymphocytes and macrophage expressed mRNA for components of the non-neuronal cholinergic system, which possessed acting sites for endogenous and exogenous mAChR agonists [30–33]. Therefore, it is likely that mAChRs on immune cells play a role in regulating the inflammatory responses. Our research findings lend further support to this notion and demonstrate that mAChRs on macrophage mediated the LPS-induced release of TNF-α, evidenced by the fact that both non-specific mAChR atropine and M3-preferring antagonist 4-DAMP, but not M1 and M2 mAChR antagonists, could attenuate LPS stimulated TNF-α production in lung macrophage. LPS-induced macrophage TNF-α production that is either attenuated by a mAChR antagonist or enhanced by a mAChR agonist leads us to presume that endogenous ACh might be involved in the modulation of the function of macrophage through paracrine and/or autocrine mechanisms. If the presumption is
true, then it was reasonable to believe that the effect of endogenous ACh could be reproduced by exogenous mAChR agonists. Our study was further confirmed by our present findings that production of TNF-α from macrophage was triggered by the mAChR agonist pilocarpine alone and was blocked by a mAChR antagonist, in particular, blockage of M3 mAChR. The present findings are consistent with other research which found that activation of lung macrophages with carbachol could induce LTB4 release and is attenuated by the M3 mAChR antagonist [33] and indicate the ability of endogenous ACh, which might serve as a stimulator in inducing inflammatory cytokine release through direct interaction with M3 mAChRs to regulate the functions of macrophage, and provide proof to the concept of an anti-inflammatory activity of anti-muscarinic agents in inflammatory disease. GPCRs and their signaling molecules have diverse and central roles in regulating macrophage function [34–36]. A recent study shows that the GPCRs are also important regulators of innate and acquired immunity [36]. The mAChRs belong to the superfamily of GPCR genes and mediate the effects of ACh [37] and also contribute to control inflammatory processes via interactions with inflammatory signaling molecules [38]. Recently, Razani-Boroujerdi S. et al. [32] reported that oxotremorine stimulated and atropine inhibited the antibody and T-cell proliferative responses. Moreover, atropine also suppressed the turpentine-induced leukocytic infiltration and tissue injury, and inhibited chemotaxis of leukocytes toward neutrophil and monocyte/lymphocyte chemoattractants. The underlying mechanisms responsible for the inflammatory process remain unclear. We now have the evidence that LPS stimulation leads to increase in TNF-α production by macrophage which is dependent on downstream signaling to the IκBα/NF-κB pathways. In addition, LPS-induced IκBα degradation to regulate TNF-α secretion could be blocked by atropine or 4-DAMP, suggesting that blockage of the M3 mAChR, produced a similar effect of PNU-282987 to influence LPS-induced degradation of IκBα seen during intratracheal instillation of LPS to mice [39].
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Thus, the present study suggests that blockage of mAChRs may lead to modulation of the inflammatory response elicited by LPS both in mice and in alveolar macrophage, and endogenous ACh, acting on mAChRs, in particular, M3 mAChR, plays a broad role in the inflammatory responses. Therefore, understanding the role of M3 mAChR may offer novel therapeutic strategies to approach inflammatory lung diseases. Funding This work was supported by the National Key Basic Research Program (2010CB529800), the National Natural Science Foundation of China (Nos. 81070010, 30873109), the Key Project of Science and Technology Commission of Shanghai Municipality (No. 09JC1408800) and the Research Fund for the Doctoral Program of Shanghai Jiao Tong University School of Medicine (BXJ201007). References [1] Wang H, Yu M, Ochani M, Amella CA, Tanovic M, Susarla S. Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation. Nature 2003;421:384-8. [2] van Westerloo DJ, Giebelen IA, Florquin S, Daalhuisen J, Bruno MJ, de Vos AF, et al. The cholinergic anti-inflammatory pathway regulates the host response during septic peritonitis. J Infect Dis 2005;191:2138-48. [3] Wang H, Liao H, Ochani M, Justiniani M, Lin X, Yang L, et al. Cholinergic agonists inhibit HMGB1 release and improve survival in experimental sepsis. Nat Med 2004;10:1216-21. [4] Giebelen IA, van Westerloo DJ, LaRosa GJ, de Vos AF, van der Poll T. Local stimulation of alpha7 cholinergic receptors inhibits LPS-induced TNF-alpha release in the mouse lung. Shock 2007;28:700-3. [5] Su X, Matthay MA, Malik AB. Requisite role of the cholinergic alpha7 nicotinic acetylcholine receptor pathway in suppressing Gram-negative sepsis-induced acute lung inflammatory injury. J Immunol 2010;184:401-10. [6] Nathanson NM. Molecular properties of the muscarinic acetylcholine receptor. Annu Rev Neurosci 1987;10:195-236. [7] Wess J. Molecular biology of muscarinic acetylcholine receptors. Crit Rev Neurobiol 1996;10:69-99. [8] Bühling F, Lieder N, Kühlmann UC, Waldburg N, Welte T. Tiotropium suppresses acetylcholine-induced release of chemotactic mediators in vitro. Respir Med 2007;101:2386-94. [9] McQueen DS, Donaldson K, Bond SM, McNeilly JD, Newman S, Barton NJ, et al. Bilateral vagotomy or atropine pre-treatment reduces experimental diesel-soot induced lung inflammation. Toxicol Appl Pharmacol 2007;219:62-71. [10] Cui Yong-Yao, Zhu Liang, Wang Hao, Advenier Charles, Devillier Philippe, Chen Hong-Zhuan. Muscarinic receptors involved in airway vascular leakage induced by experimental gastro-oesophageal reflux. Life Sci 2008;82:949-55. [11] Cui Y, Devillier P, Kuang X, Wang H, Zhu L, Xu Z, et al. Tiotropium reduction of lung inflammation in a model of chronic gastro-oesophageal reflux. Eur Respir J 2010;35:1370-6. [12] Ohta S, Oda N, Yokoe T, Tanaka A, Yamamoto Y, Watanabe Y, et al. Effect of tiotropium bromide on airway inflammation and remodelling in a mouse model of asthma. Clin Exp Allergy 2010;40:1266-75. [13] Beeh KM, Beier J. Handle with care: targeting neutrophils in chronic obstructive pulmonary disease and severe asthma? Clin Exp Allergy 2006;36:142-57. [14] Lyczak JB, Cannon CL, Pier GB. Lung infections associated with cystic fibrosis. Clin Microbiol Rev 2002;15:194-222. [15] Meduri GU. Clinical review: a paradigm shift: the bidirectional effect of inflammation on bacterial growth. Clinical implications for patients with acute respiratory distress syndrome. Crit Care 2002;6:24-9.
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