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The Proceedings of the American Thoracic Society 2:320-325 (2005)
© 2005 The American Thoracic Society

Corticosteroids

Potential ß2-Agonist and Anticholinergic Interactions in Chronic Obstructive Pulmonary Disease

Malcolm Johnson

Respiratory Science, GlaxoSmithKline, Greenford, Middlesex, United Kingdom

Correspondence and requests for reprints should be addressed to Malcolm Johnson, Ph.D., Global Director, Respiratory Science, GlaxoSmithKline, Greenford Road, Greenford, Middlesex UB6 0HE, UK. E-mail: malcolm.w.johnson{at}gsk.com


   ABSTRACT
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 ABSTRACT
INTERACTIONS BETWEEN CS AND...
 INTERACTIONS BETWEEN CS AND...
 EFFECT OF LABA ON...
 CLINICAL STUDIES
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 REFERENCES
 
Corticosteroids are often used in combination with ß2-agonist and anticholinergic bronchodilators in the treatment of chronic obstructive pulmonary disease (COPD). Corticosteroids activate the ß2-receptor gene, increasing receptor number and decreasing desensitization. Long-acting ß2-agonists prime the glucocorticoid receptor and enhance nuclear translocation via activation of CCAAT enhancer binding protein-{alpha}. Corticosteroids can also increase prejunctional auto-inhibitory M2-receptor gene expression in airway smooth muscle. There is evidence of a synergistic inhibition of cytokine and chemokine release from alveolar macrophages, epithelial cells, and mucosal glands and enhanced respiratory cytoprotection against viral and bacterial infection when a corticosteroid is combined with salmeterol. In airway smooth muscle, corticosteroids inhibit the contractile effects of acetylcholine, whereas M2-receptor antagonism increases the relaxant activity of isoproterenol. Complementary interactions between corticosteroids and long-acting ß2-agonists and between corticosteroids and anticholinergic bronchodilators may be important if these drugs are combined in the treatment of COPD.

Key Words: ß2-receptors • combination therapy • muscarinic receptors

Chronic obstructive pulmonary disease (COPD) is a multicomponent disease involving mucociliary dysfunction, airway inflammation, structural changes, and systemic effects, all of which contribute to airflow limitation. ß2-Agonist and/or anticholinergic bronchodilators and corticosteroids are often used in combination in the treatment of patients with COPD. They have different mechanisms of action and target different aspects of the underlying pathophysiology of the disease. When used in combination, they can be predicted to have a profile of activity broader than that of the drugs used as monotherapy. However, there is emerging evidence of interactions between corticosteroids (CS) and ß2-agonists, particularly the long-acting ß2-agonists (LABA), and between CS and muscarinic receptors. These interactions may provide a molecular rationale for the use of LABA/CS or anticholinergic/CS dual combinations or triple combination therapy in treating patients with COPD.


   INTERACTIONS BETWEEN CS AND ANTICHOLINERGICS
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Muscarinic Receptors
Airway smooth muscle contains M2- and M3-receptors, with M2-receptors representing 80 to 90% of the total (1). M2-receptors couple to Gi and inhibit adenylate cyclase and cAMP formation (2). They oppose the stimulatory effects of ß2-agonists and reduce the effectiveness of ß2-agonists in airway smooth muscle relaxation. M3-receptors couple to Gq and activate phospholipase C, resulting in an increase in inositol triphosphates and intracellular Ca2+, leading to activation of protein kinase C (3). For the M3-receptor, a large receptor reserve is apparent in airway smooth muscle, with activation of only 4% of receptors being necessary for a full contractile response (4). It is less clear whether a receptor reserve exists for M2-receptors. However, a reduction in muscarinic receptor reserve has been reported to increase relaxation to ß2-agonists in airway smooth muscle (5).

Vagal parasympathetic neurons provide the dominant autonomic control of airway smooth muscle contraction in the lung. They release acetylcholine onto postjunctional M3-receptors to cause bronchoconstriction. Release of acetylcholine is controlled by prejunctional M2-autoreceptors located on the postganglionic parasympathetic nerves (6). These M2-receptors inhibit release of acetylcholine, functioning as a negative feedback mechanism in human airways (7).

Mechanisms of CS Action
The target receptor for CS is the intracellular glucocorticoid receptor (GR). Under resting conditions, the inactive GR is largely located in the cytosol of the cell, associated with multichaperone proteins. The CS molecule penetrates the cell membrane and then binds to the GR through the CS-binding domain (8). This induces a conformational change in the receptor protein, dissociation of the chaperone proteins, and the formation of an active CS–GR complex. The complex may form a dimer and translocate from the cytosol to the nucleus of the cell, where it binds to specific DNA sequences (glucocorticoid response elements [GRE]) in the promoter region of target genes, leading to cofactor activation and an increase or a decrease in gene transcription. This process is termed transactivation (8). Alternatively, the active CS–GR complex, as a monomer, can interact directly with intracellular transcription factors, such as activating protein-1 or nuclear factor-{kappa}B, through a protein–protein interaction to attenuate proinflammatory processes mediated by transcription factors. This transrepression process involves recruitment of histone deacetylates and modulation of chromatin structure (9).

Effect of CS on Muscarinic Receptors
The effects of CS on muscarinic receptors may be species specific and tissue/cell specific. There have been few reports using human airways; most of the evidence comes from animal experimental models.

Receptor expression.
Basenji greyhounds treated with methylprednisolone (2 mg/kg/d for 3 d) in vivo showed a decreased number of M2 and M3 receptors in airway smooth muscle homogenates (Figure 1), as determined by radioligand binding (10). There was no change in muscarinic receptor affinities. These data agree with findings from a study in the rat with dexamethasone (11), where total muscarinic receptor number in bronchial smooth muscle was decreased by 56 to 60% after 3 d of treatment.



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  		Figure 1. Effect of corticosteroids on muscarinic receptors in airway smooth muscle. Basenji greyhounds were treated with methylprednisolone (2 mg/kg/d for 3 d). M2- and M3-receptor density was determined in airway smooth muscle homogenates by radioligand binding *p < 0.05. Adapted with permission from Reference 10.

 
In contrast, in vitro treatment of canine airway smooth muscle with methylprednisolone for 72 h had no effect on muscarinic receptors (10). It is therefore unlikely that transcription of M2- or M3-receptor genes is directly regulated by CS. CS may indirectly reduce muscarinic receptors in airway smooth muscle, for example, by attenuation of another factor controlling receptor gene expression.

In guinea pigs treated with dexamethasone (0.1 mg/kg/d for 2 d), there was a substantial increase in inhibitory M2-receptor function, decreasing airway responsiveness to electrical stimulation of the vagus (12). Dexamethasone also increased cholinesterase activity. This finding agrees with the results of an earlier study where methylprednisolone (10 mg/kg/d) increased total lung muscarinic receptors by 40% after 24 h and by 53% at 48 and 96 h (13). It is likely that this in part reflects M2-receptors on airway nerves. Indeed, in primary cultures of guinea pig airway parasympathetic neurons, dexamethasone (1 µM) significantly decreased the release of acetylcholine in response to electrical stimulation and increased M2-receptor gene expression by 5- to 10-fold (Figure 1) (12). Decreased vagally mediated reflex bronchoconstriction after CS may therefore be the result of increased neuronal M2-receptor expression and function and increased degradation of acetylcholine by cholinesterase (12). In addition, dexamethasone has been reported to protect against virally induced or antigen-induced M2-receptor dysfunction and associated hyperresponsiveness (14, 15).

If these experimental findings can be extrapolated to humans, then CS may enhance the effects of anticholinergics by influencing the differential expression of M2- and M3-receptors.

Receptor function.
In addition to effects on muscarinic receptor expression, CS may influence signal transduction pathways activated by M2- and M3-receptors. Effects of CS on G-proteins, including Gi and G-proteins linked to K+ channels, have been reported (16).

In vitro in bronchial smooth muscle, dose responses to acetylcholine were significantly shifted to the right by dexamethasone (17). In vivo in the guinea pig, dexamethasone inhibited acetylcholine-induced bronchoconstriction, and further studies in dogs showed that treatment with methylprednisolone increased sensitivity to ß2-agonist–induced bronchodilation. In the human bronchus (in which M3-receptors were inactivated), acetylcholine decreased isoproterenol and forskolin sensitivity and attenuated cAMP accumulation (18). An M2-receptor selective antagonist, methoctramine, reversed the effects of acetylcholine. Similar findings were reported in canine tracheal muscle, contracted with methacholine, where methoctramine shifted the relaxant dose response to isoproterenol to the left, whereas a selective M3-antagonist had no effect (19).

These results indicate that postjunctional M2-receptors cause indirect contraction of airway smooth muscle, including human bronchus, by reversing sympathetically mediated relaxation and therefore contribute to cholinergic functional antagonism (18). Indeed, in human cultured airway smooth muscle cells where carbachol was shown to inhibit isoproterenol-induced cAMP accumulation, methoctramine reversed the effect by blocking M2-receptors (2). The relative clinical importance of blocking postjunctional M2-receptors over blocking prejunctional auto-inhibitory M2-receptors remains to be determined.


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ß2-Receptors
All ß2-agonists exert their biologic and therapeutic effects through cell-surface ß2-receptors, which are members of the seven-transmembrane, G-protein–coupled receptor family. After ligand binding to the active site of the receptor, the {alpha}-component of the associated Gs-protein dissociates and activates adenylate cyclase (20), leading to the production of intracellular cAMP and subsequent activation of protein kinase A, which phosphorylates a number of intracellular regulatory proteins (21). More recently, it has been reported that activation of the ß2-receptor can lead to coupling to a Gi-protein, resulting in stimulation of the extracellular signal-regulated kinase and p38 mitogen-activated protein kinase (MAPK) pathways (22).

Effect of CS on ß2-Receptors
CS can modulate ß2-receptors and their function by several mechanisms: protection against desensitization and the development of tolerance, increased efficiency of receptor coupling, and protection against inflammation-induced receptor downregulation and uncoupling.

CS stimulate the transcription of ß2-receptors via binding to GRE in the 5'-noncoding promoter region of the ß2-receptor gene. Dexamethasone has been shown to increase ß2-receptor mRNA (at 2 h) and protein (at 24 h) in human lung tissue in vitro (23). This increase in transcription, which has also been shown in neutrophils and T cells, is time-dependent and dose-dependent, consistent with the later induction of receptor binding activity. However, dexamethasone has not been found to alter the half-life of ß2-receptor mRNA (23). Baraniuk and colleagues (24) reported that intranasal administration of beclomethasone dipropionate (100 µg/d for 3 d) significantly increased the density of ß2-receptors on the nasal mucosa. Systemic and inhaled CS reversed ß2-receptor downregulation after exposure to high doses of short-acting ß2-agonists (25).

The efficiency of coupling between the ß2-receptor and Gs has also been reported to be modulated by CS (26). As a result, ß2-receptor–stimulated adenylate cyclase activity and cAMP accumulation increase after CS treatment. Animals that have been depleted of CS by adrenalectomy, in contrast, lose the ability to maintain the sensitivity of the ß2-receptor–coupled adenylate cyclase system.

Other studies (27) have shown that by reducing the concentrations of proinflammatory cytokines such as IL-1ß and tumor growth factor-ß1, CS regulate the coupling of ß2-receptors to adenylate cyclase and affect receptor sensitivity. Such effects may have clinical relevance in preventing the development of tolerance to ß2-agonists in patients with COPD who are on chronic therapy.


   EFFECT OF LABA ON THE GR
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LABA exert an effect on the GR in two ways: by priming the GR for subsequent CS binding and by increasing the translocation of the GR from the cell cytosol to the nucleus.

Modulation of the GR has been shown to be mediated by phosphorylation (activation) and dephosphorylation (inactivation). Priming of the GR by LABA is achieved by MAPK-dependent phosphorylation (28). After stimulation of the ß2-receptor by salmeterol and dissociation of G{alpha}s, the residual ß{gamma}-subunit of the G-protein initiates an intracellular signaling cascade, involving the nonreceptor tyrosine kinase C-Src and the G-protein Ras, culminating in the stimulation of MAPK (29). Activation of this pathway by the LABA requires that the ß2-receptor is phosphorylated, probably by protein kinase A (28). MAPK then phosphorylates the GR at a number of proline-directed serine residues in the N-terminal region of the receptor. It is possible that an increase in negative charge at the N-terminal domain leads to a conformational change in the GR protein, leading to the "priming" event and rendering the receptor more sensitive to steroid-dependent activation (Figure 2A).




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  		Figure 2. (A) Synergistic transactivation of the GR by combined salmeterol and FP. BEAS-2B cells transfected with a GR-luciferase reporter construct were incubated with salmeterol (1 nM), FP (0.1 nM), or the combination for 24 h. GR activation was measured in terms of reporter activation. *p < 0.01. Adapted with permission from Reference 33. (B) Synchronous activation of GR by FP and activation of C/EBP-{alpha} by salmeterol through a cAMP-dependent pathway allows the formation of a GR–C/EBP-{alpha} heterodimer with enhanced GR and C/EBP-{alpha} function. Adapted with permission from Reference 32.

 
Other studies have shown that translocation of the GR from the cell cytosol to the nucleus, a fundamental step in the anti-inflammatory activity of CS, is increased by the addition of a LABA (30, 31). Eickelberg and colleagues (30) showed that treatment of human lung fibroblasts and smooth muscle cells with fluticasone propionate (FP) and salmeterol resulted in increased translocation of GR compared with FP alone. These effects have been confirmed with formoterol and budesonide (31). Electrophoretic mobility shift assays confirmed that the LABA-activated nuclear GR actively bound to the GRE consensus sequence. The mechanism of this effect has been further investigated. Salmeterol and formoterol have been shown to activate a CCAAT enhancer binding protein (C/EBP-{alpha}) in airway smooth muscle cells (32) and in epithelial cells (33). The synchronous activation of GR by CS and of C/EBP-{alpha} by LABA allows a heterodimer between GR and C/EBP-{alpha} to form (Figure 2B), increasing the activation of the receptor and nuclear translocation (32, 33).

These in vitro findings have been confirmed in vivo in patients with asthma who were treated with LABA and CS. Usmani and colleagues (34) showed that inhalation of salmeterol (50 µg) and FP (100 µg) increased the nuclear translocation of GR in sputum macrophages at 60 and 120 min significantly more than the same dose of FP alone and equivalent to a fivefold higher dose of the steroid. Whether these effects of salmeterol can be shown in the patient with COPD remains to be determined. In contrast, whereas budesonide (800 µg) also significantly increased the translocation of the GR from the cytosol into the nuclei of peripheral blood leukocytes, the combination with formoterol (24 µg) did not enhance this effect versus budesonide alone (35).

The interactions between CS and LABA result in synergistic inhibitory effects on proinflammatory cytokine and chemokine release, relevant to the pathophysiology of COPD. For example, the combination of FP (10–9 or 10–10 M) and salmeterol (10–9 M) synergistically inhibited the release of human rhinovirus-induced IL-8 and RANTES (the chemokine regulated upon activation, normal T-cell expressed and secreted) (Figure 3) from bronchial epithelial cells (36). Such an effect may be of relevance to the efficacy of combination therapy in reducing COPD exacerbations, 50% of which are virally mediated. Similarly, Pang and Knox (37) showed that tumor necrosis factor-{alpha}–stimulated release of IL-8, which is a major neutrophil chemoattractant involved in the pathogenesis of COPD, was markedly inhibited by FP but was unaffected by salmeterol. However, a combination of FP with salmeterol synergistically enhanced the inhibition induced by the CS alone. Similar results were reported in peripheral blood monocytes stimulated with cigarette smoke extract (38) and in human alveolar macrophages isolated from the bronchoalveolar lavage fluid of patients with COPD (39).



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  		Figure 3. Synergistic inhibition of virally induced RANTES release from airway epithelial cells. BEAS-2B cells were incubated with salmeterol (1 nM), FP (0.1 nM), or the combination for 1 h before infection with human rhinovirus (HRV). RANTES protein release was measured after 24 h. Adapted with permission from Reference 36.

 
Dowling and colleagues (40) reported that FP or salmeterol significantly inhibited Pseudomonas aeruginosa-induced epithelial damage in human respiratory mucosa. However, when a combination of FP and salmeterol was used, the loss of cilia from the epithelial surface was synergistically reduced compared with FP or salmeterol alone (40). The mechanism of this effect seems to involve upregulation of junctional proteins such as ZO-1 and vinculin (41), thereby increasing the integrity of the epithelium against bacterial virulence factors. This may be relevant to the recurrent bacterial infections experienced by patients with COPD.

The interactions between CS and LABA may therefore be summarized as follows: CS increase ß2-receptor synthesis, and LABA prime GR for steroid-dependent activation and enhance nuclear translocation via C/EBP-{alpha} activation. These mechanisms form a possible molecular and receptor basis for additive and synergistic interactions between the two classes of drugs if used in combination in the treatment of COPD.


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Anticholinergic/ICS Combination Therapy
A few clinical studies (42–44) have examined the effects of anticholinergics in the absence and presence of inhaled CS (ICS) in patients with COPD. One study investigated whether the bronchodilatory effects of ipratropium bromide, a muscarinic receptor nonselective antagonist, were modified by treatment with ICS or oral CS for 3 wk in patients with stable COPD. The mean FEV1 values for ipratropium bromide were higher after prednisolone (40 mg) but not after budesonide (1,600 µg). Similar findings were observed with PC20 to histamine (42). However, the combined effects of CS and anticholinergics on FEV1 and PC20 were not significantly different from the sum of their individual effects.

A second preliminary study was carried out in patients with COPD who were treated with tiotropium, an M1/M3-receptor selective antagonist, in combination with ICS for 6 months (43). Morning predose (trough) improvement in FEV1 and FVC was 73 ml and 173 ml, respectively. Dyspnea (assessed with the Transitional Dyspnea Index) was reduced with combination therapy, and the number of exacerbations decreased and health status (assessed on St. George's Respiratory Questionnaire) improved.

Another preliminary study compared the effects of salmeterol/ipratropium bromide with salmeterol/ipratropium/beclomethasone dipropionate in patients with mild to moderate COPD over 2 yr (44). FEV1 significantly increased in the dual and triple therapy groups compared with salmeterol alone. Borg dyspnea scores were improved more with triple therapy than with dual salmeterol/ipratropium treatment. Similar findings were reported for COPD exacerbations.

These studies do not adequately address the issue of whether the potential interaction of CS and muscarinic receptors is reflected in additional clinical benefit when anti-cholinergic bronchodilators and ICS are combined in the treatment of COPD. Further work is required.

LABA/ICS Combination Therapy
There have been a number of clinical studies of LABA/ICS combination therapy in COPD.

A bronchial biopsy/induced sputum study in patients with COPD (n = 140) showed that salmeterol/FP combination therapy (50/500 µg b.i.d. for 12 wk) significantly reduced CD8+ T lymphocytes, tumor necrosis factor-{alpha} mRNA(+) and interferon-{gamma} mRNA(+) cells in the airway subepithelium and reduced the percentage of neutrophils in sputum (45). These anti-inflammatory effects are greater than those reported previously for FP alone (46)

Over 1 yr, salmeterol/FP (47) and formoterol/budesonide (48) increased lung function significantly more than the LABA or ICS alone. This was paralleled by a reduction in exacerbations of COPD, in the case of salmeterol/FP, by 42% (47). In patients with more severe disease (FEV1 < 50% predicted), the combination increased prebronchodilator FEV1 and reduced rescue medication use in an apparently synergistic manner (47). Retrospective analysis of the General Practice Research Database in the United Kingdom has revealed that 3-yr survival in COPD is improved with salmeterol/FP over salmeterol or FP alone (49). However, evidence for LABA/ICS synergy in COPD is preliminary and requires further clinical studies in well characterized patients.

There are polymorphic forms of the ß2-receptor (50) and GR (51) that influence responses to ß2-agonists and ICS and may affect any interaction between the drug classes in COPD. For example, there is evidence in asthma that ß2-receptor polymorphisms, especially Arg-Arg at position 16, may influence responses to regular albuterol (52, 53). However, this does not seem to apply to LABA, such as salmeterol, in terms of lung function (53–55) or exacerbations (53). There is also preliminary evidence that ß2-receptor genotype at 16 does not influence response to salmeterol/FP combination in patients with asthma (54). For the GR, Leu 753 Phe polymorphism is associated with decreased CS sensitivity, whereas Asn 363 Ser increases ICS side-effect liability (51). Of interest is a recent observation of a highly significant interaction between a coding nonsynonymous polymorphism (Ile 772 Met) of adenylate cyclase 9 and bronchodilator response to albuterol in pediatric patients with asthma taking inhaled budesonide (56). This suggests that genotype may influence ß2-receptor signaling when ß2-agonists and ICS are coadministered. Such studies in COPD have not been carried out.


   CONCLUSIONS
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 INTERACTIONS BETWEEN CS AND...
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Corticosteroids are often used in combination with ß2-agonist and anticholinergic bronchodilators in the treatment of patients with COPD. Corticosteroids increase ß2-receptor density in the airways, and long-acting ß2-agonists prime glucocorticoid receptors and enhance nuclear translocation. There is evidence of broad-spectrum anti-inflammatory activity with combined CS and LABA in COPD. Corticosteroids increase prejunctional auto-inhibitory M2-receptors on airway parasympathetic neurons, but they decrease M2- and M3-receptors in airway smooth muscle. Corticosteroids inhibit the contractile effects of acetylcholine and vagal stimulation, and they increase the relaxant activity of ß2-agonists. These interactions may be important when CS and LABA or CS and anticholinergics are used in combination in patients with COPD.


   FOOTNOTES
 
Conflict of Interest Statement: M.J. is an employee of GlaxoSmithKline who sponsored the meeting through an Educational Grant and who market Salmeterol and Salmeterol/Fluticasone Propionate Combination for the treatment of COPD. He does not own stock in GlaxoSmithKline.

(Received in original form April 20, 2005; accepted in final form July 12, 2005)


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