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Formoterol is more effective than salmeterol in suppressing neutrophil reactivity

The research question addressed in the current study was: do the long-acting β2-adrenoreceptor agonists (LABAs) formoterol (full agonist) and salmeterol (partial agonist) possess comparable suppressive effects on human neutrophils?

Neutrophils were isolated from heparinised venous blood (5 units of preservative-free heparin per mL) from nonsmoking healthy adult volunteers by centrifugation on Histopaque-1077 cushions at 400×g for 25 min at room temperature. The resultant pellets were suspended in PBS (0.15 M, pH 7.4) and sedimented with 3% gelatine to remove most of the erythrocytes. Following centrifugation (280×g at 10°C for 10 min), residual erythrocytes in the cell pellets were removed by selective lysis by exposure to 0.83% ammonium chloride at 4 o C for 10 min. The neutrophils, which were routinely of high purity (>90%) and viability (>95%), were then resuspended to 1×10 7 cells·mL −1 in PBS, and held on ice until used. In the various assays described below, neutrophils were activated with the chemoattractant N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) (1 μM final) alone with the exception of the assay of elastase release, for which fMLP was used in combination with cytochalasin B (CB) (1 μM).
Neutrophil degranulation was measured according to the extent of release of the primary granule enzyme, elastase. Neutrophils were incubated at a concentration of 2×10 6 mL −1 in HBSS for 10 min at 37°C followed by sequential addition of the test β 2 -agonists (1-100 nM) and, 20 s later, by fMLP/CB (1/1 μM). The tubes were incubated for 10 min at 37°C then transferred to an ice bath, followed by centrifugation at 400×g for 5 min to pellet the cells, and the supernatants decanted and assayed for elastase using a micromodification of a standard colourimetric procedure [6,7]. The results are expressed as milliunits of enzyme per 10 7 cells.
Leukotriene B 4 (LTB 4 ) and cAMP were measured using competitive binding immunoassay procedures (Correlate EIA; Assay Designs Inc., Ann Arbor, MI, USA). In the case of LTB 4 , neutrophils (2×10 6 mL −1 ) were pre-incubated for 10 min at 37°C followed by addition of the test agents (10 and 100 nM) and fMLP 20 s apart. Following 5 min of incubation at 37°C, the reactions were terminated by the addition of an equal volume of ice-cold HBSS and LTB 4 measured in the cell-free supernatants. The results are expressed as picograms of LTB 4 per mL.
In the case of cAMP, following pre-incubation of neutrophils (2×10 6 mL −1 ) for 10 min at 37°C, the cells were treated with the β 2 -agonists (10 and 100 nM) for 20 s, after which the reactions were terminated as above. Following centrifugation, the supernatants were discarded and cAMP extracted from the cell pellets by addition of 0.1 M hydrochloric acid for 30 min followed by centrifugation to remove cell debris. The supernatants assayed for cAMP and the results expressed as picomoles of cAMP per mL.
The results of each series of experiments using neutrophils from four to eight different donors were analysed using the Mann-Whitney U-test for comparison of nonparametric data and are presented as the median values with interquartile ranges.
Results are shown in figure 1a and b for ROS and elastase, respectively. Treatment of neutrophils with formoterol caused statistically significant inhibition of the generation of ROS by fMLP-activated neutrophils, as well as release of elastase by cells activated with fMLP/CB at concentrations of 10-100 and 1-100 nM, respectively (p<0.05). Salmeterol had no significant effect on either ROS generation or elastase release.
Results are shown in figure 2a and b for LTB 4 and cAMP respectively. Treatment of neutrophils with formoterol, but not salmeterol, at concentrations of 10 and 100 nM resulted in significant ( p<0.05) dose-related inhibition of production of LTB 4 by fMLP-activated neutrophils. Brief treatment (20 s) of neutrophils with formoterol at both concentrations tested (10 and 100 nM) resulted in significant ( p<0.05) increases in neutrophil cAMP, which were maximal at 100 nM, while salmeterol was ineffective.
The findings of the current study demonstrate that formoterol, a full agonist of β 2 -adrenoreceptors (β 2 ARs), is significantly more potent than the partial agonist, salmeterol, with respect to suppression of neutrophil reactivity in vitro. At the concentrations tested, the inhibitory effects of formoterol on the generation of ROS and LTB 4 by chemoattractant-activated neutrophils, as well as release of elastase from these cells, were maximal at a concentration of 10 nM. This is relevant, as following inhalation of a single 24-μg dose of formoterol, the local concentration of this β 2 -agonist in the airways has been estimated to reach 11 nM [6]. The suppressive effects of formoterol on neutrophil reactivity were associated with significant increases in intracellular cAMP consistent with activation of β 2 ARs, which are expressed on human neutrophils [8]. The mechanism by which formoterol, as well as that of indacaterol, an ultra-LABA with nearly full β 2 -agonist activity, suppress neutrophil reactivity appears to be achieved by cAMP-dependent protein kinase (PKA)-mediated restoration of calcium homeostasis following receptor-mediated activation of the cells with calcium-mobilising stimuli [6,7].
In addition to neutrophils [6,7,9,10], LABAs have also been reported to target other types of β 2 AR-expressing inflammatory and structural cells, including dendritic cells [11] and airway smooth muscle cells [12]. Although the clinical significance of these anti-inflammatory properties of LABAs remains contentious, the beneficial synergistic interactions of these agents with corticosteroids may be of greater relevance. These have been reviewed in detail recently [9] and include LABA-mediated: 1) potentiation of glucocorticoid receptor anti-inflammatory gene transcription; and 2) antagonism of the LTB 4 -mediated proapoptotic effects of corticosteroids on neutrophils and monocytes. Both activities are achieved by cAMP/PKA-dependent mechanisms. In addition, corticosteroids have also been reported to augment β 2 AR signalling by increasing the numbers of β 2 ARs on target cells, as well as by increasing the efficacy of β 2 AR/G s -protein subunit coupling (reviewed by THERON et al. [9]).
In apparent contrast to the findings of the current study, STRANDBERG et al. [13] reported that exposure of neutrophils from healthy humans to lipopolysaccharide (LPS) in vitro in the presence of therapeutically relevant concentrations and higher of formoterol for 8 h resulted in significant increases in the production of the cytokine interleukin (IL)-6 and the chemokines CXCL1 (GROα) and CXCL8 (IL-8). Shorter exposure of neutrophils to the LABA (30-60 min) resulted in upregulation of expression of the chemokine receptors CXCR1 and CXCR2, while chemotaxis in response to CXCL8 was unaffected. These observations, taken together with the findings of the current study, suggest that the inflammatory activities  of neutrophils are differentially affected by formoterol and may also vary according to the type of inflammatory stimulus. This contention is supported by the finding, albeit in the case of salmeterol, that exposure of neutrophils to aqueous cigarette smoke in vitro in the presence of the LABA results in significant attenuation of production of CXCL8, while other inflammatory activities, such as production of ROS and degranulation, are unaffected [14]. More recently, and consistent with the findings of the current study, WEX et al. [15] reported that intranasal administration of aerosolised olodaterol, an ultra-LABA with nearly full agonist activity, to mice and guinea pigs significantly attenuated pulmonary neutrophilia induced by inhalation of cigarette smoke or LPS. These anti-inflammatory actions of olodaterol were sustained following repeated dosing over 4 days and were associated with significant decreases in the concentrations of an array of proinflammatory cytokines/chemokines in bronchoalveolar lavage fluid, as well as inhibition of expression of the β 2 -integrin CD11b.
With respect to possible clinical relevance of the findings of the current study, only a few large randomised trials have compared the efficacy of the F/B and S/F combinations in patients with COPD. The recent PATHOS trial retrospectively compared the efficacy of S/F with that of F/B on the rates of exacerbation during treatment of COPD patients in Sweden [16]. The results of this trial indicated that F/B inhalation therapy was more effective than S/F, while a smaller trial from Canada reported similar results to those of the PATHOS study [17]. Notwithstanding differences between these two formulations, such as efficacy of delivery to the lower airways and lipophilicity of fluticasone, the apparently superior anti-inflammatory efficacy of F/B in COPD, which has also been described in asthma [18][19][20], may be related to the potency of the interaction of formoterol with β 2 ARs on neutrophils relative to that of salmeterol.
Clearly, studies of the type described here, which have focused on circulating neutrophils from healthy individuals, could serve as a template for future studies encompassing patients with COPD. Such studies would, however, require careful attention to: 1) inclusion of airway as well as circulating neutrophils; 2) awareness of the possible existence of a neutrophil phenotype that exhibits a high level of pro-inflammatory activity under the hypoxic conditions associated with COPD [1]; 3) consideration that COPD is not a homogeneous disease entity but rather a collection of phenotypes with potentially differential effects on neutrophil reactivity; and 4) interpretational difficulties posed by the complicating effects of existing anti-inflammatory therapies.