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Published ahead of print on June 30, 2005, doi:10.1164/rccm.200503-451OC
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American Journal of Respiratory and Critical Care Medicine Vol 172. pp. 878-884, (2005)
© 2005 American Thoracic Society
doi: 10.1164/rccm.200503-451OC

Original Article

Antiinflammatory Effects of Salmeterol after Inhalation of Lipopolysaccharide by Healthy Volunteers

Nico A. Maris, Alex F. de Vos, Mark C. Dessing, C. Arnold Spek, Rene Lutter, Henk M. Jansen, Jaring S. van der Zee, Paul Bresser and Tom van der Poll

Departments of Experimental Internal Medicine, Pulmonology, and Infectious Diseases, Tropical Medicine and AIDS, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

Correspondence and requests for reprints should be addressed to N. A. Maris, M.D., Academic Medical Center, Meibergdreef 9, Room F4-222, 1105 AZ Amsterdam, The Netherlands. E-mail: n.a.maris{at}amc.uva.nl


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: Salmeterol is a {beta}2-adrenoreceptor agonist used in the treatment of obstructive pulmonary disease. Salmeterol inhibits inflammatory responses by neutrophils and mononuclear cells in vitro and in mouse models of lung inflammation in vivo.

Objective: To determine the effect of salmeterol on LPS-induced lung inflammation in humans.

Methods: Thirty-two healthy subjects were enrolled in a single-blinded, placebo-controlled study. Subjects inhaled 100 µg salmeterol or placebo (t = –0.5 h) followed by 100 µg LPS or normal saline (t = 0 h; n = 8/group). Measurements were performed in bronchoalveolar lavage fluid and purified alveolar macrophages obtained 6 h post-challenge.

Measurements and Main Results: Inhalation of LPS was associated with neutrophil influx, neutrophil degranulation (myeloperoxidase, bactericidal/permeability-increasing protein and elastase), release of cytokines (tumor necrosis factor {alpha} and interleukin 6) and chemokines (interleukin 8, epithelial cell–derived neutrophil attractant 78, macrophage inflammatory proteins 1{alpha} and 1{beta}), activation of alveolar macrophages (upregulation of HLA-DR and CD71; enhanced expression of mRNAs for 13 different mediators of inflammation), and protein leakage (all p < 0.05 vs. placebo/saline). Pretreatment with salmeterol inhibited LPS-induced neutrophil influx, neutrophil degranulation (myeloperoxidase), tumor necrosis factor {alpha} release, and HLA-DR expression (all p < 0.05 vs. placebo/LPS), while not significantly influencing other responses.

Conclusion: Salmeterol exerts antiinflammatory effects in the pulmonary compartment of humans exposed to LPS.

Key Words: cytokines • endotoxins • lung • neutrophils • salmeterol

Salmeterol is a long-acting {beta}2-adrenergic agonist that is frequently used by patients with asthma and chronic obstructive pulmonary disease because of its strong bronchodilatory properties (1). Salmeterol, like other {beta}2-agonists, induces bronchodilation via activation of {beta}2-adrenoreceptors on smooth muscle cells (2). {beta}2-receptors are also expressed by cells involved in the regulation of inflammation, in particular by neutrophils, monocytes, and macrophages (3–5). Stimulation of {beta}2-receptors on these immunocompetent cells primarily results in antiinflammatory effects. Triggering of {beta}2-receptors on neutrophils results in inhibition of oxygen release and a reduction of the adhesion of neutrophils to the vascular endothelium and airway epithelial cells (6–9). Moreover, stimulation of {beta}2-adrenergic receptors on mononuclear cells and macrophages diminishes their capacity to release proinflammatory cytokines, such as tumor necrosis factor {alpha} (TNF-{alpha}) and interleukin 1{beta} (IL-1{beta}) (3, 10–12).

Asthma and chronic obstructive pulmonary disease are associated with chronic inflammation in the respiratory tract. Knowledge of the effects of {beta}2-agonists like salmeterol on airway inflammation is therefore relevant to fully understand the mechanisms underlying its clinical effects in patients with obstructive lung diseases. We recently embarked on in vivo studies investigating the potential antiinflammatory effects of inhaled salmeterol. In a murine model of LPS-induced lung inflammation, nebulized salmeterol strongly reduced the recruitment of neutrophils to the pulmonary compartment as well as the local release of TNF-{alpha} in bronchoalveolar lavage fluid (BALF) (13). The present study expanded this murine investigation and determined the effect of a clinically relevant dose of salmeterol on lung inflammation induced by LPS inhalation in humans.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
Thirty-two nonsmoking males (age, 23.2 ± 0.6 yr) were recruited by advertising. Screening, consisting of a questionnaire, physical examination, routine blood and urine investigation, ECG, and spirometry, did not reveal any abnormality. The study was approved by the institutional ethics and research committees, and written, informed consent was obtained from all subjects before enrollment in the study.

Study Design
The subjects inhaled either placebo or salmeterol (4 x 25 [100] µg; GlaxoSmithKline, Zeist, The Netherlands) using a metered-dose inhaler attached to a volumatic (750 ml; GlaxoSmithKline); after 30 min, they inhaled either normal saline or LPS (from Escherichia coli O26:B6, 100 µg; Sigma-Aldrich, St. Louis, MO) using a large-volume reservoir delivery system as described previously (14, 15). Volunteers were randomized and blinded for the challenges they received. Four groups of eight subjects were therefore studied: (1) placebo/saline, (2) salmeterol/saline, (3) placebo/LPS, and (4) salmeterol/LPS. BAL, collection of BALF, and cell counts were performed as described in the online supplement.

Assays
Myeloperoxidase (MPO) (16), bactericidal/permeability-increasing protein (HyCult, Uden, The Netherlands), and elastase (17) were determined by ELISA. IL-1{beta}, IL-6, IL-8, IL-10, and IL-12p70 were measured using a cytometric bead array (Pharmingen, San Diego, CA). TNF-{alpha} (high-sensitivity ELISA), macrophage inflammatory protein 1{alpha} (MIP-1{alpha}), MIP-1{beta}, and epithelial cell–derived neutrophil attractant 78 were measured by ELISA (all R&D Systems, Abingdon, UK). Albumin and {alpha}2-macroglobulin were measured as described previously (18, 19).

Flow Cytometric Analysis of Alveolar Macrophages
Expression of HLA-DR, CD14, and CD71 on alveolar macrophages in BALF was determined by flow cytometric analysis using fluorochrome-conjugated mouse antihuman HLA-DR, CD14, CD15, and CD71 antibodies (Pharmingen), as described in the online supplement.

Isolation of Alveolar Macrophages
Alveolar macrophages were purified by autoMACS using CD71 microbeads (Multenyi Biotec, Bergisch Gladbach, Germany), as described in the online supplement. Differential cell counts and trypan blue staining revealed a purity and viability of isolated macrophages of greater than 95% in all groups. After isolation, alveolar macrophages were dissolved in Trizol and stored at –80°C until used for RNA isolation.

Multiplex Ligation-dependent Probe Amplification
RNA isolation and multiplex ligation-dependent probe amplification were performed as described previously (20–23). In the present study, multiplex ligation-dependent probe amplification was used to analyze mRNA expression of a set of proteins involved in inflammation (see also RESULTS) (21, 23).

Statistical Analysis
Values are expressed as means ± SEM. Statistical comparisons were made using analysis of variance followed by Tukey's multiple comparison post hoc test to establish significance between separate datasets. These analyses were performed using SPSS (version 10.1; SPSS Inc., Chicago, IL). A p value of less than 0.05 was considered to represent a statistically significant difference.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Salmeterol Inhibits LPS-induced Neutrophil Recruitment
Inhalation of LPS resulted in a profound rise in the number of total cells recovered from BALF 6 h post-challenge (Table 1; p < 0.05 vs. placebo/saline). The LPS-induced increase in total cell counts was due to a rise in the number of neutrophils; LPS did not influence the number of macrophages/monocytes or lymphocytes in BALF. Salmeterol pretreatment inhibited the recruitment of neutrophils into BALF after LPS inhalation (p < 0.05 vs. placebo/LPS), whereas it did not alter the number of macrophages and increased the lymphocyte count (p < 0.05 vs. placebo/saline). LPS inhalation also elicited rises in the concentrations of the neutrophil degranulation products MPO, bactericidal/permeability-increasing protein, and elastase (Figure 1; all p < 0.05 vs. placebo/saline). Salmeterol attenuated these LPS-induced neutrophil responses, which, in the case of MPO, reached significance (p < 0.05 vs. placebo/LPS).


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  		TABLE 1. Effect of salmeterol on cellular composition of bronchoalveolar lavage fluid

 


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  		Figure 1. Effect of salmeterol on LPS-induced neutrophil degranulation. Human volunteers inhaled 100 µg LPS or saline at t = 0. Salmeterol (100 µg) or placebo was administered at t = –30 min. Bronchoalveolar lavage fluid (BALF) was obtained 6 h after inhalation of LPS or saline, and supernatants were analyzed for myeloperoxidase (MPO; A), bactericidal/permeability increasing protein (BPI; B), and elastase (C). Data are means ± SEM of eight subjects/group. *p < 0.05 versus placebo/saline; +p < 0.05 versus placebo/LPS.

 
Salmeterol Attenuates LPS-induced TNF-{alpha} Release
To further study the effects of salmeterol on the pulmonary innate immune response induced by LPS, cytokine and chemokine expression was measured at the protein level in BALF 6 h post-challenge. TNF-{alpha} and IL-6 were upregulated after inhalation of LPS (Figure 2; p < 0.05 vs. placebo/saline), whereas IL-1{beta}, IL-10, and IL-12p70 remained undetectable. Salmeterol inhibited TNF-{alpha} release after LPS challenge (p < 0.05 vs. placebo/LPS), whereas IL-6 was not significantly influenced. LPS also elicited the release of the CXC chemokines IL-8 and epithelial cell–derived neutrophil attractant 78, and the CC chemokines MIP-1{alpha} and MIP-1{beta} into BALF (Figure 3; all p < 0.05 vs. placebo/saline). Although salmeterol tended to reduce the levels of IL-8, MIP-1{alpha}, and MIP-1{beta}, these effects did not reach statistical significance.



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  		Figure 2. Salmeterol attenuates LPS-induced tumor necrosis factor (TNF)-{alpha} release. Human volunteers inhaled 100 µg LPS or saline at t = 0. Salmeterol (100 µg) or placebo was administered at t = –30 min. BALF was obtained 6 h after inhalation of LPS or saline, and supernatants were analyzed for TNF-{alpha} (A) and interleukin 6 (IL-6; B). Data are means ± SEM of eight subjects/group. *p < 0.05 versus placebo/saline; +p < 0.05 versus placebo/LPS. nd = not detectable.

 



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  		Figure 3. Salmeterol does not influence LPS-induced chemokine release. Human volunteers inhaled 100 µg LPS or saline at t = 0. Salmeterol (100 µg) or placebo was administered at t = –30 min. BALF was obtained 6 h after inhalation of LPS or saline, and supernatants were analyzed for IL-8 (A), epithelial cell–derived neutrophil attractant 78 (ENA-78; B), macrophage inflammatory protein (MIP)-1{alpha} (C), and MIP-1{beta} (D) expression. Data are means ± SEM of eight subjects/group. *p < 0.05 versus placebo/saline. nd = not detectable.

 
Salmeterol Prevents LPS-induced Upregulation of HLA-DR on Alveolar Macrophages
To study the effect of salmeterol on LPS-induced activation of alveolar macrophages, expression of HLA-DR, CD71, and CD14 was analyzed using flow cytometry. LPS upregulated the expression of HLA-DR and CD71 on alveolar macrophages (Figure 4; both p < 0.05 vs. placebo/saline), although it did not affect CD14 expression (data not shown). Salmeterol prevented upregulation of HLA-DR after LPS challenge (p < 0.05 vs. placebo/LPS), although it did not influence CD71 expression.



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  		Figure 4. Salmeterol modulates activation of alveolar macrophages. Human volunteers inhaled 100 µg LPS or saline at t = 0. Salmeterol (100 µg) or placebo was administered at t = –30 min. BALF was obtained 6 h after inhalation of LPS or saline, and cells were labeled for HLA-DR (A) and CD71 (B), which are activation markers of alveolar macrophages. Data are means ± SEM of eight subjects/group. *p < 0.05 versus placebo/saline; +p < 0.05 versus placebo/LPS.

 
Expression of Inflammatory Genes in Alveolar Macrophages
To determine the effect of salmeterol on LPS-induced gene expression of a set of inflammatory mediators in alveolar macrophages and the influence of salmeterol thereon, multiplex ligation-dependent probe amplification was performed on RNA isolated from purified alveolar macrophages harvested 6 h after inhalation of LPS or saline (Table 2). LPS inhalation increased the expression of mRNAs for several cytokines (IL-1{alpha}, IL-1{beta}, IL-1 receptor antagonist, IL-6, IL-18) and chemokines (IL-8, monocyte chemoattractant protein 1, MIP-1{alpha}, and MIP-1{beta}; all p < 0.05 vs. placebo/saline), although it did not influence the expression of IL-15, TNF receptor 1, and macrophage migration inhibitory factor. Activation of alveolar macrophages by LPS inhalation was further reflected by increased expression of mRNAs involved in the nuclear factor-{kappa}B pathway, and mRNAs for phosphodiesterase 4B and serine proteinase inhibitor B9 (all p < 0.05 vs. placebo/saline). LPS reduced mRNA levels for glutathione S-transferase (p < 0.05 vs. placebo/saline). None of these LPS-induced effects were altered by salmeterol. Inhalation of salmeterol alone (i.e., without LPS) was associated with increased expression of mRNAs for protein-tyrosine phosphatase 4A2 and thrombospondin (both p < 0.05 vs. placebo/saline), whereas salmeterol in combination with LPS enhanced protein-tyrosine phosphatase nonreceptor type 1 expression.


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  		TABLE 2. Expression of inflammatory mRNAs in alveolar macrophages

 
Salmeterol Does Not Influence the Permeability of the Blood–Airway Barrier
To obtain insight into the effect of LPS on the permeability of the blood–airway barrier and the influence of salmeterol thereon, the quotients of albumin and {alpha}2-macroglobulin levels in BALF and serum (QAlb and QA2M, respectively) and the relative coefficient of excretion (RCE = QA2M/QAlb) were determined (Table 3) (18, 19). LPS inhalation was associated with significant increases in QAlb and QA2M (both p < 0.05 vs. placebo/saline), although it did not significantly influence the RCE. These LPS effects were not altered by salmeterol.


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  		TABLE 3. Effect of salmeterol on pulmonary protein leakage

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Several studies have documented antiinflammatory effects of {beta}2-adrenoreceptor agonists in vitro and in experimental animals in vivo. Our study is the first to perform a detailed analysis of the effects of a {beta}2-agonist on lung inflammation induced in a controlled experimental setting in humans. Salmeterol was found to inhibit several inflammatory responses elicited by inhalation of LPS, in particular the recruitment of neutrophils to the bronchoalveolar space, the local release of TNF-{alpha} and the upregulation of HLA-DR on alveolar macrophages.

Several investigators have established that inhalation of nebulized LPS results in local inflammatory responses in the alveolar compartment of healthy humans, as reflected by influx of neutrophilic granulocytes and an increase in the concentrations of cytokines in BALF (24–26). Other studies have documented neutrophil influx and elevated cytokine levels in induced sputum after inhalation of LPS (27–29). Finally, two recent investigations described local inflammatory responses, including neutrophil recruitment and cytokine and chemokine release, in lung segments of healthy humans challenged with LPS via a bronchoscope (30, 31). We here extend these earlier human studies by not only reporting inflammatory protein levels in BALF but also by determining mRNA expression of a series of inflammatory mediators in purified alveolar macrophages. We thereby documented that inhalation of nebulized LPS induced the expression of genes encoding several proteins involved in the regulation of inflammation using multiplex ligation-dependent probe amplification, an established method to concurrently quantify multiple mRNA species (see Table 2) (20–23). These findings confirm that alveolar macrophages are a major source for LPS-responsive proteins in the human lung. Gene expression profiles were not influenced by salmeterol, although inhalation of salmeterol alone or with LPS enhanced mRNA levels of protein-tyrosine phosphatase type 4A2 and protein-tyrosine phosphatase nonreceptor type 1. The biological significance of this finding remains to be established; knowledge of the function of protein-tyrosine phosphatases in the immune response is rapidly increasing (32). Notably, we only studied one time point after inhalation of LPS (6 h), which impedes a detailed kinetic analysis of gene expression profiles. On the basis of previous studies in man and mice (13, 30), the 6-h time point was chosen to obtain insight in both cytokine/chemokine release and neutrophil recruitment after pulmonary delivery of LPS. We chose not to expand the number of time points in light of the invasive procedure to obtain BALF samples and cells. A kinetic analysis involving mRNA harvesting at multiple time points is required to establish the effect of LPS inhalation and salmeterol on inflammatory gene expression in alveolar macrophages in vivo in more detail.

Salmeterol strongly reduced the influx of neutrophils into the lungs after inhalation of LPS. This finding confirms and extends our previous study in mice, in which inhaled salmeterol produced a similar effect (13). In addition, systemic administration of several {beta}2-agonists, including salmeterol, formoterol, salbutamol, and terbutaline, has been reported to diminish neutrophil recruitment to the lungs of experimental animals (13, 33–35). Although the mechanism by which salmeterol decreased neutrophil influx was not investigated in our study, it should be noted that a variety of agents that increase intracellular cAMP levels, including {beta}-receptor agonists, have been found to inhibit neutrophil chemotactic responses, suggesting that cAMP negatively regulates the migratory capacity of these cells (36, 37). Interestingly, inhalation of salmeterol also in part reduced neutrophil degranulation in the lungs, which particularly was reflected by a significant reduction in BALF MPO concentrations. Although we cannot exclude that the lower BALF levels of neutrophil degranulation products were the result of the lower neutrophil numbers, several findings point to an effect of salmeterol on neutrophil degranulation. First, {beta}-adrenergic but not {alpha}-adrenergic stimulation reduced neutrophil degranulation in vitro (38, 39). Second, intravenous infusion of epinephrine attenuated the release of elastase into the circulation of healthy humans intravenously challenged with LPS while concurrently enhancing the neutrophilic leukocytosis (40). Third, several other studies in human volunteers challenged with LPS intravenously have documented opposite and, in time, unrelated effects of various interventions on peripheral blood neutrophil counts and the plasma concentrations of neutrophil degranulation products (41–43). Our findings corroborate a clinical study in patients with mild asthma, in whom salmeterol (50 µg twice daily for 6 wk) reduced the number of neutrophils in bronchial biopsies and MPO concentrations in BALF (1).

Salmeterol reduced the release of TNF-{alpha} elicited by inhalation of LPS, confirming and extending several earlier studies. We previously showed that both inhaled and intraperitoneally administered salmeterol attenuates TNF-{alpha} release in BALF after intranasal instillation of LPS in mice (13). In addition, systemic administration of several {beta}-adrenoreceptor agonists potently inhibited the production of TNF-{alpha} during endotoxemia in mice and normal humans (10, 35, 44–46). Notably, we were unable to detect TNF-{alpha} mRNA in alveolar macrophages obtained 6 h after LPS inhalation using multiplex ligation-dependent probe amplification, a method that we earlier used successfully to demonstrate a rise in TNF-{alpha} mRNA in peripheral blood leukocytes after intravenous injection of LPS into healthy humans (21). Conceivably, the 6-h time point is too late to detect such a rise in alveolar macrophages after inhalation of a relatively low LPS dose. For ethical reasons we chose not to directly address this issue by expanding the number of volunteers and sampling time points, in particular in consideration of the invasiveness of the experimental procedures (see also above).

Salmeterol reversed the LPS-induced upregulation of HLA-DR expression on alveolar macrophages, although it did not influence CD71 expression. Notably, several compounds that increase intracellular cAMP levels have been reported to inhibit IFN-{gamma}–induced HLA-DR expression (47, 48). It is therefore conceivable that salmeterol attenuates HLA-DR expression via a cAMP-dependent mechanism. To the best of our knowledge, such an association between cAMP and CD71 has not been described.

To obtain insight into the effect of LPS on plasma protein leakage into the airway lumen and the size selectivity of the blood–airway barrier, we assessed and compared the leakage of a large protein ({alpha}2-macroglobulin, 725 kD) with that of a smaller protein (albumin, 67 kD). Therefore, we calculated the RCE (QA2M/QAlb), where Qprotein equals the protein level in BALF divided by the protein level in serum (18, 19). LPS inhalation increased both QA2M and QAlb without significantly influencing the RCE, indicating that LPS caused marked protein leakage. Despite a relative larger luminal influx of A2M compared with Alb, the size-selective permeation of the blood–airway barrier apparently did not change. This latter, unexpected finding may in part be explained by the large variance in Alb and A2M BALF levels between volunteers. Salmeterol did not influence any of these responses. In line with these results, salbutamol treatment did not influence QA2M, QAlb, or RCE in patients with asthma (19). Notably, the pulmonary response to LPS reported here differs from the response to leukotriene B4 instilled into a lung subsegment: whereas LPS induced both neutrophil influx and protein leak, leukotriene B4 merely induced neutrophil influx (49) These data suggest that the LPS inhalation model may be more reflective of the pulmonary reaction to infection than the leukotriene-B4 instillation model.

In our study, salmeterol was administered 30 min before LPS; the effects of postponed salmeterol treatment were not investigated. Such studies would provide insight into whether salmeterol is able to influence an inflammatory reaction in the lung that has already been initiated. In this respect, a recent study by McAuley and colleagues (50) is of considerable interest. These authors demonstrated that salmeterol given 30 min after the inflammatory challenge reduced lung water and lung endothelial permeability after acid aspiration–induced lung injury. Delayed treatment with salmeterol would also exclude the possibility that inhalation of this {beta}2-agonist influences the levels of some inflammatory parameters primarily through altering the distribution of LPS due to bronchodilation.

Most studies examining the effects of inhaled LPS in the human lung used LPS doses of 50 µg or less (51). We chose to administer 100 µg to obtain a more evident inflammatory reaction at the laboratory level without inducing clinically relevant adverse effects. Indeed, LPS doses greater than 100 µg have been tolerated well by healthy subjects (51), and in our previous study, inhalation of 50 µg LPS did not cause clinical signs or symptoms (52). In the present study, inhalation of 100 µg LPS did not induce clinical signs or symptoms and was not associated with significant changes in FVC and FEV1 (data not shown). We chose to use salmeterol at the highest dose administered clinically to patients with severe chronic obstructive pulmonary disease to obtain a "proof of principle" that salmeterol influences inflammatory responses in the lung.

The present study demonstrates that inhalation of LPS induces a number of inflammatory responses in the human lung, including the expression of mRNAs of inflammatory mediators in alveolar macrophages. The documented antiinflammatory effects of salmeterol, in particular the inhibition of neutrophil influx, neutrophil degranulation (MPO), TNF-{alpha} release, and alveolar macrophage HLA-DR expression, expand our knowledge about the mechanisms by which this commonly used drug produces clinical effects in patients with obstructive pulmonary disease suffering from chronic airway inflammation.


   Acknowledgments
 
The authors thank Dr. Michael Tank for help with the statistical analysis and Jennie Pater for excellent technical assistance.


   FOOTNOTES
 
Supported by a grant (3.4.99.09) from the Dutch Asthma Foundation (N.A.M.).

This article has an online supplement, which is accessible from the issue's table of contents at www.atsjournals.org

Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form March 22, 2005; accepted in final form June 22, 2005


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