点击显示 收起
Departments of Experimental Internal Medicine, Pulmonology
Infectious Diseases, Tropical Medicine and AIDS, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
ABSTRACT
Rationale: Salmeterol is a 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 e salmeterol or placebo (t = eC0.5 h) followed by 100 e 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 and interleukin 6) and chemokines (interleukin 8, epithelial celleCderived neutrophil attractant 78, macrophage inflammatory proteins 1 and 1), 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 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 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 2-agonists, induces bronchodilation via activation of 2-adrenoreceptors on smooth muscle cells (2). 2-receptors are also expressed by cells involved in the regulation of inflammation, in particular by neutrophils, monocytes, and macrophages (3eC5). Stimulation of 2-receptors on these immunocompetent cells primarily results in antiinflammatory effects. Triggering of 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 (6eC9). Moreover, stimulation of 2-adrenergic receptors on mononuclear cells and macrophages diminishes their capacity to release proinflammatory cytokines, such as tumor necrosis factor (TNF-) and interleukin 1 (IL-1) (3, 10eC12).
Asthma and chronic obstructive pulmonary disease are associated with chronic inflammation in the respiratory tract. Knowledge of the effects of 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- 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
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] e; 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 e; 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, IL-6, IL-8, IL-10, and IL-12p70 were measured using a cytometric bead array (Pharmingen, San Diego, CA). TNF- (high-sensitivity ELISA), macrophage inflammatory protein 1 (MIP-1), MIP-1, and epithelial celleCderived neutrophil attractant 78 were measured by ELISA (all R&D Systems, Abingdon, UK). Albumin and 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 eC80°C until used for RNA isolation.
Multiplex Ligation-dependent Probe Amplification
RNA isolation and multiplex ligation-dependent probe amplification were performed as described previously (20eC23). 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
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).
Salmeterol Attenuates LPS-induced TNF- 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- and IL-6 were upregulated after inhalation of LPS (Figure 2; p < 0.05 vs. placebo/saline), whereas IL-1, IL-10, and IL-12p70 remained undetectable. Salmeterol inhibited TNF- 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 celleCderived neutrophil attractant 78, and the CC chemokines MIP-1 and MIP-1 into BALF (Figure 3; all p < 0.05 vs. placebo/saline). Although salmeterol tended to reduce the levels of IL-8, MIP-1, and MIP-1, these effects did not reach statistical significance.
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.
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, IL-1, IL-1 receptor antagonist, IL-6, IL-18) and chemokines (IL-8, monocyte chemoattractant protein 1, MIP-1, and MIP-1; 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-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.
Salmeterol Does Not Influence the Permeability of the BloodeCAirway Barrier
To obtain insight into the effect of LPS on the permeability of the bloodeCairway barrier and the influence of salmeterol thereon, the quotients of albumin and 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.
DISCUSSION
Several studies have documented antiinflammatory effects of 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 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- 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 (24eC26). Other studies have documented neutrophil influx and elevated cytokine levels in induced sputum after inhalation of LPS (27eC29). 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) (20eC23). 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 2-agonists, including salmeterol, formoterol, salbutamol, and terbutaline, has been reported to diminish neutrophil recruitment to the lungs of experimental animals (13, 33eC35). 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 -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, -adrenergic but not -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 (41eC43). Our findings corroborate a clinical study in patients with mild asthma, in whom salmeterol (50 e 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- elicited by inhalation of LPS, confirming and extending several earlier studies. We previously showed that both inhaled and intraperitoneally administered salmeterol attenuates TNF- release in BALF after intranasal instillation of LPS in mice (13). In addition, systemic administration of several -adrenoreceptor agonists potently inhibited the production of TNF- during endotoxemia in mice and normal humans (10, 35, 44eC46). Notably, we were unable to detect TNF- 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- 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-eCinduced 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 bloodeCairway barrier, we assessed and compared the leakage of a large protein (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 bloodeCairway 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 aspirationeCinduced lung injury. Delayed treatment with salmeterol would also exclude the possibility that inhalation of this 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 e or less (51). We chose to administer 100 e to obtain a more evident inflammatory reaction at the laboratory level without inducing clinically relevant adverse effects. Indeed, LPS doses greater than 100 e have been tolerated well by healthy subjects (51), and in our previous study, inhalation of 50 e LPS did not cause clinical signs or symptoms (52). In the present study, inhalation of 100 e 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- 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.
This article has an online supplement, which is accessible from the issue's table of contents at www.atsjournals.org
REFERENCES
Johnson M, Rennard S. Alternative mechanisms for long-acting beta(2)-adrenergic agonists in COPD. Chest 2001;120:258eC270.
Svedmyr N. The current place of beta 2-agonists in the management of asthma. Lung 1990;168:S105eCS110.
Izeboud CA, Mocking JA, Monshouwer M, van Miert AS, Witkamp RF. Participation of ETA-adrenergic receptors on macrophages in modulation of LPS-induced cytokine release. J Recept Signal Transduct Res 1999;19:191eC202.
Maisel AS, Fowler P, Rearden A, Motulsky HJ, Michel MC. A new method for isolation of human lymphocyte subsets reveals differential regulation of beta-adrenergic receptors by terbutaline treatment. Clin Pharmacol Ther 1989;46:429eC439.
Barnes PJ. Effect of beta-agonists on inflammatory cells. J Allergy Clin Immunol 1999;104:S10eCS17.
Ottonello L, Morone P, Dapino P, Dallegri F. Inhibitory effect of salmeterol on the respiratory burst of adherent human neutrophils. Clin Exp Immunol 1996;106:97eC102.
Nials AT, Coleman RA, Johnson M, Vardey CJ. The duration of action of non-beta 2-adrenoceptor mediated responses to salmeterol. Br J Pharmacol 1997;120:961eC967.
Derian CK, Santulli RJ, Rao PE, Solomon HF, Barrett JA. Inhibition of chemotactic peptide-induced neutrophil adhesion to vascular endothelium by cAMP modulators. J Immunol 1995;154:308eC317.
Bolton PB, Lefevre P, McDonald DM. Salmeterol reduces early- and late-phase plasma leakage and leukocyte adhesion in rat airways. Am J Respir Crit Care Med 1997;155:1428eC1435.
Van der Poll T, Coyle SM, Barbosa K, Braxton CC, Lowry SF. Epinephrine inhibits tumor necrosis factor-alpha and potentiates interleukin 10 production during human endotoxemia. J Clin Invest 1996;97:713eC719.
Yoshimura T, Kurita C, Nagao T, Usami E, Nakao T, Watanabe S, Kobayashi J, Yamazaki F, Tanaka H, Nagai H. Effects of cAMP-phosphodiesterase isozyme inhibitor on cytokine production by lipopolysaccharide-stimulated human peripheral blood mononuclear cells. Gen Pharmacol 1997;29:633eC638.
Yoshimura T, Kurita C, Nagao T, Usami E, Nakao T, Watanabe S, Kobayashi J, Yamazaki F, Tanaka H, Inagaki N, et al. Inhibition of tumor necrosis factor-alpha and interleukin-1-beta production by beta-adrenoceptor agonists from lipopolysaccharide-stimulated human peripheral blood mononuclear cells. Pharmacology 1997;54:144eC152.
Maris NA, Van Der Sluijs KF, Florquin S, De Vos AF, Pater JM, Jansen HM, Van Der Poll T. Salmeterol, a 2-receptor agonist, attenuates lipopolysaccharide-induced lung inflammation in mice. Am J Physiol Lung Cell Mol Physiol 2004;286:L1122eCL1128.
Van der Veen MJ, Van der Zee JS. Aerosol recovery from large-volume reservoir delivery systems is highly dependent on the static properties of the reservoir. Eur Respir J 1999;13:668eC672.
Van der Veen MJ, Van Neerven RJ, De Jong EC, Aalberse RC, Jansen HM, Van der Zee JS. The late asthmatic response is associated with baseline allergen-specific proliferative responsiveness of peripheral T lymphocytes in vitro and serum interleukin-5. Clin Exp Allergy 1999;29:217eC227.
Bresser P, Out TA, Van Alphen L, Jansen HM, Lutter R. Airway inflammation in nonobstructive and obstructive chronic bronchitis with chronic haemophilus influenzae airway infection: comparison with noninfected patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2000;162:947eC952.
Schultz MJ, Speelman P, Hack CE, Buurman WA, Van Deventer SJ, Van Der Poll T. Intravenous infusion of erythromycin inhibits CXC chemokine production, but augments neutrophil degranulation in whole blood stimulated with Streptococcus pneumoniae. J Antimicrob Chemother 2000;46:235eC240.
Van de Graaf EA. Out, Roos CM, Jansen HM. Respiratory membrane permeability and bronchial hyperreactivity in patients with stable asthma: effects of therapy with inhaled steroids. Am Rev Respir Dis 1991;143:362eC368.
Nocker RE, Weller FR, Out TA, De Riemer MJ, Jansen HM, Van der Zee JS. A double-blind study on the effect of inhaled corticosteroids on plasma protein exudation in asthma. Am J Respir Crit Care Med 1999;159:1499eC1505.
Schouten JP, McElgunn CJ, Waaijer R, Zwijnenburg D, Diepvens F, Pals G. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res 2002;30:e57.
Spek CA, Verbon A, Aberson H, Pribble JP, McElgunn CJ, Turner T, Axtelle T, Schouten J, Van Der Poll T, Reitsma PH. Treatment with an anti-CD14 monoclonal antibody delays and inhibits lipopolysaccharide-induced gene expression in humans in vivo. J Clin Immunol 2003;23:132eC140.
Eldering E, Spek CA, Aberson HL, Grummels A, Derks IA, De Vos AF, McElgunn CJ, Schouten JP. Expression profiling via novel multiplex assay allows rapid assessment of gene regulation in defined signalling pathways. Nucleic Acids Res 2003;31:e153.
Bezzina Wettinger S, Doggen CJ, Spek CA, Rosendaal FR, Reitsma PH. High throughput mRNA profiling highlights associations between myocardial infarction and aberrant expression of inflammatory molecules in blood cells. Blood 2005;105:2000eC2006.
Sandstrom T, Bjermer L, Rylander R. Lipopolysaccharide (LPS) inhalation in healthy subjects increases neutrophils, lymphocytes and fibronectin levels in bronchoalveolar lavage fluid. Eur Respir J 1992;5:992eC996.
Jagielo PJ, Thorne PS, Watt JL, Frees KL, Quinn TJ, Schwartz DA. Grain dust and endotoxin inhalation challenges produce similar inflammatory responses in normal subjects. Chest 1996;110:263eC270.
Wesselius LJ, Nelson ME, Bailey K, O'Brien-Ladner AR. Rapid lung cytokine accumulation and neutrophil recruitment after lipopolysaccharide inhalation by cigarette smokers and nonsmokers. J Lab Clin Med 1997;129:106eC114.
Michel O, Nagy AM, Schroeven M, Duchateau J, Neve J, Fondu P, Sergysels R. DoseeCresponse relationship to inhaled endotoxin in normal subjects. Am J Respir Crit Care Med 1997;156:1157eC1164.
Michel O, Dentener M, Corazza F, Buurman W, Rylander R. Healthy subjects express differences in clinical responses to inhaled lipopolysaccharide that are related with inflammation and with atopy. J Allergy Clin Immunol 2001;107:797eC804.
Nightingale JA, Rogers DF, Hart LA, Kharitonov SA, Chung KF, Barnes PJ. Effect of inhaled endotoxin on induced sputum in normal, atopic, and atopic asthmatic subjects. Thorax 1998;53:563eC571.
O'Grady NP, Preas HL, Pugin J, Fiuza C, Tropea M, Reda D, Banks SM, Suffredini AF. Local inflammatory responses following bronchial endotoxin instillation in humans. Am J Respir Crit Care Med 2001;163:1591eC1598.
Nick JA, Coldren CD, Geraci MW, Poch KR, Fouty BW, O'Brien J, Gruber M, Zarini S, Murphy RC, Kuhn K, et al. Recombinant human activated protein C reduces human endotoxin-induced pulmonary inflammation via inhibition of neutrophil chemotaxis. Blood 2004;104:3878eC3885.
Mustelin T, Vang T, Bottini N. Protein tyrosine phosphatases and the immune response. Nat Rev Immunol 2005;5:43eC57.
Whelan CJ, Johnson M, Vardey CJ. Comparison of the anti-inflammatory properties of formoterol, salbutamol and salmeterol in guinea-pig skin and lung. Br J Pharmacol 1993;110:613eC618.
Saleh TS, Calixto JB, Medeiros YS. Anti-inflammatory effects of theophylline, cromolyn and salbutamol in a murine model of pleurisy. Br J Pharmacol 1996;118:811eC819.
Wu CC, Liao MH, Chen SJ, Chou TC, Chen A, Yen MH. Terbutaline prevents circulatory failure and mitigates mortality in rodents with endotoxemia. Shock 2000;14:60eC67.
Elferink JG, VanUffelen BE. The role of cyclic nucleotides in neutrophil migration. Gen Pharmacol 1996;27:387eC393.
Sato Y. Modulation of PMN-endothelial cells interactions by cyclic nucleotides. Curr Pharm Des 2004;10:163eC170.
Zurier RB, Weissmann G, Hoffstein S, Kammerman S, Tai HH. Mechanisms of lysosomal enzyme release from human leukocytes: II. Effects of cAMP and cGMP, autonomic agonists, and agents which affect microtubule function. J Clin Invest 1974;53:297eC309.
Bazzoni G, Dejana E, Del Maschio A. Adrenergic modulation of human polymorphonuclear leukocyte activation: potentiating effect of adenosine. Blood 1991;77:2042eC2048.
Van der Poll T. Effects of catecholamines on the inflammatory response. Sepsis 2001;4:159eC167.
Spinas GA, Bloesch D, Keller U, Zimmerli W, Cammisuli S. Pretreatment with ibuprofen augments circulating tumor necrosis factor-alpha, interleukin-6, and elastase during acute endotoxinemia. J Infect Dis 1991;163:89eC95.
Pajkrt D, Manten A, Van der Poll T. Tiel-van Buul, Jansen J, Ten Cate J, Deventer SJ. Modulation of cytokine release and neutrophil function by granulocyte colony-stimulating factor during endotoxemia in humans. Blood 1997;90:1415eC1424.
Branger J, Van den Blink B, Weijer S, Madwed J, Bos CL, Gupta A, Yong CL, Polmar SH, Olszyna DP, Hack CE, et al. Anti-inflammatory effects of a p38 mitogen-activated protein kinase inhibitor during human endotoxemia. J Immunol 2002;168:4070eC4077.
Sekut L, Champion BR, Page K, Menius JA, Connolly KM. Anti-inflammatory activity of salmeterol: down-regulation of cytokine production. Clin Exp Immunol 1995;99:461eC466.
Suberville S, Bellocq A, Fouqueray B, Philippe C, Lantz O, Perez J, Baud L. Regulation of interleukin-10 production by beta-adrenergic agonists. Eur J Immunol 1996;26:2601eC2605.
Szabo C, Hasko G, Zingarelli B, Nemeth ZH, Salzman AL, Kvetan V, Pastores SM, Vizi ES. Isoproterenol regulates tumour necrosis factor, interleukin-10, interleukin-6 and nitric oxide production and protects against the development of vascular hyporeactivity in endotoxaemia. Immunology 1997;90:95eC100.
Ivashkiv LB, Ayres A, Glimcher LH. Inhibition of IFN-gamma induction of class II MHC genes by cAMP and prostaglandins. Immunopharmacology 1994;27:67eC77.
Li G, Harton JA, Zhu X, Ting JP. Downregulation of CIITA function by protein kinase a (PKA)-mediated phosphorylation: mechanism of prostaglandin E, cyclic AMP, and PKA inhibition of class II major histocompatibility complex expression in monocytic lines. Mol Cell Biol 2001;21:4626eC4635.
Martin TR, Pistorese BP, Chi EY, Goodman RB, Matthay MA. Effects of leukotriene B4 in the human lung: recruitment of neutrophils into the alveolar spaces without a change in protein permeability. J Clin Invest 1989;84:1609eC1619.
McAuley DF, Frank JA, Fang X, Matthay MA. Clinically relevant concentrations of beta2-adrenergic agonists stimulate maximal cyclic adenosine monophosphate-dependent airspace fluid clearance and decrease pulmonary edema in experimental acid-induced lung injury. Crit Care Med 2004;32:1470eC1476.
Thorn J. The inflammatory response in humans after inhalation of bacterial endotoxin: a review. Inflamm Res 2001;50:254eC261.
Maris NA, De Vos AF, Bresser P, Van der Zee JS, Meijers JC, Lijnen HR, Levi M, Jansen HM, Van der Poll T. Activation of coagulation and inhibition of fibrinolysis in the lung after inhalation of lipopolysaccharide by healthy volunteers. Thromb Haemost 2005; 93:1036eC1040.