Characterization of leukotrienes in a pilot study of older asthma subjects
© Nyenhuis et al; licensee BioMed Central Ltd. 2010
Received: 3 June 2010
Accepted: 5 July 2010
Published: 5 July 2010
Asthma is a chronic inflammatory disorder of the lower airway that results in mucus secretion, airway edema and reversible airway obstruction . These characteristic features lead to the clinical symptoms of asthma, which include recurrent episodes of wheezing, breathlessness, chest tightness and coughing . The pathophysiology of asthma is not fully elucidated but the current paradigm involves multiple cell types (ie. eosinophils, neutrophils, mast cells, T-cells) and their mediators.
The most recent estimates of asthma prevalence in those over the age of 65 are between 4 and 8%, which may be an underestimate due to an underdiagnosis of asthma in the elderly . An increase in morbidity and mortality and a reduced response to bronchodilators in the emergency department setting have been shown in older asthmatics compared to their younger counterparts [4–7]. Elderly asthmatics also have a higher rate of severe exacerbations, emergency department visits, and hospitalizations than younger asthmatics [8, 9].
Leukotrienes are potent pro-inflammatory lipid mediators that have been shown to have a role in asthma . Leukotrienes are a product of arachidonic acid via the 5-lipoxygenase pathway. Leukotriene B4 (LTB4) is a potent chemotactic agent and activator of neutrophils and is also produced mainly by neutrophils. The presence of LTB4 in the lung results in neutrophil recruitment and activation leading to superoxide anion generation and cholinergic airway hyperresponsiveness . The cysteinyl leukotrienes (CysLTs), LTC4, LTD4, and LTE4, are known for their profound effect on the airway, including increased microvascular permeability and vasodilation, airway smooth muscle contraction, mucous secretion and impaired mucociliary clearance . CysLTs are produced by eosinophils, basophils, mast cells, macrophages and to a lesser degree by T cells and endothelial cells [10, 12]. Increased CysLT levels have been detected in the sputum of patients with asthma and have been shown to correlate with symptom severity .
Despite older asthmatics often having more severe disease and exacerbations, few studies have been done to characterize their asthma at a cell and/or molecular level. We have previously shown that functional differences exist in eosinophils from older adult asthmatics in vitro; specifically, diminished IL-5 stimulated eosinophil derived neurotoxin (EDN) release and superoxide production . In this pilot study, we sought to compare pro-inflammatory lipid mediator production, specifically leukotrienes LTB4 and CysLT, both in vitro and in vivo in young and older adult asthmatics. Identifying the inflammatory differences seen in older asthmatics may be important for the diagnosis, improving the morbidity and mortality, as well as determining optimal therapies of the disease in this growing population.
We recruited subjects in two age groups, 20 to 40 years (n = 12) and 50 to 70 years (n = 6), in a study protocol approved by the University of Wisconsin Health Sciences Institutional Review Board. Inclusion criteria included a physician diagnosis of mild to moderate asthma with a provocative concentration of methacholine causing a 20% fall in FEV1 (PC20) of < 8 mg/mL or albuterol reversibility on spirometry of ≥ 12%. Exclusion criteria included history of tobacco use > 5 pack-years or use in the previous year, prednisone use within 1 month, donation of blood (greater than 1/2 pint) in the previous 8 weeks, FEV1 < 60%, severe asthma, upper and/or lower respiratory tract infection in the previous 4 weeks, diabetes, pregnancy, or an active cardiovascular disease other than controlled hypertension.
At the study visit, medical history, spirometry with bronchodilator reversibility, sputum induction, allergy skin prick test, physician exam and phlebotomy were performed. Sputum processing was performed as described previously . LTB4, LTC4 and CysLT levels in culture supernates or the sputum samples were measured using the LTB4, LTC4 and CysLT competitive EIA kits, respectively (Cayman Chemical, Ann Arbor, MI). Heparinized venous blood was lysed in a hypotonic solution and subjected to gradient centrifugation. Neutrophils were isolated from the granulocyte pellet and were > 95% pure and > 98% viable. Eosinophils were also isolated from the granulocyte pellet by negative selection using anti-CD16, anti-CD14, and anti-CD3 magnetic beads (Miltenyi Biotechnology; Auburn,CA) as described previously . The eosinophils were typically > 99% pure and > 98% viable.
Young (n = 12)
Older (n = 6)
Age (years, range given)*
Duration of disease
3.35 (+/- 0.81)
FEV1 % Predicted
86 (+/- 14.7)
81 (+/- 16.46)
3.24 (+/- 0.71)
FVC % Predicted
100 (+/- 12.15)
89 (+/- 12.62)
Absolute Eosinophil Count (cells/mL)
204 (+/- 127.75)
114 (+/- 82.95)
Positive Allergy Testing+
Inhaled Corticosteroid Use
In order to determine whether these in vivo differences were due to an age-related diminished ability of neutrophils or eosinophils to produce leukotrienes, both purified neutrophils and eosinophils were treated with calcium ionophore to stimulate leukotriene production. As shown in Figure 2C, the neutrophil production of LTB4 from older asthma subjects, 8708 ± 5326 pg/mL, was comparable to the young subjects, 10234 ± 3078 pg/mL, p = 0.45. Eosinophil production of LTC4 from young, 5836 (3678-9512) pg/mL, and older asthma subjects, 4766 (2685-8456) pg/mL, was also similar in the two groups, p = 0.78 (Figure 2D).
To our knowledge, a characterization of leukotriene levels has not been previously performed in an older adult asthma population. In this pilot study of young and older mild-to-moderate asthmatics we found that older asthma subjects had lower in vivo levels of LTB4 and CysLT in the sputum at baseline disease. This difference in leukotriene levels was not a reflection of fewer eosinophils and neutrophils in the airway as the total number of these cells in the sputum were similar or greater in our older asthmatic group. However, young and older asthma subjects produced comparable amounts of LTB4 and LTC4in vitro when neutrophils and eosinophils, respectively, were stimulated with calcium ionophore. The observation that LPS stimulation of PBMCs resulted in less GM-CSF production in the older asthma subjects provides a potential explanation for the age-related differences in sputum leukotriene levels. Rather than an intrinsic defect in neutrophils and eosinophils, there may be less GM-CSF in the airways of older asthmatics serving as a stimulant to produce leukotrienes. However, it is possible that other age-related changes in the in vivo inflammatory milieu contribute to the diminished levels of leukotrienes in the airway.
There are several limitations to this study including the absence of a control non-asthmatic population, lack of subjects >65 years old, and few total number of subjects enrolled. Though multiple studies have consistently revealed increases in leukotriene production in asthmatic airways compared to controls, we cannot fully gauge the magnitude of our findings in the older asthmatic without non-asthmatic older controls . It is possible though that our findings would have been more profound if we had more subjects in an even older (> 65 years old) population. Furthermore, as a pilot study, we had a limited number of subjects enrolled in order to establish preliminary observations to serve as the focus of future studies.
Calcium ionophore is a potent activator of leukotriene production in eosinophils and neutrophils. It is possible that we were unable to detect small differences in leukotriene production in vitro with the use of such a potent stimulator of leukotriene production. Therefore, a less potent, physiologically-relevant stimulant (such as GM-CSF) could reveal a difference in leukotriene production in vitro.
The use of inhaled glucocorticoids by some subjects in our study may represent a confounder as glucocorticoids can have multiple effects such as a decrease in inflammatory mediators and neutrophil apoptosis . However, glucocorticoids have not been shown to affect LTB4 formation in vitro and in vivo. Also, we performed a statistical analysis of paired young and older subjects that were matched based on inhaled corticosteroid use, which continued to show statistically significant differences in the in vivo leukotriene levels (data not shown). Thus, the use of inhaled glucocorticoids alone cannot explain our findings.
The lower levels of CysLTs in the airways of older adults may have an impact on the effectiveness of CysLT receptor antagonists in the older population. Two studies to date have examined the efficacy of the CysLT receptor antagonists in an older adult population and concluded that their effectiveness might be limited or altered in older asthmatics [24, 25]. Furthermore, the neutrophil predominance found in the airway of older asthmatics may actually represent a tendency for decreased responsiveness to glucocorticoids as has been observed in the neutrophilic phenotype of severe asthma .
Our findings show that aging can result in changes in the airway environment in asthmatics, specifically an increase in airway neutrophils and decreases in both LTB4 and CysLT levels at baseline. This characterization of leukotrienes in older adult asthmatics reveals significant differences that may have clinical relevance not only in baseline asthma but also during an exacerbation of disease. Understanding the biological changes of airway inflammation in the aging population will aid in the development of future therapies and impact the increased morbidity and mortality that is associated with this phenotype of asthma.
peripheral blood mononuclear cells
granulocyte macrophage colony-stimulating factor
eosinophil derived neurotoxin
forced vital capacity
forced expiratory volume in 1 second
We thank Andrea Marquardt and Kristen Fox for assistance with laboratory procedures. We thank Jurga Zdanaviciene, our research coordinator for this study. We thank Mary Anne Kennedy, Tina Palas, and Cheri Swenson for administrative assistance. We also thank Michael Evans for his statistical assistance. This study was funded by T. Franklin Williams Scholar Program, co-sponsored by the Atlantic Philanthropies, the American Academy of Allergy, Asthma and Immunology, the John A. Hartford Foundation, and the Association of Specialty Professors (SKM), Hartford Center of Excellence (SKM), NIH P01 HL088594 (SKM), GlaxoSmithKline/American Academy of Allergy, Asthma and Immunology Allergy Fellowship Award (SMN), and Wisconsin Allergy and Immunology Research Training Program T32 AI007635 (SMN). GlaxoSmithKline had no role in study design, collection, analysis, interpretation of data, writing of the manuscript or the decision to submit the manuscript for publication.
- National Asthma Education and Prevention Program: Expert panel report 3: (Source Document). 2007, Bethesda, MD: National Heart, Lung and Blood Institute, http://www.nhlbi.nih.gov/guidelines/asthma/asthgdln.htmGoogle Scholar
- Busse W, Kraft M: Cysteinyl leukotrienes in allergic inflammation: strategic target for therapy. Chest. 2005, 127 (4): 1312-26. 10.1378/chest.127.4.1312.View ArticlePubMedGoogle Scholar
- Enright PL: Underdiagnosis and Undertreatment of Asthma in the Elderly. Chest. 1999, 116 (3): 603-613. 10.1378/chest.116.3.603.View ArticlePubMedGoogle Scholar
- Bellia V: Asthma in the elderly - Mortality rate and associated risk factors for mortality. Chest. 2007, 132 (4): 1175-1182. 10.1378/chest.06-2824.View ArticlePubMedGoogle Scholar
- Quadrelli SA, Roncoroni AM: Features of asthma in the elderly. Journal of Asthma. 2001, 38 (5): 377-389. 10.1081/JAS-100000259.View ArticlePubMedGoogle Scholar
- Marks GB, Correll PK, Williamson M: Asthma in Australia 2005. Medical Journal of Australia. 2005, 183 (9): 445-446.PubMedGoogle Scholar
- Banerji A: Prospective multicenter study of acute asthma in younger versus older adults presenting to the emergency department. Journal of the American Geriatrics Society. 2006, 54 (1): 48-55. 10.1111/j.1532-5415.2005.00563.x.View ArticlePubMedGoogle Scholar
- Oguzulgen IK: What can predict the exacerbation severity in asthma?. Allergy and Asthma Proceedings. 2007, 28 (3): 344-347. 10.2500/aap.2007.28.2949.View ArticlePubMedGoogle Scholar
- Moorman JERR, Johnson CA: National Surveillance for Asthma --- United States, 1980--2004. MMWR Surveillance Summary. 2007, 56 (8): 1-54.Google Scholar
- Busse WW: Leukotrienes and inflammation. American Journal of Respiratory and Critical Care Medicine. 1998, 157 (6): S210-S213.View ArticleGoogle Scholar
- Nagata M, Sedgwick JB, Busse WW: Differential-Effects of Granulocyte-Macrophage Colony-Stimulating Factor on Eosinophil and Neutrophil Superoxide Anion Generation. Journal of Immunology. 1995, 155 (10): 4948-4954.Google Scholar
- Hamid Q: Inflammatory cells in asthma: Mechanisms and implications for therapy. Journal of Allergy and Clinical Immunology. 2003, 111 (1, Supplement 1): S5-S17. 10.1067/mai.2003.22.View ArticlePubMedGoogle Scholar
- Pavord ID: Induced Sputum Eicosanoid Concentrations in Asthma. Am J Respir Crit Care Med. 1999, 160 (6): 1905-1909.View ArticlePubMedGoogle Scholar
- Mathur SK: Age-Related Changes in Eosinophil Function in Human Subjects. Chest. 2008, 133 (2): 412-419. 10.1378/chest.07-2114.PubMed CentralView ArticlePubMedGoogle Scholar
- Gern JE: Relationship of Upper and Lower Airway Cytokines to Outcome of Experimental Rhinovirus Infection. American Journal of Respiratory and Critical Care Medicine. 2000, 162 (6): 2226-2231.View ArticlePubMedGoogle Scholar
- Yamamoto H, Sedgwick JB, Busse WW: Differential regulation of eosinophil adhesion and transmigration by pulmonary microvascular endothelial cells. Journal of Immunology. 1998, 161 (2): 971-977.Google Scholar
- Sears MR: A Longitudinal, Population-Based, Cohort Study of Childhood Asthma Followed to Adulthood. The New England Journal of Medicine. 2003, 349 (15): 1414-1422. 10.1056/NEJMoa022363.View ArticlePubMedGoogle Scholar
- Nyenhuis SM: Airway neutrophil inflammatory phenotype in older subjects with asthma. The Journal of allergy and clinical immunology. 125 (5): 1163-1165. 10.1016/j.jaci.2010.02.015.
- Fulop T: Signal transduction and functional changes in neutrophils with aging. Aging Cell. 2004, 3 (4): 217-226. 10.1111/j.1474-9728.2004.00110.x.View ArticlePubMedGoogle Scholar
- Fortin CF: GM-CSF activates the Jak/STAT pathway to rescue polymorphonuclear neutrophils from spontaneous apoptosis in young but not elderly individuals. Biogerontology. 2007, 8 (2): 173-187. 10.1007/s10522-006-9067-1.View ArticlePubMedGoogle Scholar
- Wenzel SE: The role of leukotrienes in asthma. Prostaglandins, Leukotrienes and Essential Fatty Acids. 2003, 69 (2-3): 145-155. 10.1016/S0952-3278(03)00075-9.View ArticleGoogle Scholar
- Schleimer RP: An assessment of the effects of glucocorticoids on degranulation, chemotaxis, binding to vascular endothelium and formation of leukotriene B4 by purified human neutrophils. Journal of Pharmacology And Experimental Therapeutics. 1989, 250 (2): 598-605.PubMedGoogle Scholar
- Steiss JO: Effect of inhaled corticosteroid treatment on exhaled breath condensate leukotriene E-4 in children with mild asthma. Allergy and Asthma Proceedings. 2008, 29 (4): 371-375. 10.2500/aap.2008.29.3135.View ArticlePubMedGoogle Scholar
- Creticos P: Loss of response to treatment with leukotriene receptor antagonists but not inhaled corticosteroids in patients over 50 years of age. Annals of Allergy Asthma & Immunology. 2002, 88 (4): 401-409.View ArticleGoogle Scholar
- Korenblat PE: Effect of age on response to zafirlukast in patients with asthma in the Accolate Clinical Experience and Pharmacoepidemiology Trial (ACCEPT). Annals of Allergy Asthma & Immunology. 2000, 84 (2): 217-225.View ArticleGoogle Scholar
- Wenzel SE: Asthma: defining of the persistent adult phenotypes. The Lancet. 2006, 368 (9537): 804-813. 10.1016/S0140-6736(06)69290-8.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.