|Year : 2006 | Volume
| Issue : 2 | Page : 67-70
|Circulating LTB4 and Eotaxin-1 in stable asthmatics on inhaled corticosteroids and long-acting β 2-agonists
Mohammed A Alzoghaibi1, Ahmed S BaHammam2
1 Departments of Physiology and Medicine, College of Medicine, King Saud University, Riyadh, Saudi Arabia
2 Respiratory Unit, Department of Medicine, College of Medicine, King Saud University, Riyadh, Saudi Arabia
Ahmed S BaHammam
College of Medicine, Respiratory Unit, Department of Medicine, King Saud University,
| Abstract|| |
BACKGROUND : Leukotrienes B4 (LTB4) and eotaxin-1 are thought to play a pivotal role in the pathogenesis of asthma. This study investigates the plasma levels of LTB4 and eotaxin-1 in symptom-free asthmatics on inhaled corticosteroids (ICS) and long-acting β2 (LABA) .
MATERIALS AND METHODS : Twenty asthmatic patients treated with ICS and LABA for 3 months and 17 matched healthy subjects were recruited. LTB4 and eotaxin-1 were measured in the serum by a specific enzyme immunoassay kit.
RESULTS : Treatment resulted in significant improvement in FEV1 and disappearance of symptoms. LTB4 levels were significantly lower in the treated asthmatics compared to the healthy subjects (19.17 ± 0.8 pg/ml versus 23.34 ± 0.82 pg/ml respectively, P <0.001). However, there was no significant difference in the levels of eotaxin-1 between healthy subjects and asthmatic patients.
CONCLUSION : Asthmatics treated with ICS and LABA showed significantly lower levels of LTB4 compared to healthy subjects. Regular use of inhaled corticosteroids and long-acting β2 may help in controlling the inflammatory process in asthma. Further studies are needed to confirm these findings and assess the association between clinical and physiological parameters and circulating chemokines and cytokines.
Keywords: Leukotrines B4, asthma, inhaled corticosteroids
|How to cite this article:|
Alzoghaibi MA, BaHammam AS. Circulating LTB4 and Eotaxin-1 in stable asthmatics on inhaled corticosteroids and long-acting β 2-agonists. Ann Thorac Med 2006;1:67-70
|How to cite this URL:|
Alzoghaibi MA, BaHammam AS. Circulating LTB4 and Eotaxin-1 in stable asthmatics on inhaled corticosteroids and long-acting β 2-agonists. Ann Thorac Med [serial online] 2006 [cited 2019 Nov 21];1:67-70. Available from: http://www.thoracicmedicine.org/text.asp?2006/1/2/67/27104
Asthma is a chronic inflammatory condition characterized by inflammatory cell infiltration of the airways with activated T cells and eosinophils, reversible bronchoconstriction and bronchial hyper-responsiveness with resultant respiratory symptoms.,
Cytokines are proteins produced by different cell types in the body that modulate the function of other cell types in different tissues. Chemokines are low molecular weight, 8-10 kd, chemotactic cytokines. They represent the chemotactic signals that are produced by different cell types to initiate leukocyte infiltration into inflamed tissue.
The immunopathology of asthma is complex and involves many inflammatory cells and mediators. It is well recognized that eosinophils and neutrophils are important inflammatory cells in asthma., It has been reported that airway epithelial cells produce a large variety of a and b chemokines such as eotaxin-1, interlukin-5 (IL-5) that are capable of attracting and activating eosinophils and IL-8 that attracts neutrophils.,,, Eosinophils release basic proteins that are cytotoxic and lipid mediators such as cystinyl leukotrienes that cause airflow obstruction and bronchial epithelial damage.,,,, Activated neutrophils also release toxic substances such as reactive oxygen species (ROS) elastase and myloperoxydase. Eotaxin has been reported to be a paramount marker of asthma severity.
Leukotrienes (LTs) are potent proinflammatory mediators that participate in the pathophysiological changes in the airways of patients with asthma. LTB 4 stimulates neutrophil chemotaxis and activates those cells, leading to the release of mediators and superoxides. LTB 4 affects cells other than neutrophils -as for example, LTB 4 increases IL-6 production by human monocytes and may affect the production of other cytokines by stimulating early gene transcription in mononuclear cells.
There is no agreement with regard to the effect of corticosteroids on eotaxin expression and levels. While in vitro studies confirmed that corticosteroids decrease cytokine-induced eotaxin expression in human epithelial cells in a concentration-dependent manner,, in vivo studies demonstrated that the systemic expression of eotaxin is not significantly decreased by clinical doses of inhaled or oral steroids. On the other hand, many studies suggested that long-acting b 2 agonists (LABA), like corticosteroids, may affect the production of a wide range of chemokines and cytokines in airway smooth muscles and other cells., Collectively, it appears that there is no consensus regarding the effect of corticosteroids on plasma eotaxin levels. Additionally, no studies have addressed the effect of combined inhaled corticosteroids (ICS) and LABA on plasma eotaxin. Furthermore, the effects of combined corticosteroids and LABA on circulating LTB 4 in asthmatics have not been explored well in the literature. Therefore, we conducted this study to assess plasma levels of LTB 4 and eotaxin-1 in symptom-free asthmatics on ICS and LABA and to compare them to matched healthy subjects.
| Materials and Methods|| |
This prospective controlled study was conducted in King Khalid University Hospital between September 2003 and May 2004 wherein patients were recruited from the general respirology outpatient clinic. The study group comprised 20 (14 females and 6 males) nonsmoking asthmatic patients who were treated with ICS and LABA for 3 months. Asthmatic patients were compared to 17 (3 females and 14 males) age- and body mass index (BMI)-matched, nonsmoking healthy subjects (control group). Complete medical history and physical examination were obtained for all patients. Patients were excluded if they had a history of chronic medical, neurological or psychiatric disorders other than asthma. None of the patients or controls was on medications that may affect the levels of cytokines. At the time of recruitment, all patients had a history of intermittent wheezing and nocturnal cough with an increase of >12% in the 1-second forced expiratory volume (FEV 1 ) and an increase of more than 200 ml in the FEV 1 following the inhalation of 2.5 mg of nebulized salbutamol. Spirometry for the healthy subjects was normal. Patients received twice daily a combination of either both budesonide (320 ug/day) and frometrol (9 ug/day) (12 patients) or both fluticasone propionate (250 ug/dose) and salmetrol (50 ug/dose) (8 patients). Patients were followed every 2 weeks thereafter and the presence of symptoms and the need for rescue therapy (salbutamol) were recorded. Good control was defined as the absence of nocturnal symptoms and the need to use inhaled salbutamol once or less per week for the last 2 months of treatment. Blood samples were collected in the morning (8-10 a.m.) and immediately transferred to the laboratory for cytokine measurements. The study was approved by the ethics committee in our institute and an informed consent was obtained from all participants.
Eotaxin-1 concentrations were determined by enzyme immunoassay (EIA) using commercially available reagents: anti-human eotaxin-1 monoclonal capture antibody, biotinylated anti-human eotaxin-1 detection antibody and recombinant human eotaxin-1 (R and D systems, Minneapolis, MN). In this assay, eotaxin-1 in the serum simultaneously binds to two antibodies directed against different epitopes on the eotaxin-1 molecule. One antibody (capture antibody) coats the wells of a 96-well microplate while the other (detection antibody) is conjugated to biotin. After removal of unbound detection antibody by washing three times by tween-20 and PBS, 100 mL of streptavidin-horseradish peroxidase conjugate (streptavidin-HRP, Zymed Laboratories Inc, San Francisco, CA) was added to each well, followed by 20-min incubation. Streptavidin-HRP binds to the bound detection antibody conjugate. The plates were then washed as before. A substrate specific for HRP (tetramethylbenzidine substrate solution, 100 mL) was added to each well, followed by 30-min incubation in the dark to detect the amount of conjugate bound to the eotaxin-1 antibodies. Fifty mL of 2N sulfuric acid diluted in double-distilled H 2 O (1 ml of 2N H 2 SO 4 to 3 ml of ddH 2 O) was added to each well to stop the reaction and the plate was read at 450 nm by a 96-well microplate reader. LTB 4 was measured using the same procedure.
Data are expressed in text and figures as mean ± standard error of mean (SEM). Comparisons between asthmatic patients versus healthy subjects were made using student's two-tailed t-test. Results were considered statistically significant at P <0.05. Standard statistical software (Sigma Stat, version 3; SPSS Chicago, Illinois, USA) was used for the analysis.
| Results|| |
Asthmatic patients had a mean age of 43.7 ± 3.0 years and a body mass index (BMI) of 33.2 ± 1.8 kg/m 2, compared to 43.1 ± 7.02 years and a BMI of 33.4 ± 3.39 4 kg/m 2sub in the healthy subjects. At presentation, FEV 1 was 1.9 ± 0.1 L (66.1 ± 4.8% of predicted), a forced vital capacity (FVC) was 2.6 ± 0.1 L (73.6 ± 3.7% of predicted). After 3 months of treatment when blood samples were collected, the patients exhibited an FEV 1 of 2.3 ± 0.14 L (81.6 ± 5.9% of predicted), an FVC of 2.9 ± 0.14 L (86.6 ± 4.4% of predicted) and no symptoms. The improvement in FEV 1 and FVC were statistically significant ( P <0.001). No new medications were added to the patients' treatment during the follow-up period.
No hospital admissions were documented during the follow-up period and patients were controlled according to the criteria adopted in this study.
LTB 4 levels were significantly lower in asthmatics treated with ICS and LABA compared to healthy subjects (19.17 ± 0.8 pg/ml for asthmatic patients versus 23.34 ± 0.82 pg/ml for healthy subjects, P <0.001) [Figure - 1].
This study showed no significant difference in the concentration of circulating Eotaxin-1 between healthy subjects and asthmatic patients (107 ± 10.9 vs. 96.2 ± 11.3 pg/ml respectively) [Figure - 2]. No differences were found between patients using budesonide and frometrol (12 patients) and patients using fluticasone propionate and salmetrol (8 patients).
| Discussion|| |
In the present study, we measured Eotaxin-1, which is an eosinophil chemokine; and LTB 4 , a potent chemoattractant and activator of neutrophils in asthmatics., LTB 4 has a role in mediating neutrophil survival in vitro by inhibiting neutrophil apoptosis and may also be involved in corticosteroid-induced neutrophil survival. Recent studies of severe asthma and exacerbation of asthma suggest that LTB 4 could be important in the pathogenesis of asthma. Cai et al reported that plasma LTB 4 levels were higher in steroid-naive children with asthma during acute asthma exacerbations and even 1 month after recovery. Cosma and colleagues did not find elevated LTB 4 levels in exhaled breath condensate collected from subjects with mild intermittent asthma. We found that the levels of circulating LTB 4 in asthmatic patients on ICS and LABA were significantly lower compared to healthy subjects. To our knowledge, no study has assessed the effect of ICS on circulating LTB 4 in asthmatics. Nevertheless, studies have shown conflicting results with regard to the effects of corticosteroids on LTB 4 in bronchoalveolar lavage or exhaled LTB 4. While some investigators failed to demonstrate any effect of ICS on LTB 4 , others demonstrated that treatment with high dose ICS or oral steroids inhibits LTB 4 in the airway.,
Our findings suggest that corticosteroid treatment may play a role, at least in part, in decreasing the levels of inflammation in asthmatic patients by inhibiting the levels of neutrophil chemokine, LTB 4 . Further studies with larger number of patients with varying degrees of asthma severity are needed to confirm these findings.
The CC chemokine (cysteine-cysteine chemokine), eotaxin, plays a crucial role in eosinophil migration in tissue. Several cytokines, such as IL-8 and eotaxin, are produced by epithelial cells during the airway inflammation of asthma.,, Eotaxin levels have been shown to be elevated in bronchoalveolar lavage fluid; induced sputum and plasma of subjects with asthma and its expression are also increased in the bronchial mucosa in asthma.,,,, The direct participation of eotaxin in asthma is suggested by the localization of eotaxin messenger RNA (mRNA) and protein to the airway epithelium., Our data showed no significant difference in the serum levels of eotaxin-1 between normal controls and asthmatic patients, which is contradictory to the previous studies mentioned above. Our results might be explained by the following: First, the number of patients in our study is relatively small to detect small differences; second, treatment may need a longer duration to inhibit the levels of eotaxin-1 significantly; and third, the level of eotaxin-1 might have been higher initially and dropped after treatment, but this could not be documented as the level of eotaxin-1 was not measured at the start of the study. In vivo studies demonstrated that BAL levels and the systemic expression of eotaxin are not significantly decreased by clinical doses of inhaled or oral steroids.,
The present study has some limitations that need to be addressed. First, the levels of eotaxin and LTB 4 were not measured at the beginning of the study to allow for better assessment of the effect of ICS and LABA. Another limitation is the fact that we measured systemic eotaxin and LTB 4 only. Further studies should recruit larger number of patients with varying degrees of severity of asthma and should seek additional information about kinetics of changes in chemokine and cytokine levels with continued treatment, the association (or lack thereof) of changes in chemokine and cytokine levels with clinical and physiological parameters and the relationship between circulating and airway levels of chemokines and cytokines. Nevertheless, the primary value of this study lies in the fact that it addresses a topic that has not been well explored in the literature and provides a very good starting point for more in-detail observations by other investigators to assess the effects of ICS and LABA on circulating chemokines and cytokines.
In conclusion, asthmatics treated with ICS and LABA showed significantly lower levels of LTB 4 compared to healthy subjects. On the other hand, eotaxin levels showed no significant difference between healthy subjects and treated asthmatic patients. Further studies are needed to confirm these findings and assess the association between clinical and physiological parameters and circulating chemokines and cytokines.
| Acknowledgment|| |
This study was supported by a grant from the College of Medicine Research Center (CMRC).β
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