Brain-derived Neurotrophic Factor in Platelets and Airflow Limitation in Asthma
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美国呼吸和危急护理医学 2005年第1期
Department of Pneumology and Institute of Clinical Chemistry and Pathobiochemistry, University of Rostock, Rostock
Department of Neurology, Charitee, Berlin, Germany
ABSTRACT
Brain-derived neurotrophic factor (BDNF), a key mediator of neuronal plasticity, contributes to airway obstruction and hyperresponsiveness in a model of allergic asthma. BDNF is stored in human platelets and circulates in human plasma, but the significance of BDNF in this compartment is poorly understood. We investigated the relationship between platelet and plasma BDNF levels and pulmonary function in a cohort of 26 adult patients with recently diagnosed allergic asthma. BDNF levels in serum, platelets, and plasma were significantly increased in participants with asthma, as compared with 26 age- and sex-matched control subjects. In steroid-naive patients, but not in patients using inhaled corticosteroids, enhanced platelet BDNF levels correlated with parameters of airway obstruction and airway hyperresponsiveness to histamine. Experiments with activated peripheral blood mononuclear cells revealed that corticosteroids such as fluticasone effectively suppress BDNF secretion. In conclusion, we demonstrate that enhanced platelet BDNF is associated with airflow limitation and airway hyperresponsiveness in asthma. In addition, we provide evidence that corticosteroids suppress BDNF production by activated immune cells.
Key Words: airway hyperresponsiveness corticosteroids neurotrophins peak expiratory flow
Allergic bronchial asthma is associated with a characteristic type of airway inflammation, airway hyperresponsiveness to nonspecific stimuli, and a variable degree of airflow limitation (1). Although the immunologic network in asthma has been increasingly well defined over the last decades, the links between airway inflammation and changes in pulmonary function remain incompletely understood (2). Several animal studies have shown a dissociation between airway inflammation and airway hyperresponsiveness in models of allergic asthma (3, 4). In human asthma, new therapeutic strategies, such as antieCinterleukin-5 treatment, were highly effective in reducing eosinophilic inflammation but remained ineffective regarding clinical parameters such as airway hyperresponsiveness or the late-phase airway obstruction (5, 6).
These observations have resulted in a new view on the mechanisms of asthma: although allergic inflammation most likely represents the initial trigger of asthma, postinflammatory changes of resident cells are believed to determine persistent airway dysfunction (4). In this context, the role of vagal cholinergic and sensory nerves, which regulate airway tone and reactivity, has gained renewed interest (7eC10). Allergic inflammation induces profound changes in neuronal networks of the lung (11), including sensory hyperreactivity (12), enhanced signal transmission in local parasympathetic ganglia (13), and an exaggerated central reflex activity in the brainstem (14). A key mediator of neuronal plasticity in the adult, brain-derived neurotrophic factor (BDNF) (15, 16), has been found to be increased in bronchoalveolar lavage fluid of patients with allergic asthma (17). In an animal model, BDNF production was shown to be upregulated in cells infiltrating asthmatic airways, including macrophages and T cells (18). Notably, the highest BDNF levels in bronchoalveolar lavage fluid were found during regression of the inflammatory response, suggesting a postinflammatory role of BDNF (19). The inhibition of BDNF with specific anti-BDNF antibodies prevented changes in lung function occurring in response to allergen challenge, including airflow limitation, sensory hyperreactivity, and airway hyperresponsiveness to nonspecific stimuli (20).
The transportation of BDNF in platelets represents a unique feature of human BDNF physiology. Platelets acquire and store substantial amounts of BDNF, which results in high BDNF levels in serum. In contrast, plasma levels represent free circulating BDNF (21). Normal values for BDNF in human platelets and plasma have recently been established, including the influence of age, weight, sex, and the menstrual cycle on these BDNF concentrations (22). However, circulating and stored BDNF levels have not been systematically investigated in patients with allergic asthma. In addition, the relationship between these BDNF levels and pulmonary function remains unclear. This prospective study investigates this relationship in a clinical setting.
METHODS
Study Design
Patients with allergic asthma (9.0 ± 8.0 months since diagnosis) were recruited, and their diagnosis was established using the following criteria: recurrent attacks of wheezing, improvement of pulmonary function following inhalation of a 2-agonist, airway hyperresponsiveness (provocative dose of histamine causing a 20% fall in FEV1 [PC20] < 8 mg/ml of histamine) and a positive skin-prick test for typical allergens (pollen, animals, dust mites) (23). The study was approved by the local ethics committee. Participants gave their written informed consent. The presence of one of the following criteria led to exclusion from the study: (1) history of any other chronic disease than asthma, (2) any regular medication (except inhaled medication for asthma), (3) positive smoking history, or (4) signs of infection. According to these criteria, 26 patients and 26 age- and sex-matched control subjects without a history of wheezing or allergies (and a total serum IgE concentration of < 100 kU/L) were included. Table 1 shows the characteristics of the control and asthma group. Blood was collected between 8 A.M. and 12 P.M., and plasma and serum prepared as described (22).
Body Plethysmography and Histamine Challenge
Pulmonary function was assessed using a body plethysmograph (Jaeger, Viasys, Hoechberg, Germany). Airway hyperresponsiveness was measured with increasing doses of histamine inhaled using a Pari-Boy (Pari-Werke, Starnberg, Germany). Each dose was followed by body plethysmography. Inhalations were discontinued when there was a fall in FEV1 of 20% or more from the postsaline value. The results were expressed as the PC20, and this value was obtained from the log concentration versus the percentage of fall in FEV1 curve by linear interpolation of the last 2 points (24).
Blood Parameters and Cell Culture
Differential blood cell counts, IgE, serotonin (5-Hydroxytriptamine [5-HT]), BDNF, and transforming growth factor 1 (TGF-1) were measured as described (22). Platelet BDNF content was calculated by subtracting plasma BDNF from serum BDNF, and dividing the result by the platelet count as described (22). Monocyte-enriched (39.4 ± 12.6% CD14+ cells) human peripheral blood mononuclear cells were used for cell culture experiments (Optiprep; Greiner Bio-One, Frickenhausen, Germany). The 2 x 106 cells/ml were cultured in RPMI 1640 with 10% fetal calf serum, 100 U/ml of penicillin, and 100 e/ml of streptomycin for 24 hours and stimulated with tumor necrosis factor (TNF-; Strathmann-Biotec, Hamburg, Germany), prednisolone acetate (Jenapharm, Jena, Germany) and/or fluticasone propionate (GlaxoSmithKline, Brentford, UK). Because fluticasone was dissolved in alcohol, resulting in 0.01% alcohol in culture, 0.01% alcohol was added to the medium control. Nonviable cells were detected using 7-aminoactinomycin-D staining (Beckmann-Coulter, Fullerton, CA) (25). BDNF concentrations were corrected for the percentage of nonviable cells to exclude artifacts caused by corticosteroid-induced apoptosis.
Statistical Analysis
Data were analyzed using SPSS (SPSS Inc., Chicago, IL). Correlations were calculated using Spearman's correlation coefficient. For the comparison of cohorts, the nonparametric Mann-Whitney U test was used. Means in cell culture experiments were compared using analysis of variance (ANOVA with SPSS); p values less than 0.05 were regarded as statistically significant.
RESULTS
BDNF Concentrations in Serum, Platelets, and Plasma
In the control group, levels of BDNF in serum, platelets, and plasma (mean values: 20.8 ± 9.2 ng/ml serum, 81.8 ± 32.2 pg/106 platelets, 135.6 ± 132.4 pg/ml plasma) were in keeping with previously reported data in healthy adults (22). Significantly elevated BDNF concentrations in serum, platelets, and plasma were found in patients with allergic asthma (mean values: 30.2 ± 12.2 ng/ml serum, 129.2 ± 49.3 pg/106 platelets, 415.4 ± 409.9 pg/ml plasma; Figure 1). Differences in platelet BDNF levels were more significant than differences in serum BDNF levels (Figure 1B). Because platelet BDNF levels in women were shown to change during the menstrual cycle (22), all female participants were asked about the number of days since the first day of their last menstruation. There was no significant difference between female patients and female control subjects regarding the timing of the menstrual cycle (Table 1).
Platelet Counts and Platelet Markers
There were no differences in platelet counts between control subjects and patients (mean values: control subjects, 249.2 ± 36.9 millions/ml blood; patients, 238.3 ± 48.3 millions/ml blood; Figure 2A). To further elucidate the underlying mechanisms, we investigated serum concentrations of platelet markers (26). There were no differences between control subjects and patients regarding serum TGF-1 (mean values: controls, 30.8 ± 17.7 ng/ml serum; patients, 32.1 ± 9.9 ng/ml serum) or serotonin (5-HT) concentrations (mean values: control subjects, 171.3 ± 81.4 ng/ml serum; patients 177.9 ± 77.1 ng/ml serum; Figure 2B and 2C). The correlation of serum BDNF with serum TGF-1 in control subjects (r = 0.70, p < 0.01) was not found in patients with asthma (r = 0.12, p = 0.56).
Peripheral BDNF Concentrations and Lung Function
Baseline lung function parameters (n = 26 patients) are shown in Table 2. BDNF concentrations in serum, platelets, and plasma did not correlate with static volumes, such as total lung capacity or the residual volume, in the total cohort or in the subgroups (data not shown). There was no correlation between the corrected PEF (% predicted) or the corrected FEV1 (% predicted) and BDNF plasma levels in patients with asthma in the total cohort or in the subgroups (data not shown). In the subgroup of patients who had not been previously exposed to inhaled corticosteroids (steroid-naive), there was a negative correlation between platelet BDNF concentrations and FEV1 (% predicted; r = eC0.59, p < 0.05) and PEF (% predicted; r = eC0.51, p < 0.05; Figure 3A). In contrast, there were no significant correlations between platelet BDNF and FEV1 (% predicted; r = eC0.45, p = 0.19) or PEF (% predicted; r = 0.2, p = 0.58) in the subgroup that had already been treated with inhaled corticosteroids (Figure 3A). Concentrations of BDNF in platelets, but not in plasma, correlated with airway hyperresponsiveness to histamine (as measured by PC20) in steroid-naive patients (r = eC0.57, p < 0.05). Again, a significant correlation was absent in the subgroup using inhaled corticosteroids (r = eC0.07, p = 0.86; Figure 3B).
Effect of Fluticasone on BDNF Secretion by Mononuclear Cells
Cultured peripheral blood mononuclear cells (PBMCs), which constitutively secrete BDNF, did not differ between control subjects and patients regarding BDNF secretion, either unstimulated or after stimulation with TNF- (data not shown). The absence of a correlation between platelet BDNF levels and lung function in corticosteroid-treated patients prompted us to investigate the effect of an inhaled corticosteroid (fluticasone) on BDNF secretion by PBMCs. For comparison, parallel incubations with prednisolone were performed. For these experiments, monocyte-enriched PBMCs were isolated from 14 healthy volunteers. Suppression of basal BDNF secretion occurred only in the presence of higher concentrations of prednisolone (10eC5 M). In addition, prednisolone did not significantly reduce enhanced BDNF secretion after TNF- stimulation (Figure 4, prednisolone). In contrast, fluticasone effectively suppressed BDNF secretion of unstimulated and stimulated PBMCs, even at markedly lower concentrations than prednisolone (10eC7 and 10eC8 M; Figure 4, fluticasone).
DISCUSSION
There is substantial evidence from animal models suggesting that neurotrophins, such as BDNF, are involved in the pathogenesis of airway hyperresponsiveness and airflow limitation in individuals with asthma (8, 20). In human asthma, elevated concentrations of BDNF have been reported in bronchoalveolar lavage fluid after allergen challenge (17). This study is the first to demonstrate a relationship between BDNF stored in platelets and the lung function of patients with allergic asthma. We found an association between elevated concentrations of BDNF in platelets and parameters of airway obstruction (PEF and FEV1) and hyperresponsiveness (PC20) in steroid-naive patients with asthma. The absence of this association in patients using inhaled corticosteroids (ICSs) and the fluticasone-induced suppression of BDNF secretion reveal a new aspect of ICS action in allergic asthma.
In an animal model, the specific role of BDNF in the pathogenesis of allergic airway dysfunction has recently been characterized (20). The inhibition of endogenous BDNF reduced enhanced airway tone and neuronal hyperreactivity in allergen-challenged animals, whereas administration of recombinant BDNF was sufficient to induce these functional changes in healthy animals (20). Furthermore, BDNF did not affect or induce airway inflammation itself. Thus, BDNF may represent a mediator of persistent airway dysfunction in allergic asthma (19). However, there are several limitations to corroborate these animal model findings in human asthma, especially the relation between enhanced BDNF levels and altered airway function. Segmental allergen challenge in a single segment of the human lung represents an artificial model of allergic airway inflammation (17). Therefore, parameters obtained from bronchoalveolar lavage in a single challenged segment are not necessarily related to pulmonary function of the whole lung, as measured by body plethysmography. In addition, it is not reasonable to investigate bronchoalveolar lavage of unchallenged lung segments, because BDNF concentrations are below or near the detection limit in these lavage fluids (17). Finally, the cellular sources of BDNF in the lungs from patients with asthma and the kinetics of its production and secretion following allergen challenge are incompletely understood.
In contrast, it has been well established that substantial amounts of BDNF are stored and transported in human platelets (21). Platelet BDNF is neither produced by platelets nor by its precursors. On the contrary, BDNF is actively acquired by platelets from external sources and released by agonist stimulation. Therefore, platelets appear to be a unique BDNF transportation system in the human body (21). These findings are in line with the postulate that platelets represent a good estimate of the average secretion of BDNF in organs of the human body (27). The adult lung is an important source of BDNF (16, 28). In addition, allergic airway inflammation was shown to increase local BDNF production (17, 18). Therefore, enhanced platelet BDNF concentrations could reflect an enhanced uptake of BDNF from the inflamed lung. Our observation that enhanced platelet BDNF concentrations in patients with asthma are associated with clinical parameters of allergic airway dysfunction is therefore compatible with the idea that platelet BDNF may be an indirect marker of BDNF upregulation and its consequences in the lung.
In serum of healthy adults, there is a strong correlation of BDNF with the platelet -granule marker TGF-1 but not with the platelet dense-core granule marker 5-HT, suggesting a colocalization of BDNF and TGF-1 in platelet -granules (22). Although we found a similar correlation between BDNF and TGF-1 in the control group of our study, this correlation was absent in patients with allergic asthma. Levels of TGF-1 in serum of patients were comparable to the control group, whereas an elevation of serum BDNF levels, which varied from individual to individual, was observed in patients. Thus, the absence of a correlation between serum BDNF and TGF-1 might reflect the individual increase of BDNF concentrations in platelets.
ICSs have a beneficial and protective effect on airway hyperresponsiveness and obstruction in allergic asthma; however, the precise mechanisms are poorly understood (29). Airway hyperresponsiveness can improve within weeks after initiation of ICS treatment, even with low doses of ICS (30, 31). ICSs can reduce airway hyperreactivity in response to histamine within 3 days (32), whereas several weeks of treatment are needed to improve methacholine responsiveness (33). These differences suggest different pathways of airway hyperresponsiveness. Hyperreactivity to histamine is in part mediated by the vagal nerve (9, 34), whereas hyperreactivity to methacholine (or acetylcholine) predominantly reflects altered smooth muscle function (35). The hypothesis that BDNF might be a specific mediator of the neuronal pathway (20) is supported by the finding that the reduction of serum BDNF levels after ICS therapy is not correlated with changes of acetylcholine responsiveness of the airways (36).
The absence of a correlation between platelet BDNF levels and hyperresponsiveness to histamine in steroid-treated patients prompted us to investigate the effect of ICSs on BDNF secretion in cultures of human mononuclear cells. Monocyte-enriched mononuclear cell preparations were chosen for two reasons: (1) monocytes have been shown to be a major source of BDNF among human peripheral blood mononuclear cells (37); and (2) coculture of several types of mononuclear cells allows physiologic celleCcell interactions in vitro, which might be closer to an in vivo situation than highly purified single-cell preparations. We found a suppression of BDNF secretion by mononuclear cells following fluticasone stimulation, even at very low concentrations. Given the animal model finding showing adverse effects of BDNF on airway caliber and reactivity (20), the observed suppression of BDNF by fluticasone is compatible with the idea that a reduction of BDNF secretion might be one part of the beneficial effects of ICSs in asthma. The issue, however, whether ICSs act via a local or systemic (38) suppression of BDNF production remains open.
In conclusion, we report elevated concentrations of BDNF in platelets and plasma of patients with allergic asthma. In steroid-naive patients, we show a relationship between elevated concentrations of BDNF in platelets and clinical parameters of airway dysfunction. Finally, we provide evidence that ICSs can effectively suppress BDNF production. The significance of these findings in the context of the complex mechanisms by which corticosteroids affect airway obstruction and hyperresponsiveness remains to be studied.
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Department of Neurology, Charitee, Berlin, Germany
ABSTRACT
Brain-derived neurotrophic factor (BDNF), a key mediator of neuronal plasticity, contributes to airway obstruction and hyperresponsiveness in a model of allergic asthma. BDNF is stored in human platelets and circulates in human plasma, but the significance of BDNF in this compartment is poorly understood. We investigated the relationship between platelet and plasma BDNF levels and pulmonary function in a cohort of 26 adult patients with recently diagnosed allergic asthma. BDNF levels in serum, platelets, and plasma were significantly increased in participants with asthma, as compared with 26 age- and sex-matched control subjects. In steroid-naive patients, but not in patients using inhaled corticosteroids, enhanced platelet BDNF levels correlated with parameters of airway obstruction and airway hyperresponsiveness to histamine. Experiments with activated peripheral blood mononuclear cells revealed that corticosteroids such as fluticasone effectively suppress BDNF secretion. In conclusion, we demonstrate that enhanced platelet BDNF is associated with airflow limitation and airway hyperresponsiveness in asthma. In addition, we provide evidence that corticosteroids suppress BDNF production by activated immune cells.
Key Words: airway hyperresponsiveness corticosteroids neurotrophins peak expiratory flow
Allergic bronchial asthma is associated with a characteristic type of airway inflammation, airway hyperresponsiveness to nonspecific stimuli, and a variable degree of airflow limitation (1). Although the immunologic network in asthma has been increasingly well defined over the last decades, the links between airway inflammation and changes in pulmonary function remain incompletely understood (2). Several animal studies have shown a dissociation between airway inflammation and airway hyperresponsiveness in models of allergic asthma (3, 4). In human asthma, new therapeutic strategies, such as antieCinterleukin-5 treatment, were highly effective in reducing eosinophilic inflammation but remained ineffective regarding clinical parameters such as airway hyperresponsiveness or the late-phase airway obstruction (5, 6).
These observations have resulted in a new view on the mechanisms of asthma: although allergic inflammation most likely represents the initial trigger of asthma, postinflammatory changes of resident cells are believed to determine persistent airway dysfunction (4). In this context, the role of vagal cholinergic and sensory nerves, which regulate airway tone and reactivity, has gained renewed interest (7eC10). Allergic inflammation induces profound changes in neuronal networks of the lung (11), including sensory hyperreactivity (12), enhanced signal transmission in local parasympathetic ganglia (13), and an exaggerated central reflex activity in the brainstem (14). A key mediator of neuronal plasticity in the adult, brain-derived neurotrophic factor (BDNF) (15, 16), has been found to be increased in bronchoalveolar lavage fluid of patients with allergic asthma (17). In an animal model, BDNF production was shown to be upregulated in cells infiltrating asthmatic airways, including macrophages and T cells (18). Notably, the highest BDNF levels in bronchoalveolar lavage fluid were found during regression of the inflammatory response, suggesting a postinflammatory role of BDNF (19). The inhibition of BDNF with specific anti-BDNF antibodies prevented changes in lung function occurring in response to allergen challenge, including airflow limitation, sensory hyperreactivity, and airway hyperresponsiveness to nonspecific stimuli (20).
The transportation of BDNF in platelets represents a unique feature of human BDNF physiology. Platelets acquire and store substantial amounts of BDNF, which results in high BDNF levels in serum. In contrast, plasma levels represent free circulating BDNF (21). Normal values for BDNF in human platelets and plasma have recently been established, including the influence of age, weight, sex, and the menstrual cycle on these BDNF concentrations (22). However, circulating and stored BDNF levels have not been systematically investigated in patients with allergic asthma. In addition, the relationship between these BDNF levels and pulmonary function remains unclear. This prospective study investigates this relationship in a clinical setting.
METHODS
Study Design
Patients with allergic asthma (9.0 ± 8.0 months since diagnosis) were recruited, and their diagnosis was established using the following criteria: recurrent attacks of wheezing, improvement of pulmonary function following inhalation of a 2-agonist, airway hyperresponsiveness (provocative dose of histamine causing a 20% fall in FEV1 [PC20] < 8 mg/ml of histamine) and a positive skin-prick test for typical allergens (pollen, animals, dust mites) (23). The study was approved by the local ethics committee. Participants gave their written informed consent. The presence of one of the following criteria led to exclusion from the study: (1) history of any other chronic disease than asthma, (2) any regular medication (except inhaled medication for asthma), (3) positive smoking history, or (4) signs of infection. According to these criteria, 26 patients and 26 age- and sex-matched control subjects without a history of wheezing or allergies (and a total serum IgE concentration of < 100 kU/L) were included. Table 1 shows the characteristics of the control and asthma group. Blood was collected between 8 A.M. and 12 P.M., and plasma and serum prepared as described (22).
Body Plethysmography and Histamine Challenge
Pulmonary function was assessed using a body plethysmograph (Jaeger, Viasys, Hoechberg, Germany). Airway hyperresponsiveness was measured with increasing doses of histamine inhaled using a Pari-Boy (Pari-Werke, Starnberg, Germany). Each dose was followed by body plethysmography. Inhalations were discontinued when there was a fall in FEV1 of 20% or more from the postsaline value. The results were expressed as the PC20, and this value was obtained from the log concentration versus the percentage of fall in FEV1 curve by linear interpolation of the last 2 points (24).
Blood Parameters and Cell Culture
Differential blood cell counts, IgE, serotonin (5-Hydroxytriptamine [5-HT]), BDNF, and transforming growth factor 1 (TGF-1) were measured as described (22). Platelet BDNF content was calculated by subtracting plasma BDNF from serum BDNF, and dividing the result by the platelet count as described (22). Monocyte-enriched (39.4 ± 12.6% CD14+ cells) human peripheral blood mononuclear cells were used for cell culture experiments (Optiprep; Greiner Bio-One, Frickenhausen, Germany). The 2 x 106 cells/ml were cultured in RPMI 1640 with 10% fetal calf serum, 100 U/ml of penicillin, and 100 e/ml of streptomycin for 24 hours and stimulated with tumor necrosis factor (TNF-; Strathmann-Biotec, Hamburg, Germany), prednisolone acetate (Jenapharm, Jena, Germany) and/or fluticasone propionate (GlaxoSmithKline, Brentford, UK). Because fluticasone was dissolved in alcohol, resulting in 0.01% alcohol in culture, 0.01% alcohol was added to the medium control. Nonviable cells were detected using 7-aminoactinomycin-D staining (Beckmann-Coulter, Fullerton, CA) (25). BDNF concentrations were corrected for the percentage of nonviable cells to exclude artifacts caused by corticosteroid-induced apoptosis.
Statistical Analysis
Data were analyzed using SPSS (SPSS Inc., Chicago, IL). Correlations were calculated using Spearman's correlation coefficient. For the comparison of cohorts, the nonparametric Mann-Whitney U test was used. Means in cell culture experiments were compared using analysis of variance (ANOVA with SPSS); p values less than 0.05 were regarded as statistically significant.
RESULTS
BDNF Concentrations in Serum, Platelets, and Plasma
In the control group, levels of BDNF in serum, platelets, and plasma (mean values: 20.8 ± 9.2 ng/ml serum, 81.8 ± 32.2 pg/106 platelets, 135.6 ± 132.4 pg/ml plasma) were in keeping with previously reported data in healthy adults (22). Significantly elevated BDNF concentrations in serum, platelets, and plasma were found in patients with allergic asthma (mean values: 30.2 ± 12.2 ng/ml serum, 129.2 ± 49.3 pg/106 platelets, 415.4 ± 409.9 pg/ml plasma; Figure 1). Differences in platelet BDNF levels were more significant than differences in serum BDNF levels (Figure 1B). Because platelet BDNF levels in women were shown to change during the menstrual cycle (22), all female participants were asked about the number of days since the first day of their last menstruation. There was no significant difference between female patients and female control subjects regarding the timing of the menstrual cycle (Table 1).
Platelet Counts and Platelet Markers
There were no differences in platelet counts between control subjects and patients (mean values: control subjects, 249.2 ± 36.9 millions/ml blood; patients, 238.3 ± 48.3 millions/ml blood; Figure 2A). To further elucidate the underlying mechanisms, we investigated serum concentrations of platelet markers (26). There were no differences between control subjects and patients regarding serum TGF-1 (mean values: controls, 30.8 ± 17.7 ng/ml serum; patients, 32.1 ± 9.9 ng/ml serum) or serotonin (5-HT) concentrations (mean values: control subjects, 171.3 ± 81.4 ng/ml serum; patients 177.9 ± 77.1 ng/ml serum; Figure 2B and 2C). The correlation of serum BDNF with serum TGF-1 in control subjects (r = 0.70, p < 0.01) was not found in patients with asthma (r = 0.12, p = 0.56).
Peripheral BDNF Concentrations and Lung Function
Baseline lung function parameters (n = 26 patients) are shown in Table 2. BDNF concentrations in serum, platelets, and plasma did not correlate with static volumes, such as total lung capacity or the residual volume, in the total cohort or in the subgroups (data not shown). There was no correlation between the corrected PEF (% predicted) or the corrected FEV1 (% predicted) and BDNF plasma levels in patients with asthma in the total cohort or in the subgroups (data not shown). In the subgroup of patients who had not been previously exposed to inhaled corticosteroids (steroid-naive), there was a negative correlation between platelet BDNF concentrations and FEV1 (% predicted; r = eC0.59, p < 0.05) and PEF (% predicted; r = eC0.51, p < 0.05; Figure 3A). In contrast, there were no significant correlations between platelet BDNF and FEV1 (% predicted; r = eC0.45, p = 0.19) or PEF (% predicted; r = 0.2, p = 0.58) in the subgroup that had already been treated with inhaled corticosteroids (Figure 3A). Concentrations of BDNF in platelets, but not in plasma, correlated with airway hyperresponsiveness to histamine (as measured by PC20) in steroid-naive patients (r = eC0.57, p < 0.05). Again, a significant correlation was absent in the subgroup using inhaled corticosteroids (r = eC0.07, p = 0.86; Figure 3B).
Effect of Fluticasone on BDNF Secretion by Mononuclear Cells
Cultured peripheral blood mononuclear cells (PBMCs), which constitutively secrete BDNF, did not differ between control subjects and patients regarding BDNF secretion, either unstimulated or after stimulation with TNF- (data not shown). The absence of a correlation between platelet BDNF levels and lung function in corticosteroid-treated patients prompted us to investigate the effect of an inhaled corticosteroid (fluticasone) on BDNF secretion by PBMCs. For comparison, parallel incubations with prednisolone were performed. For these experiments, monocyte-enriched PBMCs were isolated from 14 healthy volunteers. Suppression of basal BDNF secretion occurred only in the presence of higher concentrations of prednisolone (10eC5 M). In addition, prednisolone did not significantly reduce enhanced BDNF secretion after TNF- stimulation (Figure 4, prednisolone). In contrast, fluticasone effectively suppressed BDNF secretion of unstimulated and stimulated PBMCs, even at markedly lower concentrations than prednisolone (10eC7 and 10eC8 M; Figure 4, fluticasone).
DISCUSSION
There is substantial evidence from animal models suggesting that neurotrophins, such as BDNF, are involved in the pathogenesis of airway hyperresponsiveness and airflow limitation in individuals with asthma (8, 20). In human asthma, elevated concentrations of BDNF have been reported in bronchoalveolar lavage fluid after allergen challenge (17). This study is the first to demonstrate a relationship between BDNF stored in platelets and the lung function of patients with allergic asthma. We found an association between elevated concentrations of BDNF in platelets and parameters of airway obstruction (PEF and FEV1) and hyperresponsiveness (PC20) in steroid-naive patients with asthma. The absence of this association in patients using inhaled corticosteroids (ICSs) and the fluticasone-induced suppression of BDNF secretion reveal a new aspect of ICS action in allergic asthma.
In an animal model, the specific role of BDNF in the pathogenesis of allergic airway dysfunction has recently been characterized (20). The inhibition of endogenous BDNF reduced enhanced airway tone and neuronal hyperreactivity in allergen-challenged animals, whereas administration of recombinant BDNF was sufficient to induce these functional changes in healthy animals (20). Furthermore, BDNF did not affect or induce airway inflammation itself. Thus, BDNF may represent a mediator of persistent airway dysfunction in allergic asthma (19). However, there are several limitations to corroborate these animal model findings in human asthma, especially the relation between enhanced BDNF levels and altered airway function. Segmental allergen challenge in a single segment of the human lung represents an artificial model of allergic airway inflammation (17). Therefore, parameters obtained from bronchoalveolar lavage in a single challenged segment are not necessarily related to pulmonary function of the whole lung, as measured by body plethysmography. In addition, it is not reasonable to investigate bronchoalveolar lavage of unchallenged lung segments, because BDNF concentrations are below or near the detection limit in these lavage fluids (17). Finally, the cellular sources of BDNF in the lungs from patients with asthma and the kinetics of its production and secretion following allergen challenge are incompletely understood.
In contrast, it has been well established that substantial amounts of BDNF are stored and transported in human platelets (21). Platelet BDNF is neither produced by platelets nor by its precursors. On the contrary, BDNF is actively acquired by platelets from external sources and released by agonist stimulation. Therefore, platelets appear to be a unique BDNF transportation system in the human body (21). These findings are in line with the postulate that platelets represent a good estimate of the average secretion of BDNF in organs of the human body (27). The adult lung is an important source of BDNF (16, 28). In addition, allergic airway inflammation was shown to increase local BDNF production (17, 18). Therefore, enhanced platelet BDNF concentrations could reflect an enhanced uptake of BDNF from the inflamed lung. Our observation that enhanced platelet BDNF concentrations in patients with asthma are associated with clinical parameters of allergic airway dysfunction is therefore compatible with the idea that platelet BDNF may be an indirect marker of BDNF upregulation and its consequences in the lung.
In serum of healthy adults, there is a strong correlation of BDNF with the platelet -granule marker TGF-1 but not with the platelet dense-core granule marker 5-HT, suggesting a colocalization of BDNF and TGF-1 in platelet -granules (22). Although we found a similar correlation between BDNF and TGF-1 in the control group of our study, this correlation was absent in patients with allergic asthma. Levels of TGF-1 in serum of patients were comparable to the control group, whereas an elevation of serum BDNF levels, which varied from individual to individual, was observed in patients. Thus, the absence of a correlation between serum BDNF and TGF-1 might reflect the individual increase of BDNF concentrations in platelets.
ICSs have a beneficial and protective effect on airway hyperresponsiveness and obstruction in allergic asthma; however, the precise mechanisms are poorly understood (29). Airway hyperresponsiveness can improve within weeks after initiation of ICS treatment, even with low doses of ICS (30, 31). ICSs can reduce airway hyperreactivity in response to histamine within 3 days (32), whereas several weeks of treatment are needed to improve methacholine responsiveness (33). These differences suggest different pathways of airway hyperresponsiveness. Hyperreactivity to histamine is in part mediated by the vagal nerve (9, 34), whereas hyperreactivity to methacholine (or acetylcholine) predominantly reflects altered smooth muscle function (35). The hypothesis that BDNF might be a specific mediator of the neuronal pathway (20) is supported by the finding that the reduction of serum BDNF levels after ICS therapy is not correlated with changes of acetylcholine responsiveness of the airways (36).
The absence of a correlation between platelet BDNF levels and hyperresponsiveness to histamine in steroid-treated patients prompted us to investigate the effect of ICSs on BDNF secretion in cultures of human mononuclear cells. Monocyte-enriched mononuclear cell preparations were chosen for two reasons: (1) monocytes have been shown to be a major source of BDNF among human peripheral blood mononuclear cells (37); and (2) coculture of several types of mononuclear cells allows physiologic celleCcell interactions in vitro, which might be closer to an in vivo situation than highly purified single-cell preparations. We found a suppression of BDNF secretion by mononuclear cells following fluticasone stimulation, even at very low concentrations. Given the animal model finding showing adverse effects of BDNF on airway caliber and reactivity (20), the observed suppression of BDNF by fluticasone is compatible with the idea that a reduction of BDNF secretion might be one part of the beneficial effects of ICSs in asthma. The issue, however, whether ICSs act via a local or systemic (38) suppression of BDNF production remains open.
In conclusion, we report elevated concentrations of BDNF in platelets and plasma of patients with allergic asthma. In steroid-naive patients, we show a relationship between elevated concentrations of BDNF in platelets and clinical parameters of airway dysfunction. Finally, we provide evidence that ICSs can effectively suppress BDNF production. The significance of these findings in the context of the complex mechanisms by which corticosteroids affect airway obstruction and hyperresponsiveness remains to be studied.
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