Original Article

Brain-derived Neurotrophic Factor in Platelets and

Airflow Limitation in Asthma
Marek Lommatzsch, Katharina Schloetcke, Jens Klotz, Katharina Schuhbaeck, Doerte
Zingler, Christiana Zingler, Olaf Schulte-Herbrüggen, Hartmut Gill, Peter Schuff-
Werner and Johann Christian Virchow

Department of Pneumology and Institute of Clinical Chemistry and Pathobiochemistry, University of Rostock,
Rostock; Department of Neurology, Charité, Berlin, Germany

Correspondence and requests for reprints should be addressed to Marek Lommatzsch, M.D., Abteilung für
Pneumologie, Klinik und Poliklinik für Innere Medizin, Universität Rostock, Ernst-Heydemann-Str. 6, Rostock
18057, Germany. E-mail: marek.lommatzsch@med.uni-rostock.de

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ABSTRACT
METHODS
RESULTS
DISCUSSION
REFERENCES

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 anti–interleukin-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 (7–10). 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.

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ABSTRACT
METHODS
RESULTS
DISCUSSION
REFERENCES

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 ß2-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).

View this table: TABLE 1. Characteristics of patients and control subjects

[in this window]
[in a new window]

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 ß1 (TGF-ß1) 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 µg/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.

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ABSTRACT
METHODS
RESULTS
DISCUSSION
REFERENCES

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).

Figure 1. BDNF levels in serum, platelets, and plasma.

Twenty-six patients with recently diagnosed allergic
asthma (gray bars) and 26 age- and sex-matched
healthy control subjects without a history of wheezing
or allergies (open bars) were examined. Shown are
BDNF levels in (A) serum, (B) platelets, and (C)
plasma of each group. The median (line within the
View larger version (20K): box), interquartile range (edges of the box), and the
[in this window] range of all values that are not outliers (vertical lines)
[in a new window] are shown. Outliers (all cases more distant than 1.5
interquartile ranges from the upper or lower quartile)
were omitted (there was one serum BDNF and one
plasma BDNF outlier in the asthma group, and two
plasma BDNF outliers in the control group). Asterisks
 mark significant differences between the groups.

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-ß1 (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-ß1 in control subjects (r = 0.70, p < 0.01)
was not found in patients with asthma (r = 0.12, p = 0.56).

Figure 2. Platelet counts and platelet markers. Twenty-

six patients with recently diagnosed allergic asthma
(gray bars) and 26 age- and sex-matched healthy
control subjects without a history of wheezing or
allergies (open bars) were examined. Shown are (A)
total platelet counts, (B) serum concentrations of TGF-
ß1, and (C) serotonin (5-HT) of each group. The
View larger version (22K): median (line within the box), interquartile range (edges
[in this window] of the box), and the range of all values that are not
[in a new window] outliers (vertical lines) are shown. Outliers (all cases
more distant than 1.5 interquartile ranges from the
upper or lower quartile) were omitted (there were two
5-HT outliers in the asthma group). There were no
significant differences between both groups regarding
all three parameters, as demonstrated by p values >
0.05.

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 = –0.59, p < 0.05) and PEF (%
predicted; r = –0.51, p < 0.05; Figure 3A). In contrast, there were no significant correlations
between platelet BDNF and FEV1 (% predicted; r = –0.45, 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 = –0.57,
p < 0.05). Again, a significant correlation was absent in the subgroup using inhaled
corticosteroids (r = –0.07, p = 0.86; Figure 3B).
 View this table: TABLE 2. Pulmonary function in patients with asthma
[in this window]
[in a new window]

Figure 3. Platelet BDNF and lung function. Shown are

the correlations between BDNF concentrations in
platelets (calculated as pg BDNF/106 platelets) and the
(A) PEF (% predicted) or the (B) provocative
concentration of histamine causing a 20% decrease in
FEV1 (PC20). Filled circles represent steroid-naive
View larger version (14K): patients, open circles represent patients using inhaled
[in this window] steroids. Correlations were calculated with SPSS using
[in a new window] Spearman's correlation coefficient (r); significant
correlations are marked with asterisks (*p < 0.05). The
line represents the regression line for the cohort of
steroid-naive participants with asthma.

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 (10–5 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 (10–7 and 10–8 M; Figure 4, fluticasone).

Figure 4. Effect of corticosteroids on BDNF secretion

by immune cells. Monocyte-enriched PBMCs were
isolated from 14 healthy volunteers, and 2 x 106
cells/ml cultured for 24 hours (see METHODS).
Unstimulated cells served as a medium control (M).
View larger version (27K): Cells were incubated with prednisolone acetate (P; 10–
5
[in this window] , 10 , and 10–9 M) or fluticasone propionate (F; 10–7,
–7

[in a new window] 10–8, and 10–9 M) in the presence (dark gray) or
absence (light gray) of 50 ng/ml of TNF- (T). Shown
 are incubations with 10–5 and 10–7 M of prednisolone,
and 10–7 and 10–8 M of fluticasone. BDNF
concentrations in supernatants were corrected for the
percentage of nonviable cells, and are expressed as
percentages of the medium control. Asterisks mark
significant differences between the groups (*p < 0.05).

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ABSTRACT
METHODS
RESULTS
DISCUSSION
REFERENCES

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-ß1 but not with the platelet dense-core granule marker 5-HT, suggesting a
colocalization of BDNF and TGF-ß1 in platelet -granules (22). Although we found a similar
correlation between BDNF and TGF-ß1 in the control group of our study, this correlation was
absent in patients with allergic asthma. Levels of TGF-ß1 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-ß1 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 cell–cell 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.

FOOTNOTES

Supported by the Deutsche Forschungsgemeinschaft (Grant No. VI 193-3-1).

Conflict of Interest Statement: M.L. does not have a financial relationship with a commercial
entity that has an interest in the subject of this manuscript; K.S. does not have a financial
relationship with a commercial entity that has an interest in the subject of this manuscript;
J.K. does not have a financial relationship with a commercial entity that has an interest in the
subject of this manuscript; K.S. does not have a financial relationship with a commercial
entity that has an interest in the subject of this manuscript; D.Z. does not have a financial
relationship with a commercial entity that has an interest in the subject of this manuscript;
C.Z. does not have a financial relationship with a commercial entity that has an interest in the
subject of this manuscript; O.S.-H. does not have a financial relationship with a commercial
entity that has an interest in the subject of this manuscript; H.G. does not have a financial
relationship with a commercial entity that has an interest in the subject of this manuscript;
P.S.-W. does not have a financial relationship with a commercial entity that has an interest in
the subject of this manuscript; J.C.V. has served as a lecturer and as a member of an advisory
board for GlaxoSmithKline (GSK) and has received research funding from GSK in 2004
($20,000).

Received in original form June 15, 2004; accepted in final form October 24, 2004

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