A�

rchivum Immunologiae et Therapiae Experimentalis, 1999, 4�

7,� 119–126P�

L ISSN 0004-069X

Hydrogen Peroxide in Expired Air Condensate CorrelatesPositively with Early Steps of Peripheral NeutrophilActivation in Asthmatic PatientsA

�. Antczak et al.: Neutrophil Activation in Asthmatics

ADAM AN�

TCZAK, DARIUSZ NOWAK, PIOTR BIALASIEWICZ and MAREK KASIELSKI

Department of Pneumonology and Allergology, University Medical School, Kopcinskiego 22, 90-153 Łódz, Poland

A�

bstract. We have found an increased H2O�

2 level in expired air of asthmatic patients. Neutrophils from thesesubjects generated higher amounts of superoxide radicals after challenge with phorbol esters than those fromhealthy subjects which may result from an increased activity of NADPH-oxidase. The enhanced C

�a2

+ mobilisation

in neutrophils from asthmatics could be responsible for increased production and subsequent elevated H2O

�2

c� oncentration in expired breath condensate. In this study we wished to determine whether neutr� ophils of asthmaticp atients have enhanced [Ca2+]

�i response after N-formyl-methionyl-leucyl-phenylalanine – fMLP challenge as

c� ompared with cells from healthy donors, and if so, does it correlate with H2O�

2 levels in expired air. We examined21 patients, 10 healthy individuals as a control group (mean age 34.3 ± 5.5, 6 males and 4 females) and 11a� sthmatic subjects (mean age 38.2 ± 7.2, 7 males and 4 females). The rise of [Ca2+]

�i as an early event of

neutrophil activation, was measured spectrofluorimetically with Fura-2-AM. The mean H2O�

2 level, measuredspectrofluorimetrically in the expired breath of asthmatics, was 20-fold higher than that in� healthy control (0.18± 0.20 vs. 0.01 ± 0.04 µM, p<0.05). [Ca2+]

�i increase after challenge by fMLP (∆[Ca2+]

�i)

� was much higher in

a� sthmatics than in control group (205.0 ±�

44 vs. 113.0 ±�

22 nM, p<0.05, respectively). A strong correlation waso� bserved between H2O

�2 and ∆[Ca2+]

�i and maximal velocity of increase in [Ca2

+]

�i� in asthmatics (r = 0.87, p<0.01

a� nd r = 0.64, p<0.05). We conclude that elevated H2 O

�2 level in the expired breath condensate of asthmatics can

b�e generated by activated neutrophils in the course of mucosal inflammation observed in bronchial asthma.

Key words: bronchial asthma; hydrogen peroxide in breath condensate; neutrophils; intracellular ca� lcium.

I�ntroduction

I�n several lung inflammatory diseases an elevated

hydrogen peroxide (H2� O

�2

� )� content in the expired breath

h�as been found, among which bronchial asthma seems

to be extensively studied3

�, 15, 28, 33, 41. H2O

�2 is one of the

most stable, toxic oxygen metabolites and relativelye� asy to detect. It is also volatile, and due to lack ofc� harge membrane permeable. It is cytotoxic3

�8, a high

c� oncentrations H2� O

�2

� causes cell necrosis and at lowl

�evels can induce apoptosis16. Catalase and glutathione

p eroxidase are basic enzymes regulating intracellularH2O

�2 level11, 35. Moreover, in asthmatic patients in-

c� reased level of H2O�

2 positively correlated with en-hanced level of lipid peroxidation products (thiobar-b

�ituric acid reactive species) in expired breath

c� ondensate which proves oxidant/antioxidant imbal-a� nce in the airways of these patients3

�. An intense air-

w� ay inflammation can be caused either by H2O�

2 aloneo� r newly generated hydroxyl radical (OHo� )

� 4�. An im-

p ortant feature of bronchial asthma is the influx of cir-c� ulating phagocytes including eosinophils, mast cells

a� nd neutrophils into the bronchial wall2�

6. When acti-v ated, they are capable of generation of reactive oxygenspecies, for example superoxide anion (O2

� •)� which is

then dismutated to hydrogen peroxide (H2O

�2)

� and can

p lay an important role in the development of pathophy-siological features of bronchial asthma, like enhanceda� rachidonic acid release, smooth muscle contraction,impaired β-adrenergic responsiveness and bronchialhyperresponsiveness1, 4, 22. It was proved that neutro-p hils from both adult and children asthmatic patientsc� an higher increase amounts of O2

� • and H2� O

�2

� after chal-lenge with phorbol esters and fMLP than cells onm! atched, healthy subjects2

�1, 23, 40. Moreover, the ability

o� f neutrophils isolated from asthmatics to producesuperoxide anion, correlated with the degree of airwayh

�yperresponsiveness to inhaled metacholine and his-

tamine19, 21, 25. A close relationship between the op-

sonized zymosan-induced chemiluminescence of neu-t

rophils and bronchial hyperreactivity to histamine in

a� sthmatic children can also be observed24. Furthermore,i

"n the same asthmatic children, the generation of O2

•

w� as significantly higher with than without asthma at-t

acks. This is consistent with the observation that

e� xacerbated asthma patients exhale more H2O�

2 than pa-t

ients with stable disease15, 28. Similarly, an enhanced

a� lveolar cell luminol-dependent chemiluminescence ina� sthmatic patients has been also found12.

An important source of H2� O

�2

� are phagocytes due toN

#ADPH oxidase system generating at least 3 oxygen

metabolites (O2•, H2O

�2 and OHo� )

� 6$

, 30. They are releasedi

"n extracellular fluid, and in the airways a part of H2O

�2,

w� hich has not been decomposed by antioxidante� nzymes, can be excreted with expired air. NADPHo� xidase system is activated by a variety of stimuli in-c� luding: 1,2-diacylglycerol, phosphatidic acid, inositolt

riphosphate and Ca2+ 3

�1, 39. Changes of intracellular

f%ree calcium concentration [Ca2

�+]

�i seem to be an im-

p ortant early step in signal transduction leading to ther� espiratory burst and degranulation. Increased [Ca2+]

�i

p romotes diacylglycerol-induced activation of proteinkinase C that is responsible for phosphorylation of cy-t

oplasmic subunits of NADPH oxidase. Neutrophils de-

p leted with Ca2�+ fail to produce superoxide in response

to stimulation unless they are supplied with extracellu-

l�ar Ca2+ 3

�2. Moreover, activation of the NADPH oxidase

o� ccurs when cytosolic Ca2�

+ rises18. What is very inter-e� sting, an increased release of calcium intracellularstores in neutrophils, obtained from asthmatic patientsa� fter stimulation with fMLP compared with healthysubjects was observed5

&. A hypothesis, therefore, can be

p ut forward that increased production of reactiveo� xygen species by neutrophils from asthmatic patients

m! ay be caused by enhanced Ca2�

+ mobilization. Thisw� ould lead to an incrased H2

� O�

2� generation which could

e� vaporate from the alveolar lining fluid and be detectedi

"n expired air. In this study we found a correlation be-t

ween [Ca2

�+]

�i

' rises, as an early step of neutrophil acti-v ation after fMLP challenge, and H2O

�2 in expired air

o� f asthmatic patients. This could reflect Ca2�

+ involve-ment in the generation of reactive oxygen species asinflammatory mediators in the course of bronchial as-t

hma.

Materials and Methods

Reagents. Peroxidase from horseradish type II(HRP, 200 U/mg solid), homovanillic acid (4-hydroxy-3-methoxy-phenylacetic acid), and N-formyl-methio-nyl-leucyl-phenylalanine (FMLP) were from SigmaC

�hemicals Co. (St Louis, MO, USA). Glycine, ethylene

d(iamine tetra-acetic acid (EDTA), phosphate buffered

saline (PBS, pH 7.4) and 30% H2� O

�2

� solution, glaciala� cetic acid, CaCl2, KCl, and NaCl were purchased fromP

)OCH (Gliwice, Poland). Fura-2-AM (acetoxymethyl

e� ster), ethylene glycol bis (2-aminoethyl-ether)-tetra-a� cetic acid (EGTA), Triton X-100, dimethyl sulphoxide(DMSO) were from Serva (Heidelberg, Germany).H2

� O�

2� solution (30%) was diluted 100-fold with PBS

a� nd stored at 4o� C�

in the dark. The actual H2O�

2 concen-t

ration was calculated from its absorbance at 230 nm

(E = 81 cm–1M–1)�. Aqueous solution of 1 U/ml HRP

w� ith addition of 400 µM homovanillic acid were pre-p ared freshly before the assay. FMLP, Fura-2-AM wered

(issolved in DMSO to final concentration 60 µM and

2* mM, respectively, and stored at –80o� C

� until assay. All

solutions were stored at 4o� C�

not longer than for 14 days.S

+tudy population. The study included 10 healthy

v olunteers as a control group (mean age 34.3 ± 5.5y, ear, 6 males and 4 females) and 11 asthmatic subjects(mean age 38.2 ± 7.4 year, 7 males and 4 females) who

Table 1. C-

haracteristic of study population

Healthys. ubjects

Asthmaticpatients

N�

umberA�

ge (year)S/

ex M: FF0

VC%F0

EV1%P�

EFR%F0

EV1 reversibility (%)A�

sthma duration (year)

103

14.3 ±

2 5.5

6:49

36.5 ±

2 1.2

101.5 ±2

4.7 101.3 ±

2 5.6

54.05 ±

2 3.3

–

1138.2 ±

2 7.4

75:4

92.5 ±2

6.8 67.1 ±

2 4.2*

68.1 ±2

13.1* 30.2 ±

2 48.8*

7.0 ±2

5.0

S/

ignificantly different from pulmonary function tests of healthysubjects: * p<0.001.

120 A. Antczak et al.: Neutrophil Activation in Asthmatics

h�ad not suffered from any infectious disease for the last

4 months (Table 1). They were recruited from Univer-sity Medical School out-patient clinic register. As-t

hmatic subjects were asked to stop any medication ex-

c� ept short-acting β-agonists (salbutamol or fenoterol)a� nd to report to the clinic after 4-week washout periodt

o perform lunb function tests. Only those patients who

w� ere able to refrain from antiinflammatory medication,indicated in this group, during washout were includedt

o the study. Bronchial asthma was diagnosed based on

history of wheezing dyspnea and previous documenta-t

ion of bronchodilator-induced bronchial reversibility

m! easured as more than 15% increase of FEV1 and thep resence of airway hyperreactivity after histamine chal-l

�enge test with PC20 of less than 8 mg/ml according tot

he method of COCKROFT13.

Bronchial reversibility at least 15% of the baselinea� nd the ability to stop other than β-agonist therapy wereb

�asic inclusion criteria. The duration of bronchial as-

thma was 1 to 18 years, mean 7 ± 5 years. Spirometryw� as performed with Flowscreen (Erich JaegerG

6mbH&Co., Germany) equipped with software com-

p atible to American Thoracic Society standards2�. This

study was approved by the local Ethics Committee andi

"nformed consent was obtained.

Collection of air condensate. The air condensatew� as collected in a tube installed in the polystyrenefoamed container filled with ice and salt as previouslyd

(escribed3

�3. Briefly, study subjects were asked to

b�reathe into the apparatus and then the condensate was

transferred to Eppendorf tubes. They were stored at

–80o� C�

for not longer than 7 days until H2� O

�2

� measure-m! ent. Our previous experiments have shown that sam-p les of expired breath condensate and 10–7 M H2

� O�

2� sol-

u7 tion, under these conditions, remain stable after 14d

(ays of storage. Similarly, 50 nM H2O

�2 incubated in the

d(evice for 20 min at 0o� C

�, revealed no significant

c� hanges of the ability to react with homovanillic acid12.A

�ll collections were performed between 9 and 11 a.m.

a� nd patients were asked to stop any medication 12 h be-fore the visit.

Measurement of hydrogen peroxide. The content ofH

82O

�2 in expired breath condensate was determined ac-

c� ording to the method of RU9

CH et al.3�6. Briefly, 600 µl

o� f expired breath condensate was mixed with 600 µl�

HRP solution of (1 U/ml) containing 100 µM homova-nillic acid and incubated for 60 min at 37o� C

�. After-

w� ards, the sample was mixed with 150 µl 0.1 M gly-c� ine-NaOH buffer (pH 12) with addition of 25 mMEDTA and transferred into microcuvette (PE 5200-4

:339). The homovanillic acid oxidation product as

a� measure of the amount of H2� O

�2

� was determined spec-

trofluorimetically using Perkin Elmer Luminescence

Spectrometer LS-50 (Norwalk, CT) operating in theread mode. Slit widths were set at 10 nm for bothe� mission and excitation and the integrate time was 0.1 se� xcitation was at 312 nm and emission was measureda� t 420 nm. Readings were converted into nM usingr� egression equation:

Y = 67.64 (X–X0; )

�,

w� here Y – nmol of H2O�

2 per one litre of expired breathc� ondensate, X – intensity of emission at 420 nm ex-p ressed in arbitrary units, X0

< – intensity of emissiong= iven by reference sample receiving distilled water in-stead of breath condensate, obtained from 3 series ofc� alibration experiments with 19 increasing (0.0125 to2

*5 µM

>) H2O

�2 concentrations. The confidence level was

9?5% and p value was less than 0.03 and 0.0001 for

c� onstant and regression coefficient, respectively. Thel

�inear least square estimation was used for calculation

o� f the regression equation. The lower limit of H2� O

�2

�

d(etection was 83 nM and the calibration curve was

l�inear up to a concentration 16.7 µM

> H2O

�2 concentra-

tions.

Cell preparation. A 20 ml sample of the wholeb

�lood was obtained from all subjects included in the

study between 8 and 9 a.m. Human neutrophils werei

"solated by dextran sedimentation followed by centrifu-

g= ation by Ficoll-Hypaque according to standard proce-d

(ures8. The viability of neutrophil suspensions was al-

w� ays above 96% and the purity was 98% as assessedb

�y trypan blue exclusion test and analysis of smears

p repared from each sample using Giemsa stain, respec-t

ively.

Fura-2-AM loading and measurement of cytosolicf

@ree calcium. Neutrophil Ca2

�+ response was measured

a� s previously described3�4. Briefly, human neutrophils

(5×106$/

Aml) were suspended in 6 mM Tris-HCl buffer

(pH 7.4, osmolality 283 mOsm/kg H2� O

�). Cells were

i"ncubated with 1 µM

> Fura-2-AM for 1 h at 37o� C

� in

total darkness in atmosphere containing 5% CO2

� 17.After 2 washes with buffered saline cells were resus-p ended (2×106

$/

Aml) in 6 mM Tris-HCl buffer (pH 7.4,

o� smolality 312 mOsm/kg H2O�

) and incubated for 15min at 37o� C

�. During all experiments cells were kept at

0B o� C

� in darkness for not more than 2 h to prevent the

leakage of Fura-2 and warmed up to 37o� C�

just beforeu7 se. Neutrophils were stimulated by addition of 1 µl offMLP solution (final concentration 10–7 M). [Ca2+]

�i was

d(etermined by dual wavelength measurements at

e� mission wavelength of 510 nm and excitation wave-l

�ength of 340 nm and 380 nm in the Perkin Elmer

Luminescence Spectrometer LS-50 (Norwalk, CT). Slit

A. Antczak et al.: Neutrophil Activation in Asthmatics 121

w� idths were set at 5.0 nm for emission and at 10.0 nmfor excitation. All measurements were performed at37o� C

� under constant stirring of 600 µl of neutrophil

suspension (1.2×C 106$ cells) kept in microcouvette (PE

5D200-4339). Fluorescence signals were calibrated after

lysis of neutrophils with 0.1% Triton X-100 (maximalsignal) and subsequent addition of EGTA to a finalc� oncentration of 10 nM (minimal signal). Preliminarye� xperiments have shown that 0.1% Triton X-100 hadn� ot changed the 340/380 nm ratio. Autofluorescence ofneutrophils was determined with cells loaded withDMSO alone. All operations and calculations of [Ca2+]

�i

w� ere performed with use of “The Intracellular Bio-c� hemistry Application” software (Perkin Elmer, Bea-c� onsfield, England 1990).

S+

tatistical analysis. H2O�

2 concentration was ex-p ressed as the mean ± SD. For readings that gave re-sults below the limit of sensitivity, H2O

�2 concentration

i"n expired breath condensate were assumed as 0 nM.The following parameters of neutrophil response wered

(etermined and also expressed as mean value ±

� SD:

∆E

[Ca2+]�

i – maximal increment in the [Ca2+]�i, V – maxi-

mal velocity of the increase in [Ca2�

+]�i

' , m[Ca2�

+]�i

' – maxi-mal [Ca2+]

�i after activation, ∆t

– time to reach m[Ca2+]

�i.

TF

he differences between results in the groups of healthya� nd asthmatic subjects were determined by the Stu-d

(ent’s tG -test. A p value less than 0.05 was considered

to be significant. Pearson correlation was used to deter-

mine the relations between measured variables. All cal-c� ulations were performed using Microsoft Excel ver-sion 5.0 software.

Results

O�

ur previous paper on H2� O

�2

� levels in the air con-d

(ensate from the same subject revealed that H2O

�2 con-

c� entration in expired breath remains stable for 4-dayo� bservation and that there are no significant differencesi

"n separate samples collected on the same day with 30

min intervals3�3. If patients did not rinse their mouths

w� ith distilled water before and during condensation,H2O

�2 level rose which was due to the saliva contami-

n� ation. It was proven by SZH

NAJDER et al.4�1 that H2O

�2

level in saliva is 7-fold higher than that in the expireda� ir. So in all experiments on the content of H2O

�2 in

e� xpired breath condensate the noseclip was used andrinsing mouth with distilled water was performed.

O�

nly one healthy volunteer (1 male) revealed de-t

ectable H2O

�2 content in the air condensate (0.18 µM

>).

In 9 healthy subjects the H2� O

�2

� level was below them! ethod sensitivity (83 nM) and were assumed as 0 nM.This is why the mean H2

� O�

2� concentration calculated for

w� hole group was 0.01 ±�

0.04 µM>

. The H2O�

2 level ine� xpired breath condensate of asthmatic subjects (n = 11)w� as almost 18-fold higher than that in control groupa� nd was 0.18 ±

� 0.20 µM

> (p<0.05) (Fig. 1). In 1 female

a� sthmatic no detectable H2� O

�2

� concentration was noted.There were no significant differences between male

a� nd female asthmatic subjects as far as H2O�

2 level isc� oncerned.

Both, neutrophils from healthy and asthmatic pa-t

ients responded to fMLP stimulation with increased

their [Ca2

�+]

�i

' (Fig. 2). The Ca2�+ response was higher in

neutrophils from asthmatic than from healthy donors.M

>aximal increment in [Ca2

�+]

�i (∆

E[Ca2

�+]

�i)

� and maximal

[Ca2+]�

i' after stimulation (m[Ca2+]

�i

' )� were 2-fold and

Table 2. Parameters of fMLP-induced Ca2�

+ response of neutrophilsfrom healthy volunteers and asthmatic patients

Subject

Parameters of fMLP-induced neutrophil Ca2+

response

∆ [Ca2+]Ii

(nM)m[Ca2+]

Ii

(nM)V

J

(KnM/s)

HealthyAsthmatics

113.9 ± 22.3177.8 ± 21.7*

205.0 ± 44.7292.8 ± 60.8*

8L.38 ± 2.23

4.23 ± 1.33*

∆M

[Ca2+]Ii – maximal increment in [Ca2+] i;N m[Ca2+]i – maximal

[Ca2+]i after activation, V – maximal velocity of the increase in[Ca2

�+]i' after stimulation with 10–7 M fMLP.S/

ignificantly different from neutrophils from healthy donors:*p<0.001.

Fig. 1. HO

2OP

2 concentration in expired breath condensate of asth-matic patients and healthy subjects. Individual results below thesensitivity of H2O2 method determination (83 nM) were assumedas 0 nM. The mean H2O

P2 content of breath condensate of all as-

thmatic subjects was higher than that found in control group0.18 ± 0.20 µQ M vs. 0.01 ± 0.04 µQ M (p<0.05)

122 A. Antczak et al.: Neutrophil Activation in Asthmatics

1.6-fold higher in neutrophils from asthmatic than fromhealthy subjects (205.0 ± 44.7 vs. 113.9 ± 22.3, and2

*92.8 ±

� 60.8 vs. 177.8 ±

� 21.7 nM, p < 0.001 and p < 0.001,

respectively) (Table 2). Neutrophils from asthmatics re-v ealed also a much higher maximal velocity of the in-c� rease in [Ca2+]

�i (V) as compared with healthy donors

(8.4 ± 2.0 vs. 4.0 ± 1.0 nM/s, respectively, p < 0.001)(Table 2). There was no significant difference betweenstudy groups in the time (∆

Et

) to reach m[Ca2

�+]

�i.

WR

hat is interesting, a positive correlation was found

FS

ig. 2. Mean changes of [Ca2+]i in Fura-2 loaded human PMNLaT fter stimulation with 10–7 MfMLP. Cells from asthmatic subjects(Kline A) and from healthy volunteers (line B) were suspended in

1 mM Ca2+ buffer. Arrow indicates time of agonist challenge

Fig. 3. Significant positive correlation between H2� O2

� concentrationin expired air condensate and (A) maximal [Ca2

�+]i' after activation

(Km[Ca2+]i) or (B) ∆[Ca2+]i in PMNL of asthmatic patients after

s. timulation with fMLP (r = 0.82, p < 0.002; r = 0.87, p < 0.001,respectively)

Fig. 4. Significant positive correlation between H2O2 concentrationin expired air condensate and (A) maximal velocity of the increasein [Ca2

�+]i' (V [nM/s]) or no correlation with (B) time to reach

m[Ca2+]Ii (∆t [s]) in PMNL of asthmatic patients after stimulation

with fMLP (r = 0.64, p<0.05; r = 0.19, p = 0.56, respectively)

A. Antczak et al.: Neutrophil Activation in Asthmatics 123

b�etween H2O

�2 in expired breath condensate and the

e� arly step of fMLP-induced neutrophil activation ex-p ressed as both ∆[Ca2+]

�i

' and m[Ca2+]�

i' in neutrophils of

a� sthmatic patients (r = 0.87, p<0.001, and r = 0.82 andp <0.002) (Fig. 3), respectively. There was also a posi-t

ive correlation between H2O

�2 in expired breath con-

d(ensate and maximal velocity of the increase in [Ca2

�+]

�i

(V) in neutrophils of asthmatic patients (r = 0.64,p <0.05) but no correlation between H2

� O�

2� in expired air

a� nd ∆E

t (r = 0.19, p = 0.56) (Fig. 4), respectively. No

c� orrelation was found between measured parameters inhealthy subjects.

DU

iscussion

I�n this study we have found that asthmatic subjects

have higher H2� O

�2

� level in expired breath condensate asc� ompared with healthy volunteers and that there isa� strong positive correlation between H2O

�2 in expired

a� ir and neutrophil Ca2+ response after fMLP challenge.I

�t seems quite likely that the inflammatory processes in

the respiratory tract lead to increased oxidant produc-

tion, which in turn can be detected by elevated H2

� O�

2� in

e� xpired breath. Thus H2O�

2 could reflect the presence ofa� irway inflammation. This hypothesis seems to be sup-p orted by the fact that H2

� O�

2� concentrations are lower

i"n asthmatics who use antiinflammatory medication20.

I�ncreased levels of H2O

�2 in expired breath were

found not only in bronchial asthma. Our previous re-p orts have shown that healthy cigarette smokers havei

"ncreased H2O

�2 in expired breath3

�3. SZ

HNAJDER et al.4

�1

have found increased H2� O

�2

� levels measured in theb

�reath condensate of patients with ARDS. DOHLMAN et

a� l.15 revealed relatively high H2� O

�2

� levels in expiredb

�reath condensate in pediatric patients with asthma. In-

c� reased H2O�

2 in expired air of asthmatic children wasa� lso found by JOBSIS et al.2

�0. Moreover, they also

showed that antiinflammatory therapy, as inhaled ste-r� oids, is associated with a lower exhaled H2O

�2 concen-

tration.

Increased content of H2� O

�2

� in expired breath conden-sate of asthmatic subjects is likely to be due to in-c� reased oxidant production in bronchial lining fluid andt

he overcome antioxidant potential of lower airways.

I�ncreased H2O

�2 in expired breath condensate of asthma-

tics can reflect a situation in which oxidant overloado� vertakes the capacity of antioxidant protection in thelower airways.

I�ncreased number of polymorphonuclear cells

(mainly eosinophils, but also neutrophils and macro-p hages) have been identified at autopsy of patientsd

(ying of asthma14 or by use of BAL7

V and induced spu-

tum2

�7. The most important source of H2O

�2 seems to be

e� osinophils. Differential cell counts showed significante� osinophil percentage in induced sputum as comparedw� ith controls27. On the other hand, neutrophils seem tob

�e quite active in the airways of asthmatics. Significant-

ly higher concentrations of neutrophil granule proteins(myeloperoxidase and human neutrophil lipocalin) ininduced sputum of asthmatic patients were found27.This finding indicates that neutrophils in the airways ofa� sthmatic patients are actively degranulated and it islikely that these proteins and eosinophil granule pro-t

eins, as major basic proteins, contribute to the airway

d(amage seen in asthma2

�9. Neutrophils can be primed

a� nd/or activated by several cytokines including IL-4,I

�L-8, and GM-CSF. These cytokines activate intracel-

l�ular tyrosine kinases leading to phosphorylation of sev-

e� ral intracellular proteines including those involved inC

�a2+ balance and subunits of NADPH oxidase9

W. Phos-

p horylation of these proteins may lead to enhancedo� xygen reactive species production by PMNL from as-t

hmatic patients and increased hydrogen peroxide ex-

h�alation.

WR

e have demonstrated that neutrophils from as-t

hmatic patients responded with higher [Ca2+]

�i rise after

stimulation with fMLP than control cells. The enhancedresponse to fMLP may come from increased number off

%MLP receptors on neutrophil outer membrane and/or

a� ltered intracellular signal transduction expressed bye� nhanced inositol triphosphate generation opening cal-c� ium channels of intracellular Ca2+ stores. Neutrophilsf

%rom asthmatics can be primed continuously and this is

w� hy ready to react so easily. Higher intracellular Ca2+

a� ccumulation in neutrophils of asthmatic patientsshould be also taken into account. Increased [Ca2

�+]

�i

' re-sponses in asthmatics may be an explanation of thesep henomena. As our patients refrained from any medi-c� ation (except short acting β-agonists 6 h before bloodc� ollection) especially any steroids for 3 months beforee� ntering the study, any inhibiting steroid action onmediator release from inflammatory cells should be ex-c� luded.

It is very interesting that a strong positive correla-t

ion was found between [Ca2

�+]

�i response after fMLP

c� hallenge and H2� O

�2

� concentration in exhaled air of as-t

hmatic patients. As we noted before, increased [Ca2+]

�i

is one of the intracellular triggers of NADPH oxidase.The correlation found could reflect a cause-and-effectrelationship. Increased [Ca2+]

�i in neutrophils of as-

thmatic patients may trigger enhanced reactive oxygen

species production and thus be responsible for en-h

�anced H2O

�2 level in expired breath condensate of these

p atients. Obviously, there can be other sources of H2� O

�2

�

124 A. Antczak et al.: Neutrophil Activation in Asthmatics

i"n the airways of asthmatic patients i. e. macrophageso� r eosinophils and their role in the free radical gener-a� tion in asthma remains to be fully explained10, 37.

O�

ur results indicate that asthmatic patients exhalemore H2

� O�

2� and that this phenomenon seems to be tight-

ly associated with [Ca2+]�

i increase which is an earlye� vent of the activation of neutrophils of these patientsw� hich proves phagocyte involvement in the developmento� f airway oxidant overburden in bronchial asthma.

RX

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Received in March 1998Accepted in December 1998

126 A. Antczak et al.: Neutrophil Activation in Asthmatics