Arcivum Immunologiae et Terapiae Experimentalis, 999, 47, 9 26 PL ISSN 0004-069X Hydrogen Peroxide in Expired Air Condensate Correlates Positively wit Early Steps of Periperal Neutropil Activation in Astmatic Patients A Antczak et al: Neutropil Activation in Astmatics ADAM AN TCZA, DARIUSZ NOWA, PIOTR BIALASIEWICZ and MARE ASIELSI Department of Pneumonology and Allergology, University Medical Scool, opcińskiego 22, 90-5 Łódź, Poland Abstract We ave found an increased H 2 O2 level in expired air of astmatic patients Neutropils from tese subjects generated iger amounts of superoxide radicals after callenge wit porbol esters tan tose from ealty subjects wic may result from an increased activity of NADPH-oxidase Te enanced Ca 2 + mobilisation in neutropils from astmatics could be responsible for increased production and subsequent elevated H 2 O2 concentration in expired breat condensate In tis study we wised to determine weter neutr opils of astmatic patients ave enanced [Ca 2+ ] i response after N-formyl-metionyl-leucyl-penylalanine fmlp callenge as compared wit cells from ealty donors, and if so, does it correlate wit H 2 O2 levels in expired air We examined 2 patients, 0 ealty individuals as a control group mean age 4 ± 55, 6 males and 4 females) and astmatic subjects mean age 82 ± 72, 7 males and 4 females) Te rise of [Ca 2+ ] i as an early event of neutropil activation, was measured spectrofluorimetically wit Fura-2-AM Te mean H 2 O2 level, measured spectrofluorimetrically in te expired breat of astmatics, was 20-fold iger tan tat in ealty control 08 ± 020 vs 00 ± 004 µm, p<005) [Ca 2+ ] i increase after callenge by fmlp [Ca2+ ] i ) was muc iger in astmatics tan in control group 2050 ± 44 vs 0 ± 22 nm, p<005, respectively) A strong correlation was observed between H 2 O2 and [Ca2+ + ] i and maximal velocity of increase in [Ca2 ] i in astmatics r = 087, p<00 and r = 064, p<005) We conclude tat elevated H 2 O2 level in te expired breat condensate of astmatics can be generated by activated neutropils in te course of mucosal inflammation observed in broncial astma ey words: broncial astma; ydrogen peroxide in breat condensate; neutropils; intracellular ca lcium Introduction In several lung inflammatory diseases an elevated ydrogen peroxide H 2 O 2) content in te expired breat as been found, among wic broncial astma seems to be extensively studied, 5, 28,, 4 H 2 O2 is one of te most stable, toxic oxygen metabolites and relatively easy to detect It is also volatile, and due to lack of carge membrane permeable It is cytotoxic 8, a ig concentrations H 2 O 2 causes cell necrosis and at low levels can induce apoptosis 6 Catalase and glutatione peroxidase are basic enzymes regulating intracellular H 2 O2 level, 5 Moreover, in astmatic patients increased level of H 2 O2 positively correlated wit enanced level of lipid peroxidation products tiobarbituric acid reactive species) in expired breat condensate wic proves oxidantantioxidant imbalance in te airways of tese patients An intense airway inflammation can be caused eiter by H 2 O2 alone or newly generated ydroxyl radical OH o 4 ) An important feature of broncial astma is te influx of circulating pagocytes including eosinopils, mast cells
% ) *, 0 0 0 + 4 5 20 A Antczak et al: Neutropil Activation in Astmatics and neutropils into te broncial wall 26 Wen activated, tey are capable of generation of reactive oxygen species, for example superoxide anion O 2 ) wic is ten dismutated to ydrogen peroxide H 2 O2) and can play an important role in te development of patopysiological features of broncial astma, like enanced aracidonic acid release, smoot muscle contraction, impaired β-adrenergic responsiveness and broncial yperresponsiveness, 4, 22 It was proved tat neutropils from bot adult and cildren astmatic patients can iger increase amounts of O 2 and H 2 O 2 after callenge wit porbol esters and fmlp tan cells on matced, ealty subjects 2, 2, 40 Moreover, te ability of neutropils isolated from astmatics to produce superoxide anion, correlated wit te degree of airway yperresponsiveness to inaled metacoline and istamine 9, 2, 25 A close relationsip between te opsonized zymosan-induced cemiluminescence of neutropils and broncial yperreactivity to istamine in astmatic cildren can also be observed 24 Furtermore, in te same astmatic cildren, te generation of O 2 was significantly iger wit tan witout astma attacks Tis is consistent wit te observation tat exacerbated astma patients exale more H 2 O2 tan patients wit stable disease 5, 28 Similarly, an enanced alveolar cell luminol-dependent cemiluminescence in astmatic patients as been also found 2 An important source of H 2 O are pagocytes due to # NADPH oxidase system generating at least oxygen metabolites O 2, H 2 O2 and OH o 6 ) $, 0 Tey are released in extracellular fluid, and in te airways a part of H 2 O2, wic as not been decomposed by antioxidant enzymes, can be excreted wit expired air NADPH oxidase system is activated by a variety of stimuli including:,2-diacylglycerol, pospatidic acid, inositol tripospate and Ca 2+, 9 Canges of intracellular free calcium concentration [Ca 2+ ] i seem to be an important early step in signal transduction leading to te respiratory burst and degranulation Increased [Ca 2+ ] i promotes diacylglycerol-induced activation of protein kinase C tat is responsible for posporylation of cytoplasmic subunits of NADPH oxidase Neutropils depleted wit Ca 2 + fail to produce superoxide in response to stimulation unless tey are supplied wit extracellular Ca 2+ 2 Moreover, activation of te NADPH oxidase occurs wen cytosolic Ca 2 + rises 8 Wat is very interesting, an increased release of calcium intracellular stores in neutropils, obtained from astmatic patients after stimulation wit fmlp compared wit ealty subjects was observed & 5 A ypotesis, terefore, can be put forward tat increased production of reactive oxygen species by neutropils from astmatic patients 2 may be caused by enanced Ca 2 + mobilization Tis would lead to an incrased H 2 O 2 generation wic could evaporate from te alveolar lining fluid and be detected in expired air In tis study we found a correlation between [Ca 2+ ] ' i rises, as an early step of neutropil acti- vation after fmlp callenge, and H 2 O2 in expired air of astmatic patients Tis could reflect Ca 2 + involvement in te generation of reactive oxygen species as inflammatory mediators in te course of broncial astma Materials and Metods Reagents Peroxidase from orseradis type II HRP, 200 Umg solid), omovanillic acid 4-ydroxy- -metoxy-penylacetic acid), and N-formyl-metionyl-leucyl-penylalanine FMLP) were from Sigma Cemicals Co St Louis, MO, USA) Glycine, etylene diamine tetra-acetic acid EDTA), pospate buffered saline PBS, ph 74) and 0% H 2 O 2 solution, glacial acetic acid, CaCl 2, Cl, and NaCl were purcased from POCH Gliwice, Poland) Fura-2-AM acetoxymetyl ester), etylene glycol bis 2-aminoetyl-eter)-tetraacetic acid EGTA), Triton X-00, dimetyl sulpoxide DMSO) were from Serva Heidelberg, Germany) H 2 O 2 solution 0%) was diluted 00-fold wit PBS and stored at 4 o C in te dark Te actual H 2 O2 concentration was calculated from its absorbance at 20 nm E = 8 cm M ) Aqueous solution of Uml HRP wit addition of 400 µm omovanillic acid were prepared fresly before te assay FMLP, Fura-2-AM were dissolved in DMSO to final concentration 60 µm and 2 mm, respectively, and stored at 80 o C until assay All solutions were stored at 4 o C not longer tan for 4 days Study population Te study included 0 ealty volunteers as a control group mean age 4 ± 55 year, 6 males and 4 females) and astmatic subjects mean age 82 ± 74 year, 7 males and 4 females) wo Table - Caracteristic of study population Number Age year) Sex M: F FVC% FEV% PEFR% FEV reversibility %) Astma duration year) Healty subjects 0 4 2 ± 55 6:4 965 2 ± 2 05 2 ± 47 0 2 ± 56 505 2 ± Astmatic patients 82 2 ± 74 7:4 925 2 ± 68 67 2 ± 42* 68 2 ± * 02 2 ± 488* 70 2 ± 50 Significantly different from pulmonary function tests of ealty subjects: * p<000
6 7 : = *? = 7 ; A Antczak et al: Neutropil Activation in Astmatics 2 ad not suffered from any infectious disease for te last 4 monts Table ) Tey were recruited from University Medical Scool out-patient clinic register Astmatic subjects were asked to stop any medication except sort-acting β-agonists salbutamol or fenoterol) and to report to te clinic after 4-week wasout period to perform lunb function tests Only tose patients wo were able to refrain from antiinflammatory medication, indicated in tis group, during wasout were included to te study Broncial astma was diagnosed based on istory of weezing dyspnea and previous documentation of broncodilator-induced broncial reversibility measured as more tan 5% increase of FEV and te presence of airway yperreactivity after istamine callenge test wit PC20 of less tan 8 mgml according to te metod of COCROFT Broncial reversibility at least 5% of te baseline and te ability to stop oter tan β-agonist terapy were basic inclusion criteria Te duration of broncial astma was to 8 years, mean 7 ± 5 years Spirometry was performed wit Flowscreen Eric Jaeger GmbH&Co, Germany) equipped wit software compatible to American Toracic Society standards 2 Tis study was approved by te local Etics Committee and informed consent was obtained Collection of air condensate Te air condensate was collected in a tube installed in te polystyrene foamed container filled wit ice and salt as previously described Briefly, study subjects were asked to breate into te apparatus and ten te condensate was transferred to Eppendorf tubes Tey were stored at 80 o C for not longer tan 7 days until H 2 O 2 measurement Our previous experiments ave sown tat samples of expired breat condensate and 0 7 M H 2 O 2 solution, under tese conditions, remain stable after 4 days of storage Similarly, 50 nm H 2 O2 incubated in te device for 20 min at 0 o C, revealed no significant canges of te ability to react wit omovanillic acid 2 All collections were performed between 9 and am and patients were asked to stop any medication 2 before te visit Measurement of ydrogen peroxide Te content of 8 H2O 2 in expired breat condensate was determined according to te metod of RU 9 CH et al 6 Briefly, 600 µl of expired breat condensate was mixed wit 600 µl HRP solution of Uml) containing 00 µm omovanillic acid and incubated for 60 min at 7 o C Afterwards, te sample was mixed wit 50 µl 0 M glycine-naoh buffer ph 2) wit addition of 25 mm EDTA and transferred into microcuvette PE 5200-49) Te omovanillic acid oxidation product as a measure of te amount of H 2 O was determined spec- 2 trofluorimetically using Perkin Elmer Luminescence Spectrometer LS-50 Norwalk, CT) operating in te read mode Slit widts were set at 0 nm for bot emission and excitation and te integrate time was 0 s excitation was at 2 nm and emission was measured at 420 nm Readings were converted into nm using regression equation: Y = 6764 X X 0 ), were Y nmol of H 2 O2 per one litre of expired breat condensate, X intensity of emission at 420 nm expressed in arbitrary units, X< 0 intensity of emission given by reference sample receiving distilled water instead of breat condensate, obtained from series of calibration experiments wit 9 increasing 0025 to 25 µm > ) H 2 O2 concentrations Te confidence level was 95% and p value was less tan 00 and 0000 for constant and regression coefficient, respectively Te linear least square estimation was used for calculation of te regression equation Te lower limit of H 2 O2 detection was 8 nm and te calibration curve was linear up to a concentration 67 µm > H 2 O2 concentrations Cell preparation A 20 ml sample of te wole blood was obtained from all subjects included in te study between 8 and 9 am Human neutropils were isolated by dextran sedimentation followed by centrifugation by Ficoll-Hypaque according to standard procedures 8 Te viability of neutropil suspensions was always above 96% and te purity was 98% as assessed by trypan blue exclusion test and analysis of smears prepared from eac sample using Giemsa stain, respectively Fura-2-AM loading and measurement of cytosolic @ free calcium Neutropil Ca 2 + response was measured as previously described 4 Briefly, uman neutropils 5 0 $ 6A ml) were suspended in 6 mm Tris-HCl buffer ph 74, osmolality 28 mosmkg H 2 O) Cells were incubated wit µm > Fura-2-AM for at 7 o C in total darkness in atmospere containing 5% CO 7 2 After 2 wases wit buffered saline cells were resuspended 2 0 $ 6A ml) in 6 mm Tris-HCl buffer ph 74, osmolality 2 mosmkg H 2 O) and incubated for 5 min at 7 o C During all experiments cells were kept at B o 0 C in darkness for not more tan 2 to prevent te leakage of Fura-2 and warmed up to 7 o C just before use Neutropils were stimulated by addition of µl of fmlp solution final concentration 0 7 M) [Ca 2+ ] i was determined by dual wavelengt measurements at emission wavelengt of 50 nm and excitation wavelengt of 40 nm and 80 nm in te Perkin Elmer Luminescence Spectrometer LS-50 Norwalk, CT) Slit
D E F + > M 22 A Antczak et al: Neutropil Activation in Astmatics widts were set at 50 nm for emission and at 00 nm for excitation All measurements were performed at 7 o C under constant stirring of 600 µl of neutropil suspension 2 C 0 $ 6 cells) kept in microcouvette PE 5200-49) Fluorescence signals were calibrated after lysis of neutropils wit 0% Triton X-00 maximal signal) and subsequent addition of EGTA to a final concentration of 0 nm minimal signal) Preliminary experiments ave sown tat 0% Triton X-00 ad not canged te 4080 nm ratio Autofluorescence of neutropils was determined wit cells loaded wit DMSO alone All operations and calculations of [Ca 2+ ] i were performed wit use of Te Intracellular Biocemistry Application software Perkin Elmer, Beaconsfield, England 990) Statistical analysis H 2 O2 concentration was expressed as te mean ± SD For readings tat gave re- sults below te limit of sensitivity, H 2 O2 concentration in expired breat condensate were assumed as 0 nm Te following parameters of neutropil response were determined and also expressed as mean value ± SD: [Ca 2+ ] i maximal increment in te [Ca 2+ ] i, V maximal velocity of te increase in [Ca 2+ ] ' i, m[ca 2+ ] ' i maximal [Ca 2+ ] i after activation, t time to reac m[ca 2+ ] i Te differences between results in te groups of ealty and astmatic subjects were determined by te Student s G t-test A p value less tan 005 was considered to be significant Pearson correlation was used to determine te relations between measured variables All calculations were performed using Microsoft Excel version 50 software Results Our previous paper on H 2 O 2 levels in te air condensate from te same subject revealed tat H 2 O2 con- centration in expired breat remains stable for 4-day observation and tat tere are no significant differences in separate samples collected on te same day wit 0 min intervals If patients did not rinse teir mouts wit distilled water before and during condensation, H 2 O2 level rose wic was due to te saliva contamination It was proven by SZ H NAJDER et al 4 tat H 2 O2 level in saliva is 7-fold iger tan tat in te expired air So in all experiments on te content of H 2 O2 in expired breat condensate te noseclip was used and rinsing mout wit distilled water was performed Only one ealty volunteer male) revealed detectable H 2 O2 content in te air condensate 08 µm > ) In 9 ealty subjects te H 2 O 2 level was below te metod sensitivity 8 nm) and were assumed as 0 nm Tis is wy te mean H 2 O concentration calculated for 2 Table 2 Parameters of fmlp-induced Ca 2 + response of neutropils from ealty volunteers and astmatic patients Parameters of fmlp-induced neutropil Ca 2+ response Subject [Ca I 2+ ] i m[ca I J 2+ ] i V nm) nm) nms) L Healty 9 ± 22 2050 ± 447 88 ± 22 Astmatics 778 ± 27* 2928 ± 608* 42 ± * [Ca 2+ I ] i maximal increment in [Ca 2+ ] i; N m[ca 2+ ] i maximal [Ca 2+ ] i after activation, V maximal velocity of te increase in [Ca 2 + ]i ' after stimulation wit 0 7 M fmlp Significantly different from neutropils from ealty donors: *p<000 Fig O H2O P 2 concentration in expired breat condensate of astmatic patients and ealty subjects Individual results below te sensitivity of H 2O 2 metod determination 8 nm) were assumed as 0 nm Te mean H P 2O2 content of breat condensate of all astmatic subjects was iger tan tat found in control group 08 ± 020 Q µ M vs 00 ± 004 Q µ M p<005) wole group was 00 ± 004 µm > Te H 2 O2 level in expired breat condensate of astmatic subjects n = ) was almost 8-fold iger tan tat in control group and was 08 ± 020 µm > p<005) Fig ) In female astmatic no detectable H 2 O 2 concentration was noted Tere were no significant differences between male and female astmatic subjects as far as H 2 O2 level is concerned Bot, neutropils from ealty and astmatic patients responded to fmlp stimulation wit increased teir [Ca 2+ ] ' i Fig 2) Te Ca 2 + response was iger in neutropils from astmatic tan from ealty donors Maximal increment in [Ca 2+ ] i E [Ca 2+ ] i) and maximal [Ca 2+ ] ' after stimulation m[ca 2+ ] ' ) were 2-fold and i i
S T * R A Antczak et al: Neutropil Activation in Astmatics 2 Fig 2 Mean canges of [Ca 2+ ] i in Fura-2 loaded uman PMNL after stimulation wit 0 7 MfMLP Cells from astmatic subjects line A) and from ealty volunteers line B) were suspended in mm Ca 2+ buffer Arrow indicates time of agonist callenge Fig 4 Significant positive correlation between H 2O 2 concentration in expired air condensate and A) maximal velocity of te increase in [Ca 2 + ]i ' V [nms]) or no correlation wit B) time to reac m[ca 2+ I ] i t [s]) in PMNL of astmatic patients after stimulation wit fmlp r = 064, p<005; r = 09, p = 056, respectively) Fig Significant positive correlation between H 2 O 2 concentration in expired air condensate and A) maximal [Ca 2 + ]i ' after activation m[ca 2+ ] i) or B) [Ca 2+ ] i in PMNL of astmatic patients after stimulation wit fmlp r = 082, p < 0002; r = 087, p < 000, respectively) 6-fold iger in neutropils from astmatic tan from ealty subjects 2050 ± 447 vs 9 ± 22, and 2928 ± 608 vs 778 ± 27 nm, p < 000 and p < 000, respectively) Table 2) Neutropils from astmatics revealed also a muc iger maximal velocity of te increase in [Ca 2+ ] i V) as compared wit ealty donors 84 ± 20 vs 40 ± 0 nms, respectively, p < 000) Table 2) Tere was no significant difference between study groups in te time E t) to reac m[ca 2+ ] i Wat is interesting, a positive correlation was found
% % R 24 A Antczak et al: Neutropil Activation in Astmatics between H 2 O2 in expired breat condensate and te early step of fmlp-induced neutropil activation expressed as bot [Ca 2+ ] ' i and m[ca 2+ ] ' i in neutropils of astmatic patients r = 087, p<000, and r = 082 and p<0002) Fig ), respectively Tere was also a positive correlation between H 2 O2 in expired breat condensate and maximal velocity of te increase in [Ca 2+ ] i V) in neutropils of astmatic patients r = 064, p<005) but no correlation between H 2 O 2 in expired air and E t r = 09, p = 056) Fig 4), respectively No correlation was found between measured parameters in ealty subjects U Discussion In tis study we ave found tat astmatic subjects ave iger H 2 O 2 level in expired breat condensate as compared wit ealty volunteers and tat tere is a strong positive correlation between H 2 O2 in expired air and neutropil Ca 2+ response after fmlp callenge It seems quite likely tat te inflammatory processes in te respiratory tract lead to increased oxidant production, wic in turn can be detected by elevated H 2 O 2 in expired breat Tus H 2 O2 could reflect te presence of airway inflammation Tis ypotesis seems to be supported by te fact tat H 2 O 2 concentrations are lower in astmatics wo use antiinflammatory medication 20 Increased levels of H 2 O2 in expired breat were found not only in broncial astma Our previous reports ave sown tat ealty cigarette smokers ave increased H 2 O2 in expired breat SZ H NAJDER et al 4 ave found increased H 2 O 2 levels measured in te breat condensate of patients wit ARDS DOHLMAN et al 5 revealed relatively ig H 2 O 2 levels in expired breat condensate in pediatric patients wit astma Increased H 2 O2 in expired air of astmatic cildren was also found by JOBSIS et al 20 Moreover, tey also sowed tat antiinflammatory terapy, as inaled steroids, is associated wit a lower exaled H 2 O2 concentration Increased content of H 2 O 2 in expired breat condensate of astmatic subjects is likely to be due to increased oxidant production in broncial lining fluid and te overcome antioxidant potential of lower airways Increased H 2 O2 in expired breat condensate of astmatics can reflect a situation in wic oxidant overload overtakes te capacity of antioxidant protection in te lower airways Increased number of polymorponuclear cells mainly eosinopils, but also neutropils and macropages) ave been identified at autopsy of patients dying of astma 4 or by use of BAL V 7 and induced spu- tum 27 Te most important source of H 2 O2 seems to be eosinopils Differential cell counts sowed significant eosinopil percentage in induced sputum as compared wit controls 27 On te oter and, neutropils seem to be quite active in te airways of astmatics Significantly iger concentrations of neutropil granule proteins myeloperoxidase and uman neutropil lipocalin) in induced sputum of astmatic patients were found 27 Tis finding indicates tat neutropils in te airways of astmatic patients are actively degranulated and it is likely tat tese proteins and eosinopil granule proteins, as major basic proteins, contribute to te airway damage seen in astma 29 Neutropils can be primed andor activated by several cytokines including IL-4, IL-8, and GM-CSF Tese cytokines activate intracellular tyrosine kinases leading to posporylation of several intracellular proteines including tose involved in Ca 2+ balance and subunits of NADPH oxidase W 9 Posporylation of tese proteins may lead to enanced oxygen reactive species production by PMNL from astmatic patients and increased ydrogen peroxide exalation We ave demonstrated tat neutropils from astmatic patients responded wit iger [Ca 2+ ] i rise after stimulation wit fmlp tan control cells Te enanced response to fmlp may come from increased number of fmlp receptors on neutropil outer membrane andor altered intracellular signal transduction expressed by enanced inositol tripospate generation opening calcium cannels of intracellular Ca 2+ stores Neutropils from astmatics can be primed continuously and tis is wy ready to react so easily Higer intracellular Ca 2+ accumulation in neutropils of astmatic patients sould be also taken into account Increased [Ca 2+ ] ' i responses in astmatics may be an explanation of tese penomena As our patients refrained from any medication except sort acting β-agonists 6 before blood collection) especially any steroids for monts before entering te study, any inibiting steroid action on mediator release from inflammatory cells sould be excluded It is very interesting tat a strong positive correlation was found between [Ca 2+ ] i response after fmlp callenge and H 2 O 2 concentration in exaled air of astmatic patients As we noted before, increased [Ca 2+ ] i is one of te intracellular triggers of NADPH oxidase Te correlation found could reflect a cause-and-effect relationsip Increased [Ca 2+ ] i in neutropils of astmatic patients may trigger enanced reactive oxygen species production and tus be responsible for enanced H 2 O2 level in expired breat condensate of tese patients Obviously, tere can be oter sources of H 2 O 2
Z ^ ^ g i k g l m j 4 l l j g n o ] q l u A Antczak et al: Neutropil Activation in Astmatics 25 in te airways of astmatic patients i e macropages or eosinopils and teir role in te free radical generation in astma remains to be fully explained 0, 7 Our results indicate tat astmatic patients exale more H 2 O 2 and tat tis penomenon seems to be tigtly associated wit [Ca 2+ ] i increase wic is an early event of te activation of neutropils of tese patients wic proves pagocyte involvement in te development of airway oxidant overburden in broncial astma X References ABRAHAM W M 994): Te interaction among granulocyte lipid mediators and te generation of oxygen radicals in antigen-induced airway yperresponsiveness Adv Prostaglandin Tromboxane Leukot Res, 22, 40 2 American Toracic Society 987): Standardization of spirometry 987 update Am Rev Respid Dis Y 6, 285 298 Z ANTCZA A, [ NOWA D, ROL M, SHARIATI B and \ URMA- NOWSA Z 997): Increased ydrogen peroxide and tiobarbituric acid-reactive 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