Effects of oral N-acetylcysteine on cell content and macrophage function in bronchoalveolar lavage from healthy smokers

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1 Eur Respir J 1988, 1' Effets of oral N-aetylysteine on ell ontent and marophage funtion in bronhoalveolar lavage from healthy smokers M Linden*, E Wieslander*, A Eklund**, K Larsson***, R Brattsand* Effets of oral N-aetylysteine 11 ell ontent and marophage funtion in brollhoalveolar lavage from healthy smokers M Linden, E Wieslander, A Eklund, K Larsson, R Brattsand ABSTRACT: Bronboalveolar lavage (BAL) was performed in fourteen healthy non-smokers and eleven healthy smokers In smokers BAL was performed before and after eight weeks' treatment with N-aetylysteine (NAC; 2 mg tid) CeU number, omposition and viability were determined in the BAL fluid Alveolar marophages (AMs) were ultured before examjnation of their phagoyti apaity and their ability to produe leukotriene B (L TB 4 ) BAL fluid from smokers ontained more ells than that from non-smokers (p < 1) This was mainly attributable to inreases in both proportion and absolute number of AMs (p<oooj) and to an inrease in absolute number of neutrophils {p < 5) However, there was a derease in proportion of lymphoytes in BAL fluid from smokers (p<oooi) Phagoyti apaity of adherent ells and apaity of AMs to generate L TB after stimulation with opsonized zymosan (OZy) were dereased in smokers (p < 5 and p < 1 respetively) NAC treatment of smokers did not affet ell number but resulted in an inreased proportion of lymphoytes in BA L fluid (p < 5) The phagoyti apaity of AMs was not signifiantly altered but was improved in five of eleven smokers after NAC treatment NAC also enhaned the dereased LTB 4 seretion by smokers' AMs (p < 5) We onlude that smoking leads to redued phagoyti apaity and LTB seretion of AMs and that oral NAC treatment may improve the funtion of AMs Eur Respir J, 1988, 1, * AB DRACO, Lund, Sweden The Department of T horai Mediine, Karolinska Hospital, Solna, Sweden ***The National Board of Oupational Safety and Health, Solna, Sweden Correspondene: M Linden, The Department of Clinial Researh, AB DRACO Box 34, S 221 Lund, Sweden Keywords: Alveolar marophages; bronho alveolar lavage; LTB 4 ; N-aetylysteine; phagoytosis; smokers Reeived: May 27, 1987; aepted after revision January 26, I 988 Many forms of hroni lung disease are assoiated with smoking [1) The reasons for this have not been fully established, but muh attention has been foused on smoke-indued hanges in the harater and ontent of bronhoalveolar lavage (BAL) ells suh as the alveolar marophage (AM) The AM plays a major role as a savenger of partiles and mirobes in the lung The antimirobial funtion of AMs requires a ombination of phagoytosis and generation of reative oxygen speies Furthermore, AMs reruit other phagoytes, suh as neutrophils, to the lung by seretion of hemotati fators, eg LTB 4 Studies of AMs from smokers have demonstrated a dereased [2] or normal [3, 4] phagoyti apaity Moreover, dereased metabolism of arahidoni aid, by means of lowered apaity of prostaglandin E 2 (PGE 2 ) and LTB 4 seretion by AMs from smokers, has been reently reported by LAVIOLETTE and eo-workers [5, 6] and by WIESLANDER and eo-workers [7, 8) Considering these effets of igarette smoking and its assoiation with an inreased inidene of infetious diseases in the lung [9, 1], we ompared the LTB 4 seretion and the phagoyti ativity of AMs from smokers and non-smokers in this study N-aetylysteine (NAC) has previously been reported to redue the number of exaerbations in patients with hroni bronhitis [1 1-13], a ondition assoiated with igarette smoking Although NAC is often referred to as a muolyti agent [11, 12] or a radial savenger, the reason for the benefiial effet of NAC is not lear We therefore examined whether oral NAC treatment of smokers influenes the ontent and the omposition of BAL ells and the funtion of AMs by means of their phagoyti apaity and LTB 4 seretion apaity Subjets Patients, materials and methods Fourteen healthy non-smoking volunteers (three females) with a mean age of 27 ± I yrs and eleven healthy smokers (seven females) with a mean age of 29 ± 2 yrs partiipated in the study The mean igarette onsumption was 145 ± 28 pak-years All subjets had a normal ECG and hest X-rays Five of the smokers had produtive ough in the morning All subjets gave their informed onsent and the study had the approval of the loal Ethial Committee

2 646 M LINOEN ET AL Lung funtion Lung volumes were measured in a body plethysmograph and dynami spirometry was performed with a rolling seal spirometer (Ohio Model 84) as previously desribed [14] The lung funtion was within normal limits in all partiipating subjets NAC treatment After BAL the smokers reeived oral mediation with NAC (Drao, Sweden) 2 mg tid for eight weeks One and a half hours after the final dose, a seond BAL was performed Bronhoalveolar lavage Bronhosopy was performed through the mouth with a flexible fibreopti bronhosope (Olympus Type 4B2) under loal anaesthesia with 2% lignoaine (Xyloain, Astra, Sweden) after premediation with morphine-sopolamine and phenemal [15] The bronhosope was wedged in a middle lobe bronhus and 25 ml of sterile saline solution (ph 74) at 37 C was instilled in aliquots of 5 ml After eah instillation the fluid was gently aspirated and olleted in a silionized plasti bottle kept on ie Cell olletion and haraterization The BAL fluid was filtered through a double layer of Daron nets and the volume was measured Cells were pelleted at 4 g for 5 min at + 4 C and then resuspended in 9 ml of saline One hundred /of this ell suspension was used for a total ell ount in a Burker hamber The viability of the ells was determined by means of Trypan blue exlusion Cytoentrifuge preparations were made using the Cytospin 2 (Shandon, GB) Approximately 5 x 1 4 ells suspended in 1 1 of RPMI 164 were entrifuged at 5 rpm for 3 min Cell differentials were performed on ytoentrifuge preparations stained with May-Grunwald Giemsa reagents Eight hundred ells per preparation were ounted The remaining BAL ells were repelleted by entrifugation for 5 min at 4 g This ell pellet was resuspended in 4 ml of RPMJ 164 (Flow Lab, Sweden) supplemented with gentamyin (5 g ml - 1 ) and 5% foetal alf serum The ell suspension was then kept on ie during transportation (5-7 h) Total and differential ell ounts and ell viability were also determined diretly after the transport There was no hange in ell number, ell omposition or ell viability aused by the transport Phagoytosis assay The phagoyti ativity of adherent BAL ells was determined aording to the method desribed by HED [ 16] Thus, Saharomyes ervisiae (baker's yeast) were killed by boiling in a water bath for 3 min The heat-killed yeast partiles were labelled with fluorosein isothioyanate (FITC, Sigma Chemial, St Louis, USA) in a 5 M arbonate buffer at ph 95 ontammg 1 8 yeast partiles and 1 mg FITC per ml The suspension was inubated for 6 min at 37 C and then washed four times with Hank's buffer FITC-labelled yeast partiles were resuspended in Hank's buffer (I 8 ells ml - 1 ) and stored at -2 C These partiles were opsonized with human serum (I: l, 3 m in at 37 C) just before use The BAL ells were seeded on multispot mirosope slides (Novakemi, Enskede, Sweden), 1 5 ells per slide, and inubated for 1 h at 37"C Nonadherent ells were removed by washing three times with prewarmed Hank's buffer After the washing, the adherent ells were fed with FlTC-labelled yeast partiles (25 x 1 6 of opsonized FJTC-labelled yeast partiles per slide) and inubated at 37 C for 5, 15, 3 or 6 min At the end of inubation the supernatant was deanted and phagoytosis was stopped by plaing the slides in ie-old Hank's buffer Immediately before mirosopi examination the exess of Hank's buffer was removed from the slides and a drop of Trypan blue (5 mg ml- 1 in saline) was added This dye quenhed the fluoresene of the extraellularly loated FITC-Iabelled yeast partiles, whereas the ingested ones remained fluoresent [ 17] Two hundred ells were examined in dupliate slides using fluoresene and light mirosopy, and the perentage of ingesting ells was alulated Alveolar marophage ulture The BAL ells were seeded in fresh growth medium ( I x 1 6 ells in 2 ml per well) in tissue ulture 24-well multidishes (Nun, Denmark) They were inubated at 37 C in an atmosphere of 5% C 2 and 95% air for l h The non-adherent eijs were then removed by washing three times with Ca + +- and Mg+ +-free phosphate buffered saline The adherent ells were further inubated in fresh medium (2 ml per well) overnight LTB 4 analysis After overnight ulture, AMs were inubated with human serum opsonized zymosan (OZy) suspended in Hank's buffer (I mg ml- 1 and well), or in Hank's buffer only, for 9 min at 37 C The multidish was then transferred to an ie bath and the supernatants were olleted, entrifuged at 2 g for 1 min at 4 C, deanted and stored at -7 C A l aliquot of the supernatant was analysed for LTB 4 using a radioimmunoassay kit (Amersham lot, UK) [18] DNA analysis The number of AMs in ultures was determined by measurement of DNA ontent Cells were disrupted by soniation in ie-old phosphate buffered saline (PBS) (I ml per well) and 2 Jll aliquots of the homogenates were used for determination of DNA by the bisbenzirnidazole (Hoehts 33258, Sigma Chemials, St Louis, USA) method as desribed by LABARCA and PAIGEN (19]

3 N-ACETYLCYSTEINE AND ALVEOLAR MACROPHAGE FUNCTION 647 Statistial analysis -, ;!:! - Results are presented as mean + SEM and ompared by Student's t-test for paired and unpaired observations P values < 5 are onsidered signifiant Results BALjluid reovery, ell viability and ell ounts () < z (i) iij "' Ill D E 1/) "' Ill 1/) E 1/) >- (ij r::: E,g "' s - ->< "' ->< +I +I 'D M o tloo ('loo +I +l -:!:(<) 'D V"l +I +l M V"l -ab -i\ci +t+t '! MM -r- : (<) -o tlrt! t- V"l \ a- <'1 ;a \Ci-6 - t I -r--: t- q V Q The reovered volume of BAL fluid was similar in non-smokers and smokers, before and after NAC treatment (68±2, 64 ± 3, and 67 ± 2% respetively) There was a signifiant inrease in total ell number, both before and after NAC treatment, in BAL from smokers, ompared with non-smokers {p<oooi; table 1) This was due to an inrease in the proportion and absolute number of marophages reovered from the lungs of smokers (table 1) There was no differene in proportion of neutrophils or eosinophils between smokers and non-smokers However, the absolute number of neutrophils was signifiantly inreased in smokers (p < 5) The proportion of lymphoytes in BAL was signifiantly lower in smokers than in non-smokers (p < 1) NAC treatment resulted in a signifiant inrease in 1 PHAGOCYTOSING CELLS(%) TIME (MINUTES) :: Fig I Phagoyti ativity of adherent AMs from non-smokers (n= 7; - ), and from smokers before (n= I I; 6-6 ) and after NAC treatment (n = I I; ) AMs (los ells per slide) were inubated with FITC-Labelld yeast partiles (25 x 1 6 partiles per slide) for 5, I 5, 3 and 6 m in at 37"C : p < 5 ompared with non-smokers (Student's t-test)

4 648 M LINO EN ET AL the proportion of lymphoytes (p < 5) However, the lymphoyte proportion was still signifiantly lower than in non-smokers (p < 5) When the absolute number of lymphoytes was alulated from the total ell yield, smokers had as many lymphoytes as non-smokers (table 1) In smokers the proportions and absolute numbers of AMs and neutrophils remained unhanged after NAC treatment The viability of the BAL ells was similar in all three groups; 74 ± 3% for ells from non-smokers, 8 ± 2% for ells from smokers before as well as after NAC treatment There was no relationship noted between ell viability and the funtional parameters that will be desribed Phagoyti ativity of alveolar marophages After 3 and 6 min of inubation, AMs from smokers had a signifiantly dereased phagoyti ativity, ie lower perentage of ingesting ells, ompared with AMs from non-smokers (fig 1) The mean of phagoyti ativity of smokers' AMs was not signifiantly influened by the NAC treatment (fig 1) However, the phagoyti ativity of AMs was enhaned in five of eleven treated smokers (fig 2) LTB 4 seretion by alveolar marophages LTB 4 seretion in unstimulatd AM ultures from smokers was signifiantly lower than in unstimulated AM ultures from non-smokers AMs from both smokers and non-smokers signifiantly inreased the seretion of L TB 4 when they were stimulated with OZy (results not shown) However, the inrease of LTB 4 seretion in AM ultures from smokers was signifiantly lower than the inrease in AM ultures from non-smokers (p < I; fig3) NAC treatment of smokers improved the seretion of L T B 4 by AMs (p < 5) However, this improvement did not return seretion of L TB 4 to the value found in OZy stimulated ultures of AMs from non-smokers (p < 5; fig 3) Disussion The present report provides data on effets of oral NAC treatment on ell omposition and AM funtion in BAL from healthy smokers It onfirms previous reports onerning ell omposition and numbers [2], however, the absolute number of lymphoytes was similar in smokers and non-smokers In the Fig 2 Phagoyti ativity of adherent AMs from smokers (n = 11) before ( 6-6 ) and after ( ) oral NAC treatment AMs (los ells per slide) were inubated with FITC-labelled yeast partiles (25 x 1 6 partiles per slide) for 5, 15, 3 and 6 min at 37"C

5 N-ACETYLCYSTEINE AND ALVEOLAR MACROPHAGE FUNCTION L TB 4 (pqiilq DNA} Fig 3 LTB 4 seretion into the medium of AM ultures stimulated with opsonized zymosan (I mg per ulture) for 9 min at 37 C The data given are orreted for the DNA ontent in AM ultures Open bars: AMs from non-smokers (n = 14}; horizontally striped bars: AMs from smokers before NAC treatment (n = 11 ); vertially striped bars: AMs from smokers after NAC treatment (n=ll); : p<5 ompared with non-smokers; : p<ool ompared with non-smokers; a: p < 5 ompared with smokers before NAC treatment present study oral NAC treatment resulted in a signifiant inrease in lymphoyte proportion in BAL from smokers The basis of this effet is not known Phagoyti ability of AMs is an important omponent of pulmonary learane of inhaled partiles A possible alteration of phagoyti apaity of AMs aused by igarette smoke has been investigated by many researhers [21] and ontraditory results have been reported Although most investigators did not find an altered phagoyti ativity of AMs from smokers, MARTIN and W ARR [2] reported that the ativity of smokers' AMs was less than that of nonsmokers It seems that hoie of important assay parameters, suh as test partiles, time of ultivation of the AMs, time of inubation of the AMs with test partiles, greatly influenes the results of phagoytosis assay Furthermote, the phagoyti assays used in previous studies did not always distinguish between partiles that had been ingested and partiles that had remained on the ell membrane without being engulfed The present work provides data whih indiate that smokers' AMs have redued phagoyti ativity, assayed as their apaity to ingest heat-killed yeast partiles The mean phagoyti apaity of the smokers' AMs was not signifiantly affeted by NAC treatment; it was, however, improved in five of eleven smokers Although only further experiments an ** a * larify the ation ofnac in this respet, there may be a parallel between our findings and those of NTELSON and BONDE [22] They found that Biostim PVRPV, an immunostimulating agent, enhaned the phagoyti ativity of blood monoytes from patients with hroni bronhitis The reent reports from LAVIOLETTE and eoworkers [6] and from our laboratory [7, 8] on the depressed synthesis of L TB 4 by smokers' AMs were the reason for examining whether oral NAC treatment ould affet this impaired funtion In fat, the L TB 4 prodution by ultured AMs from smokers was signifiantly improved as a result of oral NAC treatment The reason why NAC enhanes this synthesis is not known at present Aording to DNA determinations, we found no differenes in the adherene of AMs from non-smokers or smokers before and after NAC treatment Furthermore, there was no signifiant orrelation between the phagoyti abiuty of AMs to ingest yeast partiles and the stimulation of LTB 4 seretion by OZy (r=28) Thus, these two ellular events seem to be regulated by separate mehanisms This is in agreement with the data reported by RouzER et al [23], whih indiate that phagoytosis is not a prerequisite for formation of prostaglandin E, another metabolite in the arahidoni aid pathways, whereas an interation between a ligand and its reeptor is a ritial step in this proess In onlusion, sine AMs are involved in the early stages of immunologial and inflammatory proesses, alteration of their funtion may hange these proesses in the lungs of smokers The data provided by the present work as well as the report of BERGSTRAND et al [24] on the partial redution of the enhaned superoxide radial generation by smokers' AMs as a result of oral NAC treatment, suggest an influene of NAC on AMs as a possible explanation for the ation of this drug This influene may help to explain the reported apaity of NAC to redue the frequeny of exaerbations in hroni bronhitis [11-13] Aknowledgements: This investigation was supported by the Swedish Medial Researh Counil, the Swedish Assoiation against Heart and Chest Diseases and AB Drao, Lund, Sweden We thank R Hernbrand and S Klint for tehnial assistane and M Broman for seretarial assistane Referenes I Reynolds YH, Chretin Y- Respiratory trat fluids: analysis of ontent and ontemporary use in understanding lung diseases Disease-a-month, 1984, 3, Martin RR, Warr GA - Cigarette smoking and human pulmonary marophages Hosp Pra, , Harris JO, Swenson EW, Johnson JE - Human alveolar marophages: omparison of phagoyti ability, gluose utilization, and ultrastruture in smokers and non-smokers J Clin lm est, 197, 49, Hinman LM, Stevens CA, Matthay RA, Gee JBL - Elastase and lysozyme ativities in human alveolar marophages Effet of igarette smoking Am Re1 Respir Dis, 198, 121,

6 65 MLJNDEN ET AL 5 Laviolette M, Chang J, Newombe OS - liuman alveolar marophages: a lesion in arahidoni aid metabolism in igarette smokers Am Rev Respir Dis, 1981, 124, Laviolette M, Coulombe R, Piard S, Braquet P, Borgeat P - Dereased leukotrien 8 4 synthesis in smokers' alveolar marophages in vitro J Clinlnvest, 1986, 77, Wieslander E, Linden M Albertsson E Johansson U, Edholm LE, Brattsand R, Karlsson SE - In: L TB 4 prodution in ultured AM from guinea pig, rat and man Abstrat 141 at 6th Int Congress on Inflammation, Paris, Wieslander E, Linden M, Hakansson L, Eklund A, Blashke E Brattsand R, Venge P - Human alveolar marophages from smokers have an impaired apaity to serete L TB 4 but not other hemotati fators Eur J Respir Dis, in press 9 Haynes WF Jr, Krstlovi VJ, Bell ALL Jr - Smoking habit and inidene of respiratory trat infetions in a group of adolesent males Am Rev Respir Dis, 1966, 93, Parnell JL, Andersson DO, Kinnis C - Cigarette smoking and respiratory infetions in a lass of student nurses N Engl J Med, 1966, 274, Multientre Study Group - Long-term oral ysteine in hroni bronhitis A double-blind ontrolled study Eur J Respir Dis, 198, 61 (Suppl III) Boman G, Baker U, Larsson S, Melander B, Wahlander L - Oral aetylysteine redues exaerbation rate in hroni bronhitis: report of trial organized by the Swedish Soiety for Pulmonary Diseases Eur J Respir Dis, I 983, 64, British Thorai Soiety Researh Committee - Oral N aetylystine and exaerbation rates in patients with hroni bronhitis and severe airway obstrution Thorax, 1985, 4, Larsson K, Hedenstrom H, Malmberg P - Learning effet, variation during offie hours and reproduibility of stati and dynami spirometry Respiration, in press 15 Eklund A, Blashke E - Relationship between hanged alveolar-apillary permeability and angiotensin-onverting enzyme ativity in serum in saroidosis Thorax, 1986, 41, Hed J - The extintion of fluoresene by rystal violet and its use to differentiate between attahed and ingested miroorganisms in phagoytosis FEMS Mirobial Left, 1977, I, Loike JD, Silvrstein SC - A fluoresene quenhing tehnique using Trypan blue to differentiate between attahed and ingested glutaraldehyde-fixed red blood ells in phagoytosing murine marophages J /mnumol, 1983, 57, Salmon JA, Simmons PM Palmer RMJ - A radioimmunoassay for leukotriene 8 4 Prostaglandins, 1982, 24, Labara C, Paigen K - A simple, rapid and sensitive DNA assay proedure Anal Biohem, 198, 12, Reynolds YH, Merrill WW - Airway hanges in young smokers that may antedate hroni obstrutive lung disease Med Clin North Am, 1981, 65, Finh GL, Fisher GL Hays TL Golde DW - Surfae morphology and funtional studies of human alveolar marophages from igarette smokers and nonsmokers J Retiuloendothelial Se, 1982, 32, Nielsen H, Bonde J- lmmunostimulation of blood monoyte funtion by RU 4174 (Biostim '') in patients with hroni bronhitis /111 J lmmwwpharmaol, 1986, 8, Rouzer CA, Sott WA, Kempe J, Cohn ZA - Prostaglandin synthesis by marophages requires a speifi reeptor-ligand interation Pro Natl Aad Si, 198, 77, Bergstrand H, Bjornson A, Eklund A, Hernbrand R, Larsson K, Linden M, Ni1sson A - Inreased stimuli-indued superoxide radial generation in vitro by human alveolar marophages from smokers may partially be redued by N-aetylysteine treatment in vh o J Free Rad Bioi Med, 1986, 2, RESUME: Un lavage bronho-alveolaire a ete exeute hez 14 non fumeurs et hez I I fumurs, tous bien portants Chez les fumeurs, le lavage a ete realise avant et apres huit semaines de traitement a la N-aetylysteine a raison de 2 mg trois fois par jour L'on a determine le nombre de ellules, leur type l lur viabilit dans le lavage bronho-alveolaire Les marophages alveolaires ont ete ultives avant l'examen de leur apaite phagoytaire et de leur apait a produir de la leukotriene B 4 Le liquide ue lavage des fumeurs ontenait plus des ellules que elui des non fumeurs (p < 1) Cei eta it du surtout a un augmentation a la fois de la proportion et du nombre absolu de marophags alveolaires (p< OOOI) et a une augmentation du nombre absolu de neutrophiles (p < 5) Toutefois, on nota it une diminution de la proportion des lyrnphoytes dans le lavage alveolaire des fumeurs (p < 1) La apaite phagoytaire des ellules adhesives et la apaite des marophages alveolaires a produire de la L TB 4 apres stimulation ave du zymosan opsonise, s'averent diminuees hez les fumeurs (p<5 et p<ooi respetivement) Le lraitment des fumeurs a la N-aetylysteine n'influe pas le nombre de elluls, mais entraine une augmentation de la proportion des lymphoytes du lavage alveolaire (p < 5) La apaite phagoytaire des marophages alveolaires n'est pas signifiativement modifee par le traitement a la N-aetylystine, mais est ni:anmoins amlioree hez 5 des 11 fumeurs apres traitement a la N-aetylysteine La N aetylysteine stimule la sen!tion diminuee de L TB 4 par les marophags de fumeurs (p <5) Nous onluons que le tabagisme entraine une redution de la apait phagoytaire et de la seretion de LTB 4 des marophages alveolaires, et que le traitment a la N-aetylysteine peut ameliorer la fontion marophagique