Expression of functional NK 1 receptors in human alveolar macrophages: superoxide anion production, cytokine release and involvement of NF-jB pathway

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1 British Journl of Phrmcology (25) 45, & 25 Nture Pulishing Group All rights reserved 7 88/5 $3. Expression of functionl NK receptors in humn lveolr mcrophges: superoxide nion production, cytokine relese nd involvement of NF-jB pthwy Cludio Brdelli, Griele Gunell, 2 Federic Vrsldi, 3 Pietro Blo, Elis Del Boc, Ilri Seren Bernrdone, Angel Amoruso &,,4 Sndr Brunelleschi Deprtment of Medicl Sciences, School of Medicine, University of Piemonte Orientle A. Avogdro, Vi Solroli, 7, 28 Novr, Itly; 2 Deprtment of Chemistry, Food, Phrmceuticl nd Phrmcologicl Science (DISCAFF), University of Piemonte Orientle A. Avogdro, Novr, Itly; 3 Aziend Ospedlier Mggiore dell Crit`, Novr, Itly nd 4 IRCAD (Interdisciplinry Reserch Center on Autoimmune Diseses), University of Piemonte Orientle A. Avogdro, Novr, Itly Keywords: Arevitions: Sustnce P (SP) is deeply involved in lung pthophysiology nd plys key role in the modultion of inflmmtory-immune processes. We previously demonstrted tht SP ctivtes guine-pig lveolr mcrophges (AMs) nd humn monocytes, ut creful exmintion of its effects on humn AMs is still scrce. 2 This study ws undertken to estlish the role of SP in humn AM isolted from helthy smokers nd non-smokers, y evluting the presence of tchykinin NK receptors (NK-R) nd SP s ility to induce superoxide nion (O 2 ) production nd cytokine relese, s well s ctivtion of the nucler fctor-kb (NF-kB) pthwy. 3 By Western lot nlysis nd immunofluorescence, we demonstrte tht uthentic NK-R re present on humn AMs, three-fold enhnced expression eing oserved in helthy smokers. These NK-R re functionl, s SP nd NK gonists dose-dependently induce O 2 production nd cytokine relese. In AMs from helthy smokers, SP evokes n enhnced respirtory urst nd significntly incresed relese of tumor necrosis fctor- s compred to helthy non-smokers, ut hs inconsistent effects on IL- relese. The NK selective ntgonist CP 96,345 ((2S,3S)-cis-2-diphenylmethyl-N[(2- methoxyphenyl)-methyl]--zicyclo-octn-3-mine)) competitively ntgonized SP-induced effects. 4 SP ctivtes the trnscription fctor NF-kB, three-fold incresed nucler trnsloction eing oserved in AMs from helthy smokers. This effect is receptor-medited, s it is reproduced y the NK selective gonist [Sr 9 Met(O 2 ) ]SP nd reverted y CP 96, These results clerly indicte tht humn AMs possess functionl NK-R on their surfce, which re upregulted in helthy smokers, providing new insights on the mechnisms involved in tocco smoke toxicity. British Journl of Phrmcology (25) 45, doi:.38/sj.jp.7698 Pulished online 2 Mrch 25 Sustnce P; humn lveolr mcrophges; NK receptor; NF-kB ctivtion; respirtory urst; cytokine relese; TNF-; IL-; IL- AMs, lveolr mcrophges; CP96,345, (2S,3S)-cis-2-diphenylmethyl-N[(2-methoxyphenyl)-methyl]--zicyclooctn-3-mine); GR82334, ([D-Pro 9 (spiro-gmm-lctm)leu,trp ]physlemin( )); GR725, ([Pro 9 (spiro-gmm-lctm) Leu,Trp ]SP); NF-kB, nucler fctor-kb; NK-R, NK receptor; O 2, superoxide nion; PMA, phorol 2-myristte 3-cette; SP, sustnce P Introduction The neuropeptide sustnce P (SP), memer of the tchykinin receptor fmily, is involved in mny physiologicl processes, including nociception, vsodiltion, exocrine nd endocrine glnd secretion, smooth muscle contrction, cell prolifertion, nd lrgely contriutes to the locl control of the immune responses (Severini et l., 22). It induces lymphocyte prolifertion (Pyn et l., 983), enhnces immunogloulin production y cloned B lymphom cells (Pscul Author for correspondence t: Deprtment of Medicl Sciences, School of Medicine, University of Piemonte Orientle A. Avogdro, Vi Solroli, 7, 28 Novr, Itly; E-mil: sndr.runelleschi@med.unipmn.it Pulished online 2 Mrch 25 et l., 99), degrnultes rt mst cells (Mousli et l., 989), modultes eosinophil nd neutrophil ctivity (Brunelleschi et l., 99; Iwmoto et l., 993), stimultes humn peripherl monocytes to produce inflmmtory cytokines including IL-, IL-6, IL-2 nd tumor necrosis fctor- (TNF-) (Lotz et l., 988; Lvgno et l., 2). By using nturl tchykinins nd selective receptor gonists nd ntgonists, we previously demonstrted tht guine-pig lveolr mcrophges (AMs) possess NK receptors (NK-R) nd NK 2 receptors, their stimultion leding to superoxide nion (O 2 ) production nd eicosnoid relese, nd tht ovlumin-sensitized AMs demonstrte n enhnced responsiveness to NK-2R stimultion (Brunelleschi et l., 99; 992). We lso showed tht SP, s well s neurokinin A (NKA)

2 386 C. Brdelli et l NK receptors on humn lveolr mcrophges nd the selective NK 2 receptor gonist [-Al 8 ]-NKA(4 ), induces O 2 production in AMs otined from ptients with ctive srcoidosis (Brunelleschi et l., 996). The iologicl responses to SP re medited y the G protein-coupled tchykinin NK-R, lthough SP cn lso ind, with lower ffinity, NK 2 nd NK 3 tchykinin receptors (Severini et l., 22; Pennefther et l., 24). The presence of NK receptors (NK-R) on monocyte/mcrophges hs een demonstrted y evluting the effects of selective receptor gonists nd ntgonists on functionl prmeters (for exmple, Brunelleschi et l., 99; 992; 998) nd/or y moleculr iology nd protein chemistry techniques. RT PCR nd in situ hyridiztion hve een used to identify NK-R mrna expression in monocytes nd mcrophges (Ho et l., 997; Germonpre et l., 999), ut little is known out NK expression t the protein level. Mrriott & Bost (2) nd Simeonidis et l. (23) demonstrted the presence of NK-R protein in murine peritonel mcrophges nd THP- cells, respectively, ut, to our knowledge, noody hs investigted this possiility in humn AMs. Recent evidence indictes tht NK-R gene expression in THP- cells is incresed fter exposure to IL- nd TNF-: this effect is medited y the trnscription fctor NF-kB, which inds to the promoter region of the NK-R gene nd so regultes its expression (Simeonidis et l., 23). In resting cells, nucler fctor-kb (NF-kB) is retined in the cytoplsm through n ssocition with inhiitory proteins of the IkB fmily, which msk the nucler locliztion signl (Bldwin, 996). Upon stimultion, IkB is phosphorylted, uiquitinylted nd degrded, thus llowing NF-kB to trnslocte to the nucleus. Once NF-kB enters the nucleus, it inds to the promoter region of vrious genes nd induces their trnscription (Bldwin, 996). SP specificlly ctivtes NF-kB pthwy in cells of the monocyte/mcrophge linege, for exmple, humn strocytom cells, murine peritonel mcrophges nd dendritic cells (Lie et l., 997; Mrriott et l., 2), ut no informtion re ville concerning humn AMs. The present study ws undertken to estlish the role of tchykinin NK-R in humn AMs isolted from helthy smokers nd non-smokers. We demonstrte the presence of uthentic NK-R, s determined y Western lot nlysis nd immunofluorescence, nd indicte tht the NK-R expressed in humn AM re functionl, s demonstrted y the ility of SP to evoke O 2 production nd cytokine relese. We lso present direct evidence tht SP ctivtes the trnscription fctor NF-kB pthwy, so providing new insights on the mechnisms involved in neuropeptidergic control of AM responsiveness. Methods Study popultion This study nd the reserch protocol were pproved y the locl Ethicl Committee. A totl of 25 individuls, 5 mle nd femle sujects, ged etween 28 nd 76 yers, 3 smokers nd 2 non-smokers, were studied. The chrcteristics nd smoking history of the study popultion re presented in Tle, Results section. None of the sujects received medicl therpy t the time of the study. Broncho-lveolr lvge Suject Tle Sex (For M) Study popultion Age (yers) Smoker (BAL) ws minly performed for dignostic purposes to hve further vlidtion/confirmtion of the suspected disese; helthy sujects were individuls who hd no history of crdiopulmonry disese or other chronic disese, no dignosed lung diseses nd were not on mediction. In few cses, the ttriution of helthy suject to the ctegory ws done fter the BAL procedure. Isoltion of humn AMs from BAL Numer of cigrette dy Yers on smoke F 5 Yes M 54 Yes F 43 No F F 4 M 63 Yes M 5 No F F 6 F 28 Yes 7M 43 Yes M 5 No F F 9 M 46 No F F F 29 No F F M 37Yes F 35 No F F 3 M 69 No F F 4 F 56 Yes M 48 No F F 6 M 62 Yes F 7 No F F 8 F 45 Yes M 39 No F F 2 M 45 Yes M 55 No F F 22 F 76 No F F 23 F 33 Yes M 67Yes 3 25 M 58 Yes 2 33 AMs were isolted from BAL s descried (Brunelleschi et l., 996). After informed consent ws otined from ech ptient nd pretretment with prenterl tropine sulphte (.5 mg), irwys were nesthetized with 2% lidocine. A fieroptic ronchoscope ws dvnced nd wedged into the middle loe under direct visuliztion. Lvge ws crried out with 4 2 ml of prewrmed (37C) sterile sline solution in 2-ml liquots with immedite gentle vcuum (syringe) spirtion fter ech injection. The fluid so otined ws filtered through two lyers of sterile surgicl guze nd centrifuged (4 g, 3 min). The whole BAL pellet ws wshed twice in phosphteuffered slt solution (PBS), resuspended in RPMI 64 medium supplemented with 5% fetl clf serum (FCS), 2mM glutmine, mm Hepes, 5 mgml streptomycin nd 5Uml penicillin, nd plted in six-well tissue culture pltes (35 mm dimeter; Costr, U.K.). After 2 h t 37C in humidified 5% CO 2 tmosphere, nondherent cells (minly lymphocytes) were gently removed nd AMs were used for the experiments. Totl cell count nd viility evlution (Trypn lue dye exclusion test, lwys 498%) were performed on Burker hemocytometer. Differentil cell count ws crried out on Diff-Quick (Don Bxter)-stined cytospin smers, counting t lest 4 cells. The dherent cell popultion ws 499% AM. Phenotypicl nlysis ws crried out on cytocentrifuge (Cytospin, U.K.; 5 r.p.m., min) slides y

3 C. Brdelli et l NK receptors on humn lveolr mcrophges 387 employing leukocyte-specific monoclonl ntiodies for CD68, CD4 nd HLA-DR (from Becton Dickinson, U.K.). O 2 production in AMs Adherent AMs (.4 6 cells plte ) were wshed twice with PBS, incuted in RPMI 64 medium (without phenol red, no ntiiotics nd no FCS) nd chllenged with incresing concentrtions of tchykinins for 3 min. SP is the mjor endogenous lignd for NK-R, [Sr 9 Met(O 2 ) ]SP nd Pro 9 SP re selective NK gonists. In the experiments with the NK-R ntgonists CP 96,345, GR82334 ([D-Pro 9 (spirogmm-lctm)leu,trp ]physlemin(-)) nd GR725 (([Pro 9 (spiro-gmm-lctm) Leu,Trp ]SP)), AMs were preincuted for 5 min with these drugs nd then chllenged with tchykinins. The effects of tchykinins were compred with those evoked y phorol 2-mirystte 3-cette (PMA), stndrd stimulus cting s direct protein kinse C ctivtor. The O 2 production ws evluted y the superoxide dismutse (SOD)-inhiitle cytochrome c reduction, the sornce chnges eing recorded t 55 nm in Beckmn DU 65 spectrophotometer. O 2 production ws expressed s nmol cytochrome c reduced/ 6 cells/3 min, using n extinction coefficient of 2. mm (Brunelleschi et l., 2). To void interference with spectrophotometricl recordings of O 2 production, AMs were incuted with RPMI 64 without phenol red. Experiments were performed in duplicte or triplicte; control vlues (e.g., sl O 2 production in the sence of stimuli) were sutrcted from ll determintions. Relese of TNF- nd other cytokines from AMs Adherent AMs were chllenged with the selected stimuli (SP, NK selective gonists, PMA) for 24 h t 37C to ensure mximl cytokine relese. Superntnts were collected nd stored t 2C. TNF-, IL- nd IL- (the ltter ws evluted s the most importnt nti-inflmmtory cytokine) in the smples were mesured using enzyme-linked immunossy kit (Pelikine Compctt humn ELISA kit). The mesurements were performed ccording to the mnufcturer s instructions. The minimum detectle concentrtions of humn TNF-, IL- nd IL- were.4,.5 nd.3 pg ml, respectively. No crossrectivity ws oserved with ny other known cytokine. Control vlues (e.g., cytokine relese from untreted, unstimulted cells) were sutrcted from ll determintions. Results re expressed in pg ml. Immunofluorescence for NK-R in AMs Humn AMs were cultured onto geltin-coted glss slides. The cells were fixed in ice-cold 4% prformldhyde (2 min), wshed twice with PBS, permeilized with.5% Triton X- (5 min, 25C), wshed twice with PBS, nd locked with % FCS, 2% BSA, % glycine,.5% Triton X- in PBS ( h, 25C). The cells were then incuted in the presence of rit ntiody directed ginst the humn NK-R (Snt Cruz Biotechnology, U.S.A.) t dilution of : 2 in PBS overnight t 4C. After wshing, FITC-conjugted nti-rit immunogloulins ( : 3) (Dko Cytomtion, Miln, Itly) were dded (2 h, 25C). After nother wshing with PBS, nucler stining ws performed using Hoechst (.8 mgml, h, 37C) (Sigm-Aldrich, Miln, Itly). Fluorescence ws visulized using -fold mgnifiction. Western lotting of the tchykinin NK-R in AMs Suconfluent AMs ( 6 cells) were wshed twice with icecold PBS nd lysed with ml RIPA uffer (% Triton X-, % sodium deoxycolte,.% SDS, 5 mm Hepes ph 7.4, 5 mm NCl, % glycerol,.5 mm MgCl 2, mm EGTA, mm NF) contining mm N 3 VO 4 nd protese inhiitors ( mgml protinin, mgml pepsttin, 5 mgml leupeptin, mm phenylmethylsulphonyl fluoride-pmsf). Cells were plced on ice for 2 min nd scrped. Cell lystes were sonicted on ice four times for 5 s ech, clered y centrifugtion t 5, g for min t 4C nd the superntnts (cell lystes) trnsferred into new tue. If necessry, cell lystes were stored t 8C. Aout 3 mg totl extrcts were seprted on 8 % SDS PAGE nd trnsferred to nitrocellulose filters (Protrn, Perkin-Elmer Life Sciences, Boston, MA U.S.A.). Nonspecific inding sites on memrne were locked t room temperture for h in TBS-5% BSA nitrocellulose filters. Filters were proed with commercil nti-humn NK-R ntiody (NK-R (H-83): sc-5323, Snt Cruz Biotechnology, U.S.A.; rit polyclonl ntiody mpping t the C-terminus of the humn NK receptor) ( : 2 in TBS-5% BSA) for 2 h t room temperture. Proteins were visulized y using ECL Western lotting detection regents (Perkin-Elmer Life Science, Boston, MA, U.S.A.). Quntifiction of Western lots ws performed y densitometry using Quntity One, -D Anlysis softwre (Bio-Rd, U.S.A.) nd expressed s intensity dt units. Evlution of NF-kB ctivtion The ctivtion of NF-kB induced y SP, NK gonists or PMA ws evluted y mesuring the nucler migrtion (y electrophoretic moility shift ssy (EMSA)) s well s the nucler content of p5 nd p65 suunits (y ELISA nd Western lot). The methods we used re detiled elow. Preprtion of nucler nd cytosolic cellulr frctions After chllenge with stimuli, AMs (out 5 6 cells) were wshed with ice-cold PBS, scrped nd centrifuged t g for 5 min t 4C. The cell pellet ws resuspended in 3 ml of lysis uffer ( mm Hepes, ph 7.6, 6 mm KCl, mm EDTA, mm PMSF, mm dithiothreitol, mlml protese cocktil inhiitors) nd incuted on ice for 5 min. At the end of this incution, 2 ml of % NP-4 ws dded nd the tue vortexed for s. After centrifugtion t 3, g for min t 4C, superntnts (cytosolic frctions) were collected nd stored t 8C, wheres the pellets were further processed to otin nucler extrcts. The pellets were resuspended in extrction uffer (2 mm Tris HCl, ph 8, 42 mm NCl,.5 mm MgCl 2,.5 mm PMSF,.2 mm EDTA, mlml protese cocktil inhiitors, glycerol 25% v v ) nd incuted for 3 min t 4C. Nucler proteins were isolted y centrifugtion t 3, g for 5 min. The superntnt ws liquoted nd stored t 8C until used for EMSA or p5/ p6 ELISA ssys. Protein concentrtions were determined y using protein ssy (Bio-Rd, U.S.A.).

4 388 C. Brdelli et l NK receptors on humn lveolr mcrophges EMSA of NF-kB Nucler extrcts (5 mg) were incuted with 2 mg poly (di-dc) nd the g [ 32 P]ATP-lelled oligonucleotide proe (, 5, c.p.m.; Promeg) in inding uffer (5% glycerol, mm Tris HCl, ph 7.6, 5 mm KCl, mm EDTA, mm dithiothreitol) in finl volume of 2 ml for 3 min t room temperture. The NF-kB consensus oligonucleotide (5 - AGTTGAGGGGACTTTCCCAGGC-3 ) ws otined from Promeg. The nucleotide protein complex ws seprted on 5% polycrylmide gel in.5 TBE uffer ( mm Tris HCl, mm oric cid, 2 mm EDTA) t 5 V on ice. The gel ws dried nd rdioctive nds were detected y utordiogrphy. p5 nd p65/rela ssys Nucler extrcts were prepred s descried ove nd evluted for the presence of p5 nd p65/rela suunits using Trns AMt NF-kB p5 Chemi nd NF-kB p65 Chemi Trnscription Fctor Assy kits (Active Motif Europe, Belgium), ccording to the mnufcturer s instructions. Briefly, n equl mount ( mg) of nucler lyste ws dded to incution wells precoted with n oligonucleotide contining the NF-kB consensus site (5 -GGGACTTTCC-3 ) sequence, the ctive form of NF-kB contined in the cell extrct specificlly inding to this oligonucleotide. Sometimes, the cytosolic content of oth suunits ws lso mesured. These ssy kits specificlly detected ound NF-kB p65 or p5 suunits in humn extrcts; ctivities of p5 nd p65 were mesured y Rosys Anthos Lucy luminometer nd results re expressed s RLU (reltive luminescence unit). In some cses, the nucler extrcts (5 mg protein, for ech smple) were lso used to evlute p5 nd p65 suunits y Western lot. For these ssys, commercil ntiodies (nti-nf-kb p5: 797 nd nti-nf-kb p65: 797) were otined from Acm (U.K.). Nucler extrcts were chllenged for 2 h t room temperture with the ntiody t finl concentrtion of mgml. Drugs nd nlyticl regents Sustnce P, selective NK gonists nd NK ntgonists were otined from Neo-System (Strsourg, Frnce). The nti- NK-R-specific ntiody (NK-R (H-83): sc-5323) ws from Snt Cruz Biotechnology (U.S.A.). The nti-nf-kb p5 nd nti-nf-kb p65 ntiodies were otined from Acm (U.K.). PBS, RPMI 64 (with or without phenol red), BSA, glutmine, Hepes, streptomycin, penicillin, PMA, ethnol, SOD, cytochrome c, N-deoxycholte, NCl, EDTA, protese cocktil inhiitors (protinin.3 mm, esttin 3 mm, leupeptin mm), romophenol lue, glycine, glycerol, methnol nd Tween 2 were otined from Sigm (Milwukee, WI, U.S.A.). Poly(dI-dC) were otined from Phrmci (Uppsl, Sweden). Triton X- nd -mercptoethnol were from Fluk (Buchs, Switzerlnd); PMSF ws from Promeg (Mdison, WI, U.S.A.). SDS nd DMSO were from Merck (Drmstdt, Germny). BCA Protein Assy Regent kit ws from Pierce (Rockford, IL, U.S.A.). Nitrocellulose filters (Hyond) nd the enhnced chemiluminescence system were from Amershm (Buckinghmshire, U.K.). Tissue-culture pltes were purchsed from Costr Ltd (Buckinghmshire, U.K.). All cell culture regents, with the exception of fetl ovine serum, were endotoxin-free ccording to detils provided y the mnufcturer. Fetl ovine serum (lot 4G34 K, contining o EU ml ) ws from Life Technologies Inc. (Rockville, U.S.A.). TNF-, IL- nd IL- immunossy kit ws otined from CLB/Snquin, Centrl Lortory of the Netherlnds Red Cross (Netherlnds). Gel shift ssy Core system nd ll the regents for NF-kB EMSA were from Promeg Corportion (St Louis, CA, U.S.A.). Dt nd sttisticl nlysis Dt re men7s.e.m. of duplicte determintions of n independent experiments. Concentrtion response curves for SP nd NK gonists were constructed nd EC 5 vlues were interpolted from curves of est-fit. When required, sttisticl evlution ws performed y Student s t test. Results Study popultion, BAL nd phenotype of AMs In ll, 25 individuls, 5 mle nd femle sujects (men ge ¼ yers; men ge of mle nd femle sujects: nd yers, respectively, P ¼.7), were studied. A totl of 3 (eight mle nd five femle sujects) were smokers nd 2 (seven mle nd five femle sujects) were non-smokers; men ge of smokers ( yers; n ¼ 3) nd non-smokers (5.874 yers, n ¼ 2) eing very similr. The chrcteristics nd smoking history of the study popultion re listed in Tle. None of the sujects received medicl therpy t the time of the study. Totl nd differentil cell counts in BAL nd phenotype of AMs from smokers nd non-smokers re presented in Tle 2. As expected, significnt (Po.5) increse in the totl cell numer in BAL (with no significnt differences in differentil cell counts) ws oserved in smokers s compred to non-smokers. The gret mjority of AMs (967%) in helthy smokers ws CD68 þ nd high percentge (867 nd 6673%, respectively) of AM expressed lso HLA-DR nd CD4. As known, CD68 expression is relted to the presence of AM involved in the oxidtive urst, CD4 expression is relted to cytokine production y LPS receptor, wheres HLA-DR is relted to ntigen presenttion. The expression CD4 nd CD68 ws significntly (Po.5) higher in AMs collected from helthy smokers s compred to helthy non-smokers (Tle 2). NK-R expression in humn AMs We exmined the expression pttern of the NK-R gene products in humn AMs. We collected AMs from helthy smokers nd helthy non-smokers undergoing BAL for dignostic procedures, fter their informed consent. First, we performed immunofluorescence ssys in AMs isolted from helthy non-smokers nd oserved the expression of the NK- R protein loclized primrily t the cell surfce (Figure ). A phse contrst photomicrogrph of AM is shown in Figure. Immunofluorescence with nti-nk-r ntiody nd FITC-conjugted nti-rit ntiody revels green colour (Figure ), nucler stining with Hoechst (.8 mgml, 3 min, 37C) is depicted in lue (Figure c),

5 Tle 2 Totl nd differentil cell count in BAL nd AM phenotype C. Brdelli et l NK receptors on humn lveolr mcrophges 389 Sujects Totl cell ml BAL AMS (%) Lympho (%) PMNs (%) CD68+ (%) HLA-DR+ (%) CD4+ (%) Smokers (n ¼ 3) Non-smokers (n ¼ 2) Dt re given s totl cell numer ml BAL nd percentge of totl cell popultion (differentil) in BAL. AMs ¼ lveolr mcrophges; Lympho ¼ lveolr lymphocytes; PMNs ¼ lveolr neutrophils. The AM phenotype ws evluted y mesuring CD68, CD4 nd HLA- DR: positive cells re expressed s percentge of totl AMs. Denotes Po.5 vs smokers. MW J774.A HEALTHY S HEALTHY S HEALTHY NS HEALTHY NS 75 KD NK r 5 KD Figure Immunocytochemicl nlysis of the expression nd loction of NK-R in humn AMs. () Phse contrst. () Immunofluorescence of nti-nk-r polyclonl A followed y FITC-conjugted nti-rit immunogluulins (green). (c) Nucler stining with Hoechst (lue). (d) Merge of B nd C. Fluorescence ws visulized y verticl fluorescence microscopy (-fold mgnifiction; Eclipse E6, Nikon, Tokyo, Jpn). nd merge of oth visulizes NK-R on AM surfce (Figure d). We next performed Western lot in AMs isolted from helthy smokers nd non-smokers to evlute NK-R expression t the protein level (Figure 2). AMs were otined from four helthy smokers (sujects, 2, 4 nd 2) nd four helthy non-smokers (sujects 3, 5, nd 2); the experiments were performed seprtely (y using the sme protein mount, 3 mg, for ech individul ssy) nd were lwys compred with the NK-R expression in the positive control, the cultured J774.A cells ( murine mcrophge cell line). As shown in Figure 2, Western lot nlysis of J774.A cells, AMs from two helthy smokers (sujects nd 2) nd two non-smokers (sujects 3 nd 5), proed with the polyclonl nti-nk-r ntiody, revels three prominent nds of 68, 53 nd 42 kd, respectively (Figure 2). According to mnufcturer s instructions, the commercil polyclonl ntiody we used detected protein of out 68 kd. As known, NK-R possess different sites for cetyltion nd phosphoryltion nd my e present s truncted forms (Li et l., 997; Pge & Bell, 22; Cerlotto et l., 23). In our experiments, we lwys oserved positive nd t 68 kd (s indicted y the mnufcturer) nd two nds of 53 nd 42 kd (Figure 2). Such oservtions re in keeping with previous reports in which other uthors, y using noncommercil monoclonl NK-R ntiodies, detected 46 kd protein in humn ntrl tissue (Smith et l., 2), moleculr mss nd of 53 kd in the monocyte/mcrophge THP- cells (Simeonidis et l., % expression NK r J774.A S NS Figure 2 NK-R expression in humn AMs from helthy smokers (S) nd non-smokers (NS). In (), Western lot nlysis of NK-R in AM extrcts of two smokers nd two non-smokers. The lots re ssemled from different single experiments in which AMs from S or NS hve een evluted. As positive control for the presence of NK-R, the mcrophge cell line J774.A ws used (for clrity, only one lot of J774.A cells, representtive of seven others, is shown). The sme protein mount (3 mg) ws used in ech experiment with AMs from smokers nd non-smokers. An rrowhed indictes the 68-kD nd corresponding to the receptor. The migrtion of protein stndrds of known sizes is shown on the left. In (), quntittive evlution of NK-R expression y densitometry. Intensity of the specific nd of NK-R in the mcrophge J774.A cells mounted to intensity units (mens7s.e.m. of eight experiments) nd ws tken s %. Results re expressed s % expression of the positive control; men þ s.e.m. of four experiments for S nd NS. 23) or 42 kd protein in murine peritonel mcrophges nd murine microgli (Mrriott & Bost, 2; Rsley et l., 22). Interestingly, densitometric evlution of NK-R expression reveled tht AMs collected from helthy smokers demonstrted 43-fold increse s compred to AMs isolted from helthy non-smokers (Figure 2). The intensity of the specific nd of 68 kd in the positive control, the J774.A cell line, mounted to intensity units (n ¼ 8) nd ws tken s %. NK-R expression in AMs from helthy nonsmokers ws 327.5% (n ¼ 4), wheres NK-R expression in AMs from helthy smokers mounted to 9674% (n ¼ 4). NK-R re functionl in humn AMs: O 2 production nd cytokine relese Bsl vlues (O 2 production from unstimulted AMs) in smokers nd non-smokers were (n ¼ 6) nd

6 39 C. Brdelli et l NK receptors on humn lveolr mcrophges (n ¼ 5; Po.) nmol cytochrome c reduced/ 6 AMs, respectively. These vlues were sutrcted from those otined fter tchykinins or PMA chllenge to otin the net O 2 production. PMA, used t 7 M ( ner mximl concentrtion), produced (n ¼ 6) nd 77.6 (n ¼ 5; Po.5) nmol cytochrome c reduced/ 6 AMs in smokers nd nonsmokers, respectively. In the concentrtion rnge 2 6 M, SP dose-dependently evoked O 2 production in AMs from oth smokers nd non-smokers, higher production eing oserved in smokers (Figure 3). As depicted in Figure 3, mximl ctivtion y SP ws oserved t 6 M,EC 5 s eing.25 nm in smokers nd nm in non-smokers. In the presence of cocktil of inhiitors (thiorphn, cptopril nd esttin, ll t mm) of tchykinin degrding enzymes, SP effects were significntly enhnced (dt not shown). The metoliclly stle NK-R gonist [Sr 9 Met(O 2 ) ]SP, lthough less potent thn SP, evoked significnt respirtory urst in AMs collected from oth smokers nd non-smokers, EC 5 s eing out 3 nm in smokers nd nm in non-smokers (Figure 3). Pro 9 SP, the other NK selective gonist we used, cted dosedependently, lthough less ctive nd potent thn SP or [Sr 9 Met(O 2 ) ]SP (Figure 3). EC 5 s for Pro 9 SP were out nmol cytochrome C reduced/ 6 AM nmol cytochrome C reduced/ 6 AM SP Sr9 Pro9SP SMOKERS log [DRUGS] (M) SP Sr9 Pro9SP NON-SMOKERS log [DRUGS] (M) Figure 3 NK-R gonists evoke O 2 production in humn AMs isolted from helthy smokers () nd non-smokers (). Cells were chllenged with incresing concentrtions of SP, [Sr 9 Met(O 2 ) ]SP nd Pro 9 SP for 3 min. Results re mens7s.e.m. of five to six experiments in duplicte. Po.5 vs non-smokers. nm in smokers nd 3 nm in non-smokers (Figure 3). The nonpeptide NK selective ntgonist CP 96,345 t nm competitively ntgonized the effects of SP nd NK selective gonists: the dose response curve for SP ws shifted to the right out two orders of mgnitude (Figure 4) s were those for [Sr 9 Met(O 2 ) ]SP (out.5-fold; Figure 4) nd Pro 9 SP (one order of mgnitude; not shown). GR82334, reversile NK ntgonist devoid of histmine-relesing properties in rt mst cells (Guo et l., 995), nd GR725, selective NK ntgonist tht possesses GABA-relesing ctions in rt spinl cord (Hgn et l., 99), t mm competitively ntgonized the effects of SP nd NK selective gonists in AM (dt not shown). We lso evluted the relese of proinflmmtory cytokines, nmely TNF- (the most undnt cytokine in AMs) nd IL-, s well s IL- relese (the most relevnt ntiinflmmtory cytokine in AMs), fter chllenge with tchykinins or PMA. Bsl vlues (tht is the relese from nmol cytochrome C red./ 6 AM nmol cytochrome C red./ 6 AM SP SP + CP96, log[sp] M Sr9 Sr9 + CP96, log[sr 9 ] M Figure 4 The NK selective ntgonist CP96,345 competitively ntgonizes SP-induced O 2 production () nd [Sr 9 Met(O 2 ) ]SPinduced O 2 production () in AMs from helthy smokers. Cells were preincuted with CP 96,345 t nm for 5 min nd then chllenged with the NK gonists for further 3 min. Results re mens7s.e.m. of four experiments in duplicte.

7 C. Brdelli et l NK receptors on humn lveolr mcrophges 39 Tle 3 Bsl relese of cytokines in AMs collected from helthy smokers nd non-smokers Cytokine Smokers (n ¼ 6) Non-smokers (n ¼ 6) TNF- (pg ml ) IL- (pg ml ) IL- (pg ml ) Vlues re mens7s.e.m. of experiments in triplicte. Po.5 vs non-smokers. unstimulted AM) were sutrcted from ll determintions nd re listed in Tle 3. As shown in Figure 5, SP (Figure 5) nd the NK gonist [Sr 9 Met(O 2 ) ]SP (Figure 5) dosedependently induced TNF- relese from AMs, significntly higher effect eing oserved in AMs from helthy smokers s compred to non-smokers (Figure 5 nd ; Po.). CP 96,345, tested t nm, competitively ntgonized SP-induced TNF- relese in smokers AMs (Figure 5c), so confirming tht this effect is medited y NK-R ctivtion. Also PMA, t 7 M, induced higher TNF- relese in AMs from smokers (53576 pg ml ; n ¼ 8) s compred to non-smokers (3672 pg ml ; n ¼ 6; Po.5). By evluting IL- production from humn AMs, we oserved tht SP cted dose-dependently, mximl relese (out 4 pg ml ) eing detected t. mm, with no mjor difference etween smokers nd non-smokers (Figure 6). PMA, t 7 M, lso relesed similr mounts of IL- in oth smokers nd nonsmokers (Figure 6). SP, in dose-independent wy, relesed very smll mounts of the nti-inflmmtory cytokine IL- (Figure 6) y humn AMs, higher ut not significnt relese eing oserved in non-smokers. As depicted in Figure 6, PMA 7 M did not stimulte IL- secretion, while inducing IL- relese from humn AMs. SP nd NK-R gonists induce NF-kB ctivtion Previous oservtions indicte tht, in different cell models, SP cn induce the ctivtion of the trnscription fctor NF-kB. We checked this hypothesis in humn AMs y first evluting the nucler trnsloction of NF-kB y EMSA nd, to ensure etter quntittive evlution, the mounts of trnslocted p5 nd p65 suunits y n ELISA kit. As known, lthough different NF-kB forms hve een descried, the p5 p65 heterodimer is the predominnt species in mny cell types (Bldwin, 996). As positive control for the detection of NFkB ctivtion, humn AMs were stimulted y PMA, s this gent hs previously een demonstrted to induce NF-kB nucler trnsloction in humn monocytes (Lvgno et l., 24). To investigte the time- nd dose-dependent effects of SP, AMs from helthy non-smokers (Figure 7) nd smokers (Figure 7) were chllenged with two different concentrtions of SP ( 8 nd 6 M) for different times ( 2 h). As reported in Figure 7, SP-induced NF-kB ctivtion, just detectle fter h, ws mximl t 2 h nd hd out the sme intensity s PMA (Figure 7). Interestingly, AMs otined from helthy smokers (Figure 7) demonstrted constitutively (control, unstimulted AMs, lne 7) enhnced nucler trnsloction of the trnscription fctor NF-kB s compred to AMs isolted from non-smokers (Figure 7, lne 7). Accordingly, PMA- nd SP-induced nucler trnsloction ws higher in AMs from TNF-α (pg ml - ) TNF-α (pg ml - ) c TNF-α (pg ml - ) non smokers smokers log[sp] M non smokers smokers log[sr 9 ] M 5 SP SP + CP96, log[sp] M Figure 5 NK-R stimultion induces TNF- relese in humn AMs isolted from helthy smokers nd helthy non-smokers. SPinduced TNF- relese in (); [Sr 9 Met (O 2 ) ]SP-induced relese in (); reversl y CP 96,345 nm of SP-induced relese in smokers in (c). Dt re mens7s.e.m. of five to six experiments in duplicte. Po. vs non-smokers. See text for further detils. smokers (Figure 7, lnes 2: PMA 6 M, 2 nd h chllenge, respectively; lnes 3 6: SP 6 nd 8 M, or 2 h chllenge) s compred to non-smokers. The NK-R ntgonist CP96,345 ((2S,3S)-cis-2-diphenylmethyl-N[(2-methoxyphenyl) -methyl]--zicyclo-octn-3-mine)), here evluted t mm, while not ffecting per se PMA-stimulted trnsloction

8 392 C. Brdelli et l NK receptors on humn lveolr mcrophges 8 smokers non smokers NF-κB Non smokers IL-β (pg ml - ) IL- (pg ml - ) log[sp] M smokers non smokers log[sp] M PMA -7 M PMA -7 M Figure 6 Effects of SP on IL- relese () nd IL- relese () in humn AMs isolted from helthy smokers nd helthy nonsmokers. PMA-induced relese is shown for comprison. Dt re mens7s.e.m. of five to six experiments in duplicte. NF-κB Smokers Figure 7 SP induces NF-kB ctivtion in humn AMs from helthy non-smokers () nd helthy smokers () in time- nd dose-dependent mnner. AMs were stimulted with SP ( 6 nd 8 M)orPMA 6 M for or 2 h, in the presence or sence of CP 96,345. The NK-R ntgonist ws evluted t mm nd preincuted for 5 min. Nucler extrcts (5 mg) were prepred nd ssyed for NF-kB ctivity y EMSA (see text for further detils). In () (non-smoker): lne ¼ PMA 2 h; lne 2 ¼ PMA h; lne 3 ¼ SP 6 M h; lne 4 ¼ SP 6 M 2 h; lne 5 ¼ SP 8 M h; lne 6 ¼ SP 8 M 2 h; lne 7 ¼ control, unstimulted AM; lne 8 ¼ CP 96,345 þ SP 8 M 2 h; lne 9 ¼ CP þ SP 6 M 2 h; lne ¼ CP 96,345 lone; lne ¼ CP 96,345 þ PMA 2 h. In () (smoker): lne ¼ PMA 2 h; lne 2 ¼ PMA h; lne 3 ¼ SP 6 M 2 h; lne 4 ¼ SP 6 M h; lne 5 ¼ SP 8 M 2 h; lne 6 ¼ SP 8 M h; lne 7 ¼ control, unstimulted AM; lne 8 ¼ CP 96,345 þ SP 8 M 2h; lne 9 ¼ CP þ SP 6 M 2 h; lne ¼ CP 96,345 lone; lne ¼ CP 96,345 þ PMA 2 h. This experiment ws performed three times with similr results. (Figure 7, lne ) nd sl constitutive ctivity (lne ), potently reduced SP-induced effects (lnes 8 nd 9), so confirming tht SP-induced NF-kB nucler trnsloction in humn AMs is receptor-medited effect. Moreover, the NK ntgonist seemed to e less effective in reducing SP-induced nucler trnsloction in smokers, s compred to smokers. The utordiogrphs presented in Figure 7(which re representtive of two other dditionl experiments) re reltive to AMs from smokers nd non-smokers, which were otined nd contemporrily processed on the sme dy; the two gels presented s Figure 7 (non-smokers) nd Figure 7 (smokers) were run concomitntly. Competition experiments performed with -fold excess unlelled NF-kB sequence demonstrted the specificity of the induced NF-kB/DNA inding complex (not shown). In AMs collected from helthy smokers, SP effects re reproduced, to out the sme intensity, y the NK gonist [Sr 9 Met(O 2 ) ]SP, evluted t 8 nd 6 M (Figure 8). The ntgonist CP 96,345 potently reduced the effects of the NK gonist, so confirming tht NF-kB nucler trnsloction is receptor-medited effect (Figure 8). We lso performed Western lot experiments to evlute the nucler trnsloction of p5 nd p65 suunits of the NF-kB complex in AMs from oth smokers nd non-smokers. As depicted in Figure 9 (: p5 suunit nd : p65 suunit), PMA, SP nd the NK gonist [Sr 9 Met(O 2 ) ]SP induced the trnsloction of oth suunits, n incresed p5 trnsloction eing oserved in smokers (Figure 9). To ensure etter quntittive evlution, we lso ssessed the trnsloction of p65 suunit nd p5 suunit in AMs from oth smokers nd non-smokers, y using commercilly ville ELISA kit. As depicted in Figure, SP dosedependently induced p5 trnsloction (Figure ) nd p65 trnsloction (Figure ) in AMs, significntly (Po.5) enhnced effect eing oserved in smokers especilly for the p5 suunit. The NK gonist [Sr 9 Met(O 2 ) ]SP, here evluted t mm, lso incresed p5 nd p65 trnsloction, AMs from smokers depicting significnt enhnced p5 trnsloction. PMA-induced trnsloction is shown for comprison (Figure ). CP 96,345 t 6 M significntly reduced SP-induced nucler p5 nd p65 trnsloction (not shown). Results re expressed s the nucler/cytosolic rtio, which is the rtio etween the mount of p5 (or p65) in nucler extrcts nd cytosolic extrcts. Discussion These results demonstrte tht humn AMs, isolted from oth helthy smokers nd non-smokers, possess functionl NK-R. In fct, we here report the presence of NK-R protein (y Western lotting) s well s the ility of SP, the endogenous NK-R lignd, to evoke O 2 production nd

9 C. Brdelli et l NK receptors on humn lveolr mcrophges 393 NF-κB Figure 8 The NK selective gonist [Sr 9 Met(O 2 ) ]SP induces NFkB ctivtion in humn AMs from helthy smokers nd its effects re reduced y the NK-R ntgonist CP 96,345. AMs were stimulted with [Sr 9 Met(O 2 ) ]SP 8 or 6 M for 2 h, in the presence or sence of CP 96,345 t mm. The effects of SP 6 M re shown for comprison. Nucler extrcts (5 mg) were prepred nd ssyed for NF-kB ctivity y EMSA (see text for further detils). Lne ¼ control, unstimulted AM; lne 2 ¼ Sr 9 8 M; lne 3 ¼ Sr 9 6 M; lne 4 ¼ SP 6 M; lne 5 ¼ CP 96,345 þ Sr 9 6 M. This experiment ws performed three times with similr results. P5 suunit P65 suunit NS S RATIO N/C RATIO N/C p5 suunit control PMA -6 M SP -6 M SP -8 M Sr9-6 M p65 suunit non smokers smokers control PMA -6 M SP -6 M SP -8 M Sr9-6 M non smokers smokers Figure NK-R stimultion induces the trnsloction of p5 () nd p65 () suunits in humn AMs from helthy smokers nd helthy non-smokers. PMA-induced trnsloction is shown for comprison. AMs were chllenged for 2 h with the stimuli; nucler nd cytosolic extrcts were prepred nd evluted for their content in p5 nd p65 suunits. Results re expressed s the nucler/ cytosolic rtio (rtio N/C) for oth p5 nd p65 suunits. Dt re mens7s.e.m. of five experiments in duplicte. Po.5 vs nonsmokers. NS β ctin S cytokine relese in AMs, s well s to induce ctivtion of the trnscription fctor NF-kB. These effects re ll receptormedited, s they re reproduced y NK selective gonists nd reverted y NK-R ntgonists. S β ctin NS Figure 9 Western lots of p5 nd p65 suunits in AMs from oth non-smokers (NS) nd smokers (S). The nucler trnsloction of p5 is reported in (); the trnsloction of p65 is reported in (). Bet-ctin is shown for comprison. Lne ¼ control; lne 2 ¼ PMA 6 M; lne 3 ¼ SP 6 M; lne 4 ¼ SP 8 M; lne 5 ¼ Sr 9 6 M. NK-R expression hs een evluted y different uthors in different cell types, y using RT PCR technology, minly. By this pproch, humn monocytes nd monocyte-derived mcrophges were shown to express SP nd NK-R, esides producing nd relesing SP (Ho et l., 997). Moreover, n NK-R ntgonist downregulted SP mrna expression in monocyte-derived mcrophges (Li et l., 22). Li et l. (22) quntified SP mrna in different cells y rel time RT PCR nd reported lrge vriility in the level of trnscripts in monocyte-derived mcrophges, the numer of SP mrna copies/mg totl mrna detected in preprtions from four different donors rnging from 949 to 3,388 (Li et l., 22). Therefore, given the demonstrted lrge vriility in SP trnscripts (Li et l., 22) nd depending on the numer of collected AMs for the evlution of ll the other prmeters (respirtory urst, cytokine relese, NF-kB ctivtion), in our AMs preprtions, we evluted NK-R expression t the protein level, only. By using noncommercil monoclonl nti-nk-r ntiody (rised in chicken ginst the finl 5 mino cids t the C-terminus of the rt NK-R), Smith et l. (2) demonstrted the presence of NK-R in humn ntrum nd detected oth positive nd t 46 kd nd lrger moleculr mss nd of kd, representing the glycosylted form of the NK-R. Two NK-R isoforms tht differ in the length of the cytoplsmic croxyl-terminus hve

10 394 C. Brdelli et l NK receptors on humn lveolr mcrophges een reported (Fong et l., 992; Mntyh et l., 996; Li et l., 997): in the rt, the full-length nd the truncted receptor presented moleculr weights of the receptor proteins of out 8 nd 5 kd, respectively, the deglycosylted receptors eing 46-kD nd 37-kD, respectively (Li et l., 997). The truncted receptor, t vrince from the full-length one, did not undergo rpid nd long-lsting desensitiztion (Li et l., 997); cells possessing the short NK-R isoform would, therefore, e expected to hve prolonged responsiveness. By mens of noncommercil NK-R ntiodies, 42 kd protein ws detected in murine peritonel mcrophges nd microgli (Mrriott & Bost, 2; Rsley et l., 22), wheres 53 kd protein ws demonstrted in the THP- cells (Simeonidis et l., 23). Given the heterogeneity of NK-R nd the fct tht we used commercil polyclonl nti-nk-r ntiody, we detected three prominent nds of 68 (s indicted y the mnufcturer), 53 nd 42 kd in humn AMs, in ccordnce to wht oserved y others in cells of the monocyte/mcrophge linege. Interestingly, AMs from helthy smokers demonstrted 43-fold increse in NK-R expression s compred to helthy non-smokers: these results re in keeping with previous dt indicting the key role for SP nd NK-R ctivtion in tocco-induced lung toxicity in oth nimls nd humns (Dusser et l., 989; Tomki et l., 995; Wu & Lee, 999) nd further extend oservtions from Bi et l. (995), who detected n incresed NK-R expression in lung iopsies from smokers. Furthermore, we hve determined the functionl nture of these NK-R y demonstrting the ility of SP nd selective NK gonists to induce O 2 production nd cytokine relese from humn AMs. These oservtions extend our previous dt in oth humn nd guine-pig AMs (Brunelleschi et l., 99; 992; 996), demonstrting n enhnced responsiveness to tchykinins in AMs isolted from helthy smokers. By evluting the respirtory urst, we mesured significntly incresed O 2 production when AMs from smokers were chllenged with SP or selective NK gonists, EC 5 s eing.25 nm in smokers nd nm in non-smokers for SP nd 3 nm in smokers nd nm in non-smokers for [Sr 9 Met(O 2 ) ]SP. To further confirm the receptor nture of these effects, the non-peptide NK ntgonist CP 96,345 t nm shifted to the right the concentrtion response curve for oth endogenous nd synthetic NK lignds. TNF-, IL-, IL-2 nd IL-6 re frequently encountered proinflmmtory cytokines, which re involved in vriety of immunologicl functions s well s interction with different trget cells. TNF- is secreted y monocyte/mcrophges minly nd, esides exerting cytotoxic ctivity to tumor cells, hs key role in chronic inflmmtion. TNF- induces the expression of, nd enhnces cellulr responsiveness to, other cytokines nd growth fctors, nd ffect signl trnsduction pthwys leding to prolifertion. SP nd NK selective gonists dose-dependently induce TNF- relese from humn AMs, more thn douled significnt TNF- relese (Po.) eing oserved in AMs from helthy smokers. CP 96,345 t nm competitively ntgonize these effects, further confirming the receptor-medited nture of SP-induced effects in humn AMs. In the concentrtion rnge nm mm, SP lso induces IL- relese from AMs, no significnt differences eing oserved etween smokers nd non-smokers. This result ws somewht unexpected lso considering the fct (see elow) tht SP nd NK-R gonists ctivte the trnscription fctor NF-kB. As known, the regultion of TNF- nd IL- production is lrgely NF-kB-dependent, lthough evidence exists tht TNF- nd other cytokines cn lso e induced through NF-kB-independent pthwys (Bondeson et l., 999; Andrekos et l., 24). We hve no definitive explntion for SP-induced IL- relese eing similr in smokers nd nonsmokers; however, PMA, too, lthough ctivting NF-kB nd inducing cytokine relese, did not relese n enhnced mount of IL- in smokers. Moreover, AMs isolted from smokers nd chllenged with LPS relesed significntly decresed mounts of IL- s compred to non-smokers (Brown et l., 989; Ymguchi et l., 989). Brown et l. (989) concluded tht there is defect in relese ut not production of IL- from the AMs of chronic smokers. In ddition, SP exerts inconsistent effects on IL- relese, in keeping with previous oservtions on peripherl lood mononucler cells from helthy donors (Kim et l., 23). An importnt component controlling the synthesis of mny cytokines nd other proinflmmtory gene products is the trnscriptionl ctivtor NF-kB (reviewed in Bldwin, 996). Five relted mmmlin gene products prticipte in NF-kB functions (p5/nf-kb, p52/nf-kb2, p65/rel A, c-rel nd RelB), the predominnt species in mny cell types eing p5- p65 heterodimer. As known, the trnscription fctor NF-kB regultes the expression of mny proinflmmtory genes, including those of TNF- nd IL-, nd, in turn, these inflmmtory cytokines re potent inducers of NF-kB ctivtion (Bldwin, 996). SP specificlly ctivtes NF-kB in cells of the monocyte/ mcrophge linege, for exmple, humn strocytom cells, murine peritonel mcrophges nd dendritic cells (Lie et l., 997; Mrriott et l., 2), ut no informtion re ville concerning humn AM. We originlly report here tht ctivtion of NK-R y SP or [Sr 9 Met(O 2 ) ]SP dosedependently stimultes nucler trnsloction of NF-kB, s evluted y EMSA. This effect is reverted y the NK ntgonist CP 96,345. Interestingly, the entity of the effect is similr to the PMA-induced one, so indicting SP s potent ctivtor of this trnscription fctor in humn AMs. Interestingly, SP induced three-fold increse (s evluted y densitometry) in NF-kB nucler trnsloction in AMs isolted from helthy smokers s compred to non-smokers. Consistent with previous reports in which humn AMs were used (Crter et l., 998; Frver et l., 998), constitutive expression of NF-kB in the nucleus of unstimulted AMs ws lwys oserved in our experiments. By using specific NF-kB DNA-inding sequences for IL-6, IL-8 nd TNF- promoters, Crter et l. (998) demonstrted tht different NF-kB complexes re generted in AMs from helthy volunteers nd tht specific NF-kB complexes re used for the trnscription of these cytokine genes. They found tht oth p5 nd p65 proteins ound to the IL-6 sequence, wheres p5 protein ound to the TNF sequence nd p65 protein ound to the IL-8 sequence (Crter et l., 998). By Western lot ssys nd ELISA kits, we hve detected oth p65 nd p5 suunits in humn AMs. In our experiments, the p5 suunit seems to e the most undnt one in AMs from oth smokers nd non-smokers, eing more efficiently trnslocted in smokers. In fct, unstimulted AMs from helthy smokers presented more thn douled nucler trnsloction of p5 (ut out the sme for p65) s compred to AMs from non-smokers. When AMs were chllenged y

11 C. Brdelli et l NK receptors on humn lveolr mcrophges 395 PMA or NK-R gonists, further enhnced nucler trnsloction of NF-kB suunits ws oserved: SP nd [Sr 9 Met(O 2 ) ]SP were prticulrly effective on p5 trnsloction (out three-fold) nd their effects were significntly enhnced in AMs from smokers. PMA, too, ws very potent (more thn three-fold) in inducing p5 trnsloction nd lso significntly stimulted (lthough to lesser degree) p65 trnsloction in helthy smokers. On the contrry, SP nd [Sr 9 Met(O 2 ) ]SP potentited p65 nucler trnsloction, ut no significnt vritions were oserved etween smokers nd non-smokers. This oservtion deserves further investigtions ut, in our opinion, is in keeping with the dt demonstrting p5 s the mjor NF-kB suunit for the trnscription of TNF gene (Crter et l., 998). In fct, mong the cytokines we evluted, TNF- is the one relesed to greter mounts y SP nd NK gonists, wheres inconsistent effect on IL- relese re oserved. As known, IL- exerts nti-inflmmtory effects nd inhiits NF-kB ctivtion in LPS-stimulted humn AMs (Rychudhuri et l., 2). Moreover, ccording to the fct tht rective oxygen intermedites (in prticulr hydrogen peroxide) hve een proposed s second messenger molecules in the ctivtion pthwy of NF-kB nd tht ntioxidnts usully inhiit NF-kB ctivtion (Lie et l., 997), the demonstrted SP ility to induce oxy-rdicl production could ply role in SP-induced trnsloction of the trnscription fctor. To our knowledge, this is the first pper tht descries NK-R expression nd ctivtion in humn AMs s whole, lthough other different reports hve investigted, in monocyte/mcrophges of different origin, some of the points we focused here. In conclusion, this pper indictes SP s potent contriutor for tocco smoke toxicity. We thnk Pfizer (Itly) for the kind gift of the NK-R ntgonist CP 96,345. We lso re indeted with Drs Donto Colngelo nd Luigi Grzi Fresu (School of Medicine, Novr, Itly) for helpful discussion. 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