\ ì r l . 2 0 . r o . 1 . 7 5 . 1 - 7 ó . 1 ( 2 ( X ) 7 ) t \ I t . t R \ \ l t { " \ t " l ( ) L l ì \ ' \ l ' O I l \ l \ l t i \ O P \ ' l l l ( ) l ' ( X ; \ ' \ \ l ) l ' l l \ l l l \ t A ( O l ' ( X ; \
I F . I L A M M A T I O N , O X I D A T I V E S T R E S S A N D S Y S T E M I C E F F E C T S I N M I L D C H R O N I C O B S T R U C T I V E P U L M O N A R Y D I S E A S E
M . P . F O S C H I N O B A R B A R O I , G . E . C A R P A G N A N O I , A . S P A N E V E L L O I ' 2 , M.G. CAGNAZZOT and P.J. BARNESs
tlnsl!irÍe o/ Respiratoty Di,sease, Department of Medic:al and Ot:cupatbnal Scienc'es, Universily oJ' Foggit.t. tFonclazione Salvatore Maugeri, Care and Research Insîilute, Cassano delle Murye, Bari, Italy;
''"jDt:pt.
o/'Thoracic Medicine, National Heart and Lung Institute, Imperial College, London, UK Received February 23, 2007 - Accepted september 12, 2007
Chrqrnic obstructive pulmonary diseases (CoPD) is a pulmonary disease characterized by systemic abnormralities. l-he aim bf this study is to investigate inflammation and systemic effects in mild COPD' Twentlt-seven mild stable smoking related COPD patients and 15 age-matched healthy subjects were enrollc'd in the study. IL-6, TNF-0 and IL-4 in plasma, sputum and exhaled breath condensate were measuned. We also measured exhaled nitric oxide (NO) and pH in sputum and in breath condensate' Moreriver, fat-fiee mass, body mass index (BMI), respiratory muscle strength, plasma oxidative stress and 1-reactive protein (CRP) were measured. Higher concentrations were found of CRB of diacron reactiYe oxygen metabolites (DROMs) and of IL-6, TNF-0 and IL-4 either in plasma or in supernatant of induced sputum or in exhaled breath condensate of COPD subjects compared to healthy controls' Furthcrmore, higher concentrations were observed of exhaled NO and lower exhaled pH in breath condq,nsate of COpD when compared with healthy subjects. In the group of COPD patients, the subjccts with airway reversibility showed an increase of sputum eosinophils and exhaled N0, whereas the surbjects without airway obstruction reversibility showed an increase in sputum neutrophils, TNF-o and IL-6. We also fbund a trend towards a decrease in fat-free mass and respiratory muscle strength in CcJpD compared to healthy subjects and a negative correlation between these systemic indices (fat- free mass, maximal inspiratory pressure, maximal expiratory pressure) and TNF-0 concentrations in the blood, sputum and breath condensate. We conclude that mild COPD subjects present an increase in inflammatory markers in blood and in airways of similar pattern and furthermore' the neutrophilic pattern of airway inflammation observed in the group of COPD subjects without an airway obstruction revcrsibility makes it more likely that systemic features are present.
r,hronic obstructive pulmonary disease (COPD) of a wider definition of this disease (2). Several is lsually considered to be a pulmonary disease (l). systernic features have been described in COPD Horvcver, recent studies have dcrnonstrated scveral patients, including systcrnic inffammation attd e x l * - p u l m o n a r y a b n o n n a l i t i e s , p a r t i c u l a r l y i n m o r e o x i d a t i v c s t r c s s , s k c l c t a l m u s c l e d y s f u n c t i o n ( s M D ) ' scrrrely aff-ccted patients, that suggcst the necessity wcight loss, atlrcrosclcrosis and ostcoporosis (3-5)'
Kct,uttrd;;: ('OPD, l'ttrtnthodilctlor rat,cr.tibilil.r,,.s-t':;tentit al/at'ts, influnltttrlirtrt, tt'ritlutit't"slrt"s's
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r c D r t x l t r e c r l $ i l l l ( ) t l l w r i t l c l l l ) t r l l ì l \ \ l ( ) l ì t t r r t t t tl t e e t r l t ) t t g l t t lr L r L l e r
7 5 3 t " " i t ì l i J ì ì t * " ì ' ì ' ì t ' i ' " " ' " t l ' " ' " 1 ' i r r t i r r r r t t e i ; r l l r k l { r t l r c f p c r r r r l r r c \
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1'hcsc systcrlic cfTècts nray havc au advcrse cf-fcct o r r th c c l u a l i t y o f l i f b a r r c ' l r r a y contributc t o w a r d s t h c r l o r t a l i t y o f C O P D ( ( r - t ì ) . S y s t c n r i c i n f l a r n r n a t i o n antl oxidativc strcss appcar to bc risk fhctors fbr t l r c r r r e r j o r i t y o f t l r c s y s t c r r i c corrrplications i r r C O P D p a t i c n t s ( 9 ) . Scvcral hypothcscs h a v c bccrr s u g g c s t c c l t o c x p l a i n how pulrnonary i n f l a r n r n a t i o r r a n d o x i c l a t i v c s t r c s s r n a y s p i l l over into tlrc systcrrric c i r c r " r l a t i o n ( l0- I I ). Thcrc may bc a gcnetic p r e d i s p o s i t i o n i n s o m e COPD subjccts t o d c v e l o p b o t h s y s t e r n i c a n d p u l n r o n a r y i n f l a m m a t i o n ( 1 2 - 1 3 ) . However, to date the exact mechanisms through which pulmonary inflamrnation and oxidative stress r e s u l t i n s y s t e m i c e f f e c t s i n C O P D remain u n k n o w n
(2, e).
Moreover, increased reversibility to bronchodilators in COPD patients may share some of the features of asthma, such as eosinophilia ( 1 4 - 1 6 ) and increased e x h a l e d n i t r i c oxide (NO) ( l7). Recently, our group demonstrated that COPD subjects with reversible obstruction present lower oxidative stress in airways ( l8). These more reversible COPD patients may represent an intermediate group of patients between asthma and non-reversible COPD or may be COpD patients who also have concomitant asthma (19).
The aim of this study is to investigate airway inflammation and systemic effects in mild COPD when compared with a group of healthy subjects.
In particular we measured interleukin (lL)-6, tumor necrosis factor (TNF)-c and interleukin (lL)-4 in plasma, induced sputum and breath condensate, exhaled nitric oxide (NO), exhaled pH, inflammarorv sputum cells and plasmatic C reactive protein (pCR) to investigate airway and sistemic inflammation and fat-free mass, body mass indcx (BMI), rcspirator.y tnuscle strength, plasma oxidative stress to study the systemic effects of COpD.
I n s t i t u t c , U n i v c r s i t y o f ' F o g g i a , a n c l w r . i i l , . . r r i n l ì r r r n c d coriscnt was obtainccl lionr all sub-jects. l-lr,r struly waì approvecl by thc lnstitutiorral Ethics C'ornnriil,:c
N o n c o f ' t h c C ' O P D p a t i c n t s h a c l a h i s t o r - y , , t ' u s t 6 r r r a atopy or positivc skin prick tcst, of 1,vcrc strfìL.r.ing li.rr1 u r c h r o n i c c o n - q c s t i v c h c a r t lì r i l u r c . A l l p a t i c r r t s w c r c in a stablc corrditiorr at thc tir,c .l'thc str.rcly u,,,-l fì.cc lì.rrrir ac'tc cxaccfbations .f' syrrrptorr-rs of r.rpllcl r'(.iprratorv tract infòctions in thc 3 months prcccclilrg thc studlr O n l y r n i l d C O P D w c r c s c l c c t c d t o a v o i c l t h c i n f l L r c n c c on systcrnic cfl'ccts of ovcrlappcd condition., that oliqr appear in advanced stages of COpD (crrrrliovasurir disorders, respiratory fàilurc and othcrs).
Hcalthy subjects had no respiratory syrrrptorns an4 no rcspiratory tract infection for >3 months pr.ior to thc study. All COPD patients and healthy subjects enrolled were ex-srîokers frorn at least 6 months pn:cccling thc study. COPD patients were enrolled during routlnc hospital visits. A full medical history was i,,ken and general physical examination was performecl. patients then underwent lung function testing, skin prirl< test for common inhalant allergens, serum total IgE, arti:r ial blood gas analysis, laboratory tests, fat-free mass, lnaximal inspiratory pressure (PImax), maximum crpiratory pressure (PEmax), exhaled NO measurement, iriood and breath condensate and induced sputum collection. None of patients included was treated with oral stcroids and only short-acting inhaled B,-agonists were alltiwed for symptom relief.
M A T E R I A L S A N D M E T H O D S Si I t r t I.v 1 tt t 1 t r r I u Í i t s n
' l w c n t y
s c v c r t n r i k l s t a b l c c x - s r n o k c r C O p D ( 5 2 + 4 y r s , 2 | M . r , r s , r r t r l l V r | . 9 r 0 . 4 lt. 75.3+3. l , Z , o f p r c d ì c t c c l ) a r r c l
1 , 5 h c a l t l r y c x - s r r r u k c r s u l r . j c c t s ( 4 9 + 7 y r s , i Z t u t , SASRL l , l r V r 2 . 6 r 0 ( r lt, | 10.4 I ( r . l , ) 1 , o l ' l t r c d i c i c d ) w i t h n o h i s t o r y o l l r u r g rlisclsc lvcr.c strrtlictl ( . 1 ì r b l c l ) . T l r c c l i a g r r o s i s o l ( ( ) l ) [ ) w a s t r l s c c l o r r l : f ì S / A I . S ( i L r i t l c l i n c s ( l ) . A l l s u b j c c t s u , c r c r . c c n r i l c t l l ì r r t r r tl r c I ì c s p i r u t o r . y I ) i s c l s c
Stud1, de.sign
On the first day subjects underwent cliniral data collection, physical analysis, lung function, fat frec nrass and respiratory muscle strength measurements and bloo,.i tests.
On the second day exhaled NO, exhaleri breath condensate collection and sputum inductiirrr were perforrned.
Pulmonarv Íunction testinc and usses.smenl o/ rrirflow limiîation
Puhnonary fìrnction tests were performed i.rior to thc rneasurernent of exhaled breath condensatc. FEV,, F V C a n d F E V r / F V C r a t i o werc rncasurctl L , ; i n g a dry spiromctcr (Vrnax Spcctra series, Sensor t\,4cdics Corporation, Yorba Lincla, Califbrnia, USA). Thc irighcst valuc of three rnanocuvrcs was exprcsscd as a pcl.r.jlltago of the prcdicted nornral valuc. Reversiblc :iir.flow lirritation wcrc clcfìrrcd as arr irrcrcasc in FEV, of . l(X) ntl a n d / o r > l 2 r Z , f i o m b a s c l i n c - 1 0 r n i n u f ì c r in h a l a t i o n l l ' 2 ( X ) p r g o f ' s a l b u t a r n o l ( I ).
['- t r | - I i'a t, | | | ( t.\,\ t t t ( o.\ t t t. ( t I t ( | 1 I
F a t - l i c c r ' ì l a s s l v a s c l c t c r r n i n c d b y b i t r e _ . c t r . i c
1 5 s
l r r 1 . . l . In t n t u n o p r t h t t l . I ' h r r t t t r c t t l .
i r l p c r l l r r . i a r r a l y s i s ( t l o c l y s t n t | - 5 ( X ) : I ) o t r g l a s ' U K ) ( 2 ( ) ) ' llt'.r 1,,,, r I t tt'.1' ttt t tsc I a'\' t rc tl gl 11
Jlcstriiirlory tlrusclc strcngtlr wiìs itsscsscd by n.ìcasufL'lìlant ol- rlorttlt prcsstlrc al ttrltxittral static inspirator_r, arrcl cspiratory cfforts fronr rcsidr"ral voltttÎc ,,ni tot,'i lr-rng capacity, Plnrax (nlaxinral inspiriltory nrcssgrc ) irtìcl PErnax (rnaximal expiratory prcssttrc) l,g.i,rrt ,,,' obstructcd rrrouthpicce witlr a small leak to niin,n.,irc oral pressurc artifacts (Spirovis, Cosrncd S'R'L"
I t a l y ) ( 2 I ) .
lndut r',1 '\Putum
Sputurn was collected and processed according to thc nretht;tl of Spanevello et al (22)' All sputum counts and measttrctnenls were perfonned blind to the clinical tlctails. Drlinition of an adequate selected sputum was one in wÌtich therewere fewer than 2070 squarnous cells and viability > 50o . A specific enzyme immunoassay kit (Cayman ('iremical, Ann Arbor, Michigan, USA) was used to measurc'fNF-a and lL-6 concentrations in supernatant ofinduced sPuttlm.
Exhul,,l hreuth <ontJetr'sute and ussuy
Exhaletl breath condensate (EBC) was collected by using the lrcoScreen, (Jaeger, \Wurzburg, Gennany)' The subjects breathed through a mouthpiece and a two-way non-rebrealhitrg valve, which also served as a saliva trap' Thcy were lsked to breathe at a normal frequency and tidal voh.rrric. wearing a nose clip, fora period of l0 rnin' lf subjects lclt saliva in their nrouth they were instructed to swallow it ('ondensate, at least I ml, was collected as ice at -20"C, lrlnsferred to Eppendorf tubes and imrnediately storccl at -7(i"C. Sarnples were analysed within 3 months liom collectitln. To exclude saliva contamination amylase a c t i v i t y w a s a n a l y s e d i n E B C .
Meast r rc r t t r, nl ol' ex hal cd N O
A r a p i t l - r c s p o n s e c h e m i l u m i n e s c e n c e N O a n a ì y z e r (rnodcl 2bi(). Sicvers Instrunrents, Boulder, Colorado, IJSA) was Lrsccl to quarrtify NO. Two-point calibrations w c r c p c r lìrrincd daily using 5.2-parts per rnillion c u l i b r a t i o n g l : , . E x h a l c d N O ( F , N O ) w a s In e a s t t r c d l t s i n g a prcviouslv clcscribed rcstrictcd brcathing tcclrniclttc' wh ich c'r' 1.r,,ìd cxpiratory rcsistancc ancl positivc'routrr prcss[llc lo t'ltisc thc vciunr arrcl cxclucle nasal NO, ancl il colìslarìt r:r;lrinrtory flow of 45 mL/s. Thc inhalccl gas was attr[.ricrrt rril that was passcd through a fìltcr to rcducc i n h a l c t l N o r ' o n c c t l t r a t i o n s t o < 5 p a r t s p c r b i l l i o n ( p p b ) . Strh.iccts inlirrlctl to total lung capaciry, an<J exhalccl wlrilc tlrgcting ir tonstant prcssul'c of 20 nnr Ilg. Exhalations pttlcccclctl rirì1il a clcaf NO platcau of at lcast 3s clttraticltt was rcltiu cri. tì.cpcatcd cxhalations wcrc pcrl-ortnccl trntil
t h t ' c c p l i t ( c a t t s i l g l c c d r v i t h i r r 5 ' 2 i , ( 2 3 ) ' 1 1,- ó.'l' N I'- - t r t r tr d l l' - 1 tt1 ( u \ t t t'c t ì t ( tt l \
A spccilìc cl1zylllc itl.tt.utttloassay kit (C'aynlan ( ' h c m i c a l . Ann At'[ror' Michigan, USA) was ttscd to llrcasurc lL-(r, TNF-u and IL-4 concctrtrations in plasnra' supcrnatallt of incltrccd spLltulÎ and brcath corrdcnsatc' Tlic inrra-assay aucl intcr-assay variability wcrc <10'lu for cach assay atrcl tlrc cletcclion limit of the assays was l'5 pg/rnl,4 pg/ml and 20 pg/ml, rcspectively Tlre coefficicnt oi variation lbr lL-6, TNF-c, IL-4 and pH were 5'9%o;
3,3'Y,,, 4.2"1, ard 0.4'Y,, rcspcctively' Reproducibility of exhalcd lL-6, TNF-u ancl pH measurelnents were assesscd in l0 non-smokirrg nonral subiects.
pH ntcasuraments
A stable pH was achieved in all cases after deaeration/
decarbonation of supernatant of induced sputum and breath condensate specimens by bubbling with argon (350 mlhnin) for l0 min, as previously described (24)' pH was then measured within 5 rninutes ofcondensate collection by means of a pH meter (Jenway-350, Ltd Gransmore Ci.""n, UK) with a 2 to 16.00 pH range and a resolution/
accuracy on the order of 0.0 I + 0.02 pH'
Plastna reactive ox)tgen metaholites medsurenrcnÍ Systernic oxidative stress was measured by the diacron reactive oxygen metabolites (D-ROM) test (Diacron lnternational, ltaly) (25). Values of DROMS were expresscd as Carratelli Units' wlrere I U'CARR corresponds to 0.8 mg/L llrO,.
P I a:; ma C R P maas tt rcmenl
Plasrna CRP was nìcasured using the N high sensitivity CRP Kit (Dadc Bcrlring, Marburg' Germany) which has a l o w c r li r n i t o l - d e t c c t i o n o f 0 . 1 5 t n g / L .
Sl u Í i.s t i t' u I u n u I v.s i.s
Data arc expressecl as mearrs*SD. A Manrr-Wìritney tcst was uscd to colllpal'c gl'otlps, aud corrclertions bctwccn valiablcs wcrc pcrlbrmcd using Spearman's rank corrclatiott tcst. SignifìctìlìcÌc wils clcfirled as a p valuc of
< 0 . 0 - 5 .
R I l S U L T S L r r n g f i r t r t t i t t r t
All ('OPI) wcrc dcfinccl as mild according to
( ì O L D gLridclincs ( 2 ó ) . COPD subjccts showcd
l o w c r p c r c c t t t r g c s o f F E V r a n d F E V , / F V C r a t i o
conlparcd to hcaltlry controls (lì2.6+ I .5 and 65 '8+2.2
v s I 1 0 . 6 * 7 . I a r r c l l J l . 3 + L 4 ; p < 0 . 0 1 a n d p < 0 . 0 0 5 )
7 5 6
C e l l s c o u n t s i n ln d u c e d
sputum
Fig. l. Comparison betuteen non-reversible COPD, reversible COPD and healthy controls in terms of percentage o/'cells found in the induced sputum. We observed a higher percentage o.f neutrophils in non-reversible COPD than in reversible
and of eosinophils in reversible COPD than in non-reversible.
p < 0 . 0 0 5 r---1 2 0
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p l a s m a s u p e r n a t a n t
l"ig. 2. ('onct,trtt.utions ol' IL-6 in COpD strbjcc,t.s antl ltt'ttlllt.t't'.,rtrtl.s itt ltltt.trtru, .rrr)c,tlrrrtrr tsf intlttteu.sltttrttnt tr,tl <'.tlttrlt'rl ltr'<'tr!lt t'rttdt'n.strrt,. wc fittrncl alevuracl lattal.s Il,-6 irt .\'ul)(t'ttul(utl of itttltr<.ttl .\l)ultult utrd in t,.thulcd ltr'<'trtlr r. rtrtlt'rr.strlt' .f ('Ol,t) .s.trhjt,r.l.t !ltttrt itt ht,ttltlLt..
t ortlntl.:. Ili,:ltt't' (.()tt( (nlt.(tli()tt:i ol ll,_ó v,t,t.t, oh.s<,t.r,t,tl itt
t t ( ) t t t ' r ' t ' ( ' t ; i f ) l c ( ' ( ) l , l ) L . o t t t l t t t t . t , t l l o t . t , t . t , t . . s i / t l t , ( ' ( ) f l )
Fig. 3. Conc'enlrutions o/ TNF-a in COPD :;uhjec,t.s Ltrd heaIthy conlntLs in plusmu, .rupernofenÍ of int/ttc'cd sptrtrtru qncl c-rhulad brcuÍh cotttlcnsutc. I4/a .fìttrnd alcvuÍad lcrL'l.s TNl"-a in :;trltt,rnulunl of indtrt'cd .sltulttnr untl in a.rhtrl,,d ltraullr t'ondan.suta of COPD subjct'!.s thun irt haultltv t'ottttrtl.t. Ílighcr crttttattlt'uliort.s of TNI;-c trat.a ohsct't.r rl i t t n r t r r - r't, r't, r'.s i lt l t' C O P D co ttt t tu t'r' d l o t.a y t, t.,s i h l a (' O P l )
!lt_l ',J tl Itl
tìu
5L':l 4tl 3tl 2t-l 1 t l IJ
tr t- o rrtrr-rls I E e r r e r s i b l e ú F l o n r e v e r s i h l e
M a c r o p h a g e s Itle rf rop hils E o s i n n p h i l s
I n f . . t . I n ì r n u i l o D i l l h o l . I t h l r n t a r o l . 1 5 1
Rev Non rev Rev Mn rev Rev Mn rev
plasma supernatant EBC
Fig"" 4. Concentrations of IL-4 in COPD subjects and hctilthy controls in plasma, supernatanl o/ induced sputum anrl exhaled breath condensate. We./ound elevated levels IL 1 in supernatanÍ oJ'induced sputum and in exhaled brt'aÍh condensate of COPD subjects than in healthy cortlrols. Higher concentrations of IL-4 were observed in re,,ersible COPD compared lo non-reversible COPD.
p<0.05
l
I
Non rev Rev
Iúg. 5. Concentrations o/' exhaled nitric' rtxidc (NO) ir: COPD suhiecl.s and healthy rttnlrols. Elcvutcd t,tnc'enîrations o/ exhaled NO u'ara ob,ren'ed in COPD l.t,rtienls compured lo cttnlntl sultjct'Í's oncÌ in revcrsiblc
t,,mparcd lo non-reversible Cl)PD.
( i a b l e l ) . A f t e r r e v e r s i b i l i t y t c s t w i t h s a l b t t t a m o l ( - i 0 0 rncg) required for classification of ('OPD h ' y g u i d e l i n e s , l 0 C O P D ( m c a n a g c 5 3 + 3 , F ' t r V ' t J . i . 5 t l . 4 , p a c k l y e a r s 3 0 . t ì + 4 . 9 ) h a d a r c v e r s i b i l i t y , l2' and l7 had a rcversibility <12"1, (tncan agc . i 5 + 5 , F E V r t Ì 5 . 1 + l . 5 , p a c k / y c a r s 3 1 . 0 * 4 5 ' l ) .
Strnt I u tn m e u,\u ra tn a n l,\
Sputum induction was perfornrcd in all subjccts
w i t h o u t o b s c r v i n g a n y s i g n i f ì c a r r t a d v c r s c c l l ò c t o r a d c c r e a s c i r r F E V , > 2 0 " 1 ' . F o u r h c a l t l r y s u b j c c t s a u d 6 C O P D p a t i c n t s d i d n o t p r o d u c c a d e q u a t c s p u t u r ì samplcs, and thcir expectorates were discardcd.
Table ll and Fig. I show tl,e comparison between the three groups in tenn of percentage of cells found in the induced sputum.
Higher percentages were found of neutrophils and eosinophils and a reduction of rnacrophages in C O P D s u b j e c t s ( 6 6 . 4 + 8 . 8 , 4 . 6 + l . l a n d 2 0 . 9 + 4 . 1 % ) t h a n i n h e a l t h y c o n t r o l s (3 1 . 3 + 7 . 2 , 0 . 5 + 0 . 3 and 54.'7+6.5%)
A higher percentage was observed of neutrophils in non-reversible COPD than in reversible (71.0+9.5 and 59.6*8.6%, p<0.05) and of eosinophils in reversible COPD than in non-reversible (6.3+l.l a n d 1 . 5 + 0 . 3 % , p < 0 . 0 5 ) .
Elevated concentrations of IL-6 (8.54+2.02 vs 5 . 4 1 + 0 . 8 7 p g l m l ) , T N F - a ( 1 1 . 1 7 * 3 . 4 1 v s 6 . 2 2 + 0 . 8 2 pg/ml), lL-4 (60.34x1 0.66 vs 4l .41+5 .97 pglml) and lower pH (7.50*0.06 vs 7.86+0.18) were found in supematant of induced sputum of COPD subjects than in healthy controls (Figures 2-4). Higher c o n c e n t r a t i o n s o f I L - 6 ( 1 0 . 3 9 + 2 . 2 8 v s 7 . 2 1 + 0 . 7 3 pglml) and TNF-a (14.51+3.32 vs 8.37+l .32 pglml) were observed in supernatant of sputum of non- reversible and of lL-4 (71.24+5.27 vs 49.94+6.29 pgAnl) of reversible COPD patients (Figures 2-4).
Lower values of pH were found in non-reversible compared to reversible COPD (7.48+0.07 vs 7.52+0.05).
Exha l ed brcal h condensole measurcmenls B l c v a t e d c o n c e n t r a t i o n s o f l L - 6 ( 8 . 3 0 * 1 . 9 4 v s 4 . l t ì + 0 . 6 3 p g l r n l ) , T N F - a ( 9 . 5 3 + 2 . 3 5 v s 5 . 6 5 * 0 . 9 9 p g A n l ) , l L - 4 ( 5 5 . 1 5 + 7 . 8 8 v s 3 4 . 8 3 + 5 . 7 9 p g l r n l ) a n d a l o w c r p l ì ( 7 . 4 6 + 0 . 0 1 ì v s 7 . 6 1 ì + 0 . 2 3 ) w e r e f o u n d in cxhaled brcath condcnsate of COPD subjects comparcd to hcalthy controls (Table l).
f l i g h e r c o n c c n t r a t i o n s o f l L - 6 ( 9 . 4 9 + 2 . 5 5 v s 7 . 0 1 J + 1 . 0 7 p g / n r l ) a n d T N F - a ( l l . 5 l + 2 . 6 0 v s 1 . 1 2 + 0 . 6 8 p g / m l ) a n d l o w e r c o n c c n t r a t i o n s o f l L - 4 ( 4 9 . 4 9 + 5 . 5 6 v s ( r 0 . 0 7 + t J . l 3 p g / m l ) w c r c obscrvcd in exhalcd breath condcnsate o1' uorl- r c v c r s i b l e c o m p i ì r c d t o r c v e r s i b l c C O P D ( F i g . 2 - a ) . Lowcr levels of pH wcrc fburrd in exhalcd brcath condcnsatc of reversible coll'ìpared to non-revcrsi l'rl c r 0 0
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Fat-frec mass (kg) 46.42 + 4.49 5 3 . 5 + 8 . 8 < 0 . 0 5
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S p u t u m l L - 6 ( p g / r n l ) 8.54r2.02 5 . 4 1 a 0 . 8 7 <0.00 |
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Non- reversible C O P D
Healthy subjccts
Total cell count ( x 106/ml)
2 . 9 + 0 . 6 ? ? + O R 2 . 6 + 0 . 6
Macrophages (%) 23+ 4.9 t6+3.2 54.516.7
Neutroohils l%) 5 9 . 6 + 8 . 5 * 7 1 . 0 + 9 . 6 * * 3 1 . 4 + 7 . l Eosinophils (%) 6 . 3 + 1 . 2 * * 1.5+0.3 0.5 +0.2
Lvmohocvtes (%o) 0.9+0.5 2.3+0.5 t . l + 0 . 6
Eoithelial cells (%) 9 . 1 + 2 . 4 I 0 . 1 + 4 . 3 I l . l + 3 . 8 Reversible COPD vs non-reversible COPD: +p <0.05; **p <0'01
COPD (7.43+0.07 vs 7.49*0.06) (Fig. 2-4). Elevated concentrations of exhaled NO were observed in COPD patients compared to control subjects (24.91+7.94 vs 9.07+1.6a ppb) and in reversible compared to non-reversible COPD (29.59*6.41 vs 20.3+8.9 ppb) (Table I and Fig. 5).
Systemic measurements
A significant reduction of a fat free mass ( 4 6 . 4 + 2 4 . 4 9 v s 5 3 . 5 + 8 . 8 k g ) , o f P l m a x ( - 6 5 . 5 5 + 2 0 . 8 8 vs 105.8+2.4 cmHrO) and PEmax (54.78+23.52 vs
192.5+6.7 cmH2O) was observed in COPD patients compared to healthy subjects (Table I). COPD patients also showed higher concentrations of w h i t e blood cells (7.911.9 v s 5 . 1 * 1 . 3 x l 0 e / l ) , b l o o d eosinoplrils (0.41+0.26 vs 0.23+0.15 x l0'/l) and CRP ( I 1.55+ I I .l 1 vs 0.7+0.9 mg/l) than healthy subjects.
We also observed a reduction of BMI (21.0+2.4 vs 245*2.6 kgAn' ) between COPD patients and control subjects, although this did not achieve significance.
P I a,s Dt t.t (, 0 n c e n t ral io n s
H i g l r c r concentrations o f D R O M s ( 2 6 ' 7 . 8 + 3 4 . 1 2 v s 3 6 . 8 1 ( r . 0 U " C A R R ) , I L - 6 ( 9 . 1 8 + 2 . 3 1 v s 5 . 8 2 t 0 . 8 9 p g h n l ) . - l ' N F - a
( 1 1 . 2 2 + 3 . 6 7 v s 6 . 0 6 + 1 . 5 5 p g A n l ) a n d l L - 4 Q2.43+10.43 v s 3 8 . 7 1 + 5 . 3 3 p g A n l ) w e r e fo u n d in plasrra of COPD patients compared with healthy subjccrs ('hrblc I).
. The plasma levels of IL-6 and TNF-a wcre highcr r n non-rcversible C O P D ( 1 0 . 7 2 + 2 . 7 2 v s 7 . 5 7 + l "5 2
pg/ml and 13.5'1i4.49 vs 8.66+1.29 pglml) whereas the levels of IL-4 were higher in reversible COPD (7 8. 66+1 0 .22 v s 6 5 .7 2+10. 0 3 p g/ml) when comp ared to healthy subjects (Fig. 2-a).
Correlations
The relations between the fat-free mass with the inflammatory markers were studied using the Spearman's rank correlation test, finding negative correlations between fat-free mass and TNF-a in plasma (p<0.001, r:-0.83 ), TNF-a in supematant of induced sputum (p<0.001, r:-0.75), TNF-o in breath condensate (p<0.05, r:-0.44) and between Plmax, PEmax and TNF-o in plasma (p<0.01, r:-0.61; p<0.01 , r--0.64), TNF-o in supernatant of induced sputum (p<0.05, r:-0.50; p<0.05, r:- 0.48), TNF-a in breath condensate (p<0.05, r:-0.53;
p<0.05, r:-0.46). Furthermore, a strong correlation was obserued between Plmax and fat-free mass ( p < 0 . 0 1 , r : 0 . 6 7 ) .
D I S C U S S I O N
Several recent studies have investigated the inffarnmation in COPD and the possible mecl-ìanisn'ls of its systernic effects (2-5). ln agreerncnt with previous studies we found significant itrcreascs in several inflammatory biornarkcrs (CRB TNF- c x , , I L - 6 ) ( 9 , 2 7 - 3 7 ) a n d l e u k o c y t c s ( 9 , 2 7 ) i n t h e peripheral blood of a grolrp of ex-smokcr CIOPD
i ; i
