Institute of Clinica Medica University of Verona. 'Divisione di Geriatria Azienda Ospedaliera di Verona. Institute of Chimica and Microscopia Clinica,. University of ...
1/i tnfatrmtia , v.t. 2A N.. 6,
1996
NEUTROPHIL MIGRATION, OXIDATIVE METABOLISM, AND ADHESION IN ELDERLY AND YOUNG SUBJECTS D, BIASI, A. CARLETTO.] C, DELL'AGNOLA.' P, CARAMASCHI,] F. MONTESANTI.3 G, ZAVATERI.T S, ZEMINIAN.2 P, BELLAVITE.I ANd L, M, BAMBARAI d Pdtakiù Mtdi.o Utùl§iù alknnd, ttdt ^ùutt :b6!ùtk Clini.u M.ni.d Univ6ì»^ Òl va. a d 'Dittit\t. lli C?traltiu A.i. tld OV?tla!i.o tti rù)na !l\§nù. ol Chìnna on MrrÒ!..pìl Ctini.o, Unilt'sit\.j v.totd
AbttuLt Ohi.c1it.
.'...r-
sio
To elxlulre ncurtuphit
orclV\'iicdo'n..od
iu..ròn!
,Url/rl/j Wc .r,, dt xJhr
in rhc etderL!.
P\r\.rp.4.,
e p
oo
in responsc ro Mnel)- ol.onìpoLn.ls: ptrlN have bcù N)tared bodì aom htood n *in exp nnùnalrxudale (obrajned bl, slnni §kin trrirlow techtriqft) ol2s nomrxlcldù.lv nnd ol15 nomd youne.onrnìlsuhi.c6 R.ad6. No dirreE;ce \yr\ a{itrtul in PMN mig.ilion in vùo (62 9 r 2ì 3 I lo.rnd655 +91! lo!
ind
l[m
'drir PV\ h J.'r .1"d
r cJtl.Jn...o{ i e!'..F r:.(,...rr ,.rJ..bJ.l ,.tliol.n. ., .., .",. . ,u J i1e ., .. ,, J- pro.
ii brsalcondiÌroi and in rt{ìonse bSlZand pMA i0 Ìwo gn,ups.ln rLd;rty subi.cs supenì\ide pDdùdnrn. in response trì tN{Lp. ma*et\ resulÌed toscr rhrn ductìo.
..n\4ot.\ 11. l-., h .r J J' Pt'l\ .Ò .1oi. 'rri O ,,''ns.o l\'\ .e G. \ O.d.t .t b .,,0" pV\! rll.Ò a ! i.l / or Ò \'o \ n t0' P\t\. e.o. ..).1 nmsr L /.,. ,, v. , Pw\l n.-i. te. ro , . , r s o. ,. tr . .r ,," t. ' .. tr.'.ob\ .e'ldli. n -i t.,,tp., " , L L.df.. !u.ntetr_,,p .rpr,ne I. o.oi. ,f. e vl n.' ', r.o,PV\. I L J. n. o, .f. r! li
Ihis deÈd nì PVNS frcnì
ddùtr \uhjec( indicatcs rhÈ e\islekc.ra dire!ùr,ìi. disrrltu vILp Eceprorntul p.oximai ir NADpH
ol rhc \ignalraDsducùrn parhwar oxidasc a.ùvarnìn
TNTRODT]CTION Ncuttuphils (PMN) are the Iìrst cclls rhar migrate imo tissues ìn response to invading pathogens. The accùmularion of these cells ar sìres of inflammation is
i,
accompanied by modilìcatbn of lheir activity (ablliry ro .eìea§e granule con stitucnh and toxic oxygen derivatives. adhesion capaciiy elc.). Prcvious invcstigati{rÌs on human models have sho$,n that exudate PMNs are merabotically prlmed, being more responsive to various membranr dimulants, rhan blood PMNS (l-7). Thc increNcd aciivation state ol PMNS is Rle vxnl for non specitìc imnìunological surveillance agains! microbial invadcrs and also because of the potentially harÌntul l1rÌe playcd by these cells nr pathologic processes as adult respiratory distress syndronìc. poslìschemic tissue disease,
imnunocompÌexes dìsease etc. (7 10). During the last few years sonre rcpons have evaluated the PMN s lunctìons in the elderly. The results are dìscordanl. Sorne sludics havc shown thal the tunctional response of PMN§ is unaffcctcd by aging (11), while othcrs hilc pinred oul an impaimrcnt of chcmotaxis, phagocyiosis and killing (12 l4),.ì reductìon of respimi{rf burst (15 ì7) and a decrease of adhcsion (15). Thcsc srudics have been always pcrlomed in vitrc, utilizing PMNS isolated
ln rhe present study we have investigated the PMN migration in vìvo and
PMN superoxide producrion and adhcsion, in response ro a variety of compoundsi PMNs have been isolated both from blood and from a skin experimental ei{udale (obtained by Senn s skin window rechnìque) of nonnal elderly and of noroìal young conrrol subjecis.
MATERIALS AND NTETHODS Twenty live volunle6 (12 M. l3 F) olcr 65 yexrs oìd lù.rn rEe 76.56 t 7 36 yeaA, rangc 63 IOO leaÀ), RÈre :èlÈtred lccording !) SENIEUR pdocol (18) Twcn!- tìrc rolùnrcF young sex'narchedsubjc.t(.unxlc27.7712s9tcm.rangc2234yea6)rveEconsideEdascontmls. During thc I stks bcinr ùe.vrìùIion, no sùbjeth seE Ìakin8 NSAlDs. conicostemids
All the elderll sublech serc h eood nuùiÌion «arc ,Rea8€nrs The chenìotlctic peplìde N iomryl nrelhionll lcu.yl-phèilldlnine (fMLP). zyh a.d phorbol mlnnarc acctxtc {PlvfNA) Rcrc pùr.haled lrom Sigma Chenical Conpant, Sl. Louir, MÀlDri: cynìchome. fmm Boe$ùnger, Mannhcirì. cÈman!; pLnied serum bovìnè albumin and hù.ìan aìbumin lron Bèhnng lniirul, Mlrbuig, Cemny Pcroll was tiln Phar macia- Uppsala Stene 96 well mi.mÌner plales wnh flxr botonEd wèlls (Linbro tyre) weE rrum Floy hbonbn.s. Hàik s halanced laÌt !ìlutnri (HBSS) and reaeents {crc of rhc high.( pùriq availabìe Zymosan, op$nized sith a pool olnomaLhunan scn (STZ). r\ dès.nbed b, Mèr.rlf Èr ù1. (19), §r! $red in liiquots at 20rC ln ordcr to avoid conraminarion. a posiblc.ausc oI aniractual aclivation or pnning of the cells- sterile apyrugenic $lutions and dhposable pLa$icqarc scÈ ùsed ìn aìl èrFenmènts, *hi.h wcrc .ari.d out, shcnerer posrìhle. trn,lèr lùninlr n.* h@d. Realen6 wcrc prcparcd using osan
pyru8en
ir Fareror0.9? NaCl solùtions. P.?p.ati.ù. PMNS *ere ÒblainÈd rom
blood and amm skin windos cxudates $bjects aner having obtained Ìheir inlomed consent. Blood PMNS *èft prÈplEd rrom 40 nl C?11
ol
.r
N.utroohiì Nligration. Oxidrtiye Metabolisùr, nnd Àdhesion ethyleie dianine lcrnacctùt anri.oxgùlxred blood by cenlrifugarion
615
ovù Percoll endients
(19).
cclls(>95%neuÌrcphils,>95%l.rpanbluenegade)*èr.niùllysù\pendedinHBSSconraining 5 m\Iglu..se ud 0 2ri hu,ìai scnm albumnr (HCA) and kefl aÌ mom tempemlnE until use A lis ninutes beloÈ use. llox concennated lnuiio's ÒlCrclr ùnd MgSO, *èÈ rdded {o rhc.eìl àr ihc nnal conceniration of 0 5 nM and I mM Èspeciively. Exudale neuÌruphils ( >90% neulrophi\, >95% trypan blùc ncgatve) Rerè sol,ted accordinsblhè merhod dÈs.nbèd by scnn rl0). with.rodifications (:l). The volxr dnce ol nondomlnant
$spennons
.. r _J."nC
l.r.noJr.'n'i.i ,
reì.
abràlile cyliidèr oÈnr.d b! a niuing cùter (minidnl- Sainl lulien en Cenerois, France). Thè abrùsion did Dor cause bleeding as onLl the cpidcmìn qrs Èm.ved and rhÈ uet, r.Jn. sudùs sùface ol dema was èxp.\èd A b.lrshapcd, (eiLe and disposable plaslic skin chamber Rith rirulrr rdhenlc blsr (FARllalia. Verona- Italy) *as pùton the \lin ahn\i.n r hxcd sirh a ienèsrmled sticking pìasèr. The chanìb.r hxs on iÌs top a ,5 nm wide hole equipFd §ilh a Flug. One ml .r droìogoùs scnnr qas Ìhen injecled int, thè .hmrbft and lqèntl roùi hou6 laler lhe rxudatc qas collecred by aspiraln)n Thè erùdxt.rll§ *eE Ìhen cenùituged aÌ 1200 rpm, \rashed lrvìce *ith th.!ph!{è burcrcd salinc (PBS) xnd linally sustÈided in HCA and kepr rt roon tcn} peraluÉ ùtrùl usc Briore rse, the .ell suspensnìn§ weÈ $pplcnuted §irh cxcìr ùnd \1gsor as dcscribcd xbore lor blood cells. Snprroxide nnioD wa! mca:ured by lhe lupen)xide dismtxs.Oaildrie Mltub.Lilnt inhrbirxblc rcduclion ol tldc)$chr)me . (21) in ! nìi.oplxre assal slitù. Assa]- r.re pedomed drc sùtu subjè.t lnd b .onpat .dls in .rdcr ro .onpare celk 1rcnr bloon and iFm exLJate lrcnrloune andeLderly sLblÈd\ in rhc srnrdry ind usin3rhe same ni.Dplate in matched ÈpLlcate (clh. The micmplare assa) f{tr Or px)du.rion §as pcrinrired r.cordi.g ro prcviously published nreÌhods (22)- *irh the loìlLrtr ìg ùodilicarions. lhe §elh Rerc .oaied *ilh felll hovìne rètu r.
Arù\
.l
*eÈ supplenenred wirh 25 rì ol 0.6 mM .Io.hrone . in HGA containin8 0.5 mM Cacll I nìltl ltlgsor (HGAC\'I) ùd wilh 25 !1olÌhe slimullnrs dÌlured in HGAclvt ar x rÒn.È lDriùn e\rcrìt,1(inrs highcrÌhsn thaÌ EquiEd in the asa)- Th,s {$ doDc b..ausc ùa.h aaent is diluted 4 rnùes in Ìhe lì.aÌ incubrtnrn mi\rtrÉ 1sè. bclo!r). The plate sas then bruushÌ 1o 3i'C, and 50 pl or rhù PN1N srpenrnins (2 x 10'r.ll\). trrc saùncd at l?'(:. §eE xdded 10 cach weLl using a orulrich!nnel pip€t. rrd rhc pl.t.§ §eE incubared ri:17'C lnr rh. de\irtJ lime As\!ys q€rc drnt in r;tliulÈ lùtr.h c{prnnrcnlal condiiion AlrherimeinditnteJ.thepLiies*eE.iti{l}ir!nsfèrcd im. à ùrcmplxÌe Eade' (Re ler.10O SLT l-rb! In(ntrìci6l and rhc rcJùdùnor.)(o.hnrnì.. $rs 01ea$Gd il 550 nm u§ng 5.10 ,nì rs r1crcn.c \ravelength, and urln! 0.017 oplicxl densiÌy unns a! rhn,ìrrd n 1 ùnìolc oi rcd(.ed .rtochnìme . (22) ,.t&frn r,,lrJar For adhesion measuEmenb, th. platcs $crc tanstcrcd 10 an aulonralio {ashcr lEasy washer 2 SLT l-abs lnnùfrÈns) and subj.cled ro ̧o $ashins cycles lith PBs rr rhcD and
roorì rempeQtuE adhÈEnr.ell\ rer. quanrilalcd b] measurìng the meorbmne enzyme acid phoi phlra\è rnd the pcrcc agr of xdhesion sas cacuLated on rhc bÀis Òl a {andard.uflt obÌanred wnh known nunrbe^ .l PMN! (22) prtunrùic Wilcoxon ie« litr unpaircd data lNlrnn Whrney les) *a\ Srdturnr The
n.
used
nr {rlnù.al
amLvsis
RESULTS Figure 1 repons $e data conccming the values of in vivo migmtion. Thc olthe mìgration wrs 62.9 + 21.3 x 10" PMN/cm:/2,I hours in elderly gmup and 65.5 I 9.1 x 106 PMN/ctnr/24 hours iù contds. The din'erence mean
'1
't0
100
e
90
E
BO
!
70 60
.9 E
*9.1
----fr-
50 40 30
E
z
20 10 0 YOUNG
Fig, 1. DaÌa con.emmg rho vxìucs ol nì
.,
to"Pv\.. 2rT'
I,
\iy.
ELDERLY
ìig.ù{nrn. The mesD
ùfrhc ùrisunin
P\i\.
s!!
62
.ar,
9
ì n
between the mcan migration in thc ts,o g.oups was not slaiisrically signilìcant. bxl elded) subjects showed higher viri.ìrion and a morc scatered distribution of indiYidual values. Table I rcpots rhe data about superoxlde prodLrction by circulatìng and nigraled PMNS, under basaÌ condilion and nlier challenge with vadous slimuli (fMLP. STZ aùd PMA). Exudate cel1s ofboth elderly and young eroups showed rn inc.cascd supcroxidc production, in rcsponse ro |MLP. as compared with blood cells. This primlng efÈci \ras not observcd ùsìng STZ or PMA as stim
Circulating PMNs frcm elderly subjects showed superoxide production after lreatmcnt with fMLP narkcdìy lowcr than young subjects. Also the exudate cells frcm elderly subjecrs had lower activity with respect lo exudatc cells of young subjecls, but rhc difcrcncc bctwcen thc rwo §ubject s groups was nor so marked as fòrblood ceus. In facithenei increase of oxidativc mctabolism causcd by mlgmiion into the lnflammirory cxudate (ì.c. ihe priming efect) was slmilar
N€utrophil Àligraiion, Olidative M€tnbolìsm. aÌd Adhcsion
T!bl.
l
Supeoxide PFduction in Youn! (Y) ard Eld.rìy (E) Subje.s
br Ciiflldnrg (B)
and
vigratins (sw) Pr{Nl'
B NS
MLP
to rN,
sTz 0 t
\s
B
NS B
NS
NS
"Th.
v
Tahle
uès
rE expE$ed in nrmlrloi ncursphils
2, AJhrsioi .t Crrulxrng (Bl
and Nli8mling {SW) Ncùlsphrls in Youne (E) Subj€dJ
G)
and Edeny
\S
a
NS
tufLt L0 '11
B
NS NS
s'tz
B
NS
B
NS
0.1
Nrs
'The ralues
rrè.Ìptssrd
id pcn.ntrg. oladheEnt celh.
in the two grcups (fìom 9.3 to
19.,1 and from 3.6 to i 3.6 in young and clderly subjects respectively). The other values of superoxide production in response to STZ and PMA welE not dìffercnt between the l\lo gtuups. Table 2 shows the resulls conccming rdhcsion ofcìrculating and nrigrating neutrcphils under bàsal condition and after some stimuli. Erudation caused a slight increase of adhesion of both unslimuÌated and stimulaied cells. Therc was no diIìèrence between elderly and young subjects.
DISCUSSION Previous studies on PMN tunction in elderly subjecrs have shown decrcasc ofchemoùxis, phagocyrosis nnd killing after some stimnlanis (12-1,1). Recendy
678
a reduced superoxide productlon after IMLP stimulus has been dcscribcd; ii has been hlpothcsized that this detèct may be due to a reduced rcceptor expressìon
on ceÌlular surface (23-25) or to r de6ciency of crìlcium homcoslasis (17) or to an ìmpaiment ol neutrophil membrane fluiditt (26). A tunher aspect of neutrophil tunctional dclìciency in cldcrly subjects is the rùduced supe.oxide pro duclion by cells primed in vitro (27). A reduced abjllty ol adhesion to plasiic suÌface treated with proleins has been demonstraled (15). All the data have been oblaincd by Èxperiment in vitro rnd, 1o our knowledge. no studìes have been .anicd out in vivo. Moreover, in no other stùdy, thc oxidltivc nrctabolism and the adhesion have been evalLutcd s;multaneously and io the sxme experimcnlal
-Ihe
skin window rechnique aìlows us 1o evaluale the PMN nigratrcn in and to obtain cells nrigmrcd from an inlìanrmatorv tbcus: lhey are characierized by rhe chaùgcs phlsiologicall) pmvoked by phlogosis processes. Morcovcr ou. mcthod pcnnils lo conìpxre in a sìmukancous assay va.rcus tunc tional paramcters oi circulating and migrating PMNS frcnr the same suhject. In vivo migmtion of neuirophils was qu,ìntilativcly in thc nomal mnge in our elderly subjects gmup, indiclting that neither endogenous genemtion of chemotactic compounds. nor endothelial and tissue componerts involvcd in ccll mìgration. nor neutrophil nx)lcmcnt machineries are afiècted by aging process. In accordancc with prc!ìoùs ìnvestigations fn)m our and other labomtories (l 7) our r.sults show thal cxudate PMNS are metabolìcèlly primed. being more responsive 1o fMLP stimulus thxn blood ncutrophils and shos,increased adher ence in thc abscncc ofslimuliìnis. This work shows that thc priming clent occuN also i. clderly subjÈc1s. In làct the net dlfferencc oioridalivc rcsponscs to fMLP bctwcen exudate 3nd blood cells is sinrilar io the t§,o examined groups, and this is ln agreemenl with thc normal chcnDractic migraiion obseNed in vivo in
!i!o
elderly subjecls. Oùr results do noi perfecdy agree with the htÈràture dr1à: in tàct. in our study, PMN mìgralion and adhesion resulled similar in young and eldedy subjccts. PMN migratìon is r very conÌplex phenonenon. bxsed on both the lntegrily of adhesive PMN tunction of cytoskeìeton dynamics and the action of chcmi.al sùbstances; a skin window method pcmiits to cvaluatc this phenomenon ìn a global way, unÌike ln vitro lechniques that study only the chemotaxis. It is conceivable that i. vit.o evaluation of chemotaxis. that is canied oul under specìfic condilions, might unnask specìlìc defects thal arc noi relevant fòI ìn lnstead. our study falls into line with previous daia as rcgards the lower supemxjde production in elderly than in young by circulating PMNS .ìfter IMLP stimulus; wc did ùot obscne any ditrcrcnce by utilizing other stlmulì. Therefore the PMN defecl of elderly subjects seems ro bc both stinrulus spccific (fMLP
ìièùlrophil Nlig.alion, Oxidsli!. \retrùolism. and AdlEsio.
versus STZ and PMA) and tunclion specìfic Gxperoxidc production vcrsus mjsration and adhesìon). A defect ol NADPH oxidasÈ seems to be excluded- becausc the PMN rcsponse after PMA, that dircctly opcrates on prctein kinase C, and to STZ. that opcratcs ùrough disrincr rcceprors and tiììnsductbn pathways, is simila. in the elderly and young grcups. Some authors have demonstBted a diminished expression of fMLP recep tors on the PMN menrbmne in eìderly subjects (23 25). we did not measurc fMLP recepton in our sudy, but ourdak, showlng thrìt fMlP'slimulated adhe sion and in vitro migntìon are normrl, suggestthat ifadùficiency ofchemotaciic receprors is present, this is reÌevant only for some tunctions (oxidalive metab olism) and not for other§ (adhesion and migrattun). Ho$,evc.. this possibility seem§ to bc cxcluded bccause we hàve previously showed that oxidatìve metab olism actilalion and .ìdhesion have sinÌilar dependence on fMLP doses (6, 22). Accordlng to our data, the stinrulus and tuncrion specificity of blood PMN'S defect in elderly subjects appears 1c) be due to jnhibilion of some mechanism hcaled i. thc signal transduction prìthway bctwec. fMLP rcccptor lnd
NADPH oxidase. The prccisc mcchanisnì responsible forthese age related difièrences renìains to be identificd, but an hypothesis can be advanced on the basis olour prevbus lìndings using inhibito§ of Ieukocyte aclivarion. In fact. we have demoùstratcd (22) thnt adenosine, a ph)rsiological nucleotide thrt ìs known to cause lncrease of intracellular lelels ol CAMP (28, 29), inhlbils superoxide rclease induced by fMLP but has nol ellec! on STZ induccd supc()xide releèse: nDrcover. phlsiological doses ofadenosine (10 7to 10 6M) inhibir fMLP induced supemxide rclease bul not fMLP induced adhesion. Finally, we hale rEcently found (Car-
le$o el al. manuscript in prepantion) tha! penbxifylline, a CAMP regulating agent, lnhibits supemxide production by blood cells much more efectively than superoxide prcduction by exudate cells suggesting that the Iatter are morE rcsis rant to rEgulatìon. Takcn togcther, these evìdences st.ongly suggcst rhat thc àge' relrìted defect of blood neutrophiLs descnbed in this paper is due to inÌpainnent of ùe same step that iù the transduction pathway of neurrophils is afected by CAMP ùlcvation, and therefore to the activation ofprotein kìnasc A.ìnd possibly
to phospholipasc C inhibition (30. 31). The garì.g by CAMP of signal fiow through orhùr pàthwàys in diverse biologlcal processes, including lrlitogen-activdled prctein kinases involved in growrh and difièreniiation, has been recendy reevàluated (32).
lnconcÌusjon our srudy shows thal many PMN tunctions in elderly subjects, including nrìgratrc.. priming. adhcsion, activalion by opsonizcd pafticlcs do not difcr from young people, suggesting that the overlìll defence tunctìon of these cells is oot afiècted by aging. The only parameter thai we have found to be in
6ti0
nÌarked variance bctwee. lhe two groups ìs the poor supercxide producttun after
fMLP stimulus of blood cells, a defect fftnn whlch cells effcìent1y recover during thc migntion into exudaie. The spÈcilìcity of this defecr in nculrophils fiDm eldedy subjects could bc a mark of eilher an lncrcase of celÌular CAMP or of an incrcase of sensitiviry of cAMP-reÌatcd celiular rcsponses. Furrher siudies are necessary to investigate the mechanlsn ofthis ellèct of aging proccss on cellùlrr flrnctions
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3.BIÀJ.D.L IU B^IBÀR\, A CARJTTIO, \I. C{R{I'I, M C SERR{.S CHÌRÙNIIOLO, ANd P. B.[\!rÙ l!93 FacÌor specilic .hangcs ù oridxùle hursr Èsponse othuùan PNIN§ in skin window exudatcs ,1.
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17111 21
Br\r. D , L lt.
B{\rB\RA, A. CnklÈrro, P. C^R^M\s.Hr, M C. SERRA, C. S^^ror^y^so, P. Glzzo, and P. BELLA!|rE. 1993 Molecular mecha0isns otpnnìng: erudate human PMNS are primed lo lMtjT LEU PHE bnr hx!È .on rl iùfureìlular calcium and .AMP fts!Ò.ses Bu . M.1 Biot M?A l8t2?5 135. P{ù.P B.R.W GRAEFF, S J lv1{rrs, andT K HùNr. 1990 SuFosridÈ pùdùctnin by §ound PMNS. Evidence Ior in.rcùscd a.riviq oI thc NADPH oxidrse ,,lr.r. .!r,A 125:65
6. BLLL^VIE, P. A. C^RLEro. D Brasr, P CaRAMAs.Hr, F PoLr, F SrLroR\. and L. M. BANTBARA. 1994. Studies ot skin ù,ndo* c{udar. huùan neuÌiophih. Compl.x patrrns ol rdhèrèncc !J\erum.oated surlaces independence on IMLP dùses l4i"n-zri.,r 18:575 587. and S W ED\rÀRDs. l99L Nrùùophik isÒìrrèd ftom lhe slno!ial fluid ol palie.6 wìth rhcùmarord anhriln pnm,igandaction inrivo.,1,n. tlc,,.
7. N!k.o!BL, H L,R C Bu{rx\{r-r.
3. W\RD, P. 4.,I S. WaRRrN, and ( i .,oHNsoNN 1988 Oxl,gen radic!\, inflmnrarion and lissue inlury lic.1ldr. Airl. ,Url. 5:.103 .103. 9. WLrss, S J 1939 Tilsue destruction by neùtrcphils. t(. ,h,l J Mp.l 320:365 3?6. Il] FoLLÌN- P, M P W!I1^\\. B, DÈ\aLD. M, CLs({, and C, DÀHLaRLN 1991, HLùrn nrnlmphil ùìgfliion ii!) skln chanrbers À asociared §nh prodù.rbn ofNAP l/lL8 and c5a. Eur. J. Hach.1Ò|.11t1\ 16. CoRR.RAND., X.. P F. LasÀRRAcuL. rnd G. FTLLoL^. 1936 Neuùophils of healrhy ased hunìans ùrc Nnìaì. ,t/g.r. ,18.rìj DÒ 16:51 63 12. ANro\Àcr. S . E. JrRrLLo, \1. T. VENIiRA. A R GaRorALo. and L. Bo\oM! 193.1. Nonspeci6c immunit, in aging: delìciency of monocyre ànd pollno.phÒnucìearcell mediated tunc tion; Mè.h. Aglin! Dtt 24:161 315. 13. Nrq^. Y.. T. K^sÀra. Y. MryA.Hr, and T. (aNoH 1989. NcùrrcphiÌ .henoraris. phagocy tùn and paEmeÌem ol Èactire oxlaon spc.lcs,n huùrxn rging: cross secÌiooal and lonlitodinxl
l1
stodies.lil. Sr. lg:1655
1664
N€uftophil Nligrarioù, (Xidrlier \lc§bolism. aod Adùllion S.. A S{BÌoN.FLÌ-o, D DF({Rn. lnd I KARDùN 1988. Age .elaled changes in lùnctions.t pcriphcral blù.d phùgo.yts M.., ,1S.nrJ D.,. 45:221 229 ]5. ToRToFELLA. C., A OTToLFNCHÌ, P PUcÌJTs., I JIRI,J!, and S ÀNToNÀ.I. 1993. ReIa rionshipbùscù espnato']- bùEr xnd adhcsivÈne$ capacity n.ldcrly poìynDrphonù.lcar.clls 1.1. RaBArrc,
,u..li.
.4s.r?i8
16. N^cÈ1,
J
Der 69:53 63 S.P!LE.F
E.R
bactedcidal capaciÌr-
I
CHRESÌ, and
§.
H. ADLEF 1981. O{idarirc m.rxbolism aDd and rsed aduh! J C...n,/
oipllymor|h.nuclelr lcuk..r"lo ftom ]oung
Lr LrsLHrz. D. A. K B. LTDLPA,
and L. A Bo[R. 1988 The rcle oi calciunr in lhe ase nèut ph,Ì lun.(ion. 3/d1i ?1r659 665. I3 LI H\RL, G J, J X. CoFBER{\D. C FoT:RN!.R. R GAIANAII, W H!M{\s. B KTNNIS, .ni.n. tòr iùnnrno!.ron A. V!Ì,r rR HrR\J.r-r\x. nnd G G. SÈr\urN 1934. ,l!.ing D.r 28111 55 tologi.al sùdrcs in nan: ùe S!^']EUR pmtocol MtL']1 ^dnnsion 19 Vrr.arF J A . J. l. GaLLr\. W. M NnrsÈÈr. and R. K RÒoi l!86 ,rLabomÌo^ Manui .l Nenùùphn Fùn.lion Raren P6s, Nes York 10. SE._\, H. l, and \\ F .I!:Ncr l97j Nètrrophil ù,gfuròn nì helllh rnd di:crsc. S.,nr related decÌine Òl
21. B^Bror. B lvl. R S. Krp\Es. and I T. CuRNLrr.. 1971 Bnnoei.al delense mechanilms 'lhe trlductn)n bl leuk..lre\ ÒI:upèroxidc, a porcmal br.rcnridrl rlenr. J C/i,r /nr.,
22
2l
RnÌ {vrrr, P. S CÌrRLvBoro C NI-\NSoLD!. G. GnNDr\r. ùd P DRr 1992 Asiùuhamous asa] lor oxidalire nreÌabo[Ìn and idhesion .I human neutrophi]s. Eridence ior coreLalion\ and dls$cirinin rI rh. lso r.sF.ns.s. ,7 Z?xt..!n,An strlz! lll liIL laLaHLrN, 8.. K. O'tIiLLEr. aDd l. CÌ. Corrrp 1986. Ale reLaled nifeEnces in gBn
uLocyle.hemolixi! !nd degnntrlarn)n C/i, J.i. ,0:5! 62. 2.1 S.FLE\cr, L., A OrroÌrNcHr, M T V.\r!i\. A V
Por[\\\o, C ToRr.R.ua. E. Mono.lr.-pollroDh and llnrpho.ycrùrdirtd ch.noracli. iÈ!p.nnv.nc* in rhc eldc l. tOS Rn lunùnl.h tùn|hanxd.r/. 10;28:ll related alteÈ 15. Rro. K. NI ( . \'f S C!RRÌ., J PaBM^\^BHnN, ard H.I CoHEN l!92. ^er 17tB11 anons nr acrin cyÌoschLeton anl recepktrextEs\ion nì huùÌan leuktytet I A?nnd JrRrLLo, and
S
ANToNA{r. 1990
:6. RÌv\ar, B. S metrrhrur.
BERiMT!. N1 SHÌNrr/(Y rrd
GLoBEsoN. 1930 Coftcl.riotr bcÌw..n and aBc,ng in uìxn,V.d .lxf
^ ùttvrfrn \n.osn]. senrr cholesterr. lynrtho.ltc
i,,{ D{L. 12:l19 l2a
27
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23
rorphonucleir cÈlL Ìi cldcn! subjr.6 ln.n À{! Atnnnl. 4.1\l 224 CR.Nsrr\ B. N . S B Kk^!EP. C \!rnsvÀ\N rnd R HrRsHÈoRN 193:l Àdenosine r plìlsioloeicrLmodulanr of lLpn,xide rnio .!.ncmùon br_ hùnìr icnkùph'ls. J. all Ir,/
2!
CRo\sr.ÌN
30.
DELL\BbN.{,v,P DETocNr.M G(rL\(o§h(, L. \'l vr.LNLrNr,andf
B \,L DrcLM\.D Nr.H!Ls. A J Hur.È[o\. aDd NI. wrLLrai6 1990'rbe rd.iosinrrirùùophil pandox .esolved: human nenù.phil\ pÒ\sè\s bÒlh Al r J ,{2 r..cprors thar tniùìot.hcnDrax§ md inhibir Or lenenli.n, respecÌi\eLy. J. Clrn /n'?v 85:1150-
ll5l
3L
Dr vÌRcnÌo Annfrn, 1986 Cy.ìir A\'lP rnhrLilior oi phosphoinosiridc tumo\er in hunìan 'eutRìphi( Bi.?ht!. Adu Aa6:À,1l 441. BÈLL^vÌrE, P 1988 The supen)xide nimiing cnzlmaric slscn ot phrgorcllcs l,?. À.di.d7
32 IrL\ca!, R.
1916 Cating by Clclic A§IP: .xpxnded rolc inr ar sgnalrE paÌhray
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