LV
"",J Zntny
Po>z-.',È., S'^.z-z,tJ ,q
ite atnTLi[ 9 rLyr-yA..yt,s
(A,r7,",..-: , h /
[4 tsrcu- , l-t i ( t^ n , 17? e
't-'1)
,à
The neutrcphil: strucìure and
deience tunction
P. BELLAVITE, D. BARISONI, A. SOR]O, M. GOVEFNA,
F, BAZZONI, [,1.A. CASSATELLA
lstituto di Patalagia Cenetule, Uhivetsitàdi vercnae centrc Èegianale veneto
per la Studiadelle Ustiani, vercna. haly
1, INIRODUCTION
Neutrophilic granulocyles, also ca led poLymorphonuclear lelrkocytes, are mobiìe
ar_
senaLs capab e of seeking and deslroying a varieiy ol bioLog cai targels. They are one
ol lhe anost imporlant componerls olthe bodydèlence syslem and of the acuie nflam'
malory reactions n the lung and other iissues. An ouÌllne of the var ous b o oglcal ev_
ents by which neutrophils ex€rl lheir defence f!ncl ons is repofied ln Flgure 1.
When pathogenic agents are formed and/or deposiled ln a lrssue, circulatjng neulro'
phlls may be recruiieci irom the irùavascu ar cornpartrnent into the llssue by the Local
geferal on oi a variely of soluble medlators.lhese medialors are derved irom numer_
ols sou rces, lnclùding lhe nvading parasite, ce ldebr s, aclivaled p asmacomponents
and olher celltypes sLrch as endotheLial ce ls and macrophages. The acule inflammatory response may be rapidly amp iiied by the neutrophis themselves and by lhe production oi medialors that diiiuse n the bloodslream (interLeukjn-1, coLony-sÌimulaling
laciors, lumor necrosis factor, e1c.) leadlng lo a systemic body resoonse wilh synthesis oi acuie phase proieins by the liver, neuro-endocrine modllicalions, demarg nallon
oi neulrophi s Irom normal mjcrovasc!la1ure, acce erated myeloid prolllerauon ln the
marrow elc. These evenls ead lo marked eukocytosjs and prìming oi circulating neu_
irophils lo potenliale iheÌr activty n lhe subseouent ceLl! ar fuflc1ons.
CircuLallng neurophiis and endothellal ceLls ln lhe area ol lhe phlogistic reaction ex_
press membrane anchoring molecu es and leceptors lhal ,acilitaie the adhes on and
the n lial m gration ol the leukocyte through the vesselwall. Direcled cell migration ol
neuvophlls, thal are able to orlentale the movemenl in a gradienl oi chemolactlc factors, causes the accumulation oi highly resporsive phagoc)'les in lhe centle ol lhe lnflarnmat on, where they reaciwith lhe eljopalhogenìc agent, These actvaled cells have
increased numberol receptors such as for exampLe those lor formy ated bacteriaLpe}
tides, Ior the Fc porlion of immunog obulins and ior comp ement iactors, lhal enable
them to aclively eng uli Ìhe lo re gn particles, ln lhis phase, opsonìzallon and in iia dam'
age lo the lnvading microbes by components of the exlravasated plasma eliectively
cooperale wilh the neulrophi defence function, The ioreign parllce is inlernalized
wilhin cyioplasmic vacuoles which luse wilh lysosomal granules, lormlng phagolyso.
somes. Hydroi)4ic enzymes and other bacleric dal componenls aIe concomilanlLy re.
leased inio lhe phagolysosome durlng ìhe process kno\rn as " deg ran ulation '- A substanlìal amounl ol ihese Iactors may excape liom the forrn ng endocylic vesicle ("regu rg ilation du ring ieed ng') or nay be directly released in the exlerna m lle! due 1o the
membrane perlurbalion caused by solLrbLe med ators ("exocylosis'), ihus causing
damage lo lhe connective lissue and nearby cells.
OPSONIZATION
t
"+.
\, ,t'' o
N
(§..!(**
§e>
\J
ADHESION
(cDa, c018.
r.c.A.M.)
LEUKOC'TOSIS
tt
tr*
i#g?g:*
KILLING
(Oxidative merabotism,
bactencidal Bnzymes,
del€nsins,ac.)
BLOOO
Fig.1. fhe nain
VESSELWALL
CONNECTIVÉ TISSUE
lunclions ol neutrcphils in host detence.
Al lhe sarne time, a dramatlc incroa§e ol oxygen consumption occurs, wjlh activalion
ollhe hexose monophosphale shunt and generalion ol oxygen.derived free radicaisand
olher toxic dèrivatives (see seclion 2,3) that are released into lhe phagolysosome or
eventually oulside the cell. Th€ intrevecuotar pH changes iirst by rapid atkatinizelion,
due both lo protonation ol superoxide and consumplion oi hydrogen ions in the dis.
mulation reactjon, lollowed by slow and progressive acidiiicalion, due ìo activatjon o1
an Na/H. anliportèr.
All lhe above menlioned evenls are necessary and essential for the rnjcrobicidal ac1iv.
aly and tor lhe digeslion of the engulled partictes, but the same mechanisms are involved in other eiiects, i,e. tumoricjdat activily, cytoloxicily, tissue matrix injury, amDlrficarion ol lhe in'lamrnaro-y reaclron, orimrrg o{ tne 1lssu6 nea,ing processès.
Tl^e pJroose ol 1nrs reoofl is lo briery oescr;oe sorre o{ tne slruciLrat p'ope.lres ot
neulrophìls (Figure 2) and §pècilicatty to retate these propenies to lheir physiotogicaj
iunction. ln parlicular, the main neutrophit slruclures fial wal be considerèd ard fie
piasma membÉne. the cytoskelelon and lhe various tysosomal granutes. Olher cell
componenls such as nuclei, endoplasmic rsticuium and mitochondria are oi minor imponance in malu re neulrophils, whicn do notdivide, hevevery tow
É,rotein synlhesjs and
derlve mosl oi lheir energy supply irom gtycolysis. Due lo th€ targe bodyòi knowledgè
I
Th€ neutropÀll: struclur€ and del€nce functlon
t\
FECEPIOHS
TFANSPOBT
SYSIEMS
IONICCHANNELS
rV\Y,T-z--'
ll
.Y-.4.';-
-ra\:
a. \J
AZUROPHILIC
GFANULES
GOLGI
SPECIFIC
GaANULES
TEBTIABY
GFANULÉS
NUCLEUS
GLYCOGÉN
PYNOCflOSIS VACUCLES
Flg.
2.
Schenalic rcOteseòtatioo af lha ultastructute al
a
neutrcphil gnnùlacy7è.
that has been reviewed, many inlormatjons wìil be tabulated. A detailed discussion of
each oi these is nol possible in the space avarlable. but other reierences can be consulted lor further readin! []-171.
2. The plasmamembrane
The exlernal membrane of neukophils ìs a hjghly sophysticaled skucture by
which the cell communjcaies with the externalmilieu and bywhich many oi its
effector iunctions are reaiized. Both the memb.ane lipid bilayer and the pro.
teins that are there embedded have important and speciric funclions lor this
2.1 The recognitlon appaEtus
The r€cognillon apparaius of neutrophils is able of specifìc binding with a large
series of llgands, some ot which are listed in table I. Considering the various
molecules lhat interact wilh neutrophil membrane, it can be reailzed thal the
receptors are lnvolved in lhe regulalion oi cell groMh, in largeting the cells to
sndothelium of inflammed areas, in chemotaclic ori€ntation and movement. in
t-.
P, Bèllavlte 6l al.
1
labte
l.
Ligands binding lo defined neutrcphil rcceptors
Regulatory Prolein§:
lnlerleukin-1
Ìnìerieron-d
lnlerle'on-7
Tumor Necrosis Faclor
GM-CSF and G-CSF
lnreleukrn-8 (NAP)
lmmunoglobulins and comPlemenl:
Monomeric lg (lo Fc receptor-l)
lmmunocomolexes and aggregales (lo Fc
cR
rec ll and lll)
1 (C3b)
3 (c3bi) = Mac 1 (CD 1rb)
C5a
CÉmplemenl decay accelsrating iaclor
cn
Glycoproleins and caòohYdrates:
Mannose/lucose glYcoProteins
Mannose 6-PhosPhalè
Glucan
ConcanavaLin A
-
Lipopolysaccharide
Muramyldipeolide
L:pds:
Leukolriene C.,0,, 8.
Plalelet Activaling Faclor
Phorbol-my stale acelate (PK C)
Peplides:
l-Me1-Leu-Phe
Substance P
Addilional signif icanl ligands:
Translerrin
Laminin
Plasminogen
l.C.A.M.-1 and -2 (to CD11)
Beta adrenergic agonisls
Opioids
padicle uptakè and ingestion and al§o in the modulation of the cell actìvity.
The LFA.1 molecule is broadly expressed on the surface oi leukoc!'tes; it
conaists of two glycoprolein lransmembrane unils: a (CD 11a) and É (CS 18) of
170 and 95 KDa respectively. During ìhe ceil'cell contac'l LFA'1 binds to mo'
l€cules called intercellular adhesion molecu{es (l.C,A.M'1 and '2). Deiective
expression ot CD 18 Ìn some individuals causes severe disfunction of leuko'
c,4e adhesivjly and of resi§tance to inlections.
Thè
neulroohil: siruclurè and défén66 Iunctlo.
2.2 Transducllon systems
The membrane is also the sile of most of the cornponents ol ihe transduction
systems. These are the biochemical rnechanisms which perlorrn the transmission ol biological informalion bett een the membrane receptors and the various effec:or machinerìes invoLved in the funciions 6uch as movement. dègranulation, nìelaboiic acljvation, etc. The iransductìon systems are mainly,
but not exclusive y, locared ìn the plasma membrane and are composed by a
series of proteins 1,{ith enzymalic acliviìy (adenylate cyclase, kinases, proleases, pho§phoiipases and other enzymes ol lipid metabotism) or regu atory
f!nclion (channel proteins, G'protein sub!nits, anchoring proteins) and by sev.
eral other factors (rnessenqers: caici!m, inosilol phosohates, diacylglycerol,
phosphatidate, cAl/P, etc.) generated by these enzymatic mechanisms. lt is
not o!r purpose to analyse in detail the complexily ol the§e systems and their
interrelationships, but some asoects of these merit parlicular attenlion.
First ol all, many receptors (for chemolaciic peotides, lelrkotrienes, PAF) are
coupled lo cell response§ lhrough a guanine nucleolide binding protein (G.pro.
tein) simila. b!t not identìcal to the one cf the adenyiate cyclase syslem. Siu.
dÌes using perìussis toxin have proven instrumental in the understanding of Gprotèin involvement in the §limulu§.response co!piing palhways. Perlussis
loxin ADP-ribosylates lhe a-s!bunit ol some G.proteins and by this mechan.
ism inhibiìs a numbér ol responses of neutrophils. lhìs biochemical eifect
could be a ,actor o, the elevaÌed pathogenicity ot Badetella pertussis lntec.
lion.
Second, a series of marked variatìons in ihe physicochemical staie oi the lip.
ids takes place in the membrane of stimulated neutrophils. Among ìhese. a
membrane.associaled phosphoinosiliCe-specific phospholipase is activated
upon exposùre to chemolactic stim!lantS. In particuiar. phospholipa§e C hy.
drolyzes phosohatidylinosì1oi.4,5 biphosphate (PìP,) to the §econd messenger
producls inosito{ 1,4,5 triphosphate (lPJ and 1,2 diacylgiycerol. lP3 is thought
to be the main lniracelUlar messenger for release ol Cart kom intracellular
§tores, while diacylglycerol is responsible for the activalion ol protein kinase
C. lt should be pointed olrt that the stimuius.Ìnduced rise in intracellular Cart
al§o results kom an increase in pla§ha membrane permeability to extracellular Ca1". The elevation o{ intracelhlar Cart is important lor varìous subsequent events Such as: a) lriggering o, phospholipase A2, another membrane
enzyrne. that plays a pivolal role Ìn the activation of arachidonic acid cascade
with generation ol other messengers slch as arachidonic acid, thrombox.
anes, hydroxy acids and le!kolrìenes, b) activation o, calmodulin-dependent
protein kinases and also ol prolein kinase C. c) modulation ol cytosketeton
function by activaling geisolin (see below). On the other hand, evidence from
various laboratories suggest that phagocytosis and metabolic activation may
also occur in experimental conditions where the intracellular Ca?, is com.
pletely bufrered, suggesting lhat calcium.independent aclivalion pathways
may exist [18, 19]Olher stim!lus.response coupling qvents ihat are localed in lhe plasmamem'
brane a.e: a) cpening oi ionic channels lor Na', wilh subsequent membrane
depolarization; b) cyclic n!cleotide metabolism variations: exposure oi neutro'
phils 10 phagocytosable particles res!lts in a rapid doubling oi cAMP levels.
The precjse role ol lhe generation ol this second messenger in nèukophils is
unknown. Besides its possible effect ln glycogen metaboìism, like in other
cells, it may represent ai negative regulatory iactor. ln fact, agenls increa§ing
intracellular cAMP such as PGE, and dibuiiryl'cAMP cause inhibÌiion oi cellu'
lar responses, possibly due to inhibilion ol phospholipase C by cA[.4P'depen'
dent proiein kinase A: c) protein phosphorylaìion, due to the actìvity ol kinases
which are strictly assoclated wlth recepiors, such as those Jor groMh factors,
or that are translocaled flom lhe c!'losol iollowing cell stimulation, such as
protein kinase C. The inlracellular target§ of stimulus associaled phosphory'
lation processes in neutrophìls are at least 20 prolein species and they include
myosin light chain, faclors necessary ior NADPH oxìdase aciivity or activa'
tion, including a membrane prolein ol32 KDa thai we have described in pig
neuìlophils [20], cytochrome b550. However, most of neutrophil phosphopro'
leins have not been idenlilied yeÌ.
2.3 NA0PH oxidase
Another peculiar enzymatic syslem thai is located in the plasmamembrane ol
neutrophils is the NADPH oxidase. This is the enzyme that genérates the ox'
ygen-derivèd lree radicals and ìs present in the membrane of all the phago'
cyles. The slrlrcture and the activation mechanism ol NADPH oxidase have
been the object of rnany investlgations in several laboratories, including ours,
in the past Ìen years 121.341. These problems have not been Iully clariiied yet,
but ìhére is now evidence lhat this enzyme consists of an electron transport
chain where a llavoprotein, a c)'tochrome and probably othel proteins are as'
sembled in a Junctional complex. The a§sembly oi lhe various components
couLd be promoled by phosphorylation of particulal proteins, by changes of the
Iipid milieu oi the mernbrane, by G.proteins or by other unidenliiied transduc'
tion pathways [35].
Figure 3 shows a tentative model oi the actlve NADPH oxidase, which ac'
counts ior the presenl siale oi knowledge. The iirst proteìn to be considered is
the proieìn bearìng the NADPH binding §ile. This protein has been idenlified by
lracing with radioactive nonhydrolysable NADPH analogues and'has the mo'
lecular welsht oi 66 KDa. According to most aulhors, this prolein is a FADcontaining llavoproteìn, wÌth the iundamental role of accepting two electrons
,rom NAoPH on the inlernal site ol lhe membrane and o{ carrying oui one'
eleclrcn reduclion ol cytochlome b5s. However, there ìs not definile evidence
that NADPH binding site and FAD beiong to the sarne polypeplide.
A iundamental iaclor of the eleciron lran§porl system is c!'tocnrome b5ea, a
-
.:ri::- -
Th6 neuùopàil: st.uctu16 and dot€nce t!nction
peculiar b.type cyiochrome with a nurnber ol unusual prope(les, includìng a
very low oxido-reduction mid point potential C24S mV). lts molecular and lunctional features have been defined jn a better detari compared to thè fiavoprolein. Purilication studies showèd that human cytochrome bsso is a heterodimer
with two subirniÌs oi 22 KDa {n. 3 in Fig. 3) and of 68.92 KDa (n. 4 in Ftg. 3).'fhe
former ìs probably the heme-bearing s!b!nils, whfle the lafier is a gjycoproHEXOSE MONOPHOSPHATE
I
t
cYlosoL
EXTBACELLULAB
SPACE OF
PHAGO.YSOSOME
Ftg.3-
A woùing nadet ol the enzynatÉ systèn NADqH axdiase. 1-2: NAapH òinding prcEn
otetn (passibty the sane potypépndù;3: Cytochrcne bs(tow-tuotecutat wa;gft su.
buntt r': Cyrocno4e asthigr'-aatecuff weght suaunit): S. Cylosaic tactarc that ale tians(..
cated lo the nenbÈhe ourrq acttvafian ol the enzyn6; 6: Othet outatve p.atsn can@nents lhat
arc ùvotved in the enzynalc ac vtry 7: L;oids e_g_: phospàardylsetiie, pnasphahdic acÉ, u.
echidÒnic acntl
and
tava
tein whose lunction is probably to anchore the cylochrome to the membrane
and to connect it with other components. ln fact, genetic absence of the6e-92
KDa proiein, as it occurs in the X-linked fo.m ol chronic granutomatous di.
sease (CGD) causes lack ot expression also of the tow.molecular weight sublnil, of cytochrome specùum and lack of binding oi regutatory cytosotic
components to NADPH oxidase complex. we have ob§erved that jn partially
purified preparatìons ihe cytochrome b55! is p.esent in targe motar excess witÉ
respect to the flavoprotein [36, 37].
Oiher components of the system have béen recenfly identified on the basjs of
various experimental approaches: a) Sìudies in ce[kee models of oxidase ac.
tivatìon have shown that ai least i,vo proteins (n. 5 in Fig. 3) wilh motecular
I
&
§
I
weighl ol 47 and 65 KDa present in the cytosol of resting ce s partic pate in
the activation process of NADPH oxidasè. Genelic absenceof one ofthese two
proleins, aS it occurs in the autosomal recessive iorms of CGD, causes lack
or marked deliciency oi superoxide -ìroduction by neutrophils from these palients [38, 39]. Fecenlly a G.proiein that is associated with cytochrome be has
been described 1401. b) Monoclonal antibodies raised agaìnst the active oxidase complex of pig neutrophils and possessing sirong inhibilory capacity over NADPH depenéent superoxide produclion bind to sma potypeptides ot 14/
18 KDa, that have lherefore to be consicjered pùlative components of the system (n. 6 in Fig. 3) 1411. c)-ihe important role ol lipid iactors (n. 7 in Fig. 3) for
the enzymatic activity or ior maniaining the optimal coniormation ol the sys.
lem is dehonstralèd by lhe reouirement of phosphotipids exhibiled by the so.
lubillzed oxidase in order to Iunclion l42land by the fact that phosphatidic acid
ìs able 10 activate the dormant oxidase in membranes isolated Irom unstimuìated pig neulrophiis [43]. The participation o, quinones thal may shu le etec.
trons between the various components ol lhe system in the iipid environment
has been also proposèd by some authors [44, 45].
The NADPH oxidase is activated only in that region ol lhe membrane ihal is in
contact with the target [46]. 11 generates O; as the iirst oxygen reciuction prod-
ucl, but subsequent rapid reactions lead lo spontaneous and/or enzyme.catalysed lormation of several other toxic derivatives (Fig.4). The physiologic
lunction of the kee radical production is the deslruction ol invading microor
ganisms and lurnor cells. The range of microbiat largets is very wide and includes all kinds of bacteria, lungi and viruses. The most powerlul oxidizing
agent is hydroxyl radical, but other very loxic products are also generated by
ihe Hrolperoxidase/halide system (see sectlon 3). The neutrophil is also sus.
ceplible to darnage by these reaciive oxidants so lhat il rìsks to be killed along
wilh the larget. However, the phagocyles are abte to delend themselves
against oxidants, at least lo a limited exìent. The anlioxidant syslems of the
phagocytes inciude superoxide dismutase, catatase and gluÌaÌhione pe.oxidase, alocopheroi and ascorbic acid [47].
3. The cytoskeleton
The neutrophil c)loplasm contains a highly filamentous lri'dimensional network composed o, microfilaments and microtubules. These strucìures have
been ìmplicated in pseudopod formation, cell movement and intracellular
traftic ol other organellesThe microiilaments are mainly cornposed of the contractite proteins actin and
myosrn, which comprise about 20% oi cytoplasmic proteins. Actin exisls eith.
er as a giobular monomer (G.actin) or as a double-helical lilamentous polymer
Th€ n€ùtrophll: skuclur€ and deiènca luncllon
!!1s-9y!:':-a!g gl9!:ulL,ltql-"rl]3i9l
t
l
-"ldrrrbrlle.rèEepbÉ--I
u
---Br:Scgior:g:s:--moorrrcauon, ua- r rncr€4s6,
GipÉ
G.prc1oin, Pol. kinases, erc.)
I
.t
---Ési§llrrLai-
-
_NA0PB-OX0éSE
I
,,
q + "q- +H '--------'Hq
HO;
{SUPÉBCXIOE ANION BAOICAL)
(HYOBOPEBOXIL FAOICAL)
(soo)1
+
g * 10,
.
{MPOI2
HP, . Cl- +Hr --:-_--> Hp +
HCIO +
q-
+ F"
F€-'
OH'
F{-
+
"'
HrO
+
R-NHCI
-------,
q+
F€
"
Hro, --(!:$-, r"--- * on- *
+ PUFAT --..'
uProFAorcaL
HP +
+ q ------'
1HYPOCLOROUS ACID)
Hclo
------->
NH2
(HYOBOGEN PEBOXIDE)
(CHLOFAMINES)
on'
{HY0ROC,/L BADIcAL)
LIPIO BADICAL
uPoPÉRcxloE RADlcaL
A
Geoe?nar ot sùoeta\oè anian tadicat Òy stinutatèd neutrcontls and sotue stròseouenl
rcàar.,ns oat arcouce to,,c detuatives. 1: SOD: suDercxÉe disnùÈse: 2: MPO: dyetapercxi'
dasè:3: H-w: Haòe.'-vlelss rcacnans: 4: PUFA: palyunsalu.aled lalty acids.
F a.
(F.actin). The distribution between these two actin states shifts dramatically
aiter cell activation, namely the amo!nt oi G-actin decreases with an accom'
panying ìncrease in F.aclin. Moreover there is a local redistribution ol the Ìlvo
forms in ìhe exlending pseudopods during phagoc,'losis, wilh rapid polymerization/depolimerization events associated with the movement o{ lhe mem.
brane. The cytochalasins, tungal metabolites known ìo react with actin tilaments and lo inhibii their assembly, powedully inhibit phagocytosis and le
comotlon, while enhancing exocylosis.
Myosìn is an hexamercomposed ol two healy polypeptide chains and four light
T
à
{&t
P. Betlavtte et at.
polypeptide chains. The heavy chains conlain gtobular heads which bind
actin
and also possess ATPase activity. Myosin is concenlrated in the pseudopods
of migrating and phagocylosing neutrophils and contracts the actìn get by hydrolysis of ATP which provides energy lor contraction. This phenom;non m;y
be modulaled by phosphorytation ol myosin tight chains.
ln addilion to actin and myosin. neutrophils contain several aclin-binding pro
teins which regulate ihe potymerization state oj actin in the celi and its bi;ding
to the plasmamembrane. Calci!m concentralions greater than 2 x 1O-r M dissolve the actin gel by activating the protein getsotin whtch inhibits potymerization. Additional iniormation on ìhe mechanical responses of neutrophils may
be lound in other recent papers [48-51].
Other important c)losketetal struclures in neutrophils are the microlubules,
which are hollow iibers, 24 nm ìn diameter. Ìhey assemble as cylindrical polymers ol the prolein tubulin, whjch is found as a heterodimer oid and su.
É
bunits. Soluble tubulin is in equilibrìum with the assembted torm, allowrng rapid groMh and dissolution of these structures by shiilinq ot the equilibriuÀ. [.ìi-
crotlbule assembly and disassèmbty can be controlted in a variety of ways, i_e.
by cyclic nucleotides, by Ca,r and by the intraceJtutar concent;aiion of S-0.
lacloylglutathione. We have demonskated that in stiln!tated neliroohils the
concenl'ation o'S.D. acloy.g.ì.ilalnrore inc,eases aooroxtmale y tCOoo ol lne
resting concentration during the firsl iew minutes oi the activation oeriod and
that rhis nlay oe re,aleo lo strmLlation ol mtcroruoule assemoty
[52, 53].
The modulation o, microtubute functions is probabty cafiied oul by a s;ries oi
microtubule-associated proleins. The fact thal the microtubule-disrupijng
agent colchicine inhibiis phagoc).tosjs, chemotaxis, §uriace adhesion and de.
granulalìon indicates lhat these structures are important in all these ohènonena. ll is arso possible lrat mrc'otLou.es parllcioate tn senso.y perceolion oi
ihe cell's external environments: it has been suggesled that these c*oskele
lal poryrners may be caoaò.e of caorufing and uilizi"g amoient or oiologically
generated electromagnetic energy[54].
4. Cyioplasmic aranules
Nèukophils are endowed with a powerlul armameniarium ol enzymes and oiher toxic §ubstances that are accumulated in parlicular granules. thal have
been distinguished in at least three groups according to separation in density
gradienls and composiiion Oabte ). The conlent oi these granules is releaseà
,ollowing cell slimulation, so thal neutrophils in the inflammed areas are often
completely degranulaied. Besides the aelease of pre.packaged constituents.
activated neutrophiis also release products thaì are synthètized "de novo',
such as platetet activating tactor (pAF), arachidonic acid and its metabotites
produced by cyclo.oxygenase and lipoxygenase pathways, proteins such
as
lhe neuùophil: structur€
and dei€nce lunclicn
11
interleukìn-1 and granulocyte. and macrophage.colony'siìmulaling iactors.
Some of ihe components of speciiic granuies (receptors, cytochrome bss, fla'
vooroteins) are not released outside ihe cell during degranulation. but they are
incorporated in lhe plasmamembrane, where theycontribute to the neutrophil
lunction and particularly to achesion, phagocylosis and assembly ol NADPH
cxidase aciive comPlex.
fahÉ 11- Maleculat canstituenls
of gftnules af human neutrcphils
Azurophilgr:in!les
Specifìc qranules
Other gaanules
Myelcperoxidase
Calhepsins D and G
E aslase
É-glucuronidase
Cytochrome b
Gelallnase
Ccllagenase
Histamìnase
É-giucosamjnrdase
Glycosaminoglycans
Tetranectin
Laminin recepÌor
Lysozyme
Calionic proleins
Baclericidal/permeability
/inducing protein (BPl)
Deiensins
Lysozyme
Laminln receptor
C3bìreceplor
Lactolerrin
Vilam n 8,, binding
prolein
G ycosam
inogiycans
Chondroiìin suliate
Heparan sultale
p-galaclosidase
,-glycerophosphalase
ol lhe granule conslltuenls, which are lisled in table ll, possess a definiìe
toxiciiy to microorganisms. First ol all, myeloperoxidaso (MPO) which is pre'
senl in hiQh concenlralion in neutrophils (2'5% oi dry weiqhl) and confers a
greenish color to neutrophil-rich exùdales. MPO is a hemoprotein with a mo'
lecular weight of abouì 140 KDa. lts antimicrobial properties are linked to the
acility oi utilizing H,O, and halides (iodide, bromide and chloride) to generate
highly toxic agents such as hypohalous acids, long'lived oxidanls such as
chloramìnes or aldehydes, and possibly hydroxyl radicals and singlet oxygen
(see also Fì9.4).
The BPl, a protein of 59 KDa contained in azurophilic granules, exerts a nox'
ious efiect on capsulated gram-negative rods by binding to the outer mem'
brane ol the bacteria and causing an increase in lhe permeability ol lhe mem'
brane and activation of baclerial phospholipases.
l',4ost
.l
The defensìns are a family of related smalt molecular weight (<4,000 Da) bac'
lericidal proteins thal have beèn recently identified. The peptides, 32-34 ami'
noacid residues in lenght, are rich in cysteine and have a broad anìibiotic
spectrum. How ìhe delensins exerl their cytotoxic eflecl on target cells re'
mains to be clarilied.
{
l.i
Among the cathepsins, cathepsin G is a chymoirypsin-like Drotease with molecrlar weight ol about 27 KDa, and oprimum neutrat pH. lt has been shown to
have marKeo antimrcrootal acltv,ty agatnst S aureus and otner gr-m-positive
bacterialspecies.
Lactoierrin is an 80 KDa iron-binding gtycoprctein that in neutrophits is found
in speciiic granules bul il ìs present also in most secretory fluids
{it has been
actually tirstiy identiiied in miik). The role ot lactolerrin as antimiciobiat aoènt
may be .elareo lo ils iron.oinoirg capacity and nence ro ilS ao..:ly 10 aom;ele
wrÌh iro..reouiriag bacteria lor a4 essentialgroMh factor. Anotier ooss:oilhv
is lnal lactoferrin oanictoates aS iron donor (wnen Ine melat s re easèd fro;
lhe prolein by redlcing agents and by the superoxide anion itself) in the reac.
lion that generales the hydroxyi radicat (Fig. 4).
Lysozyme iS a sma (14.5 KDa) neulrophit enzyme, that is also widetv disiribuled in trssues and booy lluios, srnce it is atso colstitutively re.eased by ris.
sue macrophages. ll is strongly bactericidal ,or some grarn Dositìve bacieria
and exerts its action by cteaving the beta (1-4)glycosidic link belween N.ace.
tylglucosamine and N-acetytmuramic, resulling n the disassembly of bacterial
cellwall.
Olher possible iactors thai may contrìbute ìo the non-oxidativè aniimicrobiet
efiects are: a) olher proleases such as elastase and acid hydrolases (p-9|u.
curonidase, 6-glycerophosphalase, d-mannosidase, cathepsins B and G), Àat
can digest some of the ouler membrane proleins and polysaccharides of th6
bacierìa: b) lipid hydroiases, in particutar the phospholipid-degrading enzymes. that may pertu.b the bacterial eovelope; c) nucleases, that may degra.
dale chromosomat DNA of engulfed bacteria.
It shoLld oe pointed oul lhat these and olner granu.e constitJents may oe arao
released extracellularly when the ne0irophils are stimulated bv solubie mèmbrane.oenurbing ag-ants or when they adnere lo objecls lhar;re roo ta,9e lo
be phagocytosed ("fruslraled phagocytosis") or to surfaces that have been
coated by immuhe complexes or complement factors. Enzymes and olherfactors with potentially cyloloxic propenies are then comptexed in serum wilh
protease inhibitors and by olher proteins such as albumin and lipoproteins.
However, when the proteclive capacity of these sysiems is salurated, or loo
low due lor example to hepatic failure or inactivation by oxidative stress, a local or systemic neutrophil.mediated damage may re§ult. We have rècenfly
cafiied oul a study of the inlravascular release ol elasìase in oatients aifècteà
by ourns of va.ious oegrees of seventy. The results ol a typical and represen.
tative patientwith severe burns (uBS scorè= 104, 17.5% oi proJound iesions)
are repoded in Fig. 5. lt can be seen that ten days after the burn, a dramatic
increase in serum elastase (measured as comptex between elastase and a-1
antitrypsin) occurs. The lact that lhis increase lollows the ljrst febrite epjsode,
but does nol correlale wilh leukocyle count may be interpreled as an evidènce
that in this patiènt (and in severat others with similar behaviour)lhe teukoct.tes
The neùlrophil: structùré and dsf€nca tunc on
13
are released in the bloodstream in the first posìburn period, then they are stimulatèd to adhere to endoihelia, to migrate in the exudate and to releàse thejr
conslìtuents. lt is conceivable that these events are mediated bv interleukin-1
ano oy tuanor.necrosts lactot. wo ha!e also louno enormous amoùnIs ol elasiase in the blister ftuid ol most ot burned patients (unpubtished results). Moni-
20
F
6
I
16
12
8
_- .:_:.:/-_-__,\
9
/'\.
1tÉ
I
,._--,
100
50
'--.---.1'-----
-
\^
.-...^.
\"-^-.
----
-42
fr40
a38
c37
It+
1[,Lr,fllLL,[,,,r
30
O4YS
35
POSIEUBN
Fil 5- Moonohng ol DùtaO teurocfes (a; o/ sa.urt s,Éstas s (b). aN ot Daov tetuMhtuté tdaitu
tuaunun and dininun) (c) ia a teptasentativé oaie ans a sehaùs bun ;niuN. Etashsà ;ai
rnèas,J.eo Dy tflmunoassay as e/asÉss/6/ast se
irr,bn6, cÒ-Dlé,
"':
'l
-t4
P. B6llavli6 €r al.
toring of serum elaslase levels may be a simple and ìnformative index ol sys'
t6mia activalion of leukocytes in sevete burns, trauma and inlections'
The recognition of neutrophils as having major secretory functìons as wèll as
active phagoc,'iic capacities is in accord wilh their dual role in the inflamma'
tory process and underlin€s the importance ot siudies ol the mechanisms that
m;y conirol the function o, neutrophils 'in vivo'. One oI the most ambitious
goi,s ol this kind ol research is the possibility of biological or farmaaological
;nhancement oI phagoc,'te tunction in the lmmunocompromissd palients and,
on the other hand, the production of seloctive and non toxic inhibitors ol lhe
unnecessary damaging erfects ol neutrophìl aclivation.
:
,
ACKNOWLEDGEMENTS
This work was supported bygrants llom Mioistero Pubblica lslruzione
lrom Regione Veneìo.
(londi40%)and
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