DTIC ADA277711: Involvement of a Botulinum Toxin-Sensitive 22-kDa G Protein in Stimulated Exocytosis of Human Neutrophils

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by  Defense Technical Information Center

AD-A277 711
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4. TITLIE AND SUBTITLE S. FUNDING NUMBERS
]involvement -of- a Botulinun toxin-sesftUv&2_2At6-d
--
Protein in stinulated exocytosis of humnan neutrophi1s. WICD
I. AUTHOR(S)
Nath, Jayasree; pow1edge, Annette; Wright, r~ige l g
1. PERFORMING ORGANIZATION NAME(S) AND AOORESS(ES) 8. PERFORMING ORGANIZATION
Walter Reed A"m institute of ReseardhL REOTNME
Washington. DC 20307-5100
9. SPONSORING/ MONITORING AGENCY NAME(S) AND AOORESS(ES) 10. SPONSORING/ MONITORING
US Army Medical Research & De'velopment Cczuuaiu AGENCY REPORT NUMBER
Ft. Detrick, Frederick. MD 21702-5012
DTIC
_ __ _
11. SUPPEMENTARY NOTES EL..F.CTE
APR 05 1994
128. DISTRIBUTION / AVAILABILITY STATEMENT DISTRIBUTION CODE
-APPRYOM FOR FR1I C ILEASE:
-ISYRIJYFIC4U ILIMITED
13.A(BMSrRnAmCm 20wors) Involvement of a Botulinum Toxin-Sensitive 22-kDa G
Protein in Stimulated Exocytosis of Human Neutrophils
jaymaree Nath,' Annette Powledge, and Daniel G. Wright2
Oepartnwnst of Hematology Divisiom of Medicirie, Walter Reed A"m Itiitute of ResearcWha. shnugDtoCn2 0.3 07.5100
Studies of human peripheral blood neuitrophils (PM4s) demonstrated that botulinum neurotoxin 0 (ST-Di ADP-
ribosyfates a 22-kD& PMN G protein (CIA~) and inhibits the exocyloels of both specific and azurophilic granules
stimulated by FMLP. Furthermore, this Inhsibition of PMN exocylosis by BT.0 was found to be correlated with fte
degree of Irreversible AOI'-rlbosylation of Ca by ST.O and to require modification of at leas#tS %o f PMN C13o
2
before significant inhibition of secretion iso bserved. Although both peslussis toxin and ST-O Inhibited excocytosis
In FlMvLP-stimvulated PMNs, the inhibitory effects o(t he two toxins were found to be additive. Ptertustis toxin and
ST-O also Inhibited Ca'*/GTP/GTP~yinduced secretion ind lgitonin-permeattllrzed PMNs, but there were distinct
D~~jiC~,s. CNL~I WI) 3 dIference, between thelinhib~ltorelect, &thesotoxins nontatoSLtextxnouiu~ a
flound to AOP-rboylata primarily a 24. to 2S-k~a PMN protein, and it was not boun to inhibit Ca`- andl
GTP-lnduced secretion in permeabilized PMNs. Ultrastructural studies of 1IT-D-treated PMNi shcowed an accu-
mulation of distinct memborane-bound organelles in the periphery of the cells afltor FMLP stimulation, suggestive
of a toxion-indusced block in organelle-plassma membrane fusion. Taken together, these Findings indicate "ha ST.
0-sensitivet IS,. has a Functional role in stimulated exocytosis df PMVNsJ.k otornoaoIlfm muntology, 1994' 15S2:
Neutr qphil-G, p rotein, Botuirdnun toxin. ex ocytosi
IlkI P5RU ICBE RCOODE AI
1.SECURITY CLASSIFICATION 18. SECURITY CLASSIFICATION 19, SECURITY CLASSIFICATION 20. LIMITATION OF ABSTRAC*
OF REORT O THISPAGEOF ABSTRACTI
NSN 7S500-&*ZdUQ*SSa Standard ForM 298 (Rito :-89,
!Thc! 3 *ecmi~att ,
Involvement of a Botulinum Toxin-Sensitive 22-kDa G
__I
Protein in Stimulated Exocytosis of Human Neutrophils
__
-A
00
jayasree Nath,' Annette Powledge, and Daniel G. Wright'
Department of Heniatologv, Div ision of Medicine, Walter Reed! Army Institute ot Research, Washington, DC 20307\\c /
Studies of human peripheral blood neutrophils (PMNs) demonstrated that botulinum neurotoxin D (BT-D) ADP-
rilbosylates a 22-kDa PMN G protein (G220) and inhibits the exocytosis ot b)0th specific and azurophilic granules
stimulated by FMLP. Furthermore, this inhibition of PMN exocytosis by BT-D was found to be correlated with the
degree of irreversible ADP-ribosylation of G .,k by BT-D and to require modification of at least 851Ui of PMN G _k
b~efore significant inhibition of secretion is observed. Although both pertussis toxin and BI-D inhibited exocytosis
in F'MLP-stimulated PMNs, the inhibitory eftects ot the two toxins were found to be additive. Pertussis toxin and
BT-D also inhibited Ca>'/GIP/GTP-YS-induced secretion in digitonin-permieabilized PMNs, but there were distinct
differences between the inhibitory effects of the two toxins. In contrast to BT-D, the exotoxin botulinurn C.3 was found
to ADP-ribosylate primarily a 24- to 25-kDa PMN protein, and it was not found to inhibit Ca>-* and GTP-indUced
secretion in lpermeahilized PMNs. Ultrastructural Studies of BT-D-treated PMNs showed an accumulation of distinct
mnembrane-b)ound organelles in the periphery of the cells after FMLP stimulation, suggestive of a toxin-induced block
in organelle-plasmia membrane fusion. Taken together, these findings indicate that BT-D-sensitive G22, has a functional
role in stimulated exocytosis of PMNs. lourndl of Ifl~flufoloOy, 1994, 15 2: 13 70.
Phagocytic
cells, such as PMNs.1' synthesize pro- of mature neutrophils. the specific (or "sccondarv 't and
teins during maturation in the hone marrow t I1). azurophilic (or "primary-) granules ( It. are differentially
and store them in cytoplasmic granules (2) until regulated by calcium during both exocytosis and intracel-
the cells are appropriately stimulated to release the con- lular degranulation (7-9). Other molecular components in-
tents of these granules into an endocytic vacuole. eg.dur- v'olved in stimulus-response coupling for PMN exocytosis
ing phagocytosis of bacteria (2. 3). or to the exterior of the and degranulation have been less well characteriz~ed.
cell alter fusion with the plasmia membrane. e.g,.. during In recent years. efforts to elucidate the biochemical and
exocvtosis (4. 5). Exocytosis of PMN granules is a highly' molecular mechanisms involved in cellular membrane
regulated event that occurs within seconds of cell activa- traffic and organelle transport have focused both on ras-
tion by' soluble or particulate stimuli (2-4). As in mlost related low mnw. GTP-binding proteitns ti~e.. small G pro-
secretory cells t4. 5t. an increase in the intracellular conl- teins or GTPases) with molecular masses between 20) and
centration of' free Ca2* has been shown to be a second 30) kDa l 0-12) and on the larger. heterotrimieric G pro-
nmessenger for PMN exocvtosis (6 7). although experi- teins that have been linked to receptor protein complexes
mental evidence indicates that the two major granule types and receptor- med iated signal transduction (13. 14). The
most detinitiv'e ev idence for a functional role for small G
_________________________________________________________
kii,-',%-1 foir puhhbl,fi)ii Vigui IM1i) tI' \i ieilii for pubaion Nmme proteins in intracellular membrane and organelle transport
from genetic studies in yeast. in which mutations in
I')') Icomes
[hv' i t,o itibimi onoi tih~u mxl e.u%. %rcdmlru. im'n- di fii part h%t hei pali,\ (ifi~l the seir4 or \-pt / genes. which encode ras-related small G
lii,,orai willbM 1 t 1(, ,I I - W olvk II ndol this 1, proteins, were fou nd to inhibit fusion ot secretory vesicles
'Addiri", iorvi's ndi i ',rai nii'ur eprnt reti'qiii, iii Dr a\in vi', .fri'\ll i'parl* wkith the plasma membrane resulting in secretory defects
'nil(i iii lh-nmi o 1D)i%~h i~i of Me\lei u i' Miller ReedlV ill% Insthilt't of t 15. 1 6. Although a precise role for small G proteins in
Ri's. i I \\isi~hn~i~ni NI 2M ,1 0(1ihr 'Liv~~ el
1I-n l- )1,'jaimcnli i'm or I li'nnili Ii ii (liii ,nl,wn fllsl I iop the secretory functions of' hih re k roi el has not
1.11f io', ii I nn. Isi~ hioI o Mf j i nBe,o m MA 0I! 2 I been defined, there is substantial experimental evidence.
'lnliri's dni iris timid in thipsI miciir l'\l\. poit'iphiirai Ii oo l iii miirpiliintiti Iva obtained primarily from studies conducted wkith cell-free
nu1n l,ok iin I I P u.'nnk'jdsiiIioi 1),Ib iin u'Ih: . I oo~ Di nItinl t l kinnin i tuted systems. that inerbrane-tlowh and membrane-
pr olvan fusion events durine constitoti'.L and icgulated secretion are
oIin ringll c 11194 h% flit, Ani an fAssq~ai)iiin oll I m nloiniigists, 00)22 1 6-'4,"4 5012 00
944 4-4 --
lournal of Iniunology 1371
regulated in part by the cycling of small G proteins between Experimental Procedures
a membrane-bound and free state (17-19). Preparationo f human PMNs
PMNs are the principal effector cells in host defenses
aainst bacterial and fungal infection and are capable of PMNs were isolated from freshl} dran \enous blood ol healthy solun-
teers b%c entrifugation through Ficoll-H~paquc followked by dextran sed-
responding to a wide variety of molecular signals that trig- imentation (32). Residual er.throcMtes were remiosed hb hypotonic Isis.
ger functional responses (20). Among these functional re- and >t15% pure P.MNs %ere resuspended in HBSS without Ca2" and
LMg'. pH 7.4.
sponses. secretory events are critical to the microbicidal
and inflammatory activity of these cells (2. 3. 6). Studies Toxin treatment of PMNs
conducted in several different laboratories have shown that
In our preliminary studies with BT-D. %keu sed incubation conditions
PMN chemoattractant receptors are coupled to a PT-sen- described for pre'ious studies with rabbit neutrophils (23j. How'e.er.
sitive heterotrimeric G protein (20). and PT-induced inhi- i.hen freshly prepared human PMNs were incubated for 60 min at 37yC
bition of PMN inflammatory responses stimulated by the %kith5 M0n)g /nil of BT-D (Wako Chemicals. Richmond. VA). we did not
i obser\e an, inhibition of FNMLP-induced functional responses after the
peptide chemoattractant FMLP has provided valuable in- toxin treatment. In subsequent studies %.henP MN's at 5 x 1O"Iml %%ere
sights into the transduction of signals via these receptors incubated %kithh igher concentrations (I -5 g/mIl) of BT-D for 9O to 120
Lmin at 37 C. signiticant inhibitory effects of the toxin w.ere obsered.
(20). However. PT-insensitive G proteins have also been Therefore. for all studies described here (except \\here indicated other-
described in PMNs (21). and there is evidence for a reg- iise. as in Fi,. 4). PMNs at 5 x lli(/ml in HBSS \%ithout Ca2* and
ulatory role of these G proteins in signal-response cou- MgN'. ,ere incubated for 120 min at 37TC. in the absence or presence of
I to 5 fig/tnl of BT-D. After the incubation, both control and toxin-
piing in PMN4 (22). treated PMNs \,ere centrifuged at I(11) x Q and then resuspended in
Recently, BT-D was reported to ADP-ribosylate a ras- HBSS containing Ca-> and Me2> for assessment of FNILP-induced exo-
cytesis in intact PMNs. or. in a PB containing I(M( mM KCI. 20 mM
related 22-kDa G protein, both in rabbit peritonea neu- NaCI. I mM EGTA. 30 mM HEPES. at pH 7.0. for assessment of Ca-*/
trophils (23) and in human PMNs (24). Botulinum neuro- GTP/GTPyS-induced secretion after digitonin permeabililation of the
toxins are known to block neurotransmitter release at cells (33). The BT-D stock solution (1.0 mg/ml, in 0.2 NI NaCI %kith1 1.115
NI sodium acetate. pH 6.5) %%as used in 1:2(X) to 1:2()(1) dilutions to
peripheral cholinergic synapses producing muscular weak- achiee the required toxin concentrations used in this stud., and that did
ness and paralysis (25). and BT-D has also been shown to not affect cell 'iabilit, as assessed by trypan blue exclusion and lactic
inhibit secretory responses in bovine adrenal medullary dchydrogenase release.
cells at a step distal to the stimulus-induced increases in Digitonin permeabilization of PMNs
intracellular Ca2+ (26). Hence. it was of interest to us to
examine the effects of BT-D on stimulated exocytosis of Permeabilization of PMNs %%as achiesed b%a slight modification of a
procedure described b\ Smoten et al. (33t. Briefll. PMNs at 25 x l(t'/ml
human PMNs. Our interest in the possibility that BT- in PB wkere pre bsartmo e37de C and digitonin \tas added at 10 xg/mL
sensitive. ras-related. G proteins might have a role in stim- from a freshl. prepared stock solution of 1.01m g/rid maintained at 37-CI.
ulated PMN exocvtosis was also prompted by the obser- Permcabili/ation isas performed for 15 miin at 37C. and the PMNs %,ere
ovtst -then immediatels diluted to 5 x IO"/mIl with PB. As reported by Smolen
vation that guanine nucleotides stimulate exocytotic et al. t 33l. these digitonin-pcrn;ieabilitcd clls ioaiikained Ca'-induced
secretion in the absence of Ca2+ in a variety of permeabi- secretorN responses oif both granule tpes,. esen after the release of 30q4
to 401; of cytoplastmic lactic dehydrogenase during permeabilization.
lied secretory cells (27-29) including PMNs (30). Consistent %\itht he previous report (334. extracellular release of 03-glu-
Results of our present studies indicate that BT-D ADP- curonidase (a marker for a/'urophilic granule content) and lacloferrin or
ribosylates a 22-kDa G protein (G2,k) in human PMNs Vit B -BP (markers for specific granule contentsl in Ca2'-free PB \aN
<5% 1of2 the total, during 15 min incubation \,ith Il jtg/ml digitonin (data
and significantly inhibits exocytosis of both specific and not shosin). In our hands, incubation with Il0 ug/nil digitonin for 15 nn
azurophilic granules of PMNs stimulated by the peptide at 37'C "as found to be the most optimal pertneabiliration condition for
chemoattractant FMLP. In addition. BT-D is shown to sig- stud.ing Ca2'-induced exoc\tosis in PMNs.
nificantly inhibit the secretory responses of digitonin-per- FMLP-induced secretion in intact PMNs
meabilized PMNs. when stimulated with Ca" . Ca2+ +
For studies of FNILP-imnuced exocyttosis of PNIN granules. PNINs at
GTP. a/ITPS alone. These studies also demonstrate Oml in HBSS containing Ca" and Mg>' \ere wxarmed to 37'C in
a correlation between the degree of irreversible ADP-ri- round-bottom 12 x 75 mm polpropvletne tubes, and the PNINs "icre
bosylation of G2,k and inhibition of PMN exocytosis by treat(]d with 5 ag/nil ctochalsin B for 0 mnin at 37-C. Secretion \%as
initiated by the addition of I0 " N FMLP. and the PMNs were incubated
BT-D. Ultrastructural changes in BT-D-treated PMNs. for additional I1l nin at 37'C. The cells \kere then chilled on ice and KCI
which are consistent with an inhibitory effect of BT-D on ?ias added to a final concentration of' 0.1 NI to minimize adsorption of
PMN exocytosis. are also defined. These studies contrast exocytosed granule proteins. The samples were then transferred to Ep-
pendorf centrifuge tubes and centrifuged at 41' for 2 min at l100(cid:127)1X !,
the effects of BT-D with those of the exoenzyme BT-C3 in an Eppendorfcentrifuge. Supematants wkere carefull. aspirated. placed
(31 ). which primarily ADP-ribosylates 24- to 25-kDa on ice, and immediatetl analyzed for 13-glucuronidase and Vit B,.-BP.
PMN proteins that appear to be distinct from the 22-kDa The remainder of the satrples were stored overnight at 4'C before mea-
suremnent of lactoferin by ELISA (344. Total cellular contents of the
PMN G protein modified by BT-D. Unlike BT-D. the ex- various granule marker proteins Aere measured in parallel from an idcn-
oenzyme BT-C3 does not inhibit Ca-' and/or GTP-in- ',-al PMN samplc in I IBSS. after disruption bys onication in the presence
ot 11"T%ri ton X-IlM(. Furthermore. in each experiment. appropriate con-
duced exoc,-'osit of PMN granulcs in digitonin-permeabi- trols \kith buffer. cytochalain B,o r FILP alone. \%ere assessed routinely
lized cells. for any c.ocy tosis of PMN granules that could ha\e occurred tinder
1372ROLE OF C>,. .,N PNIN SECRETION
nntiornistmltor\ conditions. Inl studies conducted \%ith tox n-treated Electron nhicrosc-opy'
PM Ns. both control and experi itirital samples "~e re subjected to identical
incubation Conditions in the presence or absence oft the toxiin,)si. before For electron microscopic anal\ sis of' toimi-treatcd and untrecatcd PMNiý.
FMILP-iridUced secretion %%isatUs tdiCdI as descrihed above, the cells \%e re subjected to idenitical experintental conditions as,d escribed
for studs ig FMILP-st mu kited exoc tosiýs see ahos e . Foir elect ronl [iii-
croscop\. I to 2 X 10 PNINs %%ere used for each experimntcial condition.
Ca- ioduced eC,x12o, cytosis in digitonin-permeaibilized arid PMNs' \%ere incubated in HBSS \\ithout Ca" and M,". inl the pres-
PMNs etice or absence of' 5 AgemIl of' BT-t). for 120 mitt at 37 C. PNiNs "sere
then centrif-uced and resuspended tin HBS3S Cotltailiitilg a>* and Mg2'
tDigitontn-pei tieabtiti/ed PMIN' sal 5 ' IW'tit/ti in Ca'* - ree PB use re %kit h PMiN concentrat ion atl 5 x 10`/ml. FM LP-st imulIated ex~os isi
"usarmled to 37 C and secrelion %\ias induced h% addition oif' 1.5 mMI Ca> "was induced tin toxin-treated and untreated PNINs after extochalasinl b
Where itndicated. (iTP or GTP-yS suits added al 0,.1 ntM. The concentra- treatment. atsd escribed for Filenres I arid 2. The PINs, usere centrifuged
tion of' Ca'* requtired for opt imial secrctor\ responses usa s determitied at IWO0(X ge for 10Im in and Ii ed for I h in at inii ure of'4'; fiirma Ide-
from it series of' preliininar\ dose-response studies. It is noteworth\ that hyde iii 0.1 MI sodium~l phosphate buffer. pH 7.4. The tixed PMNI, %%ere
uitder our experimental conditions. preexistine intracellular f'ree Ca2. kept overtiight in 01.1 MIc acodylate buftfer (pH 7.4) at 4 C. and p0sthx~ed
ICa> ),. \%as not detectable titt these PNsN whbenl measured by at sensitive Inl V; ostniUtn tetroxide for I h at riotrn teitperaltire. The satmples usere
nitdo- I tnethod. ICa>), trieasurements \here also made after the addition then dehydrated and processed for electron rnicro~cop\ The details oft
of* extracellular Ca> to dig itonin-permeabili/ed PMNIs in PB (cotntaining the procedure have beetn described pre itiusls (36). The sections \kere
I mM EGTA .Addition of 1. 5 ti Ca'. Ml ich triggered optimtral re- poststaintedl %kitha qUeous Solutitotis of' 5' Utrati I acetatte atd lead cit ral
sponses tii the secretiotn studies. resutlted titit [ Ca' 1, of*a bout I1.3(0fA N. atid the specitrnens "sere e anlinted atnd photographed tin a Zeiss ENI It01)
It is "sorthwhi le to meciltion here that although %Ig/ATP has beeti reported tratismi sioti electrotn tiicroscope at 80I KV .
ito enhance stiniu~lated exc% tosis in per-ineabili/ed PMNIs. se did tiot
iibserxe atiN signilicant effect of' MgATP onl Ca-_- itiduteed secretor\ re-
sponses of digitotiii-permecabili/ed PMNs, itt Our prel itinar\ Studies. Itt Results
\ce\ of this tibsers atioti. MigATP \%as riot added to theexternal itediumi
durng initdutction of Ca2 -i ttduced exoc~ tosis, in di eitotitt-permeabili.'ed In view of prior reports of the intrinsic ADP-ribos\,ltrans-
PNI~s. erase activity of BT-D (23. 24. 37) and its effect on se-
ADvPit-hibbootyihliintmon t xin orC.3cretion (26). as well as reports of the ability of somte hot-
ADP-iitbhobyloaitolinn rn txinD o C3ulinumn toxins (C2 and C3) to ADP-ribosv late cellular G
BoitulitiUti toxit-itiediated At)P-ribosx latioti of' PN1N G proteins was actin selectively (38) and to block the polymerization of C
perto rmpdrm aceoeriittegs escibe prviusl 24. P~s actin to F actin (39). it was essential to deter-nine the na-
\kere sonicated in a tmediutm contairning (0.1 %1T1r is-bitifered sucrose. pH
7.2. 2 tini EDTA. atid I rnMi DTT. in the presence of at tmixture of ture of the ADP-ribosyltranslerase activity of the BT-D
proteoli~tic inhibitors tcotitaining I Agg/iil each of' PMSF. apriitinin. atid preparation used in our present studies. As shown in Fig-
leupeptit i a 4 C and centrif'uged atl IM)( x t' f~or 5 mitn to rentox ure IA and previously reported (23. 24). BT-D wvas found
uteitb ritktny ree lss.Fsoy . roeii onettrtiit uasadused to ADP-ribosvlate a 22-kDa G protein in PMN sonicates.
so ats toi add I tii 2 tug MA'N priitein per nil of the reaction MItixUtre.
At)P-ribosyIlaion "ias ciinducted in a final reactiort %olutne of' 1M JAIl. However, the possibility remnains that more than one PMN
conittaininirg IMIli nNi Tris -HCI. pH 7.4. lii Ni Et)TA. 10Itn tM thytnidintc. G6 rten of similar molecular masses. are substrates for
5 i~il %IgCI. 11.5n ii ATP. I A~NI '2P-labeled nicotitiamnide adettine
ditiucleotidle 211 -2 geCi I aiid I to 2 mng/ml PMN' protitin. BT-t) IWako BT-D-miediated ADP-rihosvlation. Results identical wkith
Chetmicals) "ias actisated xsith 21)t rni DTT f~or1 0 minita t 301C . and %%as those shown in Figure I]A were obtained in three separate
added at 25 gig/mIl to initiate At)P-ribos\Iltioti (23t. This. concenitratiotn experiments with different PMN preparations. Figure I B
of' the toxin vxas chosen frott prex iouslN reported .alties (231. When AD-iovtrnfrs actvi
ADcoPn-uricbteodM %%aiitlthsi oenx 'illustrates the comparative A Prhslrnfrs cii
Atl)t~iorit ix uas ondctd u it eoen/%t ie BT-C 3. (1.1 tghig/nl of'
at paniallN purilied preparatiotn of C'3 ai generous gift of tDr. Leonard ties of BT-D and a partially purified preparation of exoen-
Stiiih. t'inted States Artit Medical Research Institute of Infctlious D~is- zvme BT-C3 (34) in the samne PMN homogenate. It should
eases. Fort tDetrick. %II)) w4asu sed (34). All other experitiieiital condi-MNGptti.
tiiins %%ere identical. The I '-PIAD-i) ovain(tPNGopfoNe n \ b oe htteI2PAP-prirbootsevilnatsii.mb GNN
BHI-I) and Br-(3. as shown in Figure 1. AIa nd B. usere conducted \%ith shown in Figure 1. were separated by electrophoresis on
tus o different PMN sonicates. in parallel studies. The reactiontitisa per- I I r/ to 23('/ polvacrylamnide gradient vels. which gvie the
formed for611 tin atl3 7 'arid \kias stiipped with 10I; ice-cold TCA (fintal 41L
concetntration I. Samples \%ere chilled ott ice for an aidditionatl I15m .iiattd best one-dimiensional separation of 20- to 30-kDa range
the protein precipitates, \%ere collected h%c etitrif'tigation and usashed tuo proteins inl our hands. A 42-kDa I t2PIADP-ribos\ lated
more timies usith ice-cold 10'i TCA. The precipitated proteins, \%ere Iti protein \kts detected only when the reaction was con-
nally %sashed xxi th ether atid the samples Acere inmmediatel\ processed l~or
Sl)S -PAGjF onl 11' , to 23',; graidietnt gels. (Amrschaii. "Phorcast). The ducted with BT-C3 preparation and not with BT-D. This
gels %%ered ried under high %acuutr kind the 11 2 P)AIP-ribosy lated protein 42-kDa [' 2PIADP-ribosylated PMN protein is tmoust likely
banls ustettem d~tieiimtedrahput x- uin KoiikX-ina hltis. actin (38). and it raises the possibil ity that at contaminating,
C2-type toxin could also be present in the C3 preparation.
Assay of PMN granfule marker CflzyniCs, The makjor 24- to 25-kDa PMN protein that wkas modified
In our presenmt studies. rglttctirortidcts %%suas ed ais it marker for thle by BT-C3. appeared to have a dtstinctl-, dillWcrcnt moitbility
aitirophilic granules aind Vit B, '-B11 and] lactolerriti \%ere used its muarkers from that of the G.,, which wkas AI)P-ribosvlated by the
for specific graniule comntentt (1-3). All \ssArec Condticted ini dulpli- BT-D. This has been a reproducible finding in our hands.
Calte using standard proicedure,,. the details imfu sh ich has e been described
051. 13-Ghliciirtnidaise uisa vs ass d by tltc phcriiulphlttaIc iii- uc uioiltc AN is also0 shown in H-gurc IB.. BT-('3 ADP-tibos~ lated
acid miethod aiid spectrimphmmtotnetricall\ quantitated ilt 5411f ul Vit additional PMN proteins of' 20 to 30 kDa range to atm inor
B, --B11% kasm easured h\ the charcoal binditig assaý usitig cyanocobal- degree and the possibility remains that the G1, is one of'
arnin "Co. and lactoferrin \&i ts quantittatcd h,, Et .1A using it purified
humnan lactoferrin ICappel) ait the standard (351. The pi \alues \%ere Cal- these proteins. It shouild be noted that because the dynam-
culated by Students%t test. ics of' actin polymerization (and depolyrneri/ation) play at
Journal of Immunology 1,37 3
100"
A
"UI rgluc
- 130 so- 8 ,-binding
- 75 * E lactoferrin
0T
-(cid:127) 60
-50
39
*
*• 40"
-27
*
-17 20
+BT -BT 0.
PertussIs Tx Botulinum Tx
B
FIGURE 2. Inhibition of FMLP-indu(ed secretion by BT
kha and PT in intact PMNs. PMNs were incubated for 10 min at
37°C with 5 ýLg/ml cytochalasin 1) before stimulating with
- 200 10 " M FMLP. In control PMNs (n = 10), FMLP-induced
secretion varied between 20'%, and 50% for B, -binding pro-
tein, between 50'%, and 70(',, for lactoferrin, and betwveen
- 92.5 35'% and 75/,, for 0-glucuronidase (3-gluc). *p value <
0.0001.
-- 69
- 46
- 30 resent values obtained from 10 different experiments and
41 - 21.5 are highly significant (p < 0.(XX).I ) In studies conducted in
- 14.3 parallel, with the same PMN preparations, we also ob-
served a significant inhibition of PMN secretion by PT.
which varied between 40% and 70%. Interestingly, we
+BT-D +BT-C -BT never obtained a complete inhibition of FMLP-induced
3
FIGURE 1. Botulinum toxin-mediated I '-PIADP-ribosyla- secretion by PT. suggesting a PT-insensitive component of
tion of small G proteins in PMN homogenates. A, ADP-ribo- the response. In view of the results shown in Figure 2, we
sylation of a single G;, by BT-D; B, BT-D and BT-C3-me- examined the secretory responses of PMNs that were
diated ADP-ribosylation of distinctly different G proteins in a treated with both PT and BT-D. As shown in Figure 3. an
PMN homogenate. The I -'PJADP-ribosylatedp roteins were additive inhibition of FMLP-induced secretion was ob-
ele(trophoresed on a 1 I"%, to 2.3'%, polyacrylamide gradient served in PMNs incubated with both toxins, and this result
gel. Other experimental details are given in the text. The re- suggests that the two toxins act at different steps in the
suits are representative of three separate experiments, signal-transduction cascade for FMLP-stimulated exocv-
tosis in PMNs. As indicated in the legend to Figure 2. the
Vit B 2-BP release values were generally lower than the
1
lactoferrin values. This could have been due to the known
critical role in modulating PMN inflammatory responses susceptibility of Vit B,-BP to oxidative inactivation.
1
(40) including exocytosis (2-4). it was particularly impor- In preliminary studies, conducted to establish the opti-
tant to rule out the possibility of ADP-ribosylation of mal incubation conditions with BT-D (see Experint'ntal
PMN actin by the BT-D preparation that was to be used Procedures).i t was apparent to us that an incubation time
for examining its effects on PMN secretory responses. of 90 to 120 min, with I to 5 p.g/ml BT-D. was necessary
Having established the nature of the ADP-ribosylating to obtain a significant inhibition of PMN secretory re-
activity of the BT-D preparation, we next examined the sponses by BT-D. In an effort to correlate inhibition of
effect of the toxin on exocytotic secretion in PMNs. as secretion hy BT-D with the degree of irreversible ADP-
induced bI the chemotactic peptide FMLP. As a positive ribosylation of PMN G22k by the toxin. we incubated
control, we also studied the effect of PT, which is known PMNs with different concentrations of BT-D for different
to inhibit FMLP-induced secretory responses in PMNs periods of time at 37TC, and measured the time- and dose-
(41). As shown in Figure 2. a significant inhibition of exo- dependent inhibition of in vitro I'2PIADP-ribosylation of
cytosis was observed in BT-treated PMN%. The data rep- G,2" by BT-D in those PMN sonicates to quantitate the
1374 ROLE OF G,:, IN PMN SECRETION
degree of toxin-mediated, irreversible ADP-ribosylation
*
100o 1-gluc during the various preincubation conditions. As shown in
[]lactoferrin """"c"'"* Figure 4. incubation of PMNs for 90 to 120 min with I to
,
,0'"'" 5 tig/ml of BT-D was required to obtain ->85% inhibition
of subsequent in vitro [32P]ADP-ribosylation of G2.,.
These conditions were also those that were required to
:- 60 induce a significant inhibition of PMN exocytosis by
- *. BT-D. Hence. it would appear that at least 85% of G,2k
S4040 must ""' be ADP-ribosylated by the BT-D before a functional
*"effect of the toxin-mediated modification on PMN secre-
tion is detectable in FMLP-stimulated cells.
20 -,,0,,,B,,ecause a link between GTP and Ca"' has been ob-
,,, served in stimulated PMN exocytosis (21). in that GTP. or
...'., the nonhydrolyzable GTP analogue GTPyS. acts synergis-
0P tically with Ca2' in triggering secretion in permeabilized
Pertussis Tx Botulinum Tx PT + BT PMNs (21). and because there appears to be a Ca2+-inde-
FIGURE 3. Additive inhibition of FMLP-induced secretion pendent, GTPyS-inducible secretory component in exocy-
i3n7 °PCM Nwsit hb y5 P(cid:127)gT, /am3nl7d0 C BccwTyyi.tt to 5cPc Mh~haaNglaa/ssm siwinn ebrbe bbinefcfoourrbeea stsettdiimu fluoarl att1ni0ng gm iwni ittahht totic cells (27. 28. 42). it was of interest to study thc effect
10 " M FMLP. In control PMNs (n = 3), FMLP-induced of BT-D on Ca2+-induced exocytosis in digitonin-perme-
secretion varied between 50% and 70% for lactoferrin abilized PMNs. As shown in Figure 5. there was signifi-
and 350% and 75% for 3-glucuronidase. *p value < 0.07; cant inhibition of secretion of both primary and secondary
**p value < 0.05; ***p value < 0.0005. granule contents when permeabilized PMNs treated with
BT-D were stimulated with Ca0, Ca2' + GTP. or GTPyS
alone. Interestingly, extracellular release of 3-glucuroni-
dase. a marker for primary granules, appeared to be more
100.
90.
PERCENTAGF INHIBITION SHOWN GRAPHICALLY IN THE ADJOINING
80- 60 min FIGURE WAS CALCULATED FROM THE DENSITOMETRIC SCANS
OF THE RADIOACTIVE BANDS FROM THIS AUTORADIOGRAM
70- C C 60 90 ,20 f0 90 20 60 90 Q0 O T
Z 0ýý 0' 0 5 'g M1I a ..9 '6 5 0 g.901
0
-J 60-
FIGURE 4. Time- and dose-depen- 0
dent inhibition of in vitro [12PIADP- !2
ribosylation of G,k in a PMN homo- Md. 50-
genate previously incubated with a 90 min
BT-D. Details of the experimental E 40-
procedure are given in the text. 4 120 min
0 30-
20-
10-
0-
0.5 1.0 5.0
BOTULINUM TOXIN D, ig/ml
)U maIFot, 1i if Ini l Ior gs
10l -giuc .. 100' * 0-giuc
a 12 -binding C E29 -binding
C 00 tactaferrin .2 go 12
o~[ loictoferrnln
- R
A
7C 60 so
C40 40
20, a 20
Of 2+ 0.0
Digitonin-perrneabilizcd PMN4 (5 X Vm)~er eupne in a bUtter containing I 00nimM KCi. 20 mM aIInN1EAA
It0 mM HEPES, at pH 7.0. Secretion was induced I1wth e addition of 1. 5 mMl Ca", in the presence or absence oto nm.1 GTF'
or GT[)yS. W here ind icated, G P-YS was alISO Used to i nduce Sec ret ion in the absence of (a2*. Percen~tage inhibiition of sec(retionI
was calculated fromn the values obtained with control PMNs. The secretion with Ca' alone ranged b~etween 20", and .35"., for
J3-gILucuronidase. between 201l,a nd 25"' for B, .-binding protein and 20"), to 30". for lac toterrin; with Ca&' + GTIP as the
stimiulus, these values \\ere ver\ .iniilar to those obtained with Ca2' alone; Ca 2 + CTP-yS-indut ed secretion ýaried bet~een
30". anid 40",' for t3-gIucu ronidase. bet%%een 25", arnd 30".. tor B, -binding protein and 30". and 335", for lactoterrin; CITPyS-
induJced. Ca2 *-indfelendent e\o( vtosis was oniy 10 to t12"'. ahOVe buffer control. *p Value < 0.07; **/) va1lue < 0t.05 ***) aIloe
< 0.0005.
sensitive to BT treatment than exocviosis of' secondary FMILP-stimulated PMNs riot exposed to BT-D. Such ac-
granule contents. By contrast. in)P T-treated pernicabiliied cumulations of' mnembrane-bound organelles near the cell
PMNs. a signiticant inhibition of exocyt osis in response to periphery were niot difficuilt to detect and were observed in
Ca>* Ca>* + GTP. or Ca2* + GTP-yS. was also observed. >80("i of* BT-D-treated PMNs in any single preparation.
but only ot' secondlary granule contents l Fig,. 5). Further- Furthermore. as is also illustrated in Fitture 6. FMILP-stimi-
niore. unlike BT-D which inhibited Ca>+-independent. ulated PMNs treated with BT-) were relatively smooth
(;TP-yS-iiidttced Secretion. PT did not cause any signiti- and rounded in shape compared with control FMILP-stimi-
cant inhibition of GTP-yS-induced. Ca2+-indepenldent exo- ulated PMNs. wkhich had characteristic pleomiorphic shape
cN. tosi~s in permecabilized PMNs (data niot Shown). These chancLes and menmbratne rufflesI( 3. 6. 401.
differences. illustrated in Figtire 5, indicate the f'unctional
involvement of both BT-D-sensitive and PT-sensitive G Discussion
proteins in the regulation of' stimulated exocvtosis in hlt-
man PMNs. It is worthwhile it) mention here that although Models for stimlluILs-secretion coupling in a %a riety of se-
the (iTP-yS- indu~ced. Ca2*-inmdependent exocytosis was cretory cells have inclided both receptor-miediated and
only about I 0"t above buffer controls. both secretion and nonreceptor-miediated activation miechanismns (43, 441, and
its inhibition by BT-D wkere highly reproducible. These in recent years. the role of cellular G proteins as compo-
results also prouide additional evidence f'Or atd ifferential nents of these activation mechanisms in directed vesicular
regulation of the two ma11jor granule types of mnature PMNs transport and miemhrane-fusion evenits has received sub-
during stimiulated exocytosis 17. 8). We also studied thle stantial investigative attention (43. 44). Stimulated] exocv-
ctfkct of exoen/iyme BT-C3 ott Ca2'-indluced exocytosis in tosis of' PNIN granules plays a critical role iii both thle
digitonin-permecabilized PMNs. hut tfoiled to observec any host-dek'nse and inflammatory mechanisms of these cells.
inhibition of' exocvtosis by BT-C3 (data not shown). and the definition of'cellular mechanisms that are involved
L'ltrastructural studies (if B~l)-treated PMNs were con- in the regulation of exocytosis in activated PMNs remains
sistent with an inhibitory effect of* the toxin onl PMN se- an important challenge f'or research in leukocyte cell hi-
cretory responses. As illustrated in Figure 6. transmission ology. InI our present studies, wec have examnined in par-
electron imicrographs of' BT-)-treatcd PMNs that were ticular the role of' BT-D-sensitive. small mwl%G% p.r oteins
stimulated under conditions identical with those used f'or in stimlulILs-secret ion coupling of' human PMNs.
studying FMI.P-induced secretion WFigs. 2 and 3) revealed There is convincing biochemical evidence f'or tinA DP-
an accumulation of' memnbrane-bound empty organelles ribosyltransferase activityI of' BT-I) (37. 45. 46). and the
(airrvwlwhadivl and also of" PMN granule-like structures enzyme activity has been associated with the toxic coim-
(airnw~s) near the cell periphery. not seen in control. ponent of the progenitor toxin 446). Furthermiore, a 21 - to
I ~76 ROLE ()F (i.IN PIN SECREII( )N
FIGURE 6. Ultrastrut tural effects of
BT-D on exocvtotic humnan PMNs.cD
The PMNs were S~ibjected to identi-
cal experinmental conditions as dle-
scribed tor FMILP-induced secretion tj
iFig. 2). Thle cells were t" en fixed and
processed for electron-microscopv.
Other dletails are described in the
text: A, control; B, control + FMLP; C,
BT-D alone; D, BI-D + FMLP (mag-
nification x 4400); E, BI-D + FMALP-
treated PMN at a higher miagnifica-
tion x 87,000).
22-kDa Gi protein, distinct from ras G proteins. has been at BT-D-sensitive G,,, protein iti StimlUlated exoc~ tosis
shown to be ADP-ribosylated specifically by, BT-D in and the intrinsic ADP-ribosy ltransferase actiit\ of' BT-D
membrane arid cvtosolic fractions obtained fronti bovine (47. Nonetheless. a revie~k of' the existing literature pro-
adrenal gland (37. 45. 46). A BT-sensitive G'_( has also %videss uhstantial experimiental support for a functldional role
been detected in mouse brain, pancreas. and pituitary of cellular G proteins (both PT-sensitive and PT-insensi-
gland (45. 46). In addition. Knight et al, has reported an tivel in regulated secretion, at a site or sites distal to the
inhibition of" stimnulus-induced catecholamnine release by generation of' inimediate. receptor- Ilinked second niessen-
BT-D in bovine adrenal medullary cells, and the toxin- gers (2]. 27. 28. 42). For example. st-adies conducted wkith
induced blockade wvas shown to occur at a step down- permecabilized rabbit neutrophils have shown that GTP-yS
streami froni the stiniulus-dependent intracellular increase canl activate secretory processes by acting both at the level
in free Ca" (26). Subsequent studies. however, have of, second messenger generation and atl more downsirean,
raised questions about both the functional involvement of' steps that directly activate exocytotic mechanisms (21.
Journal of Immunology 1377
30). in analogous studies, synergistic effects of Ca2+ and have been shown to be associated with endocytic (I I).
GTPyS have been demonstrated in permeabilized mast exocytotic (1 2), and synaptic (50) vesicles, suggesting that
cell degranulation (27. 28. 42). the rab proteins may regulate vesicutlar trafficking in an
Our present studies confirm prior reports (23, 24) that organelle-specific manner. Furthermore. experimental ev-
human PMNs contain a G,2, protein that cau be specifi- idence for an association-dissociation cycle of a small G
cally ADP-ribosylated by BT-D (Fig. 1). Based on the protein, rab3A. during Ca 2 -dependent exocytosis of syn-
one-dimensional gel electrophoresis data, we cannot ex- aptic vesicles (the secretory organelles of nerve terminals)
clude the possibility of more than one G protein of similar has been presented (19). In addition, the dissociation of
molecular mass being ADP-ribosylated by BT-D: how- rab3A was shown to be dependent on the exocytotic fusion
ever, prior studies have purified and characterized a 22- of the vesicles and not on the stimulus-induced increase in
kDa PMN G protein, which was shown to be the substrate intracellular Ca2+ (1 9). The results of our present study do
for ADP-ribosylation by BT-D (24). Our studies also pro-
vide evidence for a functional involvement of the BT-D- nftion reGardin the echiosite of
sensitive G-, in the regulation of exocytosis in FMLP-
stimulated human PMNs (Figs. 2-4). Furthermore, the ever. they clearly indicate a functional involvement of BT-
aindvdoitlivveem iennht iboitfi on by PT and BT-D (Fig. 3) sug_ gse sts the D-sAelntshiotiuvgeh 22th-ek Dian trsimnsailcl GA pDroPt-eriinb(oss) yilntr aPnMsfNer aesxeo caycttoisviist.y
invovemet ofmultiple G proteins, at different sites, in ofB-habenclditoqsin 4.teriso-
the regulation of exocytosis in PMNs. Moreover, the data of BT-D has been called into question (47). there is con-
presented in Figure 4 demonstrate that at least 85% of G2 _,k vincing biochemical evidence in support of an ADP-ribo-
needs to be irreversibly modified by BT-D, before an in- syltransferase activity of the toxic component of the neu-
hibitory effect of the toxin on PMN secretory responses is rotoxin (37. 45. 46). The results of our present studies are
evident. This finding provides an explanation for the consistent with the reported ADP-ribosylation of G22, by
reported failure to observe inhibition of g-glucuronidase BT-D in bovine adrenal tissue (37) and in other cells in-
release from BT-D-treated PMNs under experimental con- cluding PMNs (23, 24). and also with the reported inhibi-
ditions which we found to modify only 50% to 60% of the tion of neurotransmitter release by BT-D in bovine adrenal
PMN G,,, (23). medullary cells (26). The observed correlation of inhibi-
In secretion studies with digitonin-permeabilized tion of PMN exocytosis with the degree of ADP-ribo-
PMNs. BT-D was found to significantly inhibit Ca 2+ mi- sylation of PMN G_,k by BT-D (Fig. 4) and the lack of an
nus and Ca2> + GTP-induced exocytosis of both primary inhibitory effect of exoenzyme BT-C3 on PMN secretion
and secondary granules. whereas PT only inhibited the re- in permeabilized cells (data not shown), suggest an in-
lease of secondary granule contents without affecting volvement of the BT-D sensitive G_,k in the regulation of
03-glucuronidase release, a marker for primary granules stimulated exocytosis of human PMNs. In this context. it
(Fig. 5). Furthermore. while BT-D inhibited Ca2*-inde- is worthwhile to note that the Irhol-related small G pro-
pendent. GTPyS-induced exocytosis substantially in digi- teins rac I and rac2. which are substrates for ADP-ribosyl-
tonin-permeabilized PMNs. the extracellular release of ation by BT-C3 (31). have been studied extensively in the
primary granule contents was found to be more sensitive past few years. and are believed to be involved in cytoskel-
to toxin-induced inhibition than was release of secondary etal reorganization (51) and in the activation and/or as-
granules (>80% vs 40%,). In contrast. PT treatment of sembly of NADPH oxidase complex of phagocytic cells
PMNs did not affect the Ca2*-independent. GTP- - 53fo.2
duced secretion (data not shown). In a recent report (48).
evidence was presented for the association of distinct sub- Recent evidence from several laboratories indicate that
sets of small G proteins with the primary and secondary the rab proteins, the mammalian homologues of sec4 and
granules of human PMNs. and it is tempting to speculate yptl gene products, which regulate yeast secretory vesitle
that a differential sensitivity of these PMN G proteins to fusion and endoplasmic reticulum-to-Golgi transport. re-
BT-D may distinguish the G proteins that are involved in spectively. represent a large family of ras-like small G
mediating exocytosis of one or the other granular type. proteins (or GTPases) that regulate distinct vesicular trans-
Although the nature of the accumulated membrane- port events at the level of membrane targeting and/or fu-
bound vesicles is not known at this time. ultrastructural sion (12. 44, 49). Based on evidence accumulated from
changes of BT-D-treated PMNs after activation with recent studies in cell-free systems. it is currently believed
FMLP. as shown in Figure 6. mimic those of secretion- that most organelles of the endocytic and secretory path-
defective mutant yeast strains, deficient in the ras-related ways bear distinct rab proteins on their surfaces. and that
small G protein. sec4p, in which sub-plasmalemmal accu- the rab proteins may function to ensure the accuracy of
mulations of secretory vesicles are observed (15-18). Re- vesicle targeting events (12. 44. 49). In view of the dif-
cently. the rab family of small G proteins have been iden- ferential regulation of distinct PMN granule types (7. 8). it
tified as mammalian homologues of sec4p and ypt I p (49). is conceivable that such a regulatory mechanism also ex-
and distinct rab proteins of 22- to 25-kDa molecular mass ists in the targeting and/or fusion of PMN granules during
1378 ROLE O)F G, IN PM`N SF( RETION
stimulated exocytosis. Future studies directed at charac- 20. Sn~dernian. R.. and R.J . 1,Ihngy. 1988. Phagcccsic cell,: sinnulus-
terization and subcellular localization of the BT-D-sensi- response coiupling mlechanisms. In Intlinmmeina',s. J. I. (lallin. 1. MI.
tive G.,, in stimulated and unstimulated PMNs should 2.Goldstein. and R. Snsdermian. eds. Raven Pre,,. NeseY. ork. p.3 04.
1.Barnc%%inan. MI. MI.. S. (Cickrcoft. and B. D). Gomnperts. 1986. Tvec
clarify this possibility, role% for guanine nucleotide in `4111mulusse1 cretion sequence ofiteu
trophils. Nature' 319:504,
Acknowledgments 22. Clark. R. A.. I 990. The humian neutrophil respiratory burst oxidase.
J Ilc.A.//-10
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