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Preferential expression of anti-bovine serum albumin IgE homocytotropic antibody synthesis and anaphylactic sensitivity in the neonatal rabbit
Preferential expression of anti-bovine serum albumin IgE homocytotropic antibody synthesis and anaphylactic sensitivity in the neonatal rabbit R. Neal
Pinckard,
Ph.D.,
Marilyn
Halonen,
MS.,
and Aniko
1. Meng,
M.S.
TIKSO~I, Ark.
The pkrnotypic, exprt-ssion of the gc-w-tic cccpncity to synthesize trnli-bovine srrum ctlblcmin (BSA) IgE homocytolropio antibody and inter to develop systemic ctnnphylnctic sensitivity to BK4 was studied in the neonatal rabbit. Nwborn rabbits immunized with soluble boxi,ne serum albumin were rendewd totally immunologically unresponsive to BSA. In cuntwst, ncrcborn robbits immunized with alum-precipitated B.$‘A or v.?lh soluble BS-4 in wrjunction with (I Cor.vnehctcriurn pnrvum adjutant produced only anti-HSA I.072 homocytotropic ontibotly duriag the neonatal period and throughozct adult life; thew rctbbits did ?iot produce a.nti-BSd antibody that could be detcctcd by the passive hemclgglu.tination or antigen-binding techniques. Im.munization of the T-day-old rubbit with soluble BS-4 induced immunologic unresponsiveness to BSA with the exception. of anti-US-4 IgE homocytolropic antibody thlct rws prcfewnlinlly produced when these rnbbits were adults. When alum-precipitalctl BSA or solu.ble BSA together with C. parvum was administered to the T-day-old rabbit, anti-BSA a?ltibodies rr’ere produced that vx-re detected by all of the immunologic tesl procedwes. Although anti-BSA IgE homocytotropic antibody cv~ul~l be produwd in cldull i-abbits rectit+n.g primclry or secondary injections of trl~tm~prccipilat~d 7jS.4, systemic trnctphylrrcfio sensitizntioa to BSA (let-eloped in osly .I? of Sd animals. Liowerw, of 36 adult rcthhits thnt developed ctnti-HSA Igli homocytotropic nntibotly (IS (I wsult of neonottrl czposrcre to BS.4, 31 deucloped systemic annphylaxis to BSd. 7I.e cx)nc-lrcdc thnt the gewlic potentbl to synthrsize specific, 1gE‘ homocytf>tropic fintihwly nnd to derrlop systemic antrphyloctic sensi. ti1.it.u can be phenotypicctll,y eq~ressctl in the ?tronctial robbit.
Homocytotropic or reaginia antibodies initiate the release of rasoac*tivc amines in a homologous specks by the antigen-inducctl clegranulation of tissue mast ~11s and,‘or bloofl hasophils. Although great emphasis has been plaoctl U~OII the physicochcmical characterization of homoeytotropic ant.ibocly, little is k~~ow~ about factors whic*h influence its initiation nncl /or enhancement (i.e., its phcnotypic espressiori ) . X number of ad,ju\-ants haw facilitated the induction ol’ homocyt.otropic antibody synthesis in rabbits am1 other experimental -animals to varying degrees.’ Howcvcr, thrse studiw ncrc performed esclusiwly in the atlult. animal. As it wsnlt, little is known ahout~ the tle\c~lopmcntaI biology of
302
Pinckord,
Halonen,
and Meng
:
ALLERGY CllN
IMMUNOL MAY 1972
VOLUME NUMBER
Preferential
49
5
BYA was sterilized by 0.15 p Milliporc antigens were either soluble BYA, administered together with a heat-killed the prep!irations of ABYA and the
Determination Serum of randomly i? hours,
of anti-BSA
expression
of
IgE
antibody
synthesis
303
filtration and was stored at -20” C. The immunizing :tlum.precipitatcd BYX (ABSA), or soluble BSA suspension of Corynebactertitm paruum strain 10387; C. pccrmkm adjuvant have been described previous1y.l
homocytotropic
antibody
activity
or
serum dilutions, 0.1 ml., \\crc injcctcd intracutancously into the sh:~vecl backs l.,red, 3.montll-old, male and female Californian rabbits. After a latent period of injection of 2 per rent the rabbits were: chnllengc4 with a 2.5 ml. intravenous EV:IIIS hluc tlyc in pyrogtrn-free, 11.85 per ctbnt ?T:lCl containing 50 mg. of BSA. Thirty minutes 1:1tcr ttlc rabbits wf>rc put to tl(~:~th? and the nveragc di:lmcter of bluing was determined on t III! int(brnnl sidcb of the rcficcted skin. At lc:ist two rabhitx w(‘rc used for c~:~ch hon1010g0u~ I’C.4 tlt~tcWnination ; Wch rabbit rcceivr~l no more than 24 injrctions, 0nc of which \WS a l.ontrol injfbction of normal r:illl)it strum. ,I serum wn?; (bon*icleretl llositive whcbn the dinmetcr of Illuing was grcnter than 5 nm. at two intr:lcutan(*ouS test silts.
Physicochemical
characterization
of the
anti-BSA
homocytotropic
antibody
k’our pll~sic~ochc~tlic:ll c*ritcrin were us4 to tletcrminc whcthcr the anti.BYA homocytotropic antitlotly was ns9oci:itetl with TgG; and/or IgEl. b-10, . thcsc determinations have I~cn described in ~letail ljrcvious1y.l Heat 1:ibilit.v ww :~~nscd hy incubating srra at 56” C. for 4 hours :mcl then dctcrmining tilts amount of rrGdu:~I homologous PC.4 activity when compared with a11 unhrated control. Yimil:lr innctiration studies w(‘rc performed ul)on sem that were reduced wittl O.lOhl Itler~l,torttl:lnol nncl c;~rbos~nlc~tt1yl:~te~l with 0.0131 iodoacetic acid. The anion lGn(ling prolWrtio.5 of the anti-B%% homo~~ytotropic nntfl)ody wore determinctd by stepwisr cblution from I)EAE-~llulose with the IW of 0.01 hI l~host~h:itc lmffer, pH 8.0, containing 0.015, 0.010, O.O!)O, 0.19, 0.9, and O.49hI NaC!I, respectively. JIolccular weight estimations for thv anti-ISA hon~ocgtotrol~ie nntit~olly \v(:r(: mi~dc ijp elution profiles from calibratefl SI~~~~I:II~I~S (:-L’illl 4un1ns wit11 the uw of 4lcscvu4lin~ olution :II :i How rate of 20 ml. lwr 110111~.
Determination
of the
antigen-binding
capacity
The antigen-binding c.apacity (ABC) mm tli+c~rmined by the ammonium sulfate mc~thotl ~l~~sc~ritle~l try I’arr.11 Tr:lcac-labclrd 1~~1.RH.4 w:ln l~rcparcd I)y the chloramine-T method and \\:Is u?;e(l at a concentration of 0.01 pg N to c’nsurc’ maximum sensitirit,y of the t&.1’d serum WIS I~on~idcrccl positive, WIWII :L !,,I s(‘rum Ililntion ~)ound gIcl:ltor than 1O per ccbnt of 0.01 fig N lIR.\.
Passive
hemagglutination
l’assive hemagglutinating activity viouslyl with tho use of a microtiter rcrum was considered positive when scn3itizrd sheep erythrocytes.
Experimental
(PHA) of sera was determined as described presystem (Cooke Engineering Co., Alexandria, va.). A a l/i0 dilution caused visual agglutination of the BYA-
design
I’rior to initiating experiments in the neonatal rtlhhit, the genetic potential of the (‘alifornian rabbit to responcl immunologically to BSA u-as determined. Eighteen S-monthold n~ctlr and female (Wifornian rabbits Kere given a subcutaneous injection of 10 mg. of AlW;\ (Experiment 1). The rabbits were blecl at weekly intervals thereafter, and their .Gera wcrc tested for P(:A, ABC, ant1 PHA activities. ‘I’0 (letermine the effects of neonatal exposure to HYA with or without adjuvant upon the long-term expression of anti-BYA antibody synthesis and the subsequent development of anaphylactic sensitivity, the experiments shown in Table I were performed. Yix litters of Cnlifornian rabhits were immunized within 2-L hours of birth (Exporiment Z), and 4 litters wcrc immunized 7 days following birth (Experiment 3). Each litter was dividod
304
Pinckord,
TABLE BSA
I.
with
following antibody
Halonen,
Experimental or
without
and
design
to
adiuvant
birth (Groups synthesis and --.
3A the
:
Meng
determine
within to 3C) upon development
the
the first
effects
24
hours
the Induction of systemic
of
exposing
of
life
the
(Groups
of long-term anaphylactic
; Group
MMLlNOL MAY 19?2
Ctlh
neonatal 2A
anti-BSA sensitization
to
rabblt
2C)
Birth
Day
7
j
to
or 7 days
homocytotroplc ~_
Experiment No.
AiLERCzb
Doyr 14 to 77 (Experiments 2 and .lmmuniration Day
---. 31
VOLUME NUMBER
Preferential expression of IgE antibody synthesis 305
49 5
rcwJlt. ill the: protlwticbn 01’ si.yJifi(!iJtJt ;iJJJoliJJtsof’ clvttvt;JlJlc alit i-ISA ant ilJod>. iJJ(lcctl. the ovrr-all JJrg;iti\it\- of tlw !)I cl;1\. SCJX tlenlc~JJstratr(l t hi. t hew: Jxhbits were rrr~tlcrccl imJJi~JJic~lo~i~aII~Ill~wspolisi\c to BSA. This VOJIelusion is bawl 011 x2 nJJalysis of the Jiumhor 01’ raMits in (:roup f-,A procluccing cletwtnblc anti-BSd aJJtilJod~-when c~~~JJl~arct1 to r~~sponscsof the 18 adult rabbits iJJ ISspcriJnent 1 : I’(,‘A, 1) < 0.00.1 : ABC or I’HA, p < 0.0005. In contrast to JdJhits iJJ Grout) :‘A, a significant J~uJJJheJ~ of r;Jht)its that rwcivcvl &her ABSL2 ( Group 2R) or s0ldJlc BSA tog:ctlJcr with C. ~uJ’I*uJ~L(( iroup 2C) 011the clay of birth producctl tletcct.able anti-I%4 hoJJlwyt.otJ~opic aJJtihod~. tiuring the nwnfital lwriod iIJld after the aJiimals reachctl ntlJJlthood. The homologous PC’A titers iJi Iwth (iroups 2B and ‘LC rangetl from !,i to !,L on Day 28 to ‘2; to 3/12s on Day 97. Thaw rabbits, however, (lit1 not produce dctcvtahlt~ long-tcrJu anti-H%\ nntibotl? as Jric;Jsurctl 1Jg tho nI!%Cl a~d I’Ii,J techniques. I’hpiw~heJnical characterization 0f t hca anti-13% homocytot.ropic ;lr~til)otl~ procluwtl irl Croups ‘LB ant1 2C WHS ~wrf0Jmictl on 14 to !I1 (lily wra to dctcrminc whether the I’CA activit.y wits asswi;Jt.cd with rabbit I@: antl,~or JxlJbit 1gE. The I’ollowiJJg results were obt;JiJJctl : (1) heating tlJc stw to 56” (Y. for 1 hoiirs gJ*cat.l\- rotlwed or destroyed all WA activity; (2) reduction antI alkylatiwl dcst royed all I’CA actidtg ; (3) ITA activity \VilS clutc(l cscliisivt~ly in tlicb 0.1031 Sa(‘l! pJT 8.0, fraction (~~OIldU~tilIl~~~ of 5.0 t0 7.0 mniho) fronl l.~l’:~~1F:-c~Clllll0Sc ; aJlt1 (4) the Jnoledar weight. (3r.W.) of the homcw~~totropic antihod?- \viIs t~stiIlJattv1 to he 240,000 ; the it\x~r:rge elution volunJc;‘\oitl \olunlc~ ratios ( V,/‘V,) for the four Jnarker proteiJJs, hllJllilJl SWUJI~ albumin (II.)\-. io.oC)o). lJovi;ie hcaJ’t ladic tlchyclrcqcwasc (lI.jv. 140,000), hUJllilJ1 gaIIlllla glohnlin (JI.j\-. 160,000), iIIlt1 hovinc~ liver glUtaIJli(: clt~ll~~lrogcJlasc (hL1Y. 250,000jF wc’rc 1.80, 153, 1.40, aJid 1.24, rcspcctivcly, aJJc1 tlJc%;iverage \TC,‘Y,, ratio for i he ;ltJti-BQA hoJJJwytotrol)ic antibod\- was 1.26. ‘1’hC rcslJlts of 1SsperiJnc~JJt, :3 cl~~JiioJJstr;Jtc the ilc~~~uisiti0Jlof a gJTilttlr dcgrt!c of iJJJmuJlologic cwmlwtcnw IJy tllc i-cl;Jy-old J*;Jhbit (Table TI ). Although the rill)hits ill (:roiip 3A did not I)rcIclucc aIiti-BSA hoJlioi~~t.otro~Jict antihocly 1,)
iid id!.;
l)iIy
6:3,
;I
Sigllifivallt
IlUIllb~T
Of
thW(’
IXbbitS
~1rW~llWd
tlt’tcc!table
(!!,
to
‘,{;4)
aJJti~Jocl~14 clays followiii,v ~liallt~iigc with 10 nig. of I)ay 77. IIowc~vcr~ the rabbits iii (;roup 3A (lid not protlucc anti-B% nJJt.ilJocIy that \vas tlctcctcd 1)~. the XBCY ant1 PHX tcchniqucs. In coJitrast. to (:ronp 3A, (:roul)s 313 and 31 procln~~l both hoJlJo~!-tot.rol)ic aJJtibody aJJt1aJJtihotl~~ tlrtwtcvl 1)~ the ABC aJJ(l I’IT.4 tcchniqws. ‘1%~ homologous I’CX titers iJi both (;roups 313 antl 3’ r;Jngtd from ‘,Y;to & 011 I)iI\- 28 to $4 to !Gys 011 1)ily 9 I. I’h!-sic~ocheJJJi~~l1charnc.tcri;l,7tioti of the! lioJJ~oc~tt~tropic antihood> ~~J*oclucwlby t.lJc rabbits in 1Ssprrinit~Jit 3 ww: i~lt~lltiCill to tliosc clcsorihed ill Esl)criJIJeJlt. 2. The ~\BC ant1 PHX ac+iviticls in th stw of thv rabbits in Croups 3H ;rJJ(l XY wrt’ localiztvl I)riJJJaril>. iJl the O.OlOJI ant1 0.0’1.5JI Sa(.‘l fJwtioJJs antI to it lesser cl~~grcciJl the 0.2031 I\;a(.‘I fraction t~lutecl t’r*o~n 1~l5~~1‘-ccll~lost~ ha\ing coIlc~ll~taJlWs of l.iO, 2.20, iJJJ(l 135 mmho, respectively. No anti-B% ,11W or I’HA activity could he (Icmonstratetl in thv 0.103I Na(‘l frwtions (5.0 to i.0 mmho) which containetl the homologous P(.‘il act.ivity. The dt’wts of neonatal csposurc to B8.4 with or without acliu\-aJJt up011 t,hc clP~~‘lO~~1llt~llt 0f systcniic iIlliJ~~Jl~lilSiS in tllc :1~11111 rill)l)it ilI’C SW11 in TiII)ll? 111. mti-HSA
;\l
hoIlJo~~totropic
011
306
Pinckard,
TABLE
II.
Experiments
Halonen,
Temporal 2 and
and
development 3
i
Meng
of
passive
cutaneous
anophyluctlc
ALLERGY
CLIN
actlvlty
IMMUNOL MAY 1971
IPCAI,
antlgen-
VOLUME NUMBER binding
49 5
capacity
(ABC),
Day PCA
i
l/IO !I/ 10 I,O.O05) Y/R O.OI)5)
Preferential
ond
possive
hemagglutinating
PCA
PHA
O/IO 3jlO
o/10 L’/ln
l/R
1/n
I/i
o/7
7;;
Tji ~0.01)5) L)/ti t 0.025 I
O/i i/i
2/i i/i
( 0.m) L'/ti
(
;I )
activity
Day 63 -1 ABC
28
ABC
expression
l/IO 9/10 ( 0.003) Y/S I, o.no1 )
) j
of
IgE
antibody
(PHA)
in
synthesis
the
rabbits
Day PHA
o/10 l/10
O/IO l/IO
1;s
1/n
n/7
O/T
7,‘;
i/7
(0.003) 4/ti ( <0.05)
t 0.003) I/ti I <0.05)
PCA
i
307
from
91
ABC
PHA
3/!-l $)/IO (
n/g 1,‘lO
o/9 l/10
l/S
2/s
O/i
n/7
0;;
i/i
i/i
, 0.005 I 4/G t
: 0.005) 4/c :
7/i
(ijlj
)
homologous YCA titers in these 36 rabbit.s ranged I’ronl 14 to $G, at the time of chlle11ge.
DISCUSSION
l’he present stutlics drnlonstriite that the administration of BSA to the neonatal rabbit can induce the preferential expression of anti-B% IgN homocytotropic antibody sylthcsis not 011ly during the neonatal period but throughout adult lift. Preftwntial espression of homocytotropic antibody was dependent upon the age of the neonatal rabbit and upon the adminisbration of adjutants. Injection of soluble BM wibhin the first 24 hours following birth resulted in the intluction of acquired immunologic unresponsiwncss to BSA, including IgE iillresl)onsirc~lirss ((iroup ‘LA). In contrast, the admiliistration of alum-prvvipitatctl HSA (Group 2H) or soluble BSA tog&her with C’. IM~VMUI (Croup 2C) in~lucetl anti-BSA antibody that ~oulcl be dctcvtctl 0111y by homologous PCIJ am1 11ot. by oithrr the T’IIA or ABC tcchniqurs. Tt should bc pointed out. that thv Iwgativity of the PHA ant1 ABC tcchniqucs does not ntwssarily intlicabe that TgF: homocytropic antibody can not he dctwtcd by PHA and especially by a primary binding assay such as the ABC. 011~ csplanation for this ncgativit? is that the mikimum quant.ity of homocytotropic antibody nrctle~l to induw cutaneous bluing may tw substantially less than the quantity ncetlecl to giw Imsitive PII, and ABC ksts. In ;iddition, WC ernployefl an initial 1/10 dilutiorl of s(‘l’il in the I’IIA ant1 ABC tests, whercas untlilute sera wcrc cmployecl for hoJ~wlo~ous PCA. One other possible explanation might be that the homocytutropic antibody coultl wnceivably be directed toward wntaminating proteins in our B&4 prcparat.icm. Such a prohlcm exists in any stutly in which solubles protein antigens arc employed and a highly scnsjtive biologic assay such as I~nJJ~ologo~~~ WA is used. ‘\Ve feel that such an explanation does not apply to our ~~resent stucl~- because the purit.y of the immunizing BSA antigen was grcatcr than !Kl per cent. Furthermow, the intravenous injection of as little as 500 pg of vrystallizecl BSA with a purity of 100 per writ induced positi\-e homologous PC;2 rrwdions, whcrcas 20 mg. of bovine gamma globulin (Cohn fradion 1 I), ;1
308
Pinckard,
Halonen,
ond Meng
i
AL1ERC.Y CLIN
IMMUNOL MAY 1972
VOLUME NUMBER
49 5
Preferential
expression
of
IgE
antibody
synthesis
309
spthesis of the other immunoglol)ulin classes of antibody could he expected to dtvr the character of immediate Il~vI)t!l’sensiti\it!- reactions. For ccwnple. the lack 01’ IgG “blwking” antibody coul~l pwmit small amounts ot’ ant.igen to inibiate li~I)crsciisiti\it.?reactions. The I~wscnt cspcrimrnts now I)rovitle a model \\hc*rc+y such alterations iI1 the trmporal dcwlopment of the qualitative ancI/‘or quaiititativc cxprrssioii of antibody synthesis (yin Ix stuclicil with rcspwt to cthanges in the clcvclopmcnt of imm~diatc hylwrsensitivity Statw Systemic anaphplasis in the raM)it is not commonly observd ; in those rcyorte~l illsti111(!Ps Of systemic anaphylasis, the wusc has heen awrihcd to t.hv illtravawular form;ition of ~lntigcn-antil)o(l\- wmplcsrs causing capillary emboli, ii (lwrrast~ in pulmonary arterial bloc-~1llow, and pcbriphcral c*iwulatory collal~2.‘” Ill ~0ntrast. \vc have ohscr\ecl a high frvqiit~ilcy of systrmic iitiapl~~lasis (‘I’i3l)le JTI ) in (:roups ‘LB ant1 2C ant1 3A! 3B. and 3(! when these rabbits were givcw 50 mg. of BSA intravenously on J);IJ, $11. The tlc\clqmwnt of systemic: >lll;lph~li~sis was associated otil~- with thcb l”‘escnce of tlctectahlc anti-B!+\ 1gE Ilonloc.?totroT)ic. antibody anal not with the prescnve of anti-BSA antibody as I)~~~MuY~ b\- the ABC or PIId twhniqut~s. IYtb co~~clucle from these <a thnt sJxtwic* anaphylasis in thv rahhit ciin he initiatctl l)y specific Ig% antibody in atlclition to immune w~nples~s. ,\t presrl1t the shock organ(s) ot’ the IgEmdiated systemic ana1)hylasis in the rahl)it arc not known ; howc~c~~~, preliminary c~\%lcnw in onr laboratory inclicrtcs that within 60 scwmtls following the intravtlnous injection of BSA into the unancsthctized rabbit a dramatic* fall in the III(~II adcrial 1~100~1 pressure from 100 to 110 111111. Hg t,o 30 to 50 11m. JTg owllrs illl~l persists during t.he period these animals eshihit. anaphylactic: symptoms. The Iilek of systemic: an;l1)hylactic sensitization in the rabbits from Espcrimcnt 1 is intrigiling. XI1 32 ol’ these rabbits hat1 clctcctablc anti-BSA homoc?-totropic antibody yet only 3 h;~tl aiia1bliylacG syni])tonis. Although as a group thcsc I’ilhbitS had lowr homologous I'(!i\ titers t hali tlic rabbits in Hsperimcwts 2 rlIl(l 3, 20 hat1 homologous WA titers of 1.1 or #water. Thcw tlata, tiikcll in VOII~~IWtion with the obswvations in Eslwrinients r! arici 8 that tlicre was no cwrrcl;it.ion h(bt\VWI~ the scvtlrity of the anaphylactiv symptoms ilIlt the homologons l’( ‘A titws, inclicatc that systemic anaphylasis in the raldd is clepcnclrnt U~II faders otllcr than the r+wdating Icvels of spwific TgE illltihd~. t’cd~aps the ~1nl0lll1t ali(l;‘or tlistribution of cell-11on11d 1pE antibocl>- woultl he bcttrr vritrria I’or ~n’tvlictin:: the incidence :ln(l scvcrity of systemic! an;ll)hylasis.
310
Pinckard,
Halonen,
and
Meng
:
A>:ERGf
CLIN
IMMUNOL MAY 197:,
Pinckard,
Ph.D.,
Marilyn
Halonen,
MS.,
and Aniko
1. Meng,
M.S.
TIKSO~I, Ark.
The pkrnotypic, exprt-ssion of the gc-w-tic cccpncity to synthesize trnli-bovine srrum ctlblcmin (BSA) IgE homocytolropio antibody and inter to develop systemic ctnnphylnctic sensitivity to BK4 was studied in the neonatal rabbit. Nwborn rabbits immunized with soluble boxi,ne serum albumin were rendewd totally immunologically unresponsive to BSA. In cuntwst, ncrcborn robbits immunized with alum-precipitated B.$‘A or v.?lh soluble BS-4 in wrjunction with (I Cor.vnehctcriurn pnrvum adjutant produced only anti-HSA I.072 homocytotropic ontibotly duriag the neonatal period and throughozct adult life; thew rctbbits did ?iot produce a.nti-BSd antibody that could be detcctcd by the passive hemclgglu.tination or antigen-binding techniques. Im.munization of the T-day-old rubbit with soluble BS-4 induced immunologic unresponsiveness to BSA with the exception. of anti-US-4 IgE homocytolropic antibody thlct rws prcfewnlinlly produced when these rnbbits were adults. When alum-precipitalctl BSA or solu.ble BSA together with C. parvum was administered to the T-day-old rabbit, anti-BSA a?ltibodies rr’ere produced that vx-re detected by all of the immunologic tesl procedwes. Although anti-BSA IgE homocytotropic antibody cv~ul~l be produwd in cldull i-abbits rectit+n.g primclry or secondary injections of trl~tm~prccipilat~d 7jS.4, systemic trnctphylrrcfio sensitizntioa to BSA (let-eloped in osly .I? of Sd animals. Liowerw, of 36 adult rcthhits thnt developed ctnti-HSA Igli homocytotropic nntibotly (IS (I wsult of neonottrl czposrcre to BS.4, 31 deucloped systemic annphylaxis to BSd. 7I.e cx)nc-lrcdc thnt the gewlic potentbl to synthrsize specific, 1gE‘ homocytf>tropic fintihwly nnd to derrlop systemic antrphyloctic sensi. ti1.it.u can be phenotypicctll,y eq~ressctl in the ?tronctial robbit.
Homocytotropic or reaginia antibodies initiate the release of rasoac*tivc amines in a homologous specks by the antigen-inducctl clegranulation of tissue mast ~11s and,‘or bloofl hasophils. Although great emphasis has been plaoctl U~OII the physicochcmical characterization of homoeytotropic ant.ibocly, little is k~~ow~ about factors whic*h influence its initiation nncl /or enhancement (i.e., its phcnotypic espressiori ) . X number of ad,ju\-ants haw facilitated the induction ol’ homocyt.otropic antibody synthesis in rabbits am1 other experimental -animals to varying degrees.’ Howcvcr, thrse studiw ncrc performed esclusiwly in the atlult. animal. As it wsnlt, little is known ahout~ the tle\c~lopmcntaI biology of
302
Pinckord,
Halonen,
and Meng
:
ALLERGY CllN
IMMUNOL MAY 1972
VOLUME NUMBER
Preferential
49
5
BYA was sterilized by 0.15 p Milliporc antigens were either soluble BYA, administered together with a heat-killed the prep!irations of ABYA and the
Determination Serum of randomly i? hours,
of anti-BSA
expression
of
IgE
antibody
synthesis
303
filtration and was stored at -20” C. The immunizing :tlum.precipitatcd BYX (ABSA), or soluble BSA suspension of Corynebactertitm paruum strain 10387; C. pccrmkm adjuvant have been described previous1y.l
homocytotropic
antibody
activity
or
serum dilutions, 0.1 ml., \\crc injcctcd intracutancously into the sh:~vecl backs l.,red, 3.montll-old, male and female Californian rabbits. After a latent period of injection of 2 per rent the rabbits were: chnllengc4 with a 2.5 ml. intravenous EV:IIIS hluc tlyc in pyrogtrn-free, 11.85 per ctbnt ?T:lCl containing 50 mg. of BSA. Thirty minutes 1:1tcr ttlc rabbits wf>rc put to tl(~:~th? and the nveragc di:lmcter of bluing was determined on t III! int(brnnl sidcb of the rcficcted skin. At lc:ist two rabhitx w(‘rc used for c~:~ch hon1010g0u~ I’C.4 tlt~tcWnination ; Wch rabbit rcceivr~l no more than 24 injrctions, 0nc of which \WS a l.ontrol injfbction of normal r:illl)it strum. ,I serum wn?; (bon*icleretl llositive whcbn the dinmetcr of Illuing was grcnter than 5 nm. at two intr:lcutan(*ouS test silts.
Physicochemical
characterization
of the
anti-BSA
homocytotropic
antibody
k’our pll~sic~ochc~tlic:ll c*ritcrin were us4 to tletcrminc whcthcr the anti.BYA homocytotropic antitlotly was ns9oci:itetl with TgG; and/or IgEl. b-10, . thcsc determinations have I~cn described in ~letail ljrcvious1y.l Heat 1:ibilit.v ww :~~nscd hy incubating srra at 56” C. for 4 hours :mcl then dctcrmining tilts amount of rrGdu:~I homologous PC.4 activity when compared with a11 unhrated control. Yimil:lr innctiration studies w(‘rc performed ul)on sem that were reduced wittl O.lOhl Itler~l,torttl:lnol nncl c;~rbos~nlc~tt1yl:~te~l with 0.0131 iodoacetic acid. The anion lGn(ling prolWrtio.5 of the anti-B%% homo~~ytotropic nntfl)ody wore determinctd by stepwisr cblution from I)EAE-~llulose with the IW of 0.01 hI l~host~h:itc lmffer, pH 8.0, containing 0.015, 0.010, O.O!)O, 0.19, 0.9, and O.49hI NaC!I, respectively. JIolccular weight estimations for thv anti-ISA hon~ocgtotrol~ie nntit~olly \v(:r(: mi~dc ijp elution profiles from calibratefl SI~~~~I:II~I~S (:-L’illl 4un1ns wit11 the uw of 4lcscvu4lin~ olution :II :i How rate of 20 ml. lwr 110111~.
Determination
of the
antigen-binding
capacity
The antigen-binding c.apacity (ABC) mm tli+c~rmined by the ammonium sulfate mc~thotl ~l~~sc~ritle~l try I’arr.11 Tr:lcac-labclrd 1~~1.RH.4 w:ln l~rcparcd I)y the chloramine-T method and \\:Is u?;e(l at a concentration of 0.01 pg N to c’nsurc’ maximum sensitirit,y of the t&.1’d serum WIS I~on~idcrccl positive, WIWII :L !,,I s(‘rum Ililntion ~)ound gIcl:ltor than 1O per ccbnt of 0.01 fig N lIR.\.
Passive
hemagglutination
l’assive hemagglutinating activity viouslyl with tho use of a microtiter rcrum was considered positive when scn3itizrd sheep erythrocytes.
Experimental
(PHA) of sera was determined as described presystem (Cooke Engineering Co., Alexandria, va.). A a l/i0 dilution caused visual agglutination of the BYA-
design
I’rior to initiating experiments in the neonatal rtlhhit, the genetic potential of the (‘alifornian rabbit to responcl immunologically to BSA u-as determined. Eighteen S-monthold n~ctlr and female (Wifornian rabbits Kere given a subcutaneous injection of 10 mg. of AlW;\ (Experiment 1). The rabbits were blecl at weekly intervals thereafter, and their .Gera wcrc tested for P(:A, ABC, ant1 PHA activities. ‘I’0 (letermine the effects of neonatal exposure to HYA with or without adjuvant upon the long-term expression of anti-BYA antibody synthesis and the subsequent development of anaphylactic sensitivity, the experiments shown in Table I were performed. Yix litters of Cnlifornian rabhits were immunized within 2-L hours of birth (Exporiment Z), and 4 litters wcrc immunized 7 days following birth (Experiment 3). Each litter was dividod
304
Pinckord,
TABLE BSA
I.
with
following antibody
Halonen,
Experimental or
without
and
design
to
adiuvant
birth (Groups synthesis and --.
3A the
:
Meng
determine
within to 3C) upon development
the
the first
effects
24
hours
the Induction of systemic
of
exposing
of
life
the
(Groups
of long-term anaphylactic
; Group
MMLlNOL MAY 19?2
Ctlh
neonatal 2A
anti-BSA sensitization
to
rabblt
2C)
Birth
Day
7
j
to
or 7 days
homocytotroplc ~_
Experiment No.
AiLERCzb
Doyr 14 to 77 (Experiments 2 and .lmmuniration Day
---. 31
VOLUME NUMBER
Preferential expression of IgE antibody synthesis 305
49 5
rcwJlt. ill the: protlwticbn 01’ si.yJifi(!iJtJt ;iJJJoliJJtsof’ clvttvt;JlJlc alit i-ISA ant ilJod>. iJJ(lcctl. the ovrr-all JJrg;iti\it\- of tlw !)I cl;1\. SCJX tlenlc~JJstratr(l t hi. t hew: Jxhbits were rrr~tlcrccl imJJi~JJic~lo~i~aII~Ill~wspolisi\c to BSA. This VOJIelusion is bawl 011 x2 nJJalysis of the Jiumhor 01’ raMits in (:roup f-,A procluccing cletwtnblc anti-BSd aJJtilJod~-when c~~~JJl~arct1 to r~~sponscsof the 18 adult rabbits iJJ ISspcriJnent 1 : I’(,‘A, 1) < 0.00.1 : ABC or I’HA, p < 0.0005. In contrast to JdJhits iJJ Grout) :‘A, a significant J~uJJJheJ~ of r;Jht)its that rwcivcvl &her ABSL2 ( Group 2R) or s0ldJlc BSA tog:ctlJcr with C. ~uJ’I*uJ~L(( iroup 2C) 011the clay of birth producctl tletcct.able anti-I%4 hoJJlwyt.otJ~opic aJJtihod~. tiuring the nwnfital lwriod iIJld after the aJiimals reachctl ntlJJlthood. The homologous PC’A titers iJi Iwth (iroups 2B and ‘LC rangetl from !,i to !,L on Day 28 to ‘2; to 3/12s on Day 97. Thaw rabbits, however, (lit1 not produce dctcvtahlt~ long-tcrJu anti-H%\ nntibotl? as Jric;Jsurctl 1Jg tho nI!%Cl a~d I’Ii,J techniques. I’hpiw~heJnical characterization 0f t hca anti-13% homocytot.ropic ;lr~til)otl~ procluwtl irl Croups ‘LB ant1 2C WHS ~wrf0Jmictl on 14 to !I1 (lily wra to dctcrminc whether the I’CA activit.y wits asswi;Jt.cd with rabbit I@: antl,~or JxlJbit 1gE. The I’ollowiJJg results were obt;JiJJctl : (1) heating tlJc stw to 56” (Y. for 1 hoiirs gJ*cat.l\- rotlwed or destroyed all WA activity; (2) reduction antI alkylatiwl dcst royed all I’CA actidtg ; (3) ITA activity \VilS clutc(l cscliisivt~ly in tlicb 0.1031 Sa(‘l! pJT 8.0, fraction (~~OIldU~tilIl~~~ of 5.0 t0 7.0 mniho) fronl l.~l’:~~1F:-c~Clllll0Sc ; aJlt1 (4) the Jnoledar weight. (3r.W.) of the homcw~~totropic antihod?- \viIs t~stiIlJattv1 to he 240,000 ; the it\x~r:rge elution volunJc;‘\oitl \olunlc~ ratios ( V,/‘V,) for the four Jnarker proteiJJs, hllJllilJl SWUJI~ albumin (II.)\-. io.oC)o). lJovi;ie hcaJ’t ladic tlchyclrcqcwasc (lI.jv. 140,000), hUJllilJ1 gaIIlllla glohnlin (JI.j\-. 160,000), iIIlt1 hovinc~ liver glUtaIJli(: clt~ll~~lrogcJlasc (hL1Y. 250,000jF wc’rc 1.80, 153, 1.40, aJid 1.24, rcspcctivcly, aJJc1 tlJc%;iverage \TC,‘Y,, ratio for i he ;ltJti-BQA hoJJJwytotrol)ic antibod\- was 1.26. ‘1’hC rcslJlts of 1SsperiJnc~JJt, :3 cl~~JiioJJstr;Jtc the ilc~~~uisiti0Jlof a gJTilttlr dcgrt!c of iJJJmuJlologic cwmlwtcnw IJy tllc i-cl;Jy-old J*;Jhbit (Table TI ). Although the rill)hits ill (:roiip 3A did not I)rcIclucc aIiti-BSA hoJlioi~~t.otro~Jict antihocly 1,)
iid id!.;
l)iIy
6:3,
;I
Sigllifivallt
IlUIllb~T
Of
thW(’
IXbbitS
~1rW~llWd
tlt’tcc!table
(!!,
to
‘,{;4)
aJJti~Jocl~14 clays followiii,v ~liallt~iigc with 10 nig. of I)ay 77. IIowc~vcr~ the rabbits iii (;roup 3A (lid not protlucc anti-B% nJJt.ilJocIy that \vas tlctcctcd 1)~. the XBCY ant1 PHX tcchniqucs. In coJitrast. to (:ronp 3A, (:roul)s 313 and 31 procln~~l both hoJlJo~!-tot.rol)ic aJJtibody aJJt1aJJtihotl~~ tlrtwtcvl 1)~ the ABC aJJ(l I’IT.4 tcchniqws. ‘1%~ homologous I’CX titers iJi both (;roups 313 antl 3’ r;Jngtd from ‘,Y;to & 011 I)iI\- 28 to $4 to !Gys 011 1)ily 9 I. I’h!-sic~ocheJJJi~~l1charnc.tcri;l,7tioti of the! lioJJ~oc~tt~tropic antihood> ~~J*oclucwlby t.lJc rabbits in 1Ssprrinit~Jit 3 ww: i~lt~lltiCill to tliosc clcsorihed ill Esl)criJIJeJlt. 2. The ~\BC ant1 PHX ac+iviticls in th stw of thv rabbits in Croups 3H ;rJJ(l XY wrt’ localiztvl I)riJJJaril>. iJl the O.OlOJI ant1 0.0’1.5JI Sa(.‘l fJwtioJJs antI to it lesser cl~~grcciJl the 0.2031 I\;a(.‘I fraction t~lutecl t’r*o~n 1~l5~~1‘-ccll~lost~ ha\ing coIlc~ll~taJlWs of l.iO, 2.20, iJJJ(l 135 mmho, respectively. No anti-B% ,11W or I’HA activity could he (Icmonstratetl in thv 0.103I Na(‘l frwtions (5.0 to i.0 mmho) which containetl the homologous P(.‘il act.ivity. The dt’wts of neonatal csposurc to B8.4 with or without acliu\-aJJt up011 t,hc clP~~‘lO~~1llt~llt 0f systcniic iIlliJ~~Jl~lilSiS in tllc :1~11111 rill)l)it ilI’C SW11 in TiII)ll? 111. mti-HSA
;\l
hoIlJo~~totropic
011
306
Pinckard,
TABLE
II.
Experiments
Halonen,
Temporal 2 and
and
development 3
i
Meng
of
passive
cutaneous
anophyluctlc
ALLERGY
CLIN
actlvlty
IMMUNOL MAY 1971
IPCAI,
antlgen-
VOLUME NUMBER binding
49 5
capacity
(ABC),
Day PCA
i
l/IO !I/ 10 I,O.O05) Y/R O.OI)5)
Preferential
ond
possive
hemagglutinating
PCA
PHA
O/IO 3jlO
o/10 L’/ln
l/R
1/n
I/i
o/7
7;;
Tji ~0.01)5) L)/ti t 0.025 I
O/i i/i
2/i i/i
( 0.m) L'/ti
(
;I )
activity
Day 63 -1 ABC
28
ABC
expression
l/IO 9/10 ( 0.003) Y/S I, o.no1 )
) j
of
IgE
antibody
(PHA)
in
synthesis
the
rabbits
Day PHA
o/10 l/10
O/IO l/IO
1;s
1/n
n/7
O/T
7,‘;
i/7
(0.003) 4/ti ( <0.05)
t 0.003) I/ti I <0.05)
PCA
i
307
from
91
ABC
PHA
3/!-l $)/IO (
n/g 1,‘lO
o/9 l/10
l/S
2/s
O/i
n/7
0;;
i/i
i/i
, 0.005 I 4/G t
: 0.005) 4/c :
7/i
(ijlj
)
homologous YCA titers in these 36 rabbit.s ranged I’ronl 14 to $G, at the time of chlle11ge.
DISCUSSION
l’he present stutlics drnlonstriite that the administration of BSA to the neonatal rabbit can induce the preferential expression of anti-B% IgN homocytotropic antibody sylthcsis not 011ly during the neonatal period but throughout adult lift. Preftwntial espression of homocytotropic antibody was dependent upon the age of the neonatal rabbit and upon the adminisbration of adjutants. Injection of soluble BM wibhin the first 24 hours following birth resulted in the intluction of acquired immunologic unresponsiwncss to BSA, including IgE iillresl)onsirc~lirss ((iroup ‘LA). In contrast, the admiliistration of alum-prvvipitatctl HSA (Group 2H) or soluble BSA tog&her with C’. IM~VMUI (Croup 2C) in~lucetl anti-BSA antibody that ~oulcl be dctcvtctl 0111y by homologous PCIJ am1 11ot. by oithrr the T’IIA or ABC tcchniqurs. Tt should bc pointed out. that thv Iwgativity of the PHA ant1 ABC tcchniqucs does not ntwssarily intlicabe that TgF: homocytropic antibody can not he dctwtcd by PHA and especially by a primary binding assay such as the ABC. 011~ csplanation for this ncgativit? is that the mikimum quant.ity of homocytotropic antibody nrctle~l to induw cutaneous bluing may tw substantially less than the quantity ncetlecl to giw Imsitive PII, and ABC ksts. In ;iddition, WC ernployefl an initial 1/10 dilutiorl of s(‘l’il in the I’IIA ant1 ABC tests, whercas untlilute sera wcrc cmployecl for hoJ~wlo~ous PCA. One other possible explanation might be that the homocytutropic antibody coultl wnceivably be directed toward wntaminating proteins in our B&4 prcparat.icm. Such a prohlcm exists in any stutly in which solubles protein antigens arc employed and a highly scnsjtive biologic assay such as I~nJJ~ologo~~~ WA is used. ‘\Ve feel that such an explanation does not apply to our ~~resent stucl~- because the purit.y of the immunizing BSA antigen was grcatcr than !Kl per cent. Furthermow, the intravenous injection of as little as 500 pg of vrystallizecl BSA with a purity of 100 per writ induced positi\-e homologous PC;2 rrwdions, whcrcas 20 mg. of bovine gamma globulin (Cohn fradion 1 I), ;1
308
Pinckard,
Halonen,
ond Meng
i
AL1ERC.Y CLIN
IMMUNOL MAY 1972
VOLUME NUMBER
49 5
Preferential
expression
of
IgE
antibody
synthesis
309
spthesis of the other immunoglol)ulin classes of antibody could he expected to dtvr the character of immediate Il~vI)t!l’sensiti\it!- reactions. For ccwnple. the lack 01’ IgG “blwking” antibody coul~l pwmit small amounts ot’ ant.igen to inibiate li~I)crsciisiti\it.?reactions. The I~wscnt cspcrimrnts now I)rovitle a model \\hc*rc+y such alterations iI1 the trmporal dcwlopment of the qualitative ancI/‘or quaiititativc cxprrssioii of antibody synthesis (yin Ix stuclicil with rcspwt to cthanges in the clcvclopmcnt of imm~diatc hylwrsensitivity Statw Systemic anaphplasis in the raM)it is not commonly observd ; in those rcyorte~l illsti111(!Ps Of systemic anaphylasis, the wusc has heen awrihcd to t.hv illtravawular form;ition of ~lntigcn-antil)o(l\- wmplcsrs causing capillary emboli, ii (lwrrast~ in pulmonary arterial bloc-~1llow, and pcbriphcral c*iwulatory collal~2.‘” Ill ~0ntrast. \vc have ohscr\ecl a high frvqiit~ilcy of systrmic iitiapl~~lasis (‘I’i3l)le JTI ) in (:roups ‘LB ant1 2C ant1 3A! 3B. and 3(! when these rabbits were givcw 50 mg. of BSA intravenously on J);IJ, $11. The tlc\clqmwnt of systemic: >lll;lph~li~sis was associated otil~- with thcb l”‘escnce of tlctectahlc anti-B!+\ 1gE Ilonloc.?totroT)ic. antibody anal not with the prescnve of anti-BSA antibody as I)~~~MuY~ b\- the ABC or PIId twhniqut~s. IYtb co~~clucle from these <a thnt sJxtwic* anaphylasis in thv rahhit ciin he initiatctl l)y specific Ig% antibody in atlclition to immune w~nples~s. ,\t presrl1t the shock organ(s) ot’ the IgEmdiated systemic ana1)hylasis in the rahl)it arc not known ; howc~c~~~, preliminary c~\%lcnw in onr laboratory inclicrtcs that within 60 scwmtls following the intravtlnous injection of BSA into the unancsthctized rabbit a dramatic* fall in the III(~II adcrial 1~100~1 pressure from 100 to 110 111111. Hg t,o 30 to 50 11m. JTg owllrs illl~l persists during t.he period these animals eshihit. anaphylactic: symptoms. The Iilek of systemic: an;l1)hylactic sensitization in the rabbits from Espcrimcnt 1 is intrigiling. XI1 32 ol’ these rabbits hat1 clctcctablc anti-BSA homoc?-totropic antibody yet only 3 h;~tl aiia1bliylacG syni])tonis. Although as a group thcsc I’ilhbitS had lowr homologous I'(!i\ titers t hali tlic rabbits in Hsperimcwts 2 rlIl(l 3, 20 hat1 homologous WA titers of 1.1 or #water. Thcw tlata, tiikcll in VOII~~IWtion with the obswvations in Eslwrinients r! arici 8 that tlicre was no cwrrcl;it.ion h(bt\VWI~ the scvtlrity of the anaphylactiv symptoms ilIlt the homologons l’( ‘A titws, inclicatc that systemic anaphylasis in the raldd is clepcnclrnt U~II faders otllcr than the r+wdating Icvels of spwific TgE illltihd~. t’cd~aps the ~1nl0lll1t ali(l;‘or tlistribution of cell-11on11d 1pE antibocl>- woultl he bcttrr vritrria I’or ~n’tvlictin:: the incidence :ln(l scvcrity of systemic! an;ll)hylasis.
310
Pinckard,
Halonen,
and
Meng
:
A>:ERGf
CLIN
IMMUNOL MAY 197:,