Allergen from Fasciola hepatica; IgE Antibody Production in Mice by Crude Antigen

Allergen from Fasciola hepatica; IgE Antibody Production in Mice by Crude Antigen

Setsuko TSUKIDATE and Koichiro FUJITA

月中artnヱentqr且鮎aicatZoot曙γ，

NagaSakiUniversltySch00lof脇dicine，

Ⅳaga∫a屋i，滋ゆan

Abstract: IgE antibody formation was easily demonstrated from the hosts infected with Fasciola hepatica. But, it was known to be difficult that the hosts immunized with antigen extracted from F. hepatica produced IgE antibody. IgE antibody, however, was produced relatively easily in the mice immunized with crude antigen of F. hepatica in our ex- periments. Namely, over 2.5mg of challenge antigen per rat was necessary for better reaction of PCA test and male Wistar strain of rat weighing 150g was better as a recipient animal. The best condition for the high titer of IgE antibody production was to immunize mice with 5 times stimulation of 10ƒÁ of antigen in 5 days, or with 2 times of 20ƒÁ at 20 days interval. Among adjuvants, aluminium hydroxide gel was best for the induction of IgE antibody in mice. ICR mice immunized with 20ƒÁ at 20 days interval produced over 26 titer of IgE antibody at least one week. The IgE antibody production was compared among four inbred mouse strains, two of their hybrids and six outbred strains. The inbred strains of DBA/2 and BALB/c and their hybrids, (C57BL/6•~DBA/2)F1 and (BALB/c•~DBA/2)F1 produced the highest titer of IgE antibody. On the other hand, outbred strains of ddY, ddN and ICR produced the low titer of IgE antibody.

INTRODUCTION

Parasites provide the mコSt pつtent Stimulus forimmediate type hypersensitivity．

Intradermaltestswereearlyusedtoinvestig蛹te Or t〇diagnose parasiticinfeetions．In

animalfascioliasis，theintradermaltesthasb三enuSed邑S animportant diagnostic tool．

Onoetali（1952〕studiedintradermaltestantigenextracted fromadultF．h坤aticaand

reportedthatitwasveryspecific forthecat［1e fascioliasisiTheantlgenWaS Called as

OnoAntlgen，andithasbヨ≡nuSedwidelyfoTanimalfascioliasisinourcountry．

Ontheotherhand，Shinoda（1974a〕studiedthe‡gE antibody production during the course of theinfection of the rabbits experimentallyinfected with F・h坤atica．

Accordingtohisobs巳rVation，therelativelyhightiterofIgEanLibodywas demonstrated

Received for publication，March4，1982．

Contribution No・256from the Department of MedicalZoology，NagasakiUniversity SchoolofMedicine．March4，1982−

in allinfectedrabbits fromthe5th week afterinfection，reaChed the maximumtiter on the8thweek，andmaintaineduntillthe14thweek・Shinoda（1974b〕alsoreported the

highdegreeofthepotentiatedIgEantibodyformationforbovines≡rumalbumininduced

by the Fih申aticainfection−In his studies，how・eVer，the rabbitsinjected antigen extracted from Fih申atica producedIgE antibody onlyinlow titer・The producing periodsweredetectedshortlyandtransiently；IgEantibodyofallrabbitswasterminated atthe3rdweekafterinfection− NopotentiationphenomenonofIgEantibodyforbovine s。rumalbuminwas。bservedamongtherabbitsinjectedcrudeantigenfromF・k坤atica・

Inthispaper，attemptSWeremadetodetermineandtocDnfirmvariousconditions forIgEantibodyproductionbycrudeantigenextractedfromadultF．h坤atica・

MATERIALS AND METHODS

Animals：C。nVenti。nalWistarrats。fbothsexesweighinglOOto300g were used

in this study．ConventionalDonryu strainofmaleratsweighing150g andinbred SD

rats。f b。th sexes weighing150gwerealsoused．Theserats were obtained from the

SankyoLab．Inc．，Shizuoka，Japan・

Male mice at the age of4to5weeks were us己d throughout the experiment。

OutbredmiceofddY，ddN，ICR，C3H／He，C57BL／6and DBA／2were obtainヨd from H。kurikuLab．Inc．，KanユZaWaandinbredmiceofC57BL／6（H−2b〕，BALB／c，DBA／2

（H−2d〕and C3H／He（H−2k〕，andtheir hybrids，（C57BL／6×DBA／2〕Fland（BALB／c xDBA／2〕Fl，Were Obtained from the Sユnkyo LabiInc−

crudeextra仁tOfF−h坤afica（CE〕：CEwasmade fromadultF・h坤ユticaobtained fromcattlebileductsin Kanazawaarea．Adultwormswerelyophilized，homogenized withglassandteflonhomDgenizers，disruptedbysonication（Insonator，200M・Kubota，

Tokyo〕at140Wattfor5minandextractedinphosphatebufferedsaline（PBS〕atpH7・2

for2daysinarefrigerator・The emulsionwascentrifugedat13，000×g for20min at OOC．The13，000×gsupernatantWaSdesignatedasCE・

Immunization：Mice wereimmunized2to8timeswith5to40γOf CE・Two

kindsofadjuvantswereusedintheimmunization；ICRmicewereimmunized with CE mixedwithkilledBordetellaPertuS5isorganismsoflOlOpermDuSeOraluminiumhydroxi−

de gel（alum〕of3mg per mouse・

passivecutaneousanaphylaxis（PCA〕test：PCAtestwas p巳rformed as described

（Fujita and Tsukidate，1981）− AvolumeofO・1mlofs已rialdiluted sera wasinjected

intradermallyintotheshavedbacksofrats・After48hr，theratswerechallengedwith

anintravenousinjectionoflmlof134EvansbluecontainingO・625to5mg of CE，and were killed30minlater．The diameters of the PCA reaction were measured on the

internalsurface ofth巳Skin．Areactiongreaterthan5mmwasdefinedaspositive・

Indirecthemagglutination（IHA〕test：IHAwasconducted as described for the

purificationofallergenfromDirqfilaγiaimmitiS（Fujita，1975〕t Sheepred blood cells

were treated with tannic acid（Merk〕atl：30，000for15minin a water bath at 370C and the tanned cells weres3nSitizedwithantigen（1：200diluted CE〕for15min at room temperature．TheIHA test was carried outin O．634normalrabbit serum on microtiter plates．The end point ofpositive reaction was determined as plus3defined byJacobs and Lunde（1957〕．

RESULTS

（1〕PCAγeaCtねitユノq′γeClpientγat∫．

（1−α〕J吋t㍑enCe qγcゐaiienge antなen ao∫e On f〕CAγea・Cti扇tγ．

Inordertoknow optimalconditions for PCA reaction，Challenge antigen dose was determined by uslng threeimmunized mice sera with high titer ofIgE antibody．As Shownin Tablel−a，0Ver2．5mg of challenge antlgen dose per rat was necessary for better reaction of PCA test，When male conventionalWistar strain of rat weighing150g was used asrecipient．Hereafter，PCA test was conducted with challenge antigen dose Of2．5mg per rat．

Tablel．PCA reactivlty Of recIPlent ratS

Tableニトa．Influence of challenge does on PCA reactivity

Challenge antigen does

（mg／rat＊〕

5 2．5 1．25

PCA titer（mean＋S．E．〕 25・2 ± 20・33＊ヰ 25・2 ± 20・呂3 24・2 ± 20・58

0．625

23・5 ± 2i・35

＊W■istar（conv．〕815Og．

＊＊Mean value with threeimmunized mice sera．

Tablel−b．PCA reactivity according to sex of the reciplent ratS

recIPlent rat

PCA titer（mean±S．E．〕 ^SeX

昌

早

Wistar（conv．〕    SD（BS．〕

23・8 ± 20・33＊      21・5 ± 21・0

238 ± 20■33      210 ± 21iO

＊Mean value with thTee Or fourimmunized mice sera．

Tablel−C．PCA reactivity according to strains ofthe recipient rats．

species of recipient rat

PCA titer（mean ±S．E．〕

Wistar（conv．a〕 SD（BS．8〕  Donryu（conv．占〕

243 ± 21・02＊     24・1± 21・09     250 ± 20■84

＊ Mean value with siximmunized mice sera．

（トみ）PCAγeaC加ゎaccⅣaingtotFe）rαn iageオt丘eγeC中正ntγati

Two kinds of rats，COnVentionalWistar strain andinbred SD strain，Ofboth sexes Were uSed as recipients for PCA testiAbout the same degree of reactivitywas obtained between sexesin each strain of rats，aS Shownin Tablel−b．About the same reactivity

WaSalsoobserved amongrecipientratsweighinglOOt・〇300gofm旦1e conventionalWistar

Strain，although the PCA titers of these rats were diversed alittle from24to25．From these results，itwas determined thatthe difference ofsexandage of recipient rat did notinfluence on the reactivlty Of PCA test．

（1−C〕eC』γeaCtねi抄accⅣaing to∫trai耶耳tゐeγeCi如entγat．

PCA reactivitywas compared amongWistar，SD and Donryustrain of male ratsi Aboutthe same degree of PCA reactivitywas obseTVedinthree strains of recipientrats as shownin Tablel−C，although PCA reactivity of Wistar was superior to that of SD ratin the experimentin Tablel−b．We concluded from above results thatPCAtestwas to be conductedwith male conventionalWistar strain ofrat weighing150g as recIPlent，

andchallengeantigendoseofCEwas2。5mgperrat．Thismethodwasemployedthrough−

Out．

（2〕hrEantibot＆Production ofICR miceimmuniEedwith CEi

（e−a）布μμenCe Oia〔む〟Vant∫OnなH antiみ0みpγ0血ction．

IgE antibodytiterinICR mice was compared between two kinds of adjuvants，B．

pertuSSiSandalum・Micewereimmunized with2times of25γOf CE for5days apart，

With5times oflOγ1n5days or with8times of6γ1n8days mixed with either of

adjuvants．PCA titerat14days af［er primaryantlgen Stimulation became higherin all

the miceimmunized with alum than the mice acc己Pted with B．pertu5Sis as adjuvant−

On the other hand，IHA titer was nlgherin the mice with B．pertuSSiS，aS Shownin^l■

Table 2．Hereafter，We uSed且1ilmaS adjuvant fDrIgE antibody productionin mice．

Table2．IgE antibody productioni王1ICR miceimmunized with CE

from F．h申atica mixed with B．pertusSiS Or aluminium

hydroxide gelas adjuvant

immunized with

reciprocalof

IHA titer PCA titer

25γ×2 昆諾孟＊

10γX5 昆1慧

6γ×8 昆1≡ふ

23・2 ± 20・47＊＊＊      20・4 ± 20・36 21・7 ± 20−24        2213 ± 20−71 24iO ± 20・44        22■8 ± 21・44 21・2 ± 20・13       23・9 ± 20・43 24▲2 ± 20．57        20・8 ± 20・33 21・2 ± 20i22       21・8 ± 20・42

＊Killed BrL＞detellapertusSiSOrganisms（1010／mouse〕．

＊＊Al（OH〕昌gel（3mg／mouse）．

＊＊＊Meanwith six or sevenimmunized mice seraof14days afterprlmary−

（e−ら〕なH ant拍0め∫ 夕γ0血ction accoγaing 紬tゐe tinヱe∫qr antigen∫tin7〟iation．

The production of PCA andIHA antibody was studiedin the miceimmuM nized with various times of antigen Stimulation of CE，With resultsinFig．1−

Three groups of mice wereimmunized with2times of 25γin5 days，With 5 times oflOγin5days or with8times of6γin8days．These mice were bled 14days afterprimaryantlgen Stimulation，

andPCAandIHAactivitywasdetermined．

PCA titer became highestin the mice accepted with5times oflOγ1n5days・

IHA titer was highestin the mice with 25γX2，butit did not change sD muCh accordingto the timesofantlgenStimula−

tlOn．

（e−C〕なH antiら0みprロムctio才7 aCCOγaing totゐeaαツqf一古口0∫ter 頑er pγのaり′

ヱn‡m〟nほaわOn

IgE antibody productionisknown to be produced effectively by the secondary antlgenStimulation，butthe detailIgEantibodyproductionincaseoftheimmunization

withparasiteantlg已nisnotclearly understood・Therefore，tWice repeated stimulations with5γOrlOγOrCEatvariousintervalswere glVenSixorsevenmice pergroup聾Mice w己re bled5d且ySafterboosterimmunization，andPCAactivity was assayediAs shown inFig・2，inthecaseof5γ×2immunization，tWicerepeatedstimulationsatlOdays or

20daysintervalproducedrelativelyhightiterofIgEantibodvinmice・Whereas，in the caseoflOγ×2，20daysintervalstimulatedIgEantibodyproductionstrongest・

（3〕nme co〟門e扉▲なH anti占0如pγロ血ction qfmicei

Time courseofPCAandIHAantibodyproductionwasexaminedwith fourgroups ofthemicewithtwicerep巳atedimmunizationof5γ，10γ，20γOr40γOf CE at20days

interval．The miceimmuni二led withprlmaryinjectionproducedIgE antibody transiently

at12daysafterinjection，andtheantibody activity disappearedrapidly・But，higher andlongerIgEantibodytiterswereobservedinthemiceacceptedwithboosterinJeCtion・

Among themiceimmunized，the mice with20γX20r40γX2showed relatively better

IgEantibodyproduction agalnStCE・Ontheotherhand，almostno or only poorIHA

antibodyproductionwasobservedinthesemiceonthiscondition，aS Shownin Fig・3−

（4〕なEantibodyproductionelicitedby CEin dl酔rent5trai；1SqFmice・

Preliminaryexperimentsconfirmedthataboosterimmunizationonday20gave a

丸山再∈THd HU︸叫可 Sh再p 寸一山口

再トむS 叫O hU■1︼ 叫O T巾UOトdlUむh

25

2〕−

21

mean ± SE

PCA

工H且

25 x 2   10 x 5   6 x 8 amount of antigen

＊ mean value with six or seven miceimmunize

Fig・1・PCA andIHA antibodytiterinmice

according to the times of antigen stimulation．

㍉U︸SOOq hUμ叫再 Sh巾p m 叫0

巾hUS 叫0 ︵．凹iS≠U巾む∈︶ hU︸T︸ 司Ud

2／ 巨＝∃

2⊥

23

22

21

転∃

pr土mary  十十  十十  ＋f alumb00Ster  −＋  一十  −＋

十十    十十   十十

−＋    −＋    −＋

Fig・2・IgEantibody productioninICR mice according to the day ofbooster after primaryimmuniEation．

40Y X 2 20Y X 2 ・10Y X 2 5Y：＜ 2

PCA

hU︸T︼ 叫O T巾UOJdTUUJ ♯

2β

26

2−

22

28

26

2斗

22

IHA

5  7  9 1215 18 25 27 33 35      5  7  9 12 15 18 25 27 33 35

†       †

booster booster daYS after primary antigen challenge

★ mean titer of five mice imrrLunized

Figi3・Time course of PCA andIHA antibody productionin theIC三三mice

Sequentialchallengedwith2times of CE for20daysinterval．

★       戸      l

‡￣ニ：−

5．tC57BL／′6 〉：DE泊／21F

6・（BAl月′ノc 〉て DBA／2）Fコ

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10 亡コ11ノHr。

11 c57BL′ノら

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h∂Pl⊂＝t｝Pモ nしOuSe

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recIPrOCalロf PCA tlter Of sera

ロF 5 days af亡e工、booste⊥

★ 打0．1一 打′〕i4：1nb王」ed stralTl

＝ Ⅳ0・5 −、J（つ 6：nlγbニ1d t★t Ⅳ0．7【†To lコ：ClロSe（】colロnン

★t★t mearl￣．γdlu（、and S E wlth foし1r Or 上1Ve

mlCe lmmun⊥Z（∃d

recユPrロC己10ご PCA 亡ユter elI与erヨ

○宣■14 d8）γS after pI■um8l＼

貴 L、Ol− ND．4：1nbred straln

t★ ゝ、n．う ー Nu．も：hybrl〔1

‥＝．ト0，7 − ND．12：Closed colony

★★鼻★ mean v∂1u（三 and S．E h・1ヒh four or flVe mlCe lITLmし】n12．ed

★★彙★★ all the mlCe de己d

Fig．4．PCA titers of different strains of mice immunized with20γof CEand boosted

at20days．

Fig．5，PCA titer of different strains of mice accepted with fifth repeated antigen stimulation oflOγOf CEin5daysi

betterIgEantibody responseinICRmice．Fourinbred mDuSe Strains，BALB／c，DBA／2

C57BL／6 and C3H／He，tWO Of their hybrids，（C57BL／6×DBA／2）Fland（BALB／cx

DBA／2〕Fl，and sixoutbred strains，ddY，ddN，ICR，C3H／He，C57BL／6and DBA／2，

wereimmunized with20γOf CE and boosted at20days．Their serum PCA titers were assayed at5days after bDDSterinj巳CtiDn．Among mice tested，1ittle differencesinIgE

production could be obs巳rVed，aS Shownin Fig．4．

Then，IgE antibDdyproductionwascDmPared amDngthe mice groups accepted

with fifth TePeated antlg・en S［immulation oflOγOf CEin5days・The mice s≡ra Were taken at9days afterlastimmuniEation，and PCA activitywasa∃Sユyed−In this case，

PCAresponsewasdifferentconspicuouslydep己ndentonmicestrains−TheiIlbredstrains ofDBA／2and BALB／c and their hybrids，（C57BL／6×DBA／2）Fland（BALB／cxDBA／

2〕Fl，PrOducedheigherPCAtit≡rS．Onthe otherhand，Outbred strains of ddY，ddN andICR producedlower PCA titers，aS Shownin Fig．5・

DISCUSSION

In order to get the purified allerg己n for di…唱nDSis or forinvestigation purpDSeS of fascioliasis，Preliminary experiments were conducted，and the conditions for better production ofIgE antibコdy were studiedin the mic三immunized with crude antigen

extracted from adult F．h坤atica．According tothe pユP己r Written by Shinoda（1974a），

IgE production was very poorandtransientlyinthehostsimmunizedwith F．h申atica Crudeantigen，WhereastheinfectionofF．h申aticainduced relativelyhightiterofIgE

antibody・The N軸oStrOngyluSbmSilienSiSinfectionalsoinducedhightiterofIgEanti−

bodyin the hosts，but theimmunizationwith N・braSilienSiS did notinduce anyIgE antibody（0gi1vie，1967；WilsonandBlock，1968）．Sumi（1971）reportedthattherabbits immunizedwithDirdilariaimmitisantigenmixedwithadjuvantproducedalmostnoIgE antibody，but whenlive D・immitiS WOrmS WeretranSPlantedinto pleuralcavity of rabbit，the host producedIgEantibodywithveryease．Thus，paraSiticinfectionwas Saidtobe necessary forIgEantibodyproduction（Mota，1964〕．Inthis study，however，

We demonstratedrelatively hightiter ofIgEantibodyinthe miceimmunizedwithcrude antigen of adultF．h申atica．

Certainly，it was alittle difficult for the miceimmuniz巳dwith parasite crude

antigen to produce theIgE antibody with hightiter andlonger period．But，if w・e

Selectedthe dose ofantigen，the timesandintervalof antigen stimulation，and mouse Strainproperly，We COuldinducemiceIgEantibDdyveryeasily・The system toinduce

IgE antibodyin mice by usingtheextracted parasite antigen was thoughto be very

usefulforthe furtherstudyofparasiteantlgen・

i

Andrewsand Meister（1978〕studiedthedifferencesin susceptibility toinfection WithF・h坤atica amongmouse strains・Mice ofthirtyone different strainswereintra−

peritoneallyinfected with24hr−01d flukes taken from the donor mice which had b己en

infectedorallywithapproximately500metacercaria．Themortalityratewasdetermined from day20to39afterinfection．Theinbred strains of mice showed a resistancel。Wer thanthat foundin outbrediAmonginbred strains，the mortality rate of AJmice was

lowest（4634〕，these ofAKR／A and C57BL／10were shown aslower values（59and 60％〕，these ofDBA／2and BALB／c werehigher（70and7334）and these of C3H／Tif and C57BL／6were highest（83and8434〕．The difference between mortality rates for inbredstrainsofAJmice and C57BL／6mice was high1y significant，althoughtheir

infection rates were almostlOO鳥′．However，interestlngly，these six cross−breeds of

Which one parentwas of BALB／c strain showedthe highest degree of resistance to the F・k申aticainfection，and their mortality rates were only17to3134i

Inthe presentstudy，theIgEantibody productionwascompared among different Strains of outbred，inbred and their hybrid miceimmunized with crude antigen from F．h申atica．However，nO relationship could￣be observed between the resistance to F．

hepaticainfection and theIgE antibody productionin these different strains of mice．

REFERENCES

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hePati亡a between mouse strains．Z．Parasitenkd．，56（3），305−30S・

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肝蛭のアレルゲン

系統間マウスの粗抗原に対するIgE抗体産生能とその産生条件 月舘説子・藤田紘一郎（長崎大学医学部医動物学教室〕

寄生蠕虫感染は宿主内に特異的なIgE抗体を誘導するばかりでなく,虫に無関係な不特定多 数の抗原に対するIgE抗体の産生をも増強(potentiation)する.しかし,蠕虫由来の抗原で免 疫すると,これらのIgE抗体の産生は難しいとされてきた.われわれは肝蛭のアレルゲンの性 質を知るために肝蛭粗抗原の免疫活性を調べたが,適当な条件下で粗抗原で免疫すると,マウス は比較的高タイターのIgE抗体を産生した.すなわち,PCA反応の条件としては,ウイスタ ー系雄性(150g)のラットを用い,チャレンジ抗原量は2.5mg以上とすること,またIgE抗体 産生にはマウスを粗抗原10γで5日間5回免疫する方法か,20γを20日間隔で2回免疫する方法 が良いこと,アジュバントとしては水酸化アルミニウムゲルが優れていることなどが明らかにさ れた.ICRマウスを20γ,20日間隔2回免疫すると,26以上のIgE抗体価を少なくとも1週間

以上持続した.また,inbred strain 4種,hybrids 2種,closed colony 6種のマウスについ て,IgE抗体産生能を比較検討した.inbredではDBA/2系が最もよく反応し,次いでBALB/c 系の順であった.hybridsのなかでは,このC57BL/6とDBA/2とのF1マウスが最もよく反応

し,両親より高いIgE抗体を産出した.しかしclosed colonyのdd系やICR系のマウスの IgE抗体産生能は低かった.なお,このIgE抗体産生能と肝蛭の感染に対するマウスの抵抗性

との間には,何ら有意な関係は見出せなかった.

熱帯医学 第24巻 第1号, 27‑36頁, 1982年3月