Imt~tt~i~,l,,~.,~ I.cn,'~,. 6 t 1 9 ~ x ~ 92

E I,e',~er B~omedlcal P r e , ,

I M M U N O C Y T O C H E M I C A L L O C A L I Z A T I O N O F A N T I B O D Y - P R O D U C I N G C E L L S

I N A D U L T L A M P R E Y

Michael HAGEN, Michael F. FILOSA*. and John H. YOUSON D~7'ttrlmt~t! ,'1 Z..~,I,.L,I a~t(I A'~a,.I,.,~,,tteh ( . , / / t t '~ [ t m , ' , , z t , ~,t l~..r,.nt,.. U c,! Ht/I (htl I11( I 44 ( , t na , l,t

IRece~:ed 14 N o ~ e m b e r 19x2~

I Accepted "2 Nc, , .ember 19,',,2 ~

I. Summar)

Antibod.~-producing cells were localized in the fat column of adult lamprey prex iously immunized with "O'RBC. These cells ~ere ~isualized using rabbit an- ti-lamprey antibody antiserum in the peroxidase an- ti-peroxidase I PAP) immunoc.~tochemical tech- nique. Specificit) of the rabbit anti-lampre.~ antibod 3 antiserum ~xas determined by absorbing the anti.,,erum with 'O'RBC coated ~ith a xanet.v of antibod3- and non-antibod)-containing serum frac- tion~.

2. Introduction

As a lixing representative of the most primitixe group of xertebrates, the agnathans, the lampre.~ has been considered an important link in the stud) of the ph.~ Iogenetic de~ elopment of immunit3 [ 1.2]. Previous intestigations have demonstrated lampre.~ ,mmunocompetence to a xariet.~ of particulate anti- gens [I.3-7] and ha~e characterized lamprey anti- bodies [I,5,8].

Lampre.~ lack discrete lymphoid organb but ~hen "O" cells are used as an antigen, the response has been shown to be induced and specific [5,9]. A se- condar3 response has also been demonstrated [6,9].

Lampre3 do posses~ I.~ mphoid cell aggregations along the intestinal submuco~a, underlying the pha- ryngeal epithelium, and dispersed amongst haema- topoietic ussues [10-16]. Lymphoid cells haxe been described in the haematopoieuc tissue of the larxal lampre3 lammocoete) t)phlo.,,ole, a splenic homo- Iogue, and the anterior nephrtc fold [17 19]. With the inxolution of these latter structure~ during me- tamorphosis, the major haematopoietic organ of the adult lampre.~ becomes the fat column [14,18-20]. .Although a proliferation of mononuclear cell~ ha~ prexiously been observed ~ithin the fat column in response to antigenic stimulation, plasma cell~ ha~e not been identified in this tissue [2,3. I I ].

Recentl3. cells described as plasma cells ~olely on the basis of morphological criteria haxe been report- ed in the typhlosole and anterior kidney of non-ma- munized ammocoetes [21,22], and in the endost)le [23] and intest,ne [24] of transforming individuals. During the preparation of this manuscript, anti- bod3-prodt,cing cells haxe been functionallx idenu- fled by an immunoc.~tochemical method in the t3ph- Iosole of immunized ammocoetes [25].

In this paper, rabbit antiserum against lampre.~ antibod.~ (RALA) ~as used in the PAP technique to localize the antibod.~-producing cells of the lamprey. We pre~ent e\idence that ~uch cells are in the fat col- umn of immunized adult lampre).

h, ' , ,~,.rd, lampre.', anl lbod3-produ¢ing. ¢ell~

iI~ii nlU no¢.~ tocheml~tr.,,

* To ",~ horn ¢orrespLmdence should be addre.,.,ed.

fat c o l u m n

I~)(~) Ol~lO ,";3 01100 O(I(H~ $3 (10 ~ 19,x3 El~e',ter ";,clence Publl,hCr',

3. Materials and methods

3. I..4nimal.~ Aduh lampre.~. Petromr-on marinuL were caught

87

in dip net,, during their upstream spa~ning migra- tion in the Ht, mber Rkcr, Ontario, and ~cre main- rained in ~ell-aerated aquaria ,~upphed ~lth a con- tinuous Ilo~ of dechlormated ~ater at I "~ : C.

3.2. hnmumzation and hh , , d , olle, non

Human red blood cells, t)pe O. rhesus po~itke I 'O'RBCI, ~ere washed and suspended to 50r7 in 0.15 M NaCI. Lampre~ receked pericardial and in- tramuscular miection~ of 0. I ml "O'R BC ~eekl\. All animals ~ere bled ~la cardiac puncture, and ,ert.m ~a_,, obtained b~ allotting the blood to clot prior to centrifugation. Anti-'O'R BC titre~ ~ere detected one week after the iniual in ection, and reached 2 II b,~ ~eek 7.

3.3. I~o&tion ol the htmprel' antibodl Lamprey serum fractions containing haemaggluu-

nating activih ~ere isolated to be used for absorp- tion in the immunoc.~tochemical localization ol the anubod~-producing cells, and for the production of R hLA antisera.

Haemagglutinating serum fractions ,aere obtained b.~ electrophoretic separation of immune lampre.~ se- rum on I% Noble agar (Difco Labs, Detroit, Mll or 1'7 agarose ( Biorad Labs, Richmond, CAI gels 1200 V 1.5 hi in Laurell',, barbital buffer [26]. Serum ~as also separated using tubular polyaco lamide disc gels (PAGE){ 125 V, 70 mini following the method,s ol Pollara et al. [5]. All gels ~ere cut into 0.5 cm discs for elution of the haemagglutinating activitx using 0.02 M Tris. 0.15 M NaCI, pH 8.0 [8]. Hae- magglutinating activ~t~ corresponded ~ith Cooma- sie-blt.e .,,rained protein bands immediately anodal to the origin on agarose, immediatel.~ cathodal to the origin using Noble agar, and at the top of pol.~acD I- amide gels I R, 0). Lampre~ antibod~ ~as also ob- tained through ion exchange and affinit.~ chromatog- raphy, Serum ~as passed through Cibacron-blue Sepharose-4B, to remoxe a major serum cornponent [28]. Haemagglutinating activity was found in the non-bound protein fraction. This fraction ~a~ sub- sequentb separated on a column of DEAE-Sephace[ (Pharmacia. Uppsala. S~eden) u~ing a starting buffer of 0.05 M Tris, pH 8.0. and actkit'~ was de- tected in the lirst protein peak obtained after appli- cation of a linear Tris-NaCI gradient (final gradient buffer 0.05 M Tri~. 0.5 M NaCI, pH 8.01.

~8

We also attempted to obtain a single protein frac- tion demonstrating haemagglutinating actk it5 using DEAE and CM cellulose [4]. A final method used in the isolation of the lampre~ antibodx invoked pays- ing immune lampre.~ serum through a Con A-sepha- ro~e column. Acti~ih ~a,, detected in the bound protein fraction eluted v,~th 0.01 M Tr~s 0.5 M NaCI, pH 7.5, containing 0.2 M a-meth~ Imanno~ide.

3.4. PreparatttJn olrahhit anti-/amprel' antdv,t/i tm- li~eru

New Zealand ~hite rabbit,, ~ere immunized ~ittt different haemagglutinatmg preparations to produce RALA antiserum. Initial injections ~ere adminis- tered both ,,ubcutaneousl.~ and intradermally ~ith Freund's complete adju~ant. Subsequent rejections ~cre administered subcutaneousl,, ~ithout adju~ant at monthb inter~als. Rabbits ~ere bled ",-10 days atter recei~ ing an injection. The follo~ ing proce- dure,~ were used to produce an antiserum.

.lleHu>d.4. The antibody v, as absorbed from mmlune lamprey ~erum onto "O'RBC and the ag- ghninated ceils were x~ashed and injected.

Jh,tta,d B. The antibod,, obtained after the se- quential separation of immune lamprey serum using Sepharo~¢-blue, DFAE cellulose and PAGE. ~as absorbed onto "O'R BC, and the agghltinatcd cells ~erc ~ashcd and rejected.

JleHu~d C The haemagglutinating fraction ob- tained after sequential electrophoresis of immune lampre.~ serum on agarose gels and pol~acrylamide gels ~as injected.

]-he RALA antisera ~ere characterized in t~o ~a~ : {ll by determining the ability of the antisera to block the haenmgglutinating actix it.~ of immune lamprey sera directed against "O'R BC: and (2) bx immunoelectrophoresis. Blocking experiment~ ~ere performed at room temperature using a slide assay. Rabbit antisera ~cre repeatedl.~ absorbed ~ith ~ashed and packed "O'RBC until they sho~ed no agghnination o f 'O 'RBC. Immune lampre.~ ,;era ~ere then seriall.~ diluted and titred using a 2ei "O'RBC suspension. Blocking ~as measured b~ adding equal

olumes of pre~ iousl.~ utrcd lampre~ serum to R a~- LA antiserum and incubating for I0 min prior to retitration. Immtmoelectrophoresis ~as peHormed as described b~ Wlliams and Grabar [29] u,,ing bar- bital buffer [26].

3.5. ImmunohLslo( hemcu/ te~ ]miqtw Tissue~ examined in this stud.~ included the fat col-

umn, gills, kidne3, liver and anterior intestine of aduh lamprey. Samples ~ere fixed in 4% phosphate- buffered formaldehyde (pH "/.4). Paraffin sections were cut at 5 #m.

Cell,-, containing the lampre} antibod3 were idenu- fled using the PAP immunoc.~tochenfical technique [30]..All section~ x~ere pre-treated ~i th 3c~ - normal goat serum (NGS) for 30 rain at room temperature to reduce non-specific background staining. Thex ~ere then incubated with dilutions of R A L A antise- rum (I 50 to I 10,000, in PBS ~ith IC~ NGS) for 24 h at 4 :C , then ~ashed with PBS IC~ NGS. A I 10 dilution of goat anti-rabbit IgG (Cappel) ~as then applied for 30 rain at room temperature. .After ~ashing ~ith P B S - I % NGS, diluted rabbit PAP ( I 50) (Miles-Yeda) wa~ applied for 30 min. Diami- nobenzidine was used to x isualize sites of peroxidase activity [30]. Some sections ~ere also counterstained using neutral red and haematoxylin. The xarious controls for the staining procedure are detailed in the results.

4. Results and discussion

T ~ o separate methods ~ere used to test the purit.~

of the haemagglutinating fractions: PAGE sho~ed that none of the separation technique~ described aboxe .~ielded a pure fraction, and ,mmunoelectro- phoretic anabs is using rabbit a nti-x~ hole [ampre.~ ~e- rt.m re~ealed more than one arc for all of the aboxe ant ibods-containing preparat,ons.

Antisera produced b.~ methods A. B and C pro- duced more than one precip,tin arc ~hen u,,cd in mm~unoelectrophoresis against immune and non- immune lampre~ sera. In the case of anti ,era pro- duced b} method~ A and B, thi~ ~ould indtcate that other serum proteins in addition to the antibodx had been absorbed bx the "O'RBC..Although other in- vestigator~ [ 1,8] reported that their anti-lampre.~ immunoglobulin antisera ga~e onl) one precipitin arc after immunoe[ectrophoresis against immuno- globulin-containing fractions. ~e haxe found multi- ple arcs for fractions similarly prepared and assayed (unpublished resultsl. The subunits of lamprey an- tibodie~ are kno~n to be non-co~alentl~ linked and to dissociate spontaneousb so that it i~ possible that some of the precipitin arcs obserxed are due to dis- sociated subunits [8.31].

.All 3 rabbit antisera produced x~ere capable of significantb inhibiting the agglutination reaction be- t~een "O'RBC and immtme lamprey serum, indicat- ing that the~e antisera contained ant ibody against lamprey antibody. Ahhough total inhib.tlon xsas not

Table I

Treatrnenl o1" prlmar'~ anhserum u~ed irl P A P s[alnlrl~

c l p

T.~pe ol treatment of R A L A antiserum

i"11

Titre of i m m u n e

lampre} s e rum

prior to addtlLon

of R &LA an t i se rum

131

Tllre ol llllmUn~2

ldrllpre~ ~ertml

ariel Ltddlllon

ol R A L ~ amlserum

treated a~ in

coJtln111 I 11

(41

[-at co lumn

.,4alnin~ ~Allh

treated r ~ L A

antlnerum I rom

~.'Olllllln I ] I

A R 4 L 4 a n t l , e r u n t , ab, ,orbed i~.lth "O'R BC

R 4 L 4 a t l l h e r u m ah~ot 'he, I u Iitl " O ' R B ( , , ,at~ d ~, i111

B acu ' , e t ract ion I rom Noble aga r

C non-acu ' , e I r acuon I rom Noble aga r

D. a c u t e fractJon f rom Sepharc,~e-blue

E non-aclP, e IraCtlOn l r o m Sepharo.-,e-blue

F acu ' . e I r acuon f rom D E A E cellulo,e

(i act ive Iractlc, n I r o m C on A-'gepharo.,e

H ~mmune lampre~ , e r u m

I n o n - m m m n e lampre.,. , e r u m

4096

4096

4(196

N D "

N D

I 2iklS

N D

I 4096

I .-1096

12~

1024

256

N D

N D

I 204,",

N D

I 4096

I 512

'b N [-) = nc, t done.

89

achieved, titres of ,;era from immunized lampre.~ could be reduced from 2 I~ to 22 ~ith the RALA an- ti,era. All 3 ol the a~ailable antisera were initiall~ emplo3ed in the mmmnoc.~tochemical localization of the ant ibody-producing cells. Of the 5 tissue~ exam- ined. the R ~LA antb, era reacted only ++ith cells m the fat colt, mn. ]-he fat colutnn is a rod of fattx- haematopoietic tis,~ue 13ing dorsal to the spinal col- umn [32]. It includes actixely differentiating blood cells of 1> mpho~d, mxeloid and erythroid cell line-

ages [12,14.19], and pigment cells. It appears to be poorb ',ascularized, but it doe.,, ha~e an abundance ol mature blond cells. The presence ol cells belong- mg to I.,,mphoid and m~eloid cell lineages, slow xas- cular drainage [33], and e~idence of antigen accumu- lation [3]. ~uggest that the fat coh, mn i~ a potential site for ant ibod) production in lampre).

Although all three RALA antisera stained the ,ame ceil-t) pe in the lampre.,, fat column, the antise- rum produced m response to injections o f ' O ' R B C

~r ~ ; ~ ' ~ ~,

®

?

~6

(1)

L

f

i

4-~== "

Fig , I 6 Xn t lbod) -co r l td lnu l~ cell'=, h )cahzed in the l a m p i r ) lat c ,~lumn R XL .~ a n t l , e r u m ".'.a- u , ed m the P. - \P t echn ique . Fizz, I 5 the

dnt l ,drLinl ~.~,1, t l ,cd a l [ 2()1)1] dlhl l lOl l , d 1"5(){20 ddut ton x~a, u,ed in Fig. h Fit.: I [v,o po.qtl~.el'., , tamed cel l , ( - ; and n u m e r o u , p lgnlenl

cel l , I PI are ~Nb le i41(P. ) Fig 2 an o~M. po,l t i~el~ , tamed tri l l i - I : ,ecuon a l ,o , tamed ~ l t h haematox~.lin and neutra l red Erec D~ment

g . r a m d e ~ , a r d a N o , e e n ( P G ; I¢ ;J( ; . I FJg, 3 a n d 4 R .M &-reaell~ccell, ha~mgt.xptcal r o u n d - ~ h a p e t - I , no ted t l l c r en t nuclear t oc ! , t op la , -

talc ra t io , 19"1) • I Fl~ 5 d po~ltlXel.~ Malned cell I ~11 , .~een adlacem t~) d p lgnlent cell I PI I '~( ) - I Fig 6 a ~Jnglc po~.ltl'~el) ~talncd cell q ~1

h ~ m~ ,m elongated ,.hape (¢;'11 •

90

coated with ~hole immune lampre) s e rum(Method A I ~a~ used predominantly in the immunoc.~to- chemical localization of the haemagglutinin-produc- ing cells. This antiserum ',~as chosen since it had the highest blocking capacit) of the RALA antisera test- ed. Good staining results were obtained u~ing a I 2000 or I 5000 dilution of the primary anuserurn. Since the antisera v, ere not monospecific, a ~ariet) of controls was used.

RALA antiserum v, as tested for its abilit,, to block the agglutination o f ' O ' R B C b.~ lampre) im- mune serum and to gi',e positi~el) stained cells in the fat column using the PAP technique. Removal of the antibodies against lamprey antibod~ from R A L A antiserum ~as accomplished b) absorption of the antiserum with "O'RBC coated ~ith haemag- glutinatmg fractions obtained Irom immune lampre~ serum b~ the ~arious ~eparation techniques. This procedure simultaneousb remo~ ed the staining and blocking capacities of the R A L A antiserum lTa- ble I B, D, F, G). Absorpt ion of R A L A antiserum v,~th "O'RBC pre~iously incubated ~i th the non- haemagglutinating protein fractions obtained from the separation procedures did not remo~e these properties of the R A L A antiserum 1Table I C, El. Absorpt ion of the RALA antiserum v, ith "O'RBC coated ~i th non-immune lampre) ~erum had no e[- fect on the staining or blocking abilit.~ of the antise- rum, ~ hereas absorption with immune serum-coated "O'RBC removed both the staining and blocking properties of the RALA antiserum {Table I H, IL Other controls included the use of pre-immune rab- bit serum or PBS in place of the pr imaD antiserum. In both cases, no reaction v, as observed.

The cells reacting with the R A L A antiserum were easib distinguished from surrounding cells and from pigment cells by their brov, n-gold colouration. I Figs. I and 2L The reaction product ~as restricted to the c.~toplasm of round I Figs. 3 and 4), and o',al [Fig. 2~ cells ha~ ing slightly eccentric nuclei. React- ing cells v, ere irregularly distributed throughout the fat column ( Fig. I ~ and although thex ~ere seen as- sociated with blood ~essels and large ',acuoles, the~ were ne',er present ~ithin these structures. Occa- ~ionall.~, PAP-posi t f fe cells ~ere closeb a~sociated to pigment cells ~ F~g. 51. A similiar association be- tween immunoglobulin-containing cells and pigment cells has been reported for the fish, Pleuronecte.~ l,la-

tes.~a [34]. The positively stained cells appeared pol- .~ morphic, ranged in size from 7 to 12 urn, and oc- ca~donall.~ were ~tellate v~ith Ie~ c.~toplasmic projection~ or v, ere fu,aform in ~hape I Figs. 2 and 6). The size of the nucleus and the amount of cstopla~m in the cells al~o ,,aried. Some cells had a large nu- cleus and scant~ c.~toplasm I Fig. 2p ~hile others pos- sessed a small nucleus and a moderate an~ount of c) toplasm I Figs. 4 and 61.

]-he cells that ~e ha~e localized bear a striking re- semblance to the ant ibods-containing cell> Iocahzed by Fujii in the ammocoete tsphlosole [25]. He de- scribed them as pla,ana cells belonging to t~o popu- lation~, haxing diameters o i 5 8 ~m and 12-15,um. Compar ing the micrograph~ published b5 Fu/ii ~ith our or, n, there appear,~ to be a notable difference in the numbers of antibod.~-containing cell,, in the am- mocoete typhlo~ole and the adult fat column. A,, neither Fujii% nor our ~tud~ in,,ol',ed a quantitatixe anaK ~is of the ant ibody-producing cells, further in- vestigation is required to indicate v, hether this diller- ence i~ real.

Applicat ion of the RAL & antiserum to the fat col- umn of non-immunized lampre.~ produced positi',e- staining resuhs in one of the 3 animal,, e \ ammed , but only, at a Iov, di[uuon of the primaL', antiserum (l 51)1)1. The cells that ,,tamed appeared identical to tho~e ob~er',ed in immunized lampre.~, but their fie- quenc.~ ~a.', much Io~er. The larger number of stained cells in the lat colt, ran of mmmmzed animals

max be related to the mcrea,~e in the cellular dens~D concomitant with immunization, an ob~er~auon pre~ JousT5 reported bv other m~estigator~ [2.3,14].

Ackno~'ledgements

This stud3 was supported b3 Grants A5945 and A99g I lrom the Natural Sciences and Engineering Research Council of Canada to J. H. Y. and M. F. F., re~pectixelv. The authors thank P. Sargent for her technical assistance.

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92