Immun,~h~gl' I.etter~. 6 (1983) 25 27 Else,.ier Biomedical Pres~,

HAPTEN, CARRIER RECOGNITION A N D RESPONSE BY I M M U N O C Y T E S OF THE P R ! M IT! V E V E R T E B R A T E , , 'Vo toph thahm~s v i r idescens

Laurens N. RUBEN l)~wartmeptt tt/ Bioh~gl. Reed (ollege. t~ort/att~L OR 97202. I S..-t.

(Recei;ed 20 September 19b;2) (Accepted 5 No~embcr 19~2)

I. Summary

These experiments were designed to test whether newt (Notophtha/mus viridescens) immunocytes are able to recognize the hapten, 2,4,6-trinitrophenyl (TNP), separately from a heterologous erythrocyte (RBC) carrier. While a single priming injection with an erythrocyte species which is unrelated to the car- rier of the T N P fails to stimulate amplification of the ant i-TNP response, sequential (2×) priming pro- rides about 509[ as much amplification as a single priming injection of R BC homologous with the car- rier. The anti--carrier response is not increased bv sequential priming with the unrelated RBC. Moreover, sequential priming with R BC homolo- gous with the carrier species initiates a suppression of the anti-hapten response without affecting anti- carrier activity levels. Thus the recognition and regu- lation of responses to a hapten and its carrier may be unlinked in this species.

2. Introduction

Carrier-dependent and specific amplification of an anti-hapten (TNP) response has been reported for the newt, Notophthahnus viridescens [I]. However, immunocytes of this species display less of a capacity to discriminate among related and unrelated haptens than do those of more recently evolved amphibia [2]. Interpretations of their physiological behavior have been further complicated by the finding that

&ev n~n'dw hapten carri,.:r recogmtion urodelc amphibia

individual newt immunocytcs may not be phenotyp- ically restricted to the recognition of a single epitope [-3-5]. Nevertheless. data from experiments, in which injection order and content were varied [6], have seemed to require a phenomenological explanation of carrier-dependent and specific 'help' which is analogous to that offered initially from experiments with rodents [e.g. 7,8]. These data supported the view that heterogeneous populations of immuno- cytes do exist in these primitive vertebrates which in- teract in immunological responses inxolving TNP- derivatized heterologous crythrocytes. These populations are differentially affected by antigen dose, temperature [9] and by catccholamines [10]. While the amplifying population has not yet been directly related to a thymus-dependent subset in this species, other [11 ], using thymectomy in combina- tion with anti-thymocyte serum, ha~e established this connection in a considerably larger and even more primitive amphibian, ,4 mb.vstoma mexicanum (the Mexican Axolotl). Recently, it has been found that two populations of TNP-specific immunocytes can be recognized in the spleen of Notophthahnus. One subset responds to TNP where it is conjugated to erythrocytes or lipopolysaccharide (I, PS), while the other is stimulated by TNP-conjugates of linear polysaccharides, e.g. Ficoll [ 12] or dextran [ 13].

The experiments included in this report were designed to explore the question of whether newt immunocytes can recognize a hapten as an entity distinct from its carrier. The corollary question is also approached, namely can responses to a hapten and its carrier be regulated independently of one another'?

0165 2478 83 0000 0000 $3.00 © 19~3 Eb, e,.icr Science Publishers 25

3. M a t e r i a l s a n d m e t h o d s

3. I..,tnimaZ~

l h e 175 adult newts used in these experiments were purchased from Connecticut Valley Biology Supply (Southampton, MA). The.,,' were not a genet- ically homogeneous population. The animals were maintained in dechlorinated water at 23 ~ ('. l-hey were maximally fed with beef liver t~vice a ~aeek.

3.2. Immtmi_-aliott All injections were 100 ~ul in volume and wcre

made intraperitoneally. All TNP-derivatized ervth- rocyte dosages were 20Ci. while all priming injec- tions of erythrocytes were 0.005C4. Priming injec- tions were made two days before challenge with the hapten. These conditions are optimal for demon- strating carrier-primed 'help' in this species using I IRBC and SRBC carriers [9]. When combinations were injected at the same time, the dosages were adjusted to a total injection volume of 100 ~1. lhe conjugation of I N P to the erythrocytes was achieved using previously published protocols [14].

3.3• Assar While newt immunocytes do not secrete sufficient

titers of ant ibody to use plaque-forming cell assays, the amount of IgM they do secrete allows for the use of immunocytoadherence to monitor immune responses with erythrocyte immunogens. Details of these protocols have been published elsewhere

[e.g. 4 ]. An t i - IN P-specific immunoc\ tes ,acre deter- mined bycompeti t i , ,c binding using 10 ~M I N P - glycinc. The nnmbcr of antigen-binding cells blocked lrom forming b\ I N P-glycine ,xas used as a mea- sure of the anti-hapten response. Those which ,acre not susceptible to I-N P-glvcine blocking x~ ere i udgcd to be carrier, rather than hapten-specific, lhc spleens +~ere remoxcd from nev+ts 8 da',s after I NP challenge+ the time prex iously determined to be ~ lib- in the very narrow peak of the anti-haptcn antigen- binding cell activity [9]. ( 'ells dissociated trom 3 or 4 spleens must be pooled in order to prmide popula- tions large enough to reduce sarnpling error. Spleen cell counts of 1.5 5 ;. 10 +̀ ml '+~crc used..<,ix-thou- sand cells ,acre sur,,eved for each data point. [-ach antigen binding cell (AB( ' ) cotitlted xv;.is thc equi\ a- lent of 200 A l l ( " I0 '~ spleen cells. Five trials e l each of the 5 condit ions tested v+ere run and the data were expressed as ABC 10 ~ spleen cells. S.E.M.s ha~c also been inchided.

4. R e s u l t s

1he data in l a b l c I show that a n t i - lN P-specilic antigen-binding cells will appear in the newt spleen as a consequence of priming with R BC homologous ~ith the carrier, i )ouble priming with RBC heterol- ogous ~ith the carrier will. however, stimulate about (me-half as much ant i -TNP activity as a single prim- ing w, it h homologous R B('. Anti-carrier A BC lexels

l a b l c I

l h c t l n l i n k l l l g t)l t+laptcil i l l ld Cdl l lL ' r lCO ig l l l t i t ) l l In t i l t_' nc+~ t

h + m n u n i , , a t i o n , d l u d u l c I \ P - A B ( I(#' I I R B ( - . \ B ( I(I '

, ,p lo :n eel1,," , p i t c h L't_'11", I+

• 1X I ' - I tR I+ IO 0 • 0 2~09 • I 75

% R I l ( c. I N I ~ - I I R B C 0 • () 3011 • 210

I I R B C + I N P - t l I 4 B ( f , "3 t l - <<+C:4 51ot', • 397

% R I t ( ' , N R B ( . I \ I > - t I R B ( ' 2911 . 623: 3k56 - 5,15

I I R B C . t t R B ( . I N P - I t R B ( 11)40 . t++03 3680 • 1122 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

., l h c i+ltuTlhur o i IO ~ \'1 1 g l ' - g l ) . u i n c ulhil++ulablv" \ i t ( ' .

l h e nut 'nhu'r t)l , \FI ( • t c m a m m g al t t : r I Nl>-gl+,.cm¢ c t m l p c t i t l o n .

< 20++ R B ( " m ,\l,,,.:,.cr'>, sol td iOlL

,I (j 005+ ; R B C in ..\l,.c'+ci',, , , t )h l t i t ) I1

c N 5 (tl+i;+ll ++. 3 4 ~iIlltl;a]~. l o t e a c h datLl poII)[~ ~,|',%',I,% pr't)X-ldC X. dlld[l()l) ~II+11011~ II'ILII%+

/' • o.o5).

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are not improved by the double priming procedure, however. On the other hand. double priming with homologous R BC reduces optimal TN P-ABC levels without affecting carrier-specific ABC levels. The variation in HRBC-ABC levels is so great in this particular group of tests that there is overlap of the relevant S.E.M.s. Non-overlapping data are separa- ble at the 5(,/- level of confidence. Only' priming injec- tions of H R BC raise the anti-carrier response above the level provided bv challenge with i N P - H R BC alone.

5. Discussion

l h e data suggest that newt immunocytes may be able to recognize and respond to a hapten indepen- dently of its carrier. Double priming initiated either amplification or suppression of the anti-TNP re- sponse. The predominant regulator>' activity was de- termined by the selection of priming erythrocytes which were either heterologous or homologous with the carrier species. The important aspect of these re- suits, with respect to the initial questions posed in this report, is that the anti-carrier responses were not similarly effected. The anti-hapten and carrier re- sponses may be regulated independently in this prim- itive species.

The mechanism of amplification of only the anti- TNP response by sequential priming doses of hete- rologous erythrocytes has not yet been explored. Homologous carrier-specific suppression of an anti- hapten response has been described by others [15] and has been reported previously for long-term hapten memory responses in the newt [16].

Acknowledgements

The author is grateful for support of this research (Gnmt Ai-12846) from the National Institutes of ttealth, Bethesda, MD. U.S.A., to Dr. Wesley Bul- lock of the Oregon Health Sciences University for his suggestions for approaching this issue, and to Judith Ruben for her excellent technical assistance.

References

[I] Ruben, 1.. N . ',an der Ho'.en. A. and l)utton. R. \\'. (1973) ( ' e l l lmmuno l . 6.300 3]4.

121 Edwards. B. I-. and Ruben. 1.. N. { 1976) in: I he Ph~ Iogen.~ of I and B ( 'el ls(Wright. R. K. and Cooper. [!. I . Eds.) pp. 153 159. North- l lo l land Biomedical Pres,;. Amsterdam.

[3] Ruben, l . .N andSelkcr. F. l l . I I 975 )Ad ' . . l ! xp . Meal Biol. 84,387 395.

[4] Ruhen. l , .N and Edv, ards. B.F (1977) Cell lmmunol. 33. 437 442.

[51 Ruben+ I.. N. and t:dwards. B. 1- [19781 l)c,. ( ' o m p hn- immol. 2, 175 180.

[ 6] Ruben. I,. N. and F!d',vards. B. F. [197(-,) m: I hc Ph.',logeny of 1 and B ('ells <Wright, R. K. and Cooper. F. 1., [!ds.) pp. 161 168, North-Holland Biomedical Press. Amsterdatn.

[7] I)a,.ics. A. ,1. S., I.euchars, S. F.. Wallis. V., Marchant, R. and l!lliott, t!. V. 119071 Iransplantation 5,222 231.

Jg] | 'atkolf, R and Kettman, .I. (19721 .I. h'nmunt+l, l(lg. 54 5~,. [91 Rub,,:n, I.. N. II9751 Am. Zool. 15.93 106

[101 ttodgson. R.M..Clothier . R. It+,Ruben. I . N . and Balls, M.. Fur..I. Iramunol. 8,348 351.

I IJ l ahan . A. M. and .lurd, R. I). (19X I) l)e',. ( 'omp. Immu- nol. 5.85 95

12] Ruben, I . N and Rcischel, (;. E (19Xl) l)e',. ( 'omp. hn- munol. 5. 513 51~.

13] Ruben, 1. N. and Nt;ick..1. (1982) [)c~. ( 'omp. Imnmnol 6, 491 498.

14] Rittenberg. M. B. and Pratt, K. I . (1969) lhoc. Soc. l!xp. Biol. Med. 132,575 581.

15] tler/enberg. [.. A.. lokuhisa. 1. and tler/enberg. I . A. [1981)) Nature ( l ,ondon) 285. 664 666.

16] Ruben. I . N. (19;~2) Immunologx. m press.

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