thymus

Prolactin and early T-cell development in b " h'k em ryonlc c lC en

Javier Moreno, Angeles Vicente, Ingmar Heijnen and Agustin G. Zapata

Currenth', the information available on the ontogeny of the immune- neuroendocrme network is somewhat scant. However, increasing data suggest a role for prolactin, an important immunoregulatory molecule that resembles O,tokines, in early T-cell development. In this article, Javier

Moreno and colleagues present evidence for this role using the chicken embryo as a model system.

There is increasing evidence concerning the immuno- modulatory influem.e of the pituitary gland hormone prolactin (PRL) on the adult immune system ua. PRL has effects both on humoral and cell-mediated immunity, presumably through specific PRL receptors (PRL-Rs) expressed on various t3.'pes of leucocytes u3, as well as by the production of hormone by the lymphohaemato- poietic tissues themselves 4-~.

Several observations suggest that PRL might also act early in thvmic embryonic development, influencing the ontogeny of the immune-neuroendocrine network. Specificalb; thymus involution, classically reported in the hypopituitary dwarf Snell-Bagg and ~.mes mouse strains --~, recovers after PRL administr:_:ion I°'l~. Fur- thermore, neonatal treatment of mice with anti-PRL antiserum or bromocryptine, a dopamine agonist that inhibits release of PRL from the anterior pituitary, affects the development of T cells in the thymus and spleen t-'. Unfortunateb; the obvious difficulties associ- ated with in vivo manipulation of mammalian fetuses have left many questions unresolved.

As a model system, the chicken embryo allows easy and precise access during early ontogeny. Indeed, the widespread use of this approach to study early events in the development of the lymphohaematopoietic sys- tem has shown that avian T-cel! de,'e!opment is very similar to that of mammals I;.la. Furthermore, birds have an immune-neuroendocrine network I.~ in which PRL appears to play a similar immunoregulatory role to that described for mammals 16.

Box 1. Characteristics of the DCx-embryo thymus 21

• Reduced size • Imbalanced development of cortex and medulla (increased

development of the cortex compared with the medulla) • Increased number of pycnotic cortical thymocytes • Accumulation of precursor cells in enlarged connective

tissue trabeculae • Intrathymic granu!opoiesis • Hypertrophied epithelial cells, correlating with high levels

of circulating thymic hormones (mainly thymosin 134)

The DCx embryo Many studies have used early, partially decapitated,

chicken embryos (termed DCx embryos). This model, which stems from classical endocrinology t7, involves the removal of chick embryonic prosencephalon at 33-38 h of incubation, resulting in the elimination of primordia of the pineal system, hypothalamus and pituitary gland. Previous reports by Jankovic and co-workers Is-2° have demonstrated a delayed development of the lymphoid system in DCx embryos, and this has been attributed to an imbalance in neuroendocrine activity. In addition, preliminary morphological studies 2t have also shown profound changes, both in developing thymocytes and in non-lymphoid thymic stromal cell components. Taken together, these results suggest an altered maturation of T cells in the thymus of DCx embryos. Presumably, this results from modifications in thymic colonization by lymphoid cell precursors and/or their patterns of differentiation (summarized in Box 1).

On this basis, we have used in situ immunohisto- chemistry in conjunction with flow cytometry to analyse the patter: of T-cell development in chicken DCx embryos, ana representative results are given in Figs 1 and 2. Using monoclonal antibodies (mAbs) 2-4 (Ref. 22) and CVI-His-C7 (Ref. 23) against two ceil markers expressed on a!! T-cell subsets,. CD28 and CD45 respectively, a significant decrease in the number of positive cells can be seen throughout thymic ontogeny in DCx embryos compared with normal embryos. A comparative analysis of CD4 and CD8 expression with ,-..Abs CT4 and CT8, respectively 24, showed that the percentage of CD4-CD8- double-negative (DN) cells of DCx embryos undergoes a significant increase from day 15 of embryonic life (Fig. 1). By contrast, an important decrease in the proportion of CD4+CD8 + double-positive (DP) cells occurs in the same embry- onic period. This suggests that, in DCx embryos, there is delayed T-cell development that results in an ac- cumulation of DN cells and an almost complete disap- pearance of DP cells. In agreement, CD4-CD8 I° cells, which correspond to an immature pre-DP T-cell sub- population, also increase gradually throughout the embryonic life of DCx embryos. On the other hand, no significant differences to control values are observed in

© 1994, Elsevier Science Ltd 0167-56991941507.00

",,m,,,ology Toaar 5 2 4 ~ot. lS No. ~1 1994

thxmus

the number of mature CD4 CD8 I~' cells. However, remarkable differences occur in the differentiation pat- tern of T cells expressing T-cell receptor (TCR) c~ and ~/~, as shown with mAbs TCR2 and TCRI, respec- tively2~: a gradual significant decrease occurs in the num- ber of TCR 0if3" cells; whereas the TCR ~,~" cells do not seem to be especially affected by the experimental pro- cedure {Fig. 2). These changes are summarized in Box 2.

The lack of change in the number of C D 4 CD8 h' thymocytes could be related both to the expression of CD8 on the vast majority of chicken TCR ~/~ cells 2~-2- and the existence of a CD3"CD8"TCR (TCR0) cell population, which is thought to represent a non-pro- thymocyte lineage that migrates from the periphery into the thymus early during chicken ontogeny -'s.

What signals are involved? The ability, of PRL to recover the T-cell system of DCx

embryos has been analysed in order to understand the nature of the endocrinological signals that govern T-cell maturation during chicken ontogeny. According to avail- able data on the establishment of the endocrinological axis, it appears that PRL may play a role in the early maturation of chicken thymocytes 2"2~'-~c~. By contrast, growth hormone (GH), which is expressed late during embryonic life (on day 17 of incubation) -~l, has no effect.

Treatment of the chorioallantoid membrane with a single dose of 25 ng per day of chicken PRL (diluted in 100 ~1 of PBS), from day 10 of incubation to the day of sacrifice, induced an important, but not total, recovery of the T-cell system of DCx embryos. Thus, all of the T-cell subpopulations that were reduced in number in day-17 DCx embryos (i.e. DP and TCR otlB+ cells) under- went a significant increase, whereas those that had been increased in number (i.e. immature CD4-CD8 I'' and DN cells) decreased considerably (Figs !,2). By , - nn t r . ~c t . ~Am;n i~ t , - ~ t ; . . . . . I : { Z . I - I , , , : ; ~ . , ~ - ;m : l . - , , - , - , , . . , - , t . . . . I 5VilLi HOL~ U ~ I l l l K I * O i l H i l l I i l , 11 ~ i i U O l i l ~ ¢ 1 0 l l l l l l l l l ~ I L I i L / ~ I I I

resulted in the death of chicken embryos. The finding that recovery from the DCx phenotype

by PRL treatment was incomplete could suggest either that the length of u-eatment is insufficient for full re- covery of the immune system (the drastic experimental procedure used makes it impossible to obtain DCx embryos later than 17-18 days of incubation) or that other hormona! factors, apart from PRI., are necessary. In order to test this second pos:,ibility, we analysed two new experimental conditions: (1) p;tuitary glands from day-11 embryonic chicken were graf:ed onto the chorio- allantoid membrane of day-11 DCx embryos; and (2) cephalic chick-quail chimeras were made by replac- ing the anterior mid-portion of the prosencephalon of a 33-38 h chicken embryo with the same portion of a 30-33h quail embryo brain. As mentioned above for PRL-supplemented DCx embryos, pituitary-gland- grafted DCx embryos underwent significant, but not total, recovery of the thymic T-cell subpopulations (Figs 1,2). By contrast, chick-quail chimeras demon- strated an almost complete recovery of the chicken T-cell system, showin¢ the same number of cells expressing pan T-cell markers as control animals, with consider- able recovery of DP, DN and immature CD4-CD8 I'' cell populations. Only the TCR oLB + cell compartment failed to recover (Figs 1, 2).

l_=i

13 days 15 days 17 days

Fig. 1. PLrcentage ~4 ,ti/ferent T-cell subsets ,te/ined by the expressi, m '4 CD4 ,1rid CI)8 m,decuh's in the embrvcnlic thymus m (,t) C;nl/rul chickens. (b! D(;x embryos. (c) DCx emb1"l'~s sup)died wiih prol,lctin cPRLI, cdl DCx" embl"v,s gr, lfied with embryonic pituit,11:v gl, lnds. ,1rid eel cephalic chimeras at differ,'nt .lays ¢~f incub,ltioJz. Data corresp~md to tbe me,in of at least three different experiments, h1,111 cases, Shlnd, lrd del,i, lticm teas S-IS% of the l,,lhW, except in the case r~f DCx embryos, Ivhich r, mged S-25%. GMured bars represent the fidhm,ing s~d~p¢~pld, ltimls: green, CD4- singie-positil'e (SD cells; bhle. CD8 I': SP cells; brow,, ,hmble-positil,e cells" red, CD8 I'' cells; ~'elhm: d~mble-nel~,ltil,e cells.

No significant differences were observed between the circulating PRL levels of control chickens and chimeras, as measured by radioimmunoassav (RIM, whereas pituitary-gland-grafted and PRL-suppiemented DCx embryos both exhibited lower values than those it, control chickens. This could be due to poor develop- ,nent of grafted embryonic pituitary gland on chorio- . - , l l . . , , - , t - , , ;A , - , . . , , ~ , . , - , I . , . . . . . . , .-,,:- A . . . . . t , - . . - , , - , ~ d I - , , , I . . , h : - r , ~ i . . . . ; , - . - , I {.~[ l( 3 L1 i l I* ~ / I ¢.~1 I I l%,. I l I I. ' I L I [ I l%,. I L I~ %.I(T~.. I l I ~ / I I J L I f..l 1. %,. i l ~ i i I J i . a. / i L/~(..,.~ 1%,- ~.-I I

analyses, or due to the rapid metabolism of hormone in both experimental conditions. Nevertheless, even in this situation, low levels of PRL allow considerable reconstitution of the chicken T-cell system: thus sup- porting a role for this pituitary gland hormone in early thymocyte maturation.

It has been demonstrated in mammals that PRI. induces expression of interleukin 2 receptors {iL-2Rst, both on T cells and T-cell lines ~'~2. In chickens, as in mice and rats, thvmic cell precursors express IL-2R (Ref. 33), suggesting that PRL could provide direct or

Box 2. Changes observed in T-cell development in DCx embryos cgmpared with normal animals

• Increase in the number of carlv T-cell subsets, including DN cells and immature CD4-C158 I'' cells

• Marked diminution in the number of DP cells and TCR otlB+ cells

• Few changes in the number of mature CD4-CD8 h' cells and TCR ~/~-expressing cells

Abbreviations: DN, double negative; DP, double positive; TCR, T-cell receptor.

I,,,,,,,,,,,/,,.~y-r,,,t,,y 5 2 5 v,,l. is N,,. II i,~,~4

thymus

% Cells

50

40

30

20

10

4 51;' ? ?::?: ;:!/

/lll - .... "a b a b c d e a b c d e / /

0 -*=...,,~ 13 days 15 days 17 days

Fig. 2. I'cnc,zt.z,.,,," ,,t 7- cells cxl,rcssmz -I=ccll recc H,,r ! ~-( ;Rt ~13 tpi,tk I,,irsl .rod -IX R ya , ,',h,' l,a~s, m tlv ,.ml,ry,,,,, tl:ym,,s , 4 ,u ,,,,ur,,I ,l:i,'k,,,s, ¢1,~ l)(-x" cmi~rv,,s, i,* l)(~x cml,rv,,s suPldic, t IVM: pr,,L1ctm ePRI.I, 1,t1 l)Cx eml,n',,s .~raqe,/with emlwv,,mc pitmtar 3" gla.,ts. ,u1,t le* ceph,llic chmler, ls .it ,htli're.t [Ltvs ,'! i.cuhatil.r l)ata ,'~,rresp, md t, ~ the me.l. ,,f at h'.lSt three ,htli'rc,~t CX.l,cnmcnts. h~ .11! ,.1sos. sta.,t.lrd ,h'l'i,:ti~,n lt'as 5-IV',, of the

r.lhn., cx,cpt m tl:c case ,'t 1)( 7x emt.v.s , u'l, ich r, ulge, l 5-25%.

indirect signals fi~r differentiation of chicken DN t h l , f l lOC% [e_h I . I . , . . . uurlng e4llv tliyi111C oni. _.2,cn.v. !n .,apport tff" this, (,agnerault et ,11.~4 have rt:~cntiv shown that PRI.-Rs are more strongly expresse,,! on the surface of rat DN cells than on DP cells.

T/,.lS, since there arc different waves of entry of T-cell precursors into the chick embrvonL thymus ~', the lack ~,f pRI. in D(Tx embryos w~.mld'pr,'vent, or ,'onsiderai Iv delay, the differentiation of the se_'md wave of T-cell . . . . . • l t r ' ~ l l r ~ rla.-~r . . . . - , , - h , , ~ . el-to , - I . ; . . ! . . . . ~ - I - , , , . - , . . . . . g . . . . . 1 . . . . [ l l t ~ - " " . . . . t l l t i t I L t l ~ * l ~ - " t l l ~ ~ . I I I T h . I ~ L i t i : l l l l L l 3 I I % l l l l L i d %

12 of embryonic life. In such PRl.-def,cient conditions, the cells Wo'uld not express 1I-2R and, therefi~re, would not respond t,a the 1I.-2 that is presumably produced in the embryonic thymus.

\Vc thank M.D. Cooper, C.H. Chcn, O. \'hinio and S. .]¢t,rl-,c n fiw tile gift .f nmn,chmal antib.dics, and A.E Parlour tur kindly prmidi,lg chicken prolactin. This work was ,,upPO[tcd by (A'f(ItT grant numbers PXI89-0043 and PBgl- 11,~'4 from the Spanidl Ministry of Education and Science.

1,tl'i¢r ,~l,l"em,, Angeh's Ficente, hlgnl,lr Heijnen and A,,ttstln (;. Z,1p,lt.1,1re ,it the l)ept o/ Cell Biology, F, lctd~:' ,,] Bi~ d~ Lk'3.; (:<,rap lutense Unil 'e1~it),, 28040 M,ldrid. .spain.

References l Gala, R.R. t 199 Ii Proc..~,~t-. t(xp. Bhd. Med. 198, 513 -~2- 2 Hooghe, R., Dethasc, .M., Vergani, P., .\lalm; A. and t|ooghc-Petcrs, E.I.. (I 9931 hnm,md. Tod, lV 14, 212-214 3 Matcra, I.., Belhm¢, G. and Cesano, A. ( 19911 Adl: Nell r,,ml.mn, d. I, 158- i 72 4 .Xhmtgcmlery, D.\'{(, Shen, G.K., Ulrich, E.D., Steiner, I..1.., Parridl. P.R. and Zukowski, C.E (1992) Emh~crimdogy 131, 3019-:;02(; 5, pcllcgrini, 1.., [.ebrum, J-J., Aii, S. and Kelly, P.A. (1992)

M, d. l-)l,h ~crim d. 6, 1023-103 i 6 ()'Ncal, KD., .Momgomery, I).W., Trmmg, T.XI. and 'fu-I co, 1,. (1992)Mol. ('ell. Endocrimd. 87, R 19-R23 7 Baroni, C. (1967)Experientia 23, 182-183 8 Baroni, C., Fabris, N. and Bertoli, G. (1967) Experientia 23, 1059-106 ! 9 Duquesnoy, R.,]. (1972) 1. immured. 108, 15-78-1590 10 Fabris, N., Picrpaoli, W. and Sorkin, E. (19711CIm. Exp. Immmml. 9, 227-232 I 1 Esquifino, A.I., Villanua, M.A., Szar.v, A., Yau,.I. and Bartke, A. (I 991)Acid l-)tdocrm,l. 125, 67-72 12 Russell, I).H., Mills, K.T., Talamantes, EJ. and Bern, H.A. (1988) Proc. N,ltl Ac, ld. 5ci. USA 85, 7404-7407 13 Cooper, M.D., Chen, C.H., But.v, R.P. and Thompson, (LB. 11991) Adt: Immllmd. 50, 87-117 14 Vainio, O. and l.assila, O. (1980} Crit. Ret: Poultry Biol. 2, 97-101 15 (;lick, B. (1984).1. Exp. Z, ml. 232, 671-682 16 Skwarlo-Sonta, K. (1992} hnmumd. Lett. 33, 105-121 17 Fugo, N.\V. (1940)]. Exp. Zo,I . 85, 271-298 18 Jankovic, B.D., lsakovic, K. and Knevevic, Z. (1978) Det: Comp. lmmunol. 2, 479--492 19 .lankovic, B.D., lsakovic, K. and Micic, M. (1982)in in Vii', hnmunol,g3: Histophysiology o f the l,),nlphoid System (Nieuwenhuis, P., Van der Broek, A.A. and Hanna, ,\I.G., eds), pp. 343-348, Henum Press 20 Jank,vic, B.D., Isakovic, K., Micic, M. and Knevevic, Z. ( 1981 ) ( lm. Exp. h,,munoL 18, 108-120 21 Herrad(m, P.G., Razquin, B. and Zapata, A.G. ( 1991 ) Am..]. An,it. 19 I, 57-66 22 Vainio, O., Riwar, B., Brown, M.H. and kassila, O. ( 199 i ) .1. lmnlUmd. 147, I.,9a-1599' " 23 Jeurissen, S.H., .]anse, E.M., Ekino, S., Nieuwenhuis, P., Koch, (;. and De Boer, G.E (I 988} Vet. lmnzmtol. hmmtmqnlthol. 19. 225-238 1~ [ / - L _ _ I k 4 I l l I 1 | [ 1 1 i 1 i • • I t ~ --"-II. ~ . . l l i l l l , ,~,I.,~,1., L . H C H , L . - I . . F a . , / A g C r , l . . l . , a n l l c.ooper, M.D. (1988).1. Immumd. 140, 2133-2138 25 Chen, C-I..H., Cihak, J., 1.6sch, U. and Cooper, M.D. (1988) Eitr..I. Inmmn,l . 18,539-543 26 Bucy, R.P., Chen, C-I..H., Cihak, J., l.osch, U. and Coopel; M.D. (1988) 1. hnmunol. 14 !, 2200-2205 27 (]wn, ([-I..H., Cihak, J., l.osch, U. and Cooper, M.D. i 1988i Fun 1. hllnnmol. 18,539-543 28 Buoy, ILl:, Coke.v, M., Chen, C-I..H., Char, I)., ix i)ima,in, N.M. :.nd (Moper, M.I). (1989) Eur I. hmnml, l , i 9, 144'~-1455 29 .Mc(;ann-l.cvorse, I..M., Radecki, S.V., Donoghue, D.J., Malamed, S., Foster, D.N. and Scanes, C.G. (199.3) Proc. S,,c. Exp. Bml. Med. 202, 109-113 30 Pierpaoli, W., Kopp, H.G. aqd Bianchi, E. (1976) Clm. Exp. hmmm, l . 24, 501-50(~ 31 Harvey, S, l)avidson, T.E and Chadwick, A. (1979) (;en. Comp. Endocrimd. .39, 27(1-273 32 Mukhcriee, P., Mastro, A.M. and Hvmer. W.C. (1990) Emhu'rimdogy 126, 88-94 33 Fedccka-Bruner, B., Penningcrs, J., Vaigot, P., Ldlmann, A., Martinez-A, C. and Kmemer, G. ( 1991 ) De1: Inlnlmml. i, 237-242 34 Gagnerault, M-C., Touraine, P., Savino, ~L, Kelly, P.A. and Dardenne, M. (I 993).l. hnmltmd. 150, 5676-5681 35 l.e l)ouarin, N.M. and Jotereau, E\L (I 975)J. Exp. Med. 142, 17-40

1,,,....,#,,.~. T,,,to, 5 2 6 v.#. r~ N.. !1 I<~4