Ontogeny of PHA and Con A responses in the fetal lamb

Ontogeny

of PHA and Con A Responses Fetal Lam b1 AILA

Department

of Medicul

Microbiology.

in the

LEINO

Turks

Utzi~~ersity.

SF-20520

Turku

52, Finlund

Received January 17. 1978 Twenty-three Iambs from 10 ewes at 38 to 135 days of gestation (total gestation period is 150 days) were tested for PHA and Con A responses using cells from the liver, thymus, bone marrow, and spleen. The Ficoll-Isopaque technique was applied for the separation of cells from the liver, bone marrow, and spleen. During ontogeny, the first responses both to PHA and Con A were obtained with liver cells at 38 days of gestation. With thymus cells the first responses to PHA were observed at 68 days of gestation, whereas Con A responses were not detected before the 90th day. In the spleen, both responses were first found even later, at 98 days. With the bone marrow cells, significant responses were observed only occasionally during the later part of gestation. These results indicate that (excluding the fetal liver) PHA-responsive cells appear during ontogeny before Con A-responsive cells and that mitogen responses are observed in the spleen a few weeks later than in the thymus.

INTRODUCTION

By using the cell suicide technique and mixed lymphocyte culture (MLC), Asantila and co-workers have shown that immunologically specific recognition of foreign cells by fetal lamb lymphocytes can be demonstrated almost as soon as the first lymphocytes appear during the ontogeny (1, 2). In the present work, I have studied development of fetal lamb lymphocytes relative to responses to phytohemagglutinin (PHA) and concanavalin A (Con A). A few similar studies on PHA responses by human fetal lymphocytes have already been published (3). Stites et al. (4, 5) demonstrated the first PHA responses by human fetal thymocytes at around 10 weeks of gestation, and several other investigators have observed the same a few weeks later (6-8). PHA responses by human fetal thymocytes increase toward midpregnancy, after which they decline and remain at the same level to the adult stage of life (8). PHA-responsive cells appear in the spleen and peripheral blood of human fetuses later than in the thymus (4, 5, 9). Cells from bone marrow and liver of human fetuses were not found, with a few exceptions, to respond to PHA (4, 10). The early ontogeny of Con A responses has been studied by Papiernik (11) in the mouse. According to Papiemik. the mouse thymus harbors Con A-responsive cells only during the first 3 weeks after the birth; in the spleen Con A-responsive ceils appear about 2 weeks later than in the thymus. The purpose of the work presented in this paper has been to study, by using serum-free cultures, the development of PHA and Con A-responses in the liver, thymus, bone marrow, and spleen of the fetal lamb. I Supported by grants from the Sigrid Juselius Foundation and the Turku University Foundation and by a contract with the Association of Finnish Life Insurance Companies. 0090-1229/78/0111-0006$01.00/0 Copyright 0 1978 by Academx Press. Inc. All right? of reproduction in any form reserved

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Fetuses. Sheep of the Finnsheep strain were bred randomly under controlled conditions, allowing the date of conception to be established within 24 hr. Twenty-three fetal lambs from 10 different ewes were used. The ewes were killed at appropriate intervals and the fetuses were delivered by hysterotomy. The ages of the fetuses varied between 38 and 135 days (total gestation period is 150 days). The liver, thymus, and spleen were dissected and cut with scissors into small pieces in Medium 199 with Hanks’ base. Bone marrow cells were obtained from femurs by flushing medium through the bones. Single-cell suspensions from each organ were made with aid of a Tenbroek-type homogenizer. A Ficoll-Isopaque gradient (12) was applied for further purification of liver, bone marrow, and spleen cells, using a density of 1.082 glcm3. Cell cultures. Serum-free suspensions of 5 x 10’ separated cells were cultured in microplates in 100 ~1 of medium RPM1 1640 (Gibco) supplemented with 100 U/ml penicillin and 100 pg/ml streptomycin. The total culture period at 37°C with 5% CO, was 66 hr. In preliminary experiments with fetal cells from the liver, thymus, and bone marrow, PHA concentrations from 6.25 to 25.0 pg/well and Con A concentrations from 0.1 to 0.4 pg/well were found to give the optimal responses. On this basis, a PHA concentration of 12.5 pg/well and a Con A concentration of 0.2 pg/well were chosen for the final experiments. 5-[1ZSI]Iodo-2’-deoxyuridine ([‘2SI]UdR, 0.25 $X./well, sp act, 90- 110 mCi/mg. The Radiochemical Centre, Amersham, England) together with 5-fluoro-2’deoxyuridine (Fluka, Buchs, Swizerland) at a final concentration of 1O-6 M was added as a quantity of 20 pi/well 16 to 18 hr before harvesting (13). The harvesting was carried out with a multiple cell culture harvester (Skatron, Lierbyen, Norway), and the radioactivity was determined in a Wallac-type gammacounter. The mean counts per minute of triplicate cultures are presented. Stimulation indices are calculated as the ratio of counts per minute in the stimulated culture to those in the control culture. RESULTS Liver. Fetal liver cells (isolated with a Ficoll-Isopaque technique) at 38 days of gestation are the first cells during ontogeny which respond to PHA and Con A; the stimulation indices of the three fetal lambs at this stage of development were 3.9, 10.3, and 5.0 to PHA and 3.7, 9.4, and 2.6 to Con A (Fig. 1). The responses remained quite low until 68 days of gestation. At 78 days the responses, expressed in counts per minute, were at a maximum, even though the stimulation indices remained lower than those obtained at earlier stages of gestation; the indices to PHA of the two 78-day-old fetuses were 1.2 and 2.1, and to Con A were 3.5 and 1.1. The responses observed thereafter (at 98 days) were again low, both in counts per minute and as stimulation indices (Fig. 1). Thymus. Thymus cells in sufftcient numbers were first obtained at 68 days of gestation. Cells from one of the three fetuses showed a significant response to PHA; stimulation indices for these three fetuses were 3.1, 2.0, and 30.6 (Fig. 2). Expressed as counts per minute, PHA responses seemed to increase thereafter and reached a maximum at 90 days of gestation (Fig. 2). Stimulation indices for

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FIG. I. PHA and Con A responses by liver cells of fetal lambs at different stages of gestation. Each group of three columns represents one fetus (a mean value of triplicate cultures is given). Fetuses of the same age are siblings.

PHA of the two 90-day-old fetuses were 3.6 and 5.8. A decline occurred thereafter and at 135 days no PHA response by thymocytes was detectable. Con A responses by thymocytes seem to develop in the fetal lamb later than PHA responses. The first Con A response by thymocytes was observed in one of the two 90-day-old fetuses; the indices were 2.8 and 0.5 (Fig. 2). At 90 to 122 days of gestation the Con A indices for fetal thymocytes varied from 1.2 to 32.2, but at 135 days no Con A response by thymocytes could be observed. Bone murrow. Cultures of fetal bone marrow lymphocytes were characterized by a high spontaneous incorporation of [rz51]UdR and low mitogen responses when expressed in counts per minute. No clear tendency for an increase or

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FIG. 2. PHA and Con A responses by thymus cells of fetal lambs at different stages of gestation. Each group of three columns represents one fetus (a mean value of triplicate cultures is given). Fetuses of the same age are siblings.

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decrease in the stimulation indices during ontogeny in a manner similar to that found for thymus or liver cells was observed. Extremely weak PHA responses first occurred at 68 days of gestation (stimulation indices of 2.2, 1.9, and 1.3) and Con A responses occurred at 78 days of gestation (stimulation index of 1.7), but even higher indices were observed only occasionally thereafter (Fig. 3). Splern.In the fetal lamb, spleen was detectable macroscopically at 58 days of gestation, but sufftcient quantities of cells were not obtained before the 68th day of gestation. At this stage fetal spleen cells did not respond to PHA or Con A; the first clear responses were observed at 98 days of gestation, when PHA indices were 2.9, 2.5, 3.1, and 1.9 and Con A indices were 7.3, 4.9, 3.1, and 4.2, respectively (Fig. 4). Spleen cultures of the older fetuses were not available because of a technical failure.

DISCUSSION The results presented above indicate that first proliferative responses to PHA and Con A during ontogeny of the fetal lamb are obtained with liver cells at 38 days of gestation. With thymus and bone marrow ceils the first responses were observed at 68 days when these cells weakly respond to PHA but not to Con A. Even though liver cells at 38 days of gestation respond weakly to Con A, it is apparent that in the other organs, particularly in the thymus, PHA-responsive cells develop during ontogeny earlier than Con A-responsive cells. After midpregnancy, however, Con A responses are more dominant than PHA responses; this was the case with liver and spleen cells (Figs. 1 and 4). These findings are consistent with the ontogenetic appearance of PHA- and Con A-responsive cells in the mouse (11) and with the suggestion that cellular subpopulations responsible for these responses’are at least partially separate populations (14- 17). Thymus in the fetal lamb becomes lymphoid around the 45th day of gestation. The first mitogenic responses by fetal liver cells, however, were already observed earlier. The same order of appearance was demonstrated for responses against allogeneic and xenogeneic cells both in sheep (1, 2) and in man (2, 4, 18,

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FIG. 3. PHA and Con A responses by bone marrow cells of fetal lambs at differeht stages of gestation. Each group of three columns represents one fetus (a mean value of triplicate cultures is given). Fetuses of the same age are siblings.

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FIG. 4. PHA and Con A responses by spleen cells of fetal lambs at different stages of gestation. Each group of three columns represents one fetus (a mean value of triplicate cultures is given). Fetuses of the same age are siblings.

19). It was further demonstrated, however, by using cell suicide techniques, that these prethymic responses by fetal liver cells are not based on immunologically specific recognition of stimulating cells (1, 2, 18, 19). Proliferative responses of prethymic liver cells against foreign cells are thought to be based either on the multipotentiality of the responding T-cell precursors or on the proliferation of other cells than those belonging to the T-cell lineage. Whether mitogenic responses by fetal liver cells observed at different stages of ontogeny are different from those obtained with other fetal cell populations cannot be answered on the basis of the present experiments. Spleen cell responses to PHA and Con A were first observed at 98 days of gestation, which is considerably later than the appearance of the same responses in the cells from fetal liver or thymus. This order of appearance seems logical, since it has been demonstrated, both in man and in mouse, that mitogenic responses of spleen cells do not appear before the respective response has been observed with thymus cells (4, 5, 9, ll), indicating migration of thymus cells to the spleen. On the basis of the present findings it is obvious that mitogenic responses of fetal lymphocytes in sheep develop roughly at the same time as the specific recognition of foreign cells appears (1, 2). This is probably earlier than the appearance of effector cells for the cell-mediated immunity, since, according to Silverstein et al. (20), 64- to 67-day-old fetal lambs are not capable of rejecting allogeneic skin grafts. The rejection capacity was demonstrated at 77 days of gestation (20). Likewise, Niederhuber et al. (21) have demonstrated that fetal lambs at 70 to 130 days of gestation reject allogeneic skin grafts. Granberg and co-workers have shown that neonatal lymphocytes, both in sheep and man, can be specifically sensitized in vitro to produce cell-mediated lympholysis of allogeneic lymphocytes, although the capacity is weaker than that of adult lymphocytes (22, 23). When during ontogeny that in vitro capacity for the effector function appears still remains to be investigated. ACKNOWLEDGMENTS I thank Dr. Paavo Toivanen for help and encouragement during this study, Dr. Esko Salonen for performing the hysterotomies, and Dr. Esa Soppi and Mrs. Hillevi Tolkkb for technical assistance.

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REFERENCES 1. Asantila, T., and Toivanen, 2. Toivanen, P., Asantila, T., and J. W. Streilein, Eds.), 3. Stites, D. P., Caldwell, J..

P.. J. fmtnunol. 117, 555, 1976. and Vahala, J., In “Immune-Aspects of the Spleen” (J. R. Battisto pp. 13-24, Elsevier/North-Holland, Amsterdam, 1976. Carr, M. C., and Fudenberg. H. H., C/in. /mmurro/. Illll)l~(l~~pnrho/.

4, 519. 1975.

4. Stites. D. P.. Cart’. M. C.. and Fudenberg, H. H.. Cell. fmrnuaol. 11, 257. 1974. 5. Stites. D. P.. Carr, M. C., and Fudenberg, H. H., /II “Proceedings of the Seventh Leucocyte Culture Conference” (F. Daguillard, Ed.), p. 231. Academic Press, New York, 1973. 6. Pegrum. G. D., Ready, D.. and Thompson, E., Bit. J. Htrc~~tmd. 15, 371, 1968. 7. Kay, H. E. M., Doe, I., and Hockley, A., /~rzrn~no/o,~~ 18. 393. 1970. 8. Papiernik, M., Blood 36, 470, 1970. 9. August. C. S., Berkel. A. E., Driscoll. S., and Merler. E., Peditrr. Rcs. 5, 539, 1971. 10. Stites, D. P.. Carr. M. C.. and Fudenberg, H. H.. Proc, Nat. Acud. Sci. USA 69, 1440, 1972. I I. Papiernik, M., Cell. hrmunol. 22, 384, 1976. 12. Boyum. A., Scund. J. Clin. Ltrh. Inwst. 21, Suppl. 97. 77, 1968. 13. Asantila, T., and Toivanen, P.. J. Itnmunol. Methods 6, 73. 1974. 14. Stobo, J. D.. Tmnsplant. Re\l. 11, 60, 1972. 15. Rawson, A. J., and Huang, T. C.. Cell. Immu~wl. 17. 310, 1975. 16. Niederhuber, J. E., Frelinger. J. A., Dine, M. S., Schoffner. P.. Dugan, E., and Shreffler, D. C., J. Exp.

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F., Hadden, J. W., Hadden, E. M., and Good, R. A., Itrr. Arch. 1976. Asantila, T., Vahala, J., and Toivanen, P.. I/,lmurloRP,felic,s 1, 272, 1974. Asantila, T., Vahala, J., and Toivanen, P., I,,l/nrcNoXrrlrti~.s 1, 407, 1974. Silverstein, A. M., Prendergast. R. A., and Kraner. K. L.. J. Exp. Med. 119, 955, 1964. Niederhuber, J. E., Shermeta. D.. Turcotte. J. G., and Gicas, P. W.. Trunsp/antcrtion 12, Allerg.v

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Granberg, C., Manninen. K., and Toivanen, P., C/in. Immunol. Itnmunopathol. 23. Granberg. C.. and Toivanen. P.. Sctrr~tl. J. Itn~nurrol. 6, 1186, 1977. 22.

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