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Effect of thymosin on PHA and Con A responses by fetal lamb lymphocytes
CLINICAL
IMMUNOLOGY
Effect
AND
IMMUNOPATHOLOGY
of Thymosin on PHA and Con A Responses Fetal Lamb Lymphocytes] AILA
Departments
13, 136-145 (1979)
of Medical
LEINO~
Microbiology Turh
AND
by
ESA SOPPI
and Atlatomy. 52. Finland
Turks
University.
SF-20520
Received November 14, 1978 The effects of thymosin fraction 5 on the mitogen responses of fetal lamb lymphocytes in serum-free cultures were studied. The cells from liver, thymus, spleen, and bone marrow of 12 fetal lambs of four different ewes at 48-98 days of gestation (total gestation is 150 days) were used. Cells from the liver, bone marrow, and spleen were separated by Ficoll-Isopaque density-gradient centrifugation. At 48 to 68 days of gestation, fetal liver cells had only a slight capacity to respond to PHA and Con A mitogens; however, these responses could be induced by thymosin. Later on, when the fetal lymphocytes including those from liver, thymus, spleen. and bone marrow started to respond to mitogens (from Day 78 onward), the effect of thymosin became negligible.
INTRODUCTION
The thymus plays a crucial role in the development and maintenance of cellmediated immunity, producing large numbers of lymphocytes, at least some of which leave the gland and form T-lymphocyte populations in the periphery. On the other hand, the thymic epithelial cells produce humoral factors which affect the functional maturation and differentiation of lymphocytes ( 1-7). Thymic factors have been shown to promote the differentiation of human and murine spleen and bone marrow lymphocytes into more mature T cells as judged by the appearance of specific T-cell surface markers and mitogenic responses (S- 12). Stutman et al. (13) have reported that 19- to 2 l-day fetal liver cells in mice are sensitive to the humoral activity of thymus and that this property is still present a few days after birth. Komuro and Boyse (1973) have shown in mice that treatment of fetal liver lymphocytes with thymosin as early as Day 14 of gestation induces the appearance of Thy-l- and TL-positive cells. Furthermore, Globerson et al. have shown that a partially purified calf thymus extract matures murine fetal liver cells so that they can induce graft-versus-host reaction ( 14). Studies carried out in our laboratory by Asantila et al. (15- 18) have demonstrated that fetal lymphocytes are capable of specifically recognizing foreign cells almost immediately when the thymus has become lymphoid (i.e., at 12 weeks of gestation in man and at 58 days of gestation in sheep). Also, cells responding to phytohemagglutinin (PHA) appear in the sheep thymus during the first weeks of lymphoid development in utero, and concanavalin A (Con A)-responding cells about 2 weeks later (19). These findings have prompted us to investigate how early during the ontogeny fetal T-cell precursors in the sheep are susceptible to thymic I Supported by a contract with Association of Finnish Life Insurance Companies. 1 Address correspondence to: Mrs. Aila Leino, M.S.. Department of Medical Microbiology, University, SF-20520 Turku 52, Finland. 136 0090-1229/79/060136-10$01.00/0 Copyright All rights
@ 1979 by Academic Press. Inc. of reproduction in any form reserved
Turku
EFFECT
OF
THYMOSIN
ON
FETAL
LAMB
LYMPHOCYTES
137
humoral factors. For this purpose we have studied the effect of thymosin fraction 5 on the mitogenic responses of fetal liver, thymus, spleen, and bone marrow cells at different stages of gestation in fetal lambs. MATERIALS AND METHODS Fetuses. Twelve fetal lambs from four ewes of the Finnsheep strain were used. The ewes were killed at appropriate intervals and the fetuses were delivered by hysterotomy. The ages of the fetuses varied between 48 and 98 days (total gestation period is 150 days). Preparation of cells. Liver, thymus, and spleen of the fetuses were excised, cut in pieces, and placed in medium TC 199 (Orion, Helsinki, Finland). Bone marrow cells were obtained from femora by flushing medium through the bones. Singlecell suspensions were made with the aid of a Tenbroeck-type homogenizer. Ficoll -1sopaque gradient separation was applied for further purification of liver, bone marrow, and spleen cells, using a density of 1082. Preincubarion with thymosin. Thymosin fraction 5 (Lot BPM 390) prepared from calf thymuses was generously supplied by Dr. Allan Goldstein. Cells (10 x 106/ml) were preincubated before mitogenic stimulation with different concentrations (0.001, 0.01, 0.1, 1.0, 10, 100, 200 &ml) of thymosin in medium RPM1 1640 (Grand Island Biological Co., Grand Island, N.Y.) without any serum at 37°C for 2 hr and washed twice with large amounts of the medium. The controls were preincubated with medium RPM1 1640 and washed as the test cells before the culture. Mitogen-stimulated cultures. The culture system of fetal lymphocytes was based on the method described by Eskola ef al. (20). After thymosin treatment, serum-free suspensions of 5 x lo5 separated cells were cultured in 100 pi/ml of RPM1 1640 in microtiter plates (Cooke Microtiter M-24AR Dynatech, Zug, Switzerland) for a total period of 66 hr at 37°C with 5% CO,. In a pilot study, concentrations of phytohemagglutinin (PHA M, Difco Laboratories, Detroit, Mich.) from 31.25 to 1000 pg/ml and concanavalin A (Con A, Pharmacia Fine Chemicals, Uppsala, Sweden) from 0.25 to 8 pg/ml were tested. On the basis of these results. concentrations of 125 &ml for PHA and 2 pg/rnl for Con A were used throughout the study. After incubation for 48 hr, 0.25 &i of ‘?“I-labeled 5-iodo-2’deoxyuridine ([‘251]UdR, specific activity 90- 110 mCi/mg, The Radiochemical Centre, Amersham, England) was added in a volume of 20 @‘ml with 5-fluoro-2’deoxyuridine (FUdR, Fluka, Buchs, Switzerland) to increase the uptake of [1251]UdR (21). The final concentration of FUdR was 10-fi M. Harvesting was performed 64 hr after the start of the cultures. Radioactivity was measured in a Wallac-type gamma counter (Wallac, Turku, Finland). All cultures were performed in triplicate, and the results were calculated as the arithmetic mean of the observed counts per minute (cpm). Mitogen-stimulated incorporation was defined as the difference between the incorporation in cultures incubated with and without mitogen. Statistics. Student’s I test for matched pairs was employed. RESULTS Effect of Thymosin Preincubation on Mitogen Responses by Fetal Liver Cells Effect on PHA responses. Preincubation with thymosin increased PHA responses by liver cells of 48- and 68-day-old fetuses, i.e., to about the midway of
48
48 48 68
68 68 78 78 98 98 98 98
1
2 3 4
5 6 7 8 9 10 11 12
613 475 907 12685 1222 5842 1983 11987
(3.9) (4.2) (1.2) (2.1) (1.3) (3.4) (1.5) (4.3)
3936 (13.0) 714 (8.1) 103 (1.3)
1854 (1.7)
-
LIVER
(4.1) (4.7) (2.3)
1408 (5.6) 945 (3.8)
7608 2578 513
(2.6)
0.001
4647
OF FETAL
-
CELLS
(2.8)
AFTER
in parentheses
(5.5) (3.4) (1.3) (2.3) (1.3) (2.9) (1.9) (6.6)
7394 (3.6) 2830 (2.6) 865 (3.4)
4368
0.01
TO PHA
1431 651 1482 15660 1380 5070 4260 12489
0 Mean cpm of triplicate cultures are given. h Net cpm: stimulated minus unstimulated. Figures fold or greater increases caused by thymosin.
Age (days)
No.
Fetuses
RESPONSE
TABLE
1
(3.7) (1.7) (4.5)
give
1090 4812 2871 14004
stimulation
(1.3) (2.5) (2.0) (6.4)
1114 (4.4) -36 (0.9)
8037 2004 1355
41.56 (2.9)
0.1
PHA-stimulated (thymosin
PRETREATMENT
DIFFERENT
(3.2)
(2.4)
1407 5001 728 8094
(1.4) (2.3) (1.1) (3.4)
960 (5.1) 210 (2.2)
6497 (3.7) 325.5 (1.7) 1270 (5.5)
3682
IO
CONCENTRATIONS
(stimulatediunstimulated).
(1.6) (2.5) (1.4) (2.2) (1.2) (4.3)
2300 16652 1450 4571 1154 9012
indices
(4.5)
1269
8420 (3.9) 4829 (2.1) 1300 (3.6)
4301
1.0
cultures* &ml)
WITH
Figures
651 48 2719 12679 -519 5095 5405 8751
in italics
(3.6) (1.3) (1.9) (2.1) (0.9) (3.5) (2.2) (2.9)
6312 (4.0) 9068 (2.1) 724 (3.8)
35.51 (2.7)
100
OF THYMOSIN~
(2.3)
represent
92 (1.5) 17 (1.1)
5110 (4.0) 2538 (1.6) 192 (1.7)
2653
200
two-
$ 21
5 u
L z 0
EFFECT
OF
THYMOSIN
ON
FETAL
LAMB
LYMPHOCYTES
139
the gestation (Table 1). With the three 48-day-old fetuses considered as a group, the values obtained with all the thymosin concentrations used (0.001-200 pi/ml) were significantly different from the control values (P < 0.05 by the Student’s t test for matched pairs). Likewise, 0.001 and 0.01 &ml of thymosin induced significant increases in the group of three 68-day-old fetuses (P < 0.05). This effect of thymosin was observed first of all on net cpm (stimulated minus unstimulated); a twofold or greater increase in net cpm was obtained with thymosin concentration as low as 0.001 &ml (fetus Nos. 1 and 3-6, Table 1). With cells from a fetus with an already existing PHA response by liver cells (No. 2 in Table l), a twofold increase was obtained first with a thymosin concentration of 0.1 pg/ml. Although the effect of thymosin on PHA responses by fetal liver cells was clearly observed as an increase in net cpm, it was only rarely demonstrable as an increase of stimulation indices, indicating that thymosin also increases cell proliferation in cultures without the mitogen. After midpregnancy, no statistically significant changes (P > 0.05) were obtained in the groups of 78- and 98-day-old fetuses, because thymosin had an effect only occasionally on the cells of these fetuses, and even then only at high concentrations (fetus Nos. 7 and 11 in Table 1). Effect on Corz A responses. At first glance the effect of thymosin on Con A responses by fetal liver cells (Table 2) seems to be similar to its effect on PHA responses. It appears, however, that the effect of thymosin is strongest when the spontaneous response to Con A is weak or absent. Fetal liver cells of 48- to 68-day-old fetuses (Nos. 2-5 in Table 2) did not yet respond to Con A, but preincubation of these cells with different concentrations of thymosin resulted in twofold or greater increases in net cpm. With the cells of older fetuses, similar increases were observed if the cells did not spontaneously respond to Con A (fetus Nos. 9 and 11 in Table 2). Exceptions were presented by fetus Nos. 6 and 7. Fetal liver cells of these two fetuses did not respond spontanously to Con A, and responses could not be induced by preincubation with thymosin. On the other hand, fetal liver cells of four fetuses (Nos. 1, 8, 10, and 12) responded to concanavalin A, and preincubation with thymosin had no effect on these cells. As seen for the effect of thymosin on PHA responses, its effect on Con A responses was also observed in net cpm and only occasionally in stimulation indices. However, due to great individual variations, the differences observed do not reach, neither for net cpm nor stimulation indices, statistical significance when fetuses of each age are considered a group (P > 0.05 by Student’s f test for matched pairs). Effect of Thymosin Preincubation on Mitogen Responses by Cells from Fetal Thymus, Spleen. and Bone Marrow* Thymus. There was considerable variation in the capacity of thymus cells of five fetal lambs to respond to PHA and Con A, both in net cpm and in stimulation indices (Tables 3 and 4). For instance, fetus No. 7 at 78 days of gestation did not spontaneously respond to PHA, but small increases both in net cpm and in stimulation indices were induced by pretreatment with thymosin (Table 3). Cells from another fetus of the same age (No. 8) showed a high spontaneous proliferation without a significant PHA response, and this response was not influenced by thymosin. Thymus cells of three 98-day-old fetuses showed low responses to both mitogens, but only in one of them (No. 11) was the response increased by thymosin. As an example of the variability, the effect of thymosin on Con A responses in
Age
68 68 68 78
78 98 98 98 98
4 5 6 7
8 9 10 11 12
27331 809 3550 -553 8711
-136 32 126 441
(3.5) (1.2) (2.4) (0.9) (3.4)
(0.6) (1.2) (1.8) (1.1)
9001 (3.9) 966 (2.3) 81 (2.3)
-
0.001
LIVER
-105 (0.7) 318 (2.0) 146 (1.4)
5310 (2.8) 4788 (3.3) 296 (1.8)
OF FETAL
CELLS
A AFTER
(4.1) (1.3) (2.1) (2.1) (6.1)
(1.1) (2.3) (0.9) (1.1)
in parentheses
36440 1278 2818 5563 11238
30 400 -27 273
4997 (3.1) 3685 (2.7) 456 (1.4)
0.01
TO CON
n Mean cpm of triplicate cultures are given. b Net cpm; stimulated minus unstimulated. Figures fold or greater increases caused by thymosin.
48 48 48
(days)
1 2 3
No.
Fetuses
RESPONSE
TABLE
(3.2) (2.3) (0.9)
0.1
(thymosin
stimulation
609 (1.1) 3179 (2.0) 1475 (1.5) 9756 (4.7)
give
2
A-stimulated
567 (2.5) -120 (0.6) 74 (1.3)
4259 3713 -104
Con
PREATREATMEKT
DIFFERENT
1.0
indices
41525 2622 2618 162 11920
-2047
(2.6) (2.5) (0.8)
1655 1569 640 4143
(1.4) (1.4) (1.1) (2.2)
143 (1.5) -211 (0.5) -19 (0.8)
3377 3806 -729
10
CONCENTRATIONS
(stimulatediunstimulated).
(4.8) (1.8) (1.7) (1.0) (5.4)
(0.5)
677 (2.4) 13-o (1.4)
3942 (3.2) 4533 (2.5) 309 (1.1)
&ml)
culturesb
WITH
Figures
38109 367 2307 132 3300
7 40 -96 176
3671 3643 4035
in italics
(4.2) (1.1) (2.1) (1.0) (1.7)
(1.0) (1.2) (0.4) (1.1)
(2.7) (2.6) (1.7)
100
OF THYMOSIN”
(0.5) (0.7) (0.8)
(2.5) (2.8) (0.8)
represent
-138 -64 -31
3417 3476 -661
200
two-
B z
z
8
c
r
-0~1
(L’ZZ) (C’Z) (L’f) ZIZEI LIZ01 ZL6S Lf9ZI 088 6ZP
St%f 698 LZZ9
wasalda
(6’1) (O'f) (L’I) (f’t4 (S’S) (P’f) ts.sz) 696f (8'1).1199 (0'1) t(1.1) 9892. (E’Z) 162
sx[q
ZZOZI 6655 SOLL PO68
PS9P
(VI) (Z’Z) (O’Z) (Z.f) (S’f) (1.5)
(Z’PI) (1.5) (P’Z) 6999 ISZ9 9ow 69101 91L 6Sf
9LIS 1651 OZLP
‘(pale~nru!lsun/paie~n~!ls)
f69P DZL 6LI
SOTS 6L6S ST1 9SfZ OfZ
u! sa.~nl!~
(0.81)
(L’I) (O’Z) (Z’S) (Z’S)
(L.91) (1.2) (VI)
(S'PI) (8'1) (Z'I) (Z’I) (6’1)
saxpu!
(E’LI)
(8.1) (Z’Z) (5.2) K’Z)
WI) (P’S) (O'Z)
~u!sowdq~ dq pasnes SaseamI! Ja)ea.Id10 PlOJ
86 8L 8L
ZI II 01 6 8 L
II 8 L
snwdq~
uaqds
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IE8P 068 IIPS
86 86 86 86 8L 8L
ZI II 6 8 L
u! sa.Inl!~
(1’81) (9'1) (9.z)
6LtZI ILv9 18LL LPOOI LS9 OEZ
86 86 86 8L 8L
ai\!8 sasaqluaed
09pP 6PS OELS
(6’1) (I’f) K’s) (6.5) (6.Z) K'Z)
L18P 56 PZP IISf VCZ-
uo!w[n~U!ls
LOLS
(P.8) (f.1) (P’Z)
6568 LSZS SO88 SESOI 6Sf 6-
(L.81) (9.5) (9.Z) (Z'I) CL.01
NOa LLEZI SZLS P68L 5198
(L’I) (Z’Z) (L’Z) (L’Z) t1.Z) (0'1)
60t7t 281 6ff S8SP68f
MOJJWJ
9EZS z9z zos
(f’8) (9’S) (8'1) (8’0) (P’Z)
-4%
98
98 98
11 12
II
78 98 98
8 9 IO
78
98 78
12 7
78
98
II
8
78 98
7
78
8 9
(days)
7
No.
-309
(0.5)
(2.3) (2.5) (2.2) (3.3)
2815 (1.8) 2665 (2.8) 646 (3.3)
loo (1.3) 33420 (7.3) 20361 (4.9) 6565 (3.1) 42226 (4.2)
177 (2.2)
22221 409 69 631
-
AND BONE MARROW
(1.9) (10.2) (1.2) (1.0)
(4.3)
3605 (1.9) -401 (0.7) 116 (1.2)
4205 (2.0) 32229 (3.7)
17207
16 (1.0) 32709 (6.3)
360 371 107 2
-93 (0.7) 9778 (1.5)
0.01
WITH DIFF~RENI-
(6.6) (4.5) (2.1) (3.9)
396 (2.1)
32687 18565 4868 44621
-146 (0.7) 24978 (417.2) 544 (2.4)
0.1
(0.7)
(0.7) (1.2)
483 (2.8)
(0.2)
(2.5) (3.2)
-238 (0.6) 146 (1.8)
5037
47636 (4.2)
7709
171 (1.9) 37143 (10.8) 22542 (3.5)
23.754 (3.8) 174 (2.1) 144 (1.8)
-272
-58 (0.7) 14128 (1.9)
100
OF THYMOSIK”
Figures in italics represent two-
(5.1)
3861 (2.1) -904 (0.2) 450 (2.1)
25758
7061 (2.3) 48917 (3.8)
32645 (5.4)
90 (1.2) 254414 (3.8) 417 (2.4)
10
CONCENTRATIOW
(1.3) (8.2) (4.1) (2.1) (3.2)
86 33809 20885 5540 35962
(3.9) (2.2) 79 (2.1)
-65 16575 -132 20400 459
1.0
Con A-stimulated cultures0 (thymosin &ml)
TABLE 4 A AFTER PREATREAYMEW
CELLS 2.0 CON
I’ Mean cpm of triplicate cultures are given. b Net cpm; stimulated minus unstimulated. Figures in parentheses give stimulation indices (stimulated/unstimulated). fold or greater increases caused by thymosin.
Bone marrow
Spleen
Thymus
Cell type
SPLEEN,
Fetuses
RESPONSE OF FETAL THYMLIS.
s -2
% 0
5 0
K
EFFECT
OF THYMOSIN
ON
FETAL
LAMB
LYMPHOCYTES
143
net cpm was the same with concentrations of 0.1 to 100 pg./ml, whereas 0.1 pugirnl had the smallest effect on unstimulated cultures, resulting in an enormous thymosin effect when expressed as a stimulation index. Spleen and bone marrow. Pretreatment with thymosin did not affect the mitogen responses of spleen and bone marrow cells (Tables 3 and 4). Spleen cells responded weakly to PHA and Con A at the age of 78 days; at 98 days the responses appeared somewhat higher, particularly to Con A. Bone marrow cells of only three fetuses (78-98 days) were studied; low spontaneous responses to PHA and Con A were recorded; and thymosin pretreatment had no effect (Tables 3 and 4). DISCUSSION The effect of thymosin seems to be strongest on fetal cells from stages before midpregnancy. Both PHA and Con A responses by liver cells of 48- and 68-dayold fetal lambs were increased by a small concentration of thymosin (0.001 pg/ml), suggesting that weakly responding cells mature as a result of pretreatment with thymosin. In older fetuses (78-98 days), the mitogen responses were already developed, not only in the fetal liver cells but also in the thymus, spleen, and bone marrow cells. At that time the effect of thymosin was weaker, and on the few occasions when it becomes manifest, higher concentrations of thymosin were required than at earlier stages of gestation. For fetal liver cells, no uniform optimal concentration of thymosin exists, and concentrations throughout the whole range used (0.001-200 &ml) were equally effective. It is possible that autologous thymus of the fetuses, which is lymphoid after the age of 40-45 days, may already produce humoral factors affecting fetal T-cell precursors in early stages of fetal development, and that the more these factors are available, the higher concentrations of thymosin are required to increase the cells’ capacity to respond to PHA and Con A. However, if mitogen responses have not yet developed they can be increased with small concentrations of thymosin, even during the later part of gestation (for instance fetus Nos. 9 and 11 in Table 2). On the other hand, since thymosin fraction 5 consists of more than 30 different components, and neither of those 2 purified so far (thymosin Q~,(Y,) is alone responsible for all the effects (22), it is possible that cells of fetal lambs at different stages are susceptible to different components, resulting in variation in the optimal concentrations. In spite of the complex nature of thymosin fraction 5, this preparation has high specificity. It has been shown that control fractions prepared from spleen (12, 23-25) or from other sources (24) in the same manner as thymosin are unable to produce the effects of thymosin in various experimental conditions including mitogen responses. Spontaneous proliferation of fetal liver cells is increased by preincubation with thymosin. particularly with cells from the early stages of gestation, when the effect of thymosin is also strong in mitogen-stimulated cultures. This might be explained by thymosin-initiated maturation which continues during the whole culture period and results in an increased spontaneous proliferation. Thymosininitiated maturation during the preincubation may also explain some of the discrepancies observed in different studies, with different incubation times, concern-
144
LEINO
AND SOPPI
ing the time required for maturation of adult and fetal cells to more immunocompetent lymphocytes (8, 9, 12, 26). Conflicting views have been presented regarding the pre- or postthymic nature of target cells for the humoral thymic function. According to Komuro and Boyse (8) the target cells are prethymic, whereas Stutman et al. (13) and Rotter and Trainin (27) consider them postthymic, and Goldstein et al. (28) find prethymic, thymic, and postthymic cells susceptible to thymosin. In the present work, the effect of thymosin was strongest on fetal liver cells obtained during the first half of gestation. The thymus was already lymphoid at the time when the first liver cells were studied (at 48 days of gestation), and these may be prethymic or postthymic cells. Thus, no definite answer to this question can be provided on the basis of the data described here. ACKNOWLEDGMENTS We wish to thank Dr. Paavo Toivanen for constructive and invaluable criticism, Mrs. Hillevi Tolkko for technical assistance, and Dr. Esko Salonen for the hysterotomies.
REFERENCES 1. Stutman, O., and Good, R. A., In “Contemporary Topics in Immunobiology” (A. .I. S. Davies and R. L. Carter, Eds.), Vol. 2, pp. 299-319. Plenum, New York, 1973. 2. Trainin, N., and Small, M., In “Contemporary Topics in Immunobiology” (A. .I. S. Davies and R. L. Carter, Eds.), Vol. 2, pp. 321-337. Plenum, New York, 1973. 3. Goldstein, A. L., and White, A., In “Contemporary Topics in Immunohiology” (A. J. S. Davies and R. L. Carter, Eds.), Vol. 2, pp. 339-350. Plenum, New York, 1973. 4. Friedman, H. (Ed.), Ann. N. Y. Acad. Sci. 249, 5. 1975. 5. Bach, J.-F., and Carnaud, C., Progr. Allergy 21, 342, 1976. 6. Bach, J.-F., Transplant. Proc. 8, 243, 1976. 7. Goldstein, G.,ln “Progress in Immunology III” (T. E. Mandel, C. Cheers, C. S. Hosking, I. F. C. McKenzie, and G. J. V. Nossal, Eds.). pp. 390-396. North-Holland, Amsterdam, 1977. 8. Komuro, K., and Boyse, E. A., Lancer 1, 740, 1973. 9. Touraine, J. L., Incefy, G. S., Touraine. F.. Rho. Y. M., and Good, R. A., C/in. Exp. Immunol. 17, 151, 1974. 10. Lonai, P., Mogilner, B., and Rotter, V., Eur. J. Immunol. 3, 21, 1973. 11. Rotter, V., and Trainin. N., Cell. Immunol. 16, 413, 1975. 12. Touraine, J. L., Hadden, J. W., and Good, R. A., Proc. Nat. Acad. Sci. U.S.A. 74, 3414, 1977. 13. Stutman, O., Yunis, E. J., and Good, R. A., J. Exp. Med. 132, 601. 1970. 14. Globerson, A., Umiel, T., and Friedman, D., Ann. N.Y. Acad. Sci. 249, 248, 1975. 15. Asantila, T., Vahala, J., and Toivanen, P., immunogenetics 1, 272, 1974. 16. Asantila, T.. Vahala, J., and Toivanen, P., Immunogenetics 1, 407, 1974. 17. Asantila, T., and Toivanen, P.. J. Immurzol. 117, 555, 1976. 18. Toivanen, P., Asantila, T., and Vahala, J., In “Immuno-Aspects of the Spleen” (J. R. Battisto and J. W. Streilein, Eds.), pp. 13-24. North-Holland, Amsterdam, 1976. 19. Leino. A., C/in. Zmmunol. Immunopothol. 11, 6. 1978. 20. Eskola. J., Soppi, E., Viljanen, M., and Ruuskanen, O., Zmmunol. Commun. 4, 297, 1975. 21. Asantila, T., and Toivanen, P., J. Imnntnol. Meth. 6, 73. 1974. 22. Goldstein, A. L., Thurman, G. B., Low, T. L. K.. Rossio, J. L.. and Trivers. G. E., Reticuloendothel. Sot. 23, 253. 1978. 23. Touraine, J. L., Touraine, F., Incefy. G. S.. Goldstein, A. L., and Good, R. A., In “The Biological Activity of Thymic Hormones” (D. W. van Bekkum, Ed.), np. 31-35. Kooyker Scientific, Rotterdam, 1975. 24. Bach, J.-F., Dardenne, M., Goldstein, A. L., Guha, A., and White, A., Proc. Not. Acud. Sci. U.S.A. 68, 2734, 1971.
EFFECT
OF THYMOSIN
ON FETAL
LAMB
LYMPHOCYTES
14.5
25. Asherson, G. L., Zembaia, M., Mayhew, B., and Goldstein, A., Eur. J. Immunol. 6, 699, 1976. 26. Mukhopadhyay, N., Fernbach, D. J., Mumford, D. M., and South, M.-A.. C/in. Immurrol. fmmunopathol. 10, 59, 1978. 27. Rotter, V., and Trainin, N., Israel J. Med. Sci. 13, 363, 1977. 28. Goldstein, A. L., Thurman, G. B., Cohen, G. H., and Hooper, J. A., In “Molecular Approaches to Immunology” (E. E. Smith and D. W. Ribbons, Eds.), pp. 243-262. Academic Press, New York,
1975.
IMMUNOLOGY
Effect
AND
IMMUNOPATHOLOGY
of Thymosin on PHA and Con A Responses Fetal Lamb Lymphocytes] AILA
Departments
13, 136-145 (1979)
of Medical
LEINO~
Microbiology Turh
AND
by
ESA SOPPI
and Atlatomy. 52. Finland
Turks
University.
SF-20520
Received November 14, 1978 The effects of thymosin fraction 5 on the mitogen responses of fetal lamb lymphocytes in serum-free cultures were studied. The cells from liver, thymus, spleen, and bone marrow of 12 fetal lambs of four different ewes at 48-98 days of gestation (total gestation is 150 days) were used. Cells from the liver, bone marrow, and spleen were separated by Ficoll-Isopaque density-gradient centrifugation. At 48 to 68 days of gestation, fetal liver cells had only a slight capacity to respond to PHA and Con A mitogens; however, these responses could be induced by thymosin. Later on, when the fetal lymphocytes including those from liver, thymus, spleen. and bone marrow started to respond to mitogens (from Day 78 onward), the effect of thymosin became negligible.
INTRODUCTION
The thymus plays a crucial role in the development and maintenance of cellmediated immunity, producing large numbers of lymphocytes, at least some of which leave the gland and form T-lymphocyte populations in the periphery. On the other hand, the thymic epithelial cells produce humoral factors which affect the functional maturation and differentiation of lymphocytes ( 1-7). Thymic factors have been shown to promote the differentiation of human and murine spleen and bone marrow lymphocytes into more mature T cells as judged by the appearance of specific T-cell surface markers and mitogenic responses (S- 12). Stutman et al. (13) have reported that 19- to 2 l-day fetal liver cells in mice are sensitive to the humoral activity of thymus and that this property is still present a few days after birth. Komuro and Boyse (1973) have shown in mice that treatment of fetal liver lymphocytes with thymosin as early as Day 14 of gestation induces the appearance of Thy-l- and TL-positive cells. Furthermore, Globerson et al. have shown that a partially purified calf thymus extract matures murine fetal liver cells so that they can induce graft-versus-host reaction ( 14). Studies carried out in our laboratory by Asantila et al. (15- 18) have demonstrated that fetal lymphocytes are capable of specifically recognizing foreign cells almost immediately when the thymus has become lymphoid (i.e., at 12 weeks of gestation in man and at 58 days of gestation in sheep). Also, cells responding to phytohemagglutinin (PHA) appear in the sheep thymus during the first weeks of lymphoid development in utero, and concanavalin A (Con A)-responding cells about 2 weeks later (19). These findings have prompted us to investigate how early during the ontogeny fetal T-cell precursors in the sheep are susceptible to thymic I Supported by a contract with Association of Finnish Life Insurance Companies. 1 Address correspondence to: Mrs. Aila Leino, M.S.. Department of Medical Microbiology, University, SF-20520 Turku 52, Finland. 136 0090-1229/79/060136-10$01.00/0 Copyright All rights
@ 1979 by Academic Press. Inc. of reproduction in any form reserved
Turku
EFFECT
OF
THYMOSIN
ON
FETAL
LAMB
LYMPHOCYTES
137
humoral factors. For this purpose we have studied the effect of thymosin fraction 5 on the mitogenic responses of fetal liver, thymus, spleen, and bone marrow cells at different stages of gestation in fetal lambs. MATERIALS AND METHODS Fetuses. Twelve fetal lambs from four ewes of the Finnsheep strain were used. The ewes were killed at appropriate intervals and the fetuses were delivered by hysterotomy. The ages of the fetuses varied between 48 and 98 days (total gestation period is 150 days). Preparation of cells. Liver, thymus, and spleen of the fetuses were excised, cut in pieces, and placed in medium TC 199 (Orion, Helsinki, Finland). Bone marrow cells were obtained from femora by flushing medium through the bones. Singlecell suspensions were made with the aid of a Tenbroeck-type homogenizer. Ficoll -1sopaque gradient separation was applied for further purification of liver, bone marrow, and spleen cells, using a density of 1082. Preincubarion with thymosin. Thymosin fraction 5 (Lot BPM 390) prepared from calf thymuses was generously supplied by Dr. Allan Goldstein. Cells (10 x 106/ml) were preincubated before mitogenic stimulation with different concentrations (0.001, 0.01, 0.1, 1.0, 10, 100, 200 &ml) of thymosin in medium RPM1 1640 (Grand Island Biological Co., Grand Island, N.Y.) without any serum at 37°C for 2 hr and washed twice with large amounts of the medium. The controls were preincubated with medium RPM1 1640 and washed as the test cells before the culture. Mitogen-stimulated cultures. The culture system of fetal lymphocytes was based on the method described by Eskola ef al. (20). After thymosin treatment, serum-free suspensions of 5 x lo5 separated cells were cultured in 100 pi/ml of RPM1 1640 in microtiter plates (Cooke Microtiter M-24AR Dynatech, Zug, Switzerland) for a total period of 66 hr at 37°C with 5% CO,. In a pilot study, concentrations of phytohemagglutinin (PHA M, Difco Laboratories, Detroit, Mich.) from 31.25 to 1000 pg/ml and concanavalin A (Con A, Pharmacia Fine Chemicals, Uppsala, Sweden) from 0.25 to 8 pg/ml were tested. On the basis of these results. concentrations of 125 &ml for PHA and 2 pg/rnl for Con A were used throughout the study. After incubation for 48 hr, 0.25 &i of ‘?“I-labeled 5-iodo-2’deoxyuridine ([‘251]UdR, specific activity 90- 110 mCi/mg, The Radiochemical Centre, Amersham, England) was added in a volume of 20 @‘ml with 5-fluoro-2’deoxyuridine (FUdR, Fluka, Buchs, Switzerland) to increase the uptake of [1251]UdR (21). The final concentration of FUdR was 10-fi M. Harvesting was performed 64 hr after the start of the cultures. Radioactivity was measured in a Wallac-type gamma counter (Wallac, Turku, Finland). All cultures were performed in triplicate, and the results were calculated as the arithmetic mean of the observed counts per minute (cpm). Mitogen-stimulated incorporation was defined as the difference between the incorporation in cultures incubated with and without mitogen. Statistics. Student’s I test for matched pairs was employed. RESULTS Effect of Thymosin Preincubation on Mitogen Responses by Fetal Liver Cells Effect on PHA responses. Preincubation with thymosin increased PHA responses by liver cells of 48- and 68-day-old fetuses, i.e., to about the midway of
48
48 48 68
68 68 78 78 98 98 98 98
1
2 3 4
5 6 7 8 9 10 11 12
613 475 907 12685 1222 5842 1983 11987
(3.9) (4.2) (1.2) (2.1) (1.3) (3.4) (1.5) (4.3)
3936 (13.0) 714 (8.1) 103 (1.3)
1854 (1.7)
-
LIVER
(4.1) (4.7) (2.3)
1408 (5.6) 945 (3.8)
7608 2578 513
(2.6)
0.001
4647
OF FETAL
-
CELLS
(2.8)
AFTER
in parentheses
(5.5) (3.4) (1.3) (2.3) (1.3) (2.9) (1.9) (6.6)
7394 (3.6) 2830 (2.6) 865 (3.4)
4368
0.01
TO PHA
1431 651 1482 15660 1380 5070 4260 12489
0 Mean cpm of triplicate cultures are given. h Net cpm: stimulated minus unstimulated. Figures fold or greater increases caused by thymosin.
Age (days)
No.
Fetuses
RESPONSE
TABLE
1
(3.7) (1.7) (4.5)
give
1090 4812 2871 14004
stimulation
(1.3) (2.5) (2.0) (6.4)
1114 (4.4) -36 (0.9)
8037 2004 1355
41.56 (2.9)
0.1
PHA-stimulated (thymosin
PRETREATMENT
DIFFERENT
(3.2)
(2.4)
1407 5001 728 8094
(1.4) (2.3) (1.1) (3.4)
960 (5.1) 210 (2.2)
6497 (3.7) 325.5 (1.7) 1270 (5.5)
3682
IO
CONCENTRATIONS
(stimulatediunstimulated).
(1.6) (2.5) (1.4) (2.2) (1.2) (4.3)
2300 16652 1450 4571 1154 9012
indices
(4.5)
1269
8420 (3.9) 4829 (2.1) 1300 (3.6)
4301
1.0
cultures* &ml)
WITH
Figures
651 48 2719 12679 -519 5095 5405 8751
in italics
(3.6) (1.3) (1.9) (2.1) (0.9) (3.5) (2.2) (2.9)
6312 (4.0) 9068 (2.1) 724 (3.8)
35.51 (2.7)
100
OF THYMOSIN~
(2.3)
represent
92 (1.5) 17 (1.1)
5110 (4.0) 2538 (1.6) 192 (1.7)
2653
200
two-
$ 21
5 u
L z 0
EFFECT
OF
THYMOSIN
ON
FETAL
LAMB
LYMPHOCYTES
139
the gestation (Table 1). With the three 48-day-old fetuses considered as a group, the values obtained with all the thymosin concentrations used (0.001-200 pi/ml) were significantly different from the control values (P < 0.05 by the Student’s t test for matched pairs). Likewise, 0.001 and 0.01 &ml of thymosin induced significant increases in the group of three 68-day-old fetuses (P < 0.05). This effect of thymosin was observed first of all on net cpm (stimulated minus unstimulated); a twofold or greater increase in net cpm was obtained with thymosin concentration as low as 0.001 &ml (fetus Nos. 1 and 3-6, Table 1). With cells from a fetus with an already existing PHA response by liver cells (No. 2 in Table l), a twofold increase was obtained first with a thymosin concentration of 0.1 pg/ml. Although the effect of thymosin on PHA responses by fetal liver cells was clearly observed as an increase in net cpm, it was only rarely demonstrable as an increase of stimulation indices, indicating that thymosin also increases cell proliferation in cultures without the mitogen. After midpregnancy, no statistically significant changes (P > 0.05) were obtained in the groups of 78- and 98-day-old fetuses, because thymosin had an effect only occasionally on the cells of these fetuses, and even then only at high concentrations (fetus Nos. 7 and 11 in Table 1). Effect on Corz A responses. At first glance the effect of thymosin on Con A responses by fetal liver cells (Table 2) seems to be similar to its effect on PHA responses. It appears, however, that the effect of thymosin is strongest when the spontaneous response to Con A is weak or absent. Fetal liver cells of 48- to 68-day-old fetuses (Nos. 2-5 in Table 2) did not yet respond to Con A, but preincubation of these cells with different concentrations of thymosin resulted in twofold or greater increases in net cpm. With the cells of older fetuses, similar increases were observed if the cells did not spontaneously respond to Con A (fetus Nos. 9 and 11 in Table 2). Exceptions were presented by fetus Nos. 6 and 7. Fetal liver cells of these two fetuses did not respond spontanously to Con A, and responses could not be induced by preincubation with thymosin. On the other hand, fetal liver cells of four fetuses (Nos. 1, 8, 10, and 12) responded to concanavalin A, and preincubation with thymosin had no effect on these cells. As seen for the effect of thymosin on PHA responses, its effect on Con A responses was also observed in net cpm and only occasionally in stimulation indices. However, due to great individual variations, the differences observed do not reach, neither for net cpm nor stimulation indices, statistical significance when fetuses of each age are considered a group (P > 0.05 by Student’s f test for matched pairs). Effect of Thymosin Preincubation on Mitogen Responses by Cells from Fetal Thymus, Spleen. and Bone Marrow* Thymus. There was considerable variation in the capacity of thymus cells of five fetal lambs to respond to PHA and Con A, both in net cpm and in stimulation indices (Tables 3 and 4). For instance, fetus No. 7 at 78 days of gestation did not spontaneously respond to PHA, but small increases both in net cpm and in stimulation indices were induced by pretreatment with thymosin (Table 3). Cells from another fetus of the same age (No. 8) showed a high spontaneous proliferation without a significant PHA response, and this response was not influenced by thymosin. Thymus cells of three 98-day-old fetuses showed low responses to both mitogens, but only in one of them (No. 11) was the response increased by thymosin. As an example of the variability, the effect of thymosin on Con A responses in
Age
68 68 68 78
78 98 98 98 98
4 5 6 7
8 9 10 11 12
27331 809 3550 -553 8711
-136 32 126 441
(3.5) (1.2) (2.4) (0.9) (3.4)
(0.6) (1.2) (1.8) (1.1)
9001 (3.9) 966 (2.3) 81 (2.3)
-
0.001
LIVER
-105 (0.7) 318 (2.0) 146 (1.4)
5310 (2.8) 4788 (3.3) 296 (1.8)
OF FETAL
CELLS
A AFTER
(4.1) (1.3) (2.1) (2.1) (6.1)
(1.1) (2.3) (0.9) (1.1)
in parentheses
36440 1278 2818 5563 11238
30 400 -27 273
4997 (3.1) 3685 (2.7) 456 (1.4)
0.01
TO CON
n Mean cpm of triplicate cultures are given. b Net cpm; stimulated minus unstimulated. Figures fold or greater increases caused by thymosin.
48 48 48
(days)
1 2 3
No.
Fetuses
RESPONSE
TABLE
(3.2) (2.3) (0.9)
0.1
(thymosin
stimulation
609 (1.1) 3179 (2.0) 1475 (1.5) 9756 (4.7)
give
2
A-stimulated
567 (2.5) -120 (0.6) 74 (1.3)
4259 3713 -104
Con
PREATREATMEKT
DIFFERENT
1.0
indices
41525 2622 2618 162 11920
-2047
(2.6) (2.5) (0.8)
1655 1569 640 4143
(1.4) (1.4) (1.1) (2.2)
143 (1.5) -211 (0.5) -19 (0.8)
3377 3806 -729
10
CONCENTRATIONS
(stimulatediunstimulated).
(4.8) (1.8) (1.7) (1.0) (5.4)
(0.5)
677 (2.4) 13-o (1.4)
3942 (3.2) 4533 (2.5) 309 (1.1)
&ml)
culturesb
WITH
Figures
38109 367 2307 132 3300
7 40 -96 176
3671 3643 4035
in italics
(4.2) (1.1) (2.1) (1.0) (1.7)
(1.0) (1.2) (0.4) (1.1)
(2.7) (2.6) (1.7)
100
OF THYMOSIN”
(0.5) (0.7) (0.8)
(2.5) (2.8) (0.8)
represent
-138 -64 -31
3417 3476 -661
200
two-
B z
z
8
c
r
-0~1
(L’ZZ) (C’Z) (L’f) ZIZEI LIZ01 ZL6S Lf9ZI 088 6ZP
St%f 698 LZZ9
wasalda
(6’1) (O'f) (L’I) (f’t4 (S’S) (P’f) ts.sz) 696f (8'1).1199 (0'1) t(1.1) 9892. (E’Z) 162
sx[q
ZZOZI 6655 SOLL PO68
PS9P
(VI) (Z’Z) (O’Z) (Z.f) (S’f) (1.5)
(Z’PI) (1.5) (P’Z) 6999 ISZ9 9ow 69101 91L 6Sf
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‘(pale~nru!lsun/paie~n~!ls)
f69P DZL 6LI
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(L’I) (O’Z) (Z’S) (Z’S)
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(S'PI) (8'1) (Z'I) (Z’I) (6’1)
saxpu!
(E’LI)
(8.1) (Z’Z) (5.2) K’Z)
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86 8L 8L
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uaqds
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86 86 86 86 8L 8L
ZI II 6 8 L
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(1’81) (9'1) (9.z)
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86 86 86 8L 8L
ai\!8 sasaqluaed
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(6’1) (I’f) K’s) (6.5) (6.Z) K'Z)
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(P.8) (f.1) (P’Z)
6568 LSZS SO88 SESOI 6Sf 6-
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NOa LLEZI SZLS P68L 5198
(L’I) (Z’Z) (L’Z) (L’Z) t1.Z) (0'1)
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(f’8) (9’S) (8'1) (8’0) (P’Z)
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98
98 98
11 12
II
78 98 98
8 9 IO
78
98 78
12 7
78
98
II
8
78 98
7
78
8 9
(days)
7
No.
-309
(0.5)
(2.3) (2.5) (2.2) (3.3)
2815 (1.8) 2665 (2.8) 646 (3.3)
loo (1.3) 33420 (7.3) 20361 (4.9) 6565 (3.1) 42226 (4.2)
177 (2.2)
22221 409 69 631
-
AND BONE MARROW
(1.9) (10.2) (1.2) (1.0)
(4.3)
3605 (1.9) -401 (0.7) 116 (1.2)
4205 (2.0) 32229 (3.7)
17207
16 (1.0) 32709 (6.3)
360 371 107 2
-93 (0.7) 9778 (1.5)
0.01
WITH DIFF~RENI-
(6.6) (4.5) (2.1) (3.9)
396 (2.1)
32687 18565 4868 44621
-146 (0.7) 24978 (417.2) 544 (2.4)
0.1
(0.7)
(0.7) (1.2)
483 (2.8)
(0.2)
(2.5) (3.2)
-238 (0.6) 146 (1.8)
5037
47636 (4.2)
7709
171 (1.9) 37143 (10.8) 22542 (3.5)
23.754 (3.8) 174 (2.1) 144 (1.8)
-272
-58 (0.7) 14128 (1.9)
100
OF THYMOSIK”
Figures in italics represent two-
(5.1)
3861 (2.1) -904 (0.2) 450 (2.1)
25758
7061 (2.3) 48917 (3.8)
32645 (5.4)
90 (1.2) 254414 (3.8) 417 (2.4)
10
CONCENTRATIOW
(1.3) (8.2) (4.1) (2.1) (3.2)
86 33809 20885 5540 35962
(3.9) (2.2) 79 (2.1)
-65 16575 -132 20400 459
1.0
Con A-stimulated cultures0 (thymosin &ml)
TABLE 4 A AFTER PREATREAYMEW
CELLS 2.0 CON
I’ Mean cpm of triplicate cultures are given. b Net cpm; stimulated minus unstimulated. Figures in parentheses give stimulation indices (stimulated/unstimulated). fold or greater increases caused by thymosin.
Bone marrow
Spleen
Thymus
Cell type
SPLEEN,
Fetuses
RESPONSE OF FETAL THYMLIS.
s -2
% 0
5 0
K
EFFECT
OF THYMOSIN
ON
FETAL
LAMB
LYMPHOCYTES
143
net cpm was the same with concentrations of 0.1 to 100 pg./ml, whereas 0.1 pugirnl had the smallest effect on unstimulated cultures, resulting in an enormous thymosin effect when expressed as a stimulation index. Spleen and bone marrow. Pretreatment with thymosin did not affect the mitogen responses of spleen and bone marrow cells (Tables 3 and 4). Spleen cells responded weakly to PHA and Con A at the age of 78 days; at 98 days the responses appeared somewhat higher, particularly to Con A. Bone marrow cells of only three fetuses (78-98 days) were studied; low spontaneous responses to PHA and Con A were recorded; and thymosin pretreatment had no effect (Tables 3 and 4). DISCUSSION The effect of thymosin seems to be strongest on fetal cells from stages before midpregnancy. Both PHA and Con A responses by liver cells of 48- and 68-dayold fetal lambs were increased by a small concentration of thymosin (0.001 pg/ml), suggesting that weakly responding cells mature as a result of pretreatment with thymosin. In older fetuses (78-98 days), the mitogen responses were already developed, not only in the fetal liver cells but also in the thymus, spleen, and bone marrow cells. At that time the effect of thymosin was weaker, and on the few occasions when it becomes manifest, higher concentrations of thymosin were required than at earlier stages of gestation. For fetal liver cells, no uniform optimal concentration of thymosin exists, and concentrations throughout the whole range used (0.001-200 &ml) were equally effective. It is possible that autologous thymus of the fetuses, which is lymphoid after the age of 40-45 days, may already produce humoral factors affecting fetal T-cell precursors in early stages of fetal development, and that the more these factors are available, the higher concentrations of thymosin are required to increase the cells’ capacity to respond to PHA and Con A. However, if mitogen responses have not yet developed they can be increased with small concentrations of thymosin, even during the later part of gestation (for instance fetus Nos. 9 and 11 in Table 2). On the other hand, since thymosin fraction 5 consists of more than 30 different components, and neither of those 2 purified so far (thymosin Q~,(Y,) is alone responsible for all the effects (22), it is possible that cells of fetal lambs at different stages are susceptible to different components, resulting in variation in the optimal concentrations. In spite of the complex nature of thymosin fraction 5, this preparation has high specificity. It has been shown that control fractions prepared from spleen (12, 23-25) or from other sources (24) in the same manner as thymosin are unable to produce the effects of thymosin in various experimental conditions including mitogen responses. Spontaneous proliferation of fetal liver cells is increased by preincubation with thymosin. particularly with cells from the early stages of gestation, when the effect of thymosin is also strong in mitogen-stimulated cultures. This might be explained by thymosin-initiated maturation which continues during the whole culture period and results in an increased spontaneous proliferation. Thymosininitiated maturation during the preincubation may also explain some of the discrepancies observed in different studies, with different incubation times, concern-
144
LEINO
AND SOPPI
ing the time required for maturation of adult and fetal cells to more immunocompetent lymphocytes (8, 9, 12, 26). Conflicting views have been presented regarding the pre- or postthymic nature of target cells for the humoral thymic function. According to Komuro and Boyse (8) the target cells are prethymic, whereas Stutman et al. (13) and Rotter and Trainin (27) consider them postthymic, and Goldstein et al. (28) find prethymic, thymic, and postthymic cells susceptible to thymosin. In the present work, the effect of thymosin was strongest on fetal liver cells obtained during the first half of gestation. The thymus was already lymphoid at the time when the first liver cells were studied (at 48 days of gestation), and these may be prethymic or postthymic cells. Thus, no definite answer to this question can be provided on the basis of the data described here. ACKNOWLEDGMENTS We wish to thank Dr. Paavo Toivanen for constructive and invaluable criticism, Mrs. Hillevi Tolkko for technical assistance, and Dr. Esko Salonen for the hysterotomies.
REFERENCES 1. Stutman, O., and Good, R. A., In “Contemporary Topics in Immunobiology” (A. .I. S. Davies and R. L. Carter, Eds.), Vol. 2, pp. 299-319. Plenum, New York, 1973. 2. Trainin, N., and Small, M., In “Contemporary Topics in Immunobiology” (A. .I. S. Davies and R. L. Carter, Eds.), Vol. 2, pp. 321-337. Plenum, New York, 1973. 3. Goldstein, A. L., and White, A., In “Contemporary Topics in Immunohiology” (A. J. S. Davies and R. L. Carter, Eds.), Vol. 2, pp. 339-350. Plenum, New York, 1973. 4. Friedman, H. (Ed.), Ann. N. Y. Acad. Sci. 249, 5. 1975. 5. Bach, J.-F., and Carnaud, C., Progr. Allergy 21, 342, 1976. 6. Bach, J.-F., Transplant. Proc. 8, 243, 1976. 7. Goldstein, G.,ln “Progress in Immunology III” (T. E. Mandel, C. Cheers, C. S. Hosking, I. F. C. McKenzie, and G. J. V. Nossal, Eds.). pp. 390-396. North-Holland, Amsterdam, 1977. 8. Komuro, K., and Boyse, E. A., Lancer 1, 740, 1973. 9. Touraine, J. L., Incefy, G. S., Touraine. F.. Rho. Y. M., and Good, R. A., C/in. Exp. Immunol. 17, 151, 1974. 10. Lonai, P., Mogilner, B., and Rotter, V., Eur. J. Immunol. 3, 21, 1973. 11. Rotter, V., and Trainin. N., Cell. Immunol. 16, 413, 1975. 12. Touraine, J. L., Hadden, J. W., and Good, R. A., Proc. Nat. Acad. Sci. U.S.A. 74, 3414, 1977. 13. Stutman, O., Yunis, E. J., and Good, R. A., J. Exp. Med. 132, 601. 1970. 14. Globerson, A., Umiel, T., and Friedman, D., Ann. N.Y. Acad. Sci. 249, 248, 1975. 15. Asantila, T., Vahala, J., and Toivanen, P., immunogenetics 1, 272, 1974. 16. Asantila, T.. Vahala, J., and Toivanen, P., Immunogenetics 1, 407, 1974. 17. Asantila, T., and Toivanen, P.. J. Immurzol. 117, 555, 1976. 18. Toivanen, P., Asantila, T., and Vahala, J., In “Immuno-Aspects of the Spleen” (J. R. Battisto and J. W. Streilein, Eds.), pp. 13-24. North-Holland, Amsterdam, 1976. 19. Leino. A., C/in. Zmmunol. Immunopothol. 11, 6. 1978. 20. Eskola. J., Soppi, E., Viljanen, M., and Ruuskanen, O., Zmmunol. Commun. 4, 297, 1975. 21. Asantila, T., and Toivanen, P., J. Imnntnol. Meth. 6, 73. 1974. 22. Goldstein, A. L., Thurman, G. B., Low, T. L. K.. Rossio, J. L.. and Trivers. G. E., Reticuloendothel. Sot. 23, 253. 1978. 23. Touraine, J. L., Touraine, F., Incefy. G. S.. Goldstein, A. L., and Good, R. A., In “The Biological Activity of Thymic Hormones” (D. W. van Bekkum, Ed.), np. 31-35. Kooyker Scientific, Rotterdam, 1975. 24. Bach, J.-F., Dardenne, M., Goldstein, A. L., Guha, A., and White, A., Proc. Not. Acud. Sci. U.S.A. 68, 2734, 1971.
EFFECT
OF THYMOSIN
ON FETAL
LAMB
LYMPHOCYTES
14.5
25. Asherson, G. L., Zembaia, M., Mayhew, B., and Goldstein, A., Eur. J. Immunol. 6, 699, 1976. 26. Mukhopadhyay, N., Fernbach, D. J., Mumford, D. M., and South, M.-A.. C/in. Immurrol. fmmunopathol. 10, 59, 1978. 27. Rotter, V., and Trainin, N., Israel J. Med. Sci. 13, 363, 1977. 28. Goldstein, A. L., Thurman, G. B., Cohen, G. H., and Hooper, J. A., In “Molecular Approaches to Immunology” (E. E. Smith and D. W. Ribbons, Eds.), pp. 243-262. Academic Press, New York,
1975.