Phase dependent proliferative response of lymphoid and erythroid cells in the pregnant mouse

AND COMPARATIVE IMMUNOLOGY, VOl. 4, pp. 737-746, 1980 0145-305X/80/040737-10502.00/0 Printed in the USA. Copyright (c) 1980 Pergamon Press Ltd. All rights reserved. DEVELOPMENTAL

PHASE DEPENDENT PROLIFERATIVE RESPONSE ERYTHROID CELLS IN THE PREGNANT MOUSE

OF LYMPHOID AND

Ragnar Mattsson, Anita Mattsson and Kerstin Lindahl-Kiessling Institute of Zoophysiology, University of Uppsala, Box 560 S-751 22 Uppsala, Swedeq

ABSTRACT

Spontaneous mitotic activity in different maternal lymphoid organs was evaluated in pregnant CBA mice by in vivo and/or in vitro administration of -H-thymidine. Thym~d~ne uptake in thymus, spleen and inguinal lymph nodes increased transiently at the time of implantation, a response perhaps induced by a preimplantation peak of luteinizing hormone (LH). At mid-pregnancy there was a secoqd, more pronounced increase in splenic uptake of 5H-thymidine, which may be due partly to a hormonal response induced by chorionic gonadotrophin (CG). Both B and T cells were involved in the proliferative response of spleen cells, but the B/T activity ratio increased in late pregnancy was also accompanied by formation of splenic erythroblasts. INTRODUCTION

Splenic enlargement occurs in pregnant mice around mid-gestation (1-4), and there are data indicating that lymphoid cells are under proliferation (4). Some authors have noted a marked change in splenic histology, but there is also an increase in the number of plasma cells after the lOth day of mouse gestation, indicating activation of B lymphocytes (3,4). Fabris and coworkers have shown that spleeq cells from pregnant mice respond to sheep red blood cells (SRBC) to a much higher extent than spleen cells of virgin control mice after in vivo administration of the antigen (5). They suspected that chorionic gonadotrophin (CG) played a major role in altering the reactivity of maternal immunocompe tent cells and succeeded in imitating the condition of spleen cell reactivity during pregnancy by giving virgin mice daily injections of human CG (HCG) (6). Thus, in one way or another, gonadotrophins and their release of steroid hormones seem to be involved in the regulation of maternal lymphocyte activity. Pregnant mice show depressed cell mediated immuqity (in vitro) (6), as well as reduced in vitro blastic transformation in response to mitogene (7, 8). However, it is well known that in vitro responses following induction of mitotic activity is strongly dependent upon cell concentration. Therefore, reduced lymphocyte reactivity observed 737

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may simply mirror an increase in the proportion of erythroblasts in the spleen cell suspensions used, as splenic erythropoiesis is increased in pregnant mice (2). In this investigation~ we have analysed the pattern of changes in mitotic activity in different maternal lymphoid organs during the mouse pregnancy to clarify some of the underlying regulation mechanisms involved in the regulation of immunological fuqction during pregnaqcy. At the same time~ we analysed the degree of splenic erythropoiesis as well as spontaneous mitotic activity of splenic B and T lymphocytes at different phases of pregnancy. The possible role of gonadotrophins, luteinizing hormone (LH) and HCG, in the induction of the pregnancy specific alteration of the spleen cell population was also investigated. MATERIALS

AND METHODS

Mice CBA mice (100 days old) were purchased from Anticimex, Stockholm, Sweden° All mice were healthy and received food (staqdard food pellets, Ewes Ltd. Sthlm. Sweden) and tad water daily. Experimental

procedure:

pregnant

mice

CBA female mice were allowed to mate with males of the same strain. Groups of five pregnant and 2-5 virgin mice were investigated on day 2, 4~ 8, 12 and 16 (day 1 = the day when the vaginal plug is first

seen), Mitotic

activity

in vivo

The in vivo uptake of 3H-thymidine (Amersham, Engl.) by spleen, outsqueezed spleen cells, thymus, liver and inguinal lymph nodes was investigated by liquid scintillation technique. All mice were injected i.v. with 0.5 ~Ci 3H-thymidine/g body weight (50 ~Ci/ml, 40 Ci/mol) I hour before killed. The organs were weighed and dissolved in 2 ml 0.3 M KOH (24 hours~ 50°C)~ neutralized with HC1, mixed with oCS scintillation fluid (Nuclear Chicago) and counted in a liquid scintillation counter. Sections of the spleens were previously transferred to cold RPMI 1640 (Flow ltrs.) for further investigations. Before adding PCS to the dissolved orgaqs samples were traqsferred to plastic test tubes for protein analysis (Lowry et al., 9). The uptake of H-thymidine was then evaluated for protein content. Preparation

of spleen

cell suspensions

The spleen sections were transferred to 6 cm Petri dishes (A/S Nunc, Denmark) containing cold RPMI 1640. The cells were squeezed out by a pair of pincettes, and the cell suspension transferred to a 10 ml plastic centrifuge tube (Heger AB, Sweden). Tissue fragments were •allowed to sediment for 5 minutes. The supernatant was then transferred to a new tube 9 and the number of nucleated spleen cells was counted in a hemocytometer after suitable dilution in Turks staining reagent (0.2% gentian violet in 6% acetic acid).

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Preparation

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of B cell suspensions

Nucleated spleen cells (I0xi06) were resuspended in 0.5 ml RPMI 1640. 50 pl AKR-anti-CBA-g serum was added to the tube, which was allowed to stand for 30 minutes at room temperature. The cells were then washed once (200 g/10 min) and resusoended in 0.5 ml fresh medium. Guinea pig serum (complement) (50 pl) was added to the tube, which was theq incubated for 30 minutes at 37°C; g-positive cells should be killed by this treatment. The proportion of g-positive cells (T-cells) was estimated from a count of the relative proportion of dead cells after 3 minutes incubation in 0.1% trypan blue (Schmid & Co, Stuttgart, Germany) in RPMI 1640. CBA thymocytes were used as positive controls. The B-cell suspension was then washed and r~suspended in the culture medium at a final concentration of 1.0xi0 viable cells/ml. Preparation

of T cell suspensions

Nucleated spleen cells (20x106) were resuspended in 2 ml RPMI 1640 containing 2 mM L-glutamine and 5% heat inactivated pooled human AB serum. Cells were then incubated for 30 minutes at 37°C in a nylon wool column. Nonadherent cells (T-cells) were eluted into a plastic tube by passing 5 ml of warm culture medium through the column. Nylon wool columns were prepared as follows. Nylon wool (0.15 g) was packed in a 5 ml syringe (Sherwood medial industries ltd, Crawley Sussex, England) to a final volume of 3 ml. The column was preincubated at 37Uc with the medium described above for at least an hour before adding the cell suspension. The accuracy of the separation was confirmed by incubatioq with the anti-g serum and complement as described above. Mitotic

activity

in vitro

Spontaneous mitotic activity of separated and nonseparated spleen cells was determined by assessing tritium-labelled thymidine. Spleen cells were prepared as described above and cultured in RPMI 1640 supplemented with 2 mM L-glutamine, 20 mM HEPES (Sigma), 50 ~g/ml gentamycin and 5% pooled, h e a t - i n a c t i v a t e d human AB serum (final concentrations follow). The cell concentration was adjusted to Ixi06 viable nucleated cells/ml and cells were cultured in 0.5 ml volumes in 10xi00 mm culture tubes (Corning N Y) at 37°C in humidified air supplemented with CO 2 to a final concentration of 6%. 25 pl 3H-thymidine (50 pCi/ml, 650 Ci/mol) was immediately added to each culture tube, and cells harvested with distilled water 18 hours later by use of cell harvester (Lierbyen, Norway). Incorporated activity was assessed by liquid scintillation spectrometry. Staining

of erythroblasts

To recognize erythroblasts (nucleated cells containing haemoglobin) the Lepehne peroxidase staining (10) was used. In summary, slides were fixed in methanol for 10 minutes and then allowed to react in Lepehne reagent (2 parts of a 0.6% solution of benzidine in 96% ethanol mixed with 5 parts of a 6% solution of perhydrol in 70% ethanol) for 5 minutes. Slides were then washed in tap water, airdried and then stained finally in a 4% dilution in distilled water of original Fluka Giemsa solution.

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Hormonal

PROLIFERATION IN PREGNANT MICE

treatment

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of virgin mice

Groups of 6-10 CBA female mice were injected i.v. with either 10 IU HCG (2570 IU/mg; SIGMA), 10 IU LH (3460 IU/mg; SIGMA) or sterile salineo RESULTS Mitotic

activity

in vivo

The in vivo uptake of 3H-thymidine by spleen, thymus~ inguinal lymph nodes and liver clearly increased on the 4th day of pregnaqcy (the day for implantation) (Fig. I)o On the 8th day the mitotic activity drop~ed in all organs except in the liver, which showed a fairly constant H-thymidine uptake throughout pregnancy. On the 12th day of gestation (the day for the m~ximal splenic enlargement) there was a marked increase in splenic thymidine uptake, and a slight increase in activity of the inguinal lymph nodes. On the 16th day, activity of spleen and lymph nodes again dropped, while activity in the now involuted thymus increased. The in vivo uptake of 3H-thymidine in free spleen cells has been assayed during pregnancy by autoradiography and scintillation photometry (Fig. 2). Both techniques revealed the same results concerning mitotic activity, with typical peaks on the 4th and 12th day of gestation. Mitotic

activity

in vitro

The spontaneous mitotic activity of nonseparated- B-enriched and Tenriched spleen cell suspensions assayed in vitro is shown in Fig. 3o These graphs show ~he same pattern as those obtained after in vivo administratioq of H-thymidine 9 confirming the phase-depende-nnt sponse; the first peak at the implantation phase and the most marked peak at mid-gestation. B- and T-cells respond in the same way, although the activity of B-cells exceeded that of T-cells and both curves seem to diverge in late pregnancy. Splenic

erythroblasts

The prooortion of erythroblasts in the total number of nucleated spleen cells of pregnant mice showed a clear correlation between erythroblast coqcentration and mitotic activity of spleen cells, with the most prominent increase at mid-gestation (Fig. 4). Only haemoglobin containing mononuclear erythroblasts were observed. Hormonal

treatment

of virgin mice

Mice treated with LH and HCG were investigated 1 and 4 days after injection of the hormones. At day 1 spleen cells from both LH and HCG treated mice showed a 2-fold increase in spontaneous mitotic activity assayed in vitro. On the 4th day, spleen cells of HCG treated mice still showed a 2-fold increase in mitotic activity while the activity of cells from LH treated mice dropped to normal values. The number of splenic erythroblasts in HCG treated mice (4 days after injection) was comparable with proportions observed in 4 day pregnant mice (nonsigni-

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d~.,O~ot.

30

20

10

FIG. 1 The in vivo uptake of 3H-thymidine by spleen, thymus, inguinal lymph nodes and liver (referred to the protein content of each organ) is plotted against day after coitus in syngeneically pregnant CBA mice. Open circles = spleen, filled circles = inguinal lymph nodes, open triangles = liver, filled triangles = thymus. Each point represents the mean of five observations. dpm.m~gpot. 5O

40

o% 30

12

20

8

4

0

4

8

12

ffi

day~t~coitus

FIG. 2 The in vivo uptake of 3H-thymidine by free spleen cells, and the proportion of the total number of outsqueezed spleen cells incorporating radioactivity is plotted against day after coitus in syngeneically pregnant CBA mice. Each point represents the mean of ten observations ± standard deviation. Open circles = the in vivo uptake of 3H-thymidine, filled circles = percentage of labelled cells.

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dpm =10"3 12

C)

,~

8

FIG.

1'2

16 day after coitus

3

The in vitro spontaneous uptake of 3H-thymidine in nonseparated, Tenriched and 3-enriched spleen cells from syngeneically pregnant CBA mice is plotted against day afte~ coitus. Spleen cells were cultured in triplicate cultures (0.5 x 10- viable cells/culture) and harvested after 18 hours of incubation. Each point represents the mean of 5 observations (5 x 3 cultures). Open circles = nonseparated cells, filled circles = B-cells~ open triangles = T-cells.

o/o 6 q

0

4

8

12

1'6 day after coitus

FIG. 4 The proportion of Lepehne-positive erythroblasts of the total number of nucleated cells in spleen cell preparations from syngeneically pregnant CBA mice plotted against day after coitus. Each point represents the mean of 10 observations ± staqdard deviation.

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ficant increase in LH-treated mice). In addition, HCG-treated mice showed a slight increase in splenic wet weight 4 days after injection. DISCUSSION During mouse pregnancy there are two critical endocrinological changes necessary for maintenance of gestation, I) the preimplantation peaks of progesterone and LH (11) and 2) the switch from pituitary LH secretion to chorionic gonadotrophin (structurally very similar to LH) secretion at mid-pregnancy (11, 12). The data reported above shows that mitotic activity in both spleen and inguinal lymph nodes increased during these two phases of pregnancy. Mitotic activity of the thymus also seemed to be affected, at least on the 4th day of pregnancy. In addition, injectioq of both LH and HCG increased the 3H-thymidine uptake in spleen cells. All these data indicate a hormone induced nonspecific activation of spleen cells, and probably also cells in other lymphoid organs, during two phases of pregnancy; implantation and mid-gestation. Action of the gonadotrophins LH and CG could be explained by their potency of releasing androgens and other steroid hormones known to activate erythropoiesis. There are, however, reasons to suggest that LH and CG via particular steroid hormones also enhance lymphocyte activity (6). A marked increase in spleen size in pregnant mice on the 12th day of pregnancy has been reported by several authors (1-4), and splenic enlargement was aoparently dependent only upon erythroid hyperplasia (2, 3). Our own experiments clearly show increase in the numbers of splenic erythroblasts at mid-pregnancy, but also, at the time of implantation although this has not been confirmed. However, we do not agree with the hypothesis that proliferation of erythroid cells is the only event modulating the size and histology of the spleen, for our earlier investigations have shown that lymohoid cells also are involved in the splenic response (4). It mu~t be noted that Lepehnepositive erythroblasts have no uptake of H-thymidine, but nevertheless, some of the mitotic activity might originate from dividing proerythroblasts. Our results concerning spontaneous mitotic activity of splenic B- and T-cells (8-enriched and T-enriched cell suspensions) indicate particular activation of B-cells, especially in late pregnancy. This observation is further supported by the results of Fabris that SRBC-immunized pregnant mice produce increased numbers of PFC at mid-pregnancy (5). The degreB of splenic enlargement in pregnant mice is dependent on litter size (2~ 4) and the activity of both lymphoid and erythroid cells seems to be correlated with the spleen size (2, 4, 5). Thus, one would expect that compounds of foetal origin contribute to modulating maternal spleen cell activity. In this context, CG is produced by placental cells and the maternal CG level is probably correlated positively with increased litter size. Another physiologic change in pregqant mice, which is also dependent on litter size, is the increase in body weight and thus, dilution of the blood. To maintain homeostasis, decrease in hematocrit values would activate erythrepoiesis, while dilution of serum immunoglobulins (Ig) would enhance lymphocyte activity and Ig secretion. Therefore, the maternal B cell activity would be expected to be elevated particularly in late pregnancy.

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The discussion above convinces us that peak mitotic activity at implantatioq is induced by endocrinological changes only, while the mid-pregnancy peak is caused partly by CG secretion and partly by a blood dilution effect. Finally, we would like to outline a hypothesis on the biological implications of the immunological and histological changes in maternal lymehoid organs during pregnancy, which we and others have demonstrated previously. According to available data no induction of maternal tolerance against paternal transplantation antigens occurred during gestation (13-15). Furthermore, several reports support our own finding, that maternal immune-reactivity is stimulated rather thaq suppressed (15, 16). Protection of the foetus against maternal immunological aggressiveness seems to emanate from the foeto-placental unit itself (13, 17). Therefore, maternal immunological aggressiveness might be a good @election mechanism killing off any foetal unit which has net developed an appropriate defence mechanism.

BONE MARROW Hormones released by plac~enta~

Pro-T-cells / ~. THYMUS

\

Proliferation. Formation of Tcells with randomly chosen receptor affinities.

\ \

\ activation \

\

\ \ \

Antigenic environment

~

Pro-B-cells BURSA EQU, Proliferation. Formation of Bcells with randomly chosen receptor affinities. /

CENTRAL LYMPHOID TISSUE

j"

T+ B cells collected in -~ SPLEEN, LYMPHNODES and ~" ... OTHER LYMPHOID TISSUE ~' > | selection Proliferation of cell clones carrying receptors with high affinity for the antigen.

PERIPHERAL LYMPHOID TISSUE

FIG. 5 Theoretical maternal lymphocyte mobilization mechanism induced after implantation. Hormones released by the placenta inhibit activity of central lymphoid tissue and simultaneously activate lymphocyte proliferation in peripheral lymphoid tissue°

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On the other hand involution of the thymus in pregnant mammals is a well known phenomenon supposedly induced by an elevated level of steroid hormones. Histological investigations have shown that cortical lymphocytes are primarily affected (18, 19), hence lymphocyte numbers produced in the thymus in the latter part of pregnancy should be markedly reduced. Consequently alleged increases in the immunological capacity can be assigned only to peripheral lymphoid organs. This conclusion is in keeping with our findings that the splenic lymphocyte production increases drastically until day 12. Other authors have shown that both weight and thymidine uptake is increased in a great number of lymph nodes during mouse gestation (18). It remains to be explained how thymic involution and stimulation of peripheral lymphoid organs can be induced by both the same stimulus, e.g. the foeto-placental unit. One has to postulate that central aqd peripheral lymphoid tissues react differently in an acute physiological stress situation. The ensuing physiological advantage would be a tempory increase in numbers of lymphocytes reacting specifically to prevailing antigens at the expense of new, uncommitted thymic lymphocytes. We therefore would like to think that the peripheral and central lymphoid organs are influenced as scheduled in Fig. 5 ACKNOWLEDGEMENTS This work was aided by grants from the Fortia fond, Pharmacia (to Ragnar Mattsson) and from the Swedish Natural Science Research Council grant no. 2159-015 (to Kerstin Lindahl-Kiessling).

REFERENCES I. DAVIS, W.H., BEER, J.R. and COOK, E.F. Effects spleen in mice° D. Mammalogy 42, 53-56, 1961

of pregnancy

on the

2. FOWLER, J.H. and NASH, DoJ. Erythropoiesis in the spleen and bone marrow of the pregnant mouse. Develop. Biol. 18, 331-353, 1968 3. MARONI, E.S. and DeSOUSA, M.A.B. The lymphoid organs during pregnancy in the mouse. A comparison between a syngeneic and allogeneic mating. Clino Exp. Immunol. 13, 107-124, 1973 4. MATTSSON, R.~ NILSSON, B. and LINDAHL-KIESSLING, K. An investigation of splenic enlargement in pregnant mice. DCI accepted for publication July 1979 5. FABRIS, N. Immunological Experientia 29, 610-612,

reactivity 1973

during pregnancy

in the mouse.

6. FABRIS, N., PIANTANELLI, L. and MUZZIOLI, M. Differential effect of pregnancy or gestagens on humoral and cellmediated immunity. Clin. exp. Immunol. 28, 306-314, 1977 7. RUPPERT, B.L. and RICHIE, E.R. Phytohemagglutinin response of murine spleen cells during pregnancy and inhibition of normal phytohemagglutinin response by pregnancy or post-partum serum. Exp. Hemat. 5, 59-68, 1977

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8. FABRIS, N. aqd SERRI, F. Immunological reactivity during pregnancy in the mouse. Immunology in Obstetrics and Gynaecology, Eds. Centaro, A. and Caretti, N. pp 183-187. Exerpta Medica, Amsterdam 1974 9. LOWRY, D.H., ROSENBROUGH, N.J., FARR, A.L. and RANDALL, R.J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275, 1951 10. UNDRITZ, E. Lepehne peroxidase reaction for erythrocytes and erythroblasts. Sandoz Atlas of Haematology. pp. 35. Sandoz ltd. Basle, 1973 11. MURR, S.M., BRADFORD, G.E. and GESCHWIND, I.I. Plasma luteinizing hormone, follicle-stimulating hormone and prolactin during pregnancy in the mouse. Endocrinology 94, 112-116, 1974 12. WIDE, L. and WIDE, Mo Chorionic gonadotrophin in the mouse from implantation to term. J. Reprod. Fert. 57, 5-9, 1979 13. LANMAN, I.T. Transplantation immunity in mammalian Mechanisms of fetal protection against immunologic J. Pediat. 66, 525-540, 1965 14. BEER, A.E. and BILLINGH&M, R.E. Immunobiology duction. Adv. Immunol. 14, 1-84, 1971

pregnancy: rejection.

of mammalian

repro-

15. MARONI, E.S. and PARROTT, D.M. Progressive increase in cell mediated immunity against paternal transplantation antigens in paroeus mice after multiple pregnancies. Clin. Exp. Immunol. 13, 253-262, 1973 16. CARR, M.C., STITES, D.P. and FUDENBERG, H.H. Cellular aspects of the human fetal maternal relationship. II. In vitro response of gravida lymphocytes to phytohemagglutinin. Cell. Immunol. 8, 448-454, 1973 17. JONES, B.M. and KEMP, R.B. Self-isolation of the foetal trophoblast. Nature (London) 221, 829-831, 1969 18. MCLEAN, J.M., MOBLEY, J.G. and SIBBS, A.C.C. Changes in the thymus spleen and lymph nodes during pregnancy and lactation in the rat. J. Anat. 118, 223-230, 1974 19. MILLAR, K.G., MILLS, Po and BAINES, M.G. A study of the influence of pregnancy on the thymus gland of the mouse. Am. J. Obstet. Gynecol. 117:7, 913-918, 1973