Kinetics of lymphoproliferative responses of lymphocytes harvested from the uterine draining lymph nodes during pregnancy in rats

Joutnal o/Reprodut tt ve hmnunoh)gy, 20 ( 1991 ) 93-- I 01 Elsevier SclentLfic Publishers Ireland Ltd

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Kinetics of lymphoproliferative responses of lymphocytes harvested from the uterine draining lymph nodes during pregnancy in rats Miljenko Kapovic and Daniel Rukavina Department o/ Phystology and Immunology, Faculty of Medwme, University o/RIjeka, Olge Ban 22, 51000 Rijeka (Yugoslavta) (Accepted for publication 4 December 1990)

Summary Paraaortic lymph node (PALN) cells were harvested from xirgm and pregnant rats bearing syngeneic or allogeneic fetuses at all stages of pregnancy mcluding the preimplantation period. The specific and non-specific alloreactivity of these cells was analyzed in mixed lymphocyte reactivity (MLR) against mitomycin-C treatedpaternal strain or unrelated cells. Mitogen stimulation of the cell utihzed PHA, ConA and PWM. Cells bearing T cell markers were labeled m an indirect assay using the monoclonal antibodies W3/25 and MRC OX 8. Specific alloreactivlty is strongly suppressed in the pre-lmplantation and implantation stages of pregnancy. Specific and non-specific alloreacttvities were enhanced at mid-pregnancy and normalized by the end of pregnancy. Reactivity to polyclonal mitogens is enhanced at midpregnancy, and the CD4+/CD8 + ratio ts very low during all phases of pregnancy.

Key words: pregnancy in rats," alloreactivity; CD4+/CD8 + ratio," paraaortic lymph nodes.

Correspondence to MIIjenko Kapovlc, Department of Physiology and Immunology, Faculty of Medlcme, Umverslty of R1jeka, Olge Ban 22, 51000 Rijeka, Yugoslavia. 0165-0378/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

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Introduction

At the time of nidation in rodents there is a marked enlargement and increase in cellularity of the lymph nodes draining the uterus, PALNparaaortic lymph nodes (Beer and Billingham, 1974). This phenomenon has been studied at the cellular and molecular level in mice and involves the production of nanogram quantities of cytokine (T cell suppressor factor) that profoundly suppresses maternal reactivity to paternal antigens (Ribbing et al., 1988; Hoversland and Beaman, 1990). Monoclonal antibodies to this cytokine when given during nidation totally prevents pregnancy. The appearance of this factor in the lymph nodes requires the presence of the fertilized embryo as well as pregnancy levels of estradiol and progesterone. The gene for this cytokine J6B7 has been cloned and the molecular weight of the peptide is known (Beaman, 1990; Lee et al., 1990). We reported previously in both primiparous and multiparous mice pregnant by syngeneic or allogeneic males a marked decrease in the alloreactivity of lymphocytes harvested from the draining lymph nodes only at the periimplantation period. Increased levels of alloreactivity were seen at midgestation and a normalization by the end of pregnancy (Doric and Rukavina, 1987). Similar results in rats have been reported by others (Bauminger and Peleg, 1978). Conflicting data in this regard exist in the literature. This may be because most investigators have studied maternal immunoreactivity at one specific stage of pregnancy or after allogeneic pregnancy. Few have reported a sequential analysis of immunoreactivity at all stages of pregnancy as we did in this study. The present investigation was designed to examine specific and nonspecific alloreactivities of lymphocytes from PALN obtained from syngeneically and allogeneically pregnant rats during all phases of pregnancy. The results demonstrated significant changes in the specific alloreactivity depending on the phase of pregnancy from which they were obtained. Materials and Methods

Rats Female rats of the AO(RT1 u) strain were mated to AO or DA (RT1 ~) males, and Y-59 (RT1 c) donors provided target cells for non-specific MLR assays. All rats used in this study were from our colony in the Department of Physiology and Immunology, University of Rijeka, Yugoslavia and were used at the age of 3--5 months. Pregnancies AO females were caged and mated with AO males (syngeneic pregnancy)

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or DA males (allogeneic pregnancy). The day of spermatozoa detection in the vaginal smear was designated as day 1 of gestation.

Controls Three month virgin AO females served as controls.

Lymphoid cell suspensions Right and left paraaortic lymph nodes and spleens were excised aseptically. Lymphoid cells were released by pressing the tissue through a nylon sieve in R P M I 1640 medium and washed three times. Cell viability was determined by trypan blue exclusion test.

Mixed lymphocyte reaction (MLR) Responder cells were P A L N cells from syngeneically or allogeneically pregnant females and virgin controls. P A L N cells were stimulated with mitomycin-C treated spleen cells of paternal strain origin (DA) or unrelated third party strain males (Y-59). P A L N cells were stimulated on days 3, 7, 10, 14, 17 and 21 of gestation. Cells were washed three times in RPMI 1640 medium supplemented by 100 U/ml penicillin, 100 ~g/ml streptomycin, 0.3 mg/ml L-glutamine and sodium bicarbonate, 20% heat inactivated and absorbed human AB serum, 5 x 10-5 M 2-mercaptoethanol and 20 mM HEPES buffer. Responder cells were counted and adjusted to 3 × 106/ml. Target spleen cells were adjusted to 6 x 106/ml, treated with 20 ~g/ml mitomycin-C at 37°C for 30 min and washed three times. Cell suspensions were distributed in a 96-well culture cluster dish. Cultures were maintained at 37°C in 5% CO 2 humid atmosphere for 4 days, subsequently pulsed with 1 t~Ci [3H]thymidine per well (sp. act. 10 mCi/mmol; Radiochemical Centre, Amersham, U.K.) and harvested on day 5 via a multiple automatic sample harvester (DELTA-300).

Mitogen stimulation cultures P A L N cells (1 × 105 cells per well) were cultured in flat-bottom microplates with Lectins. P H A (Welcome) was used at a final concentration of 0.5 ~g/ml, Con-A (Pharmacia) at a final concentration of 10 t~g/ml and PWM (Sigma) was used at 10 #g/ml. After 48 h of incubations at 37°C in 5% CO 2 humidified air [3H]thymidine (1 t~Ci/well) was added for an additional period of 18 h, and cells harvested as described.

Immunofluorescence staining Cells bearing relevant markers were labeled in an indirect assay with mouse monoclonal antibodies (mAb) W3/25 and M R C OX 8 (Dr. A.F. Williams, M R C Cellular Immunology Unit, Oxford, U.K.). The W3/25 mAb

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marks CD4 + cells (helper/inducer set) and the MRC OX 8 mAb detects CD8 + cells (cytotoxic/suppressor set). PALN cells were resuspended in RPMI 1640 medium containing 2% fetal calf serum at a concentration of 1 × 106 cells/ml. After centrifugation for 10 min at 400 × g, cells were incubated with 50/~1 hybridoma supernatant for 30 min at 4°C. The cells were washed twice in RPMI 1640 medium containing 2% FCS plus 0.01% NaN 3. After centrifugation, cells were incubated with 0.1 ml FITC-conjugated IgG (H + L) goat anti-mouse serum (Cappel, Cochranville, PA) and resuspended in medium. The cells were incubated 30 min at 4°C and washed three times in RPMI 1640 and subsequently resuspended in fresh medium containing 0.2% BSA, 0.01% NaN 3 and 20 mM EDTA. Fluorescence intensity was measured optically with a fluorescent microscope using a double blind coding system.

Statistical procedures Statistical analysis utilized Student's t-test. Results

Effect o f pregnancy on the P A L N lymphocyte response to allogeneic cells The reactivity of the regional lymph node cells harvested during syngeneic

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(AO x AO) and allogeneic (AO x DA) pregnancies in rats were evaluated. The representative data of specific lymphocyte responses to paternal antigens are shown in Fig. 1. We observed a specific suppression of alloreactivity in the pre-implantation (3rd day) and peri-implantation period (7th day) of allogeneic pregnancy versus both syngeneically pregnant and virgin controls (P < 0.05). However, in the mid pregnancy PALN cells from allogeneically pregnant females showed a two-fold increase of alloreactivity (P < 0.001) but at the end of allogeneic pregnancy the reactivity returned to normal levels. There were no significant changes during syngeneic pregnancy as compared to virgin controls. A similar increase of alloreactivity during mid pregnancy by PALN cells was found in the MLR with third party, mitomycin-C treated cells (AO Y-59) (Fig 2.). However, the increase in reactivity was found for cells from both syngeneically and allogeneically pregnant AO females. We did not observe any depressed reactivity during the period of blastocyst implantation in syngeneic or allogeneic pregnant females, but we detected a suppression of alloreactivity during the last phase of allogeneic pregnancy. Effect o f pregnancy on P A L N cell response to polyclonal mitogens Kinetic results of the non-specific response of cells from local lymph nodes showed an enhancement of non-specific reactivity to all polyclonal mitogens

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Fig. 2. Non-specific alloreactivity against third party cells Y-59 (RTY) of paraaortic lymph node cells in A O ( R T I u) females sensitized by syngeneic (AO x AO) ( o o ) o1 allogeneic (AO x DA-RTP') ( o - - - o ) pregnancy.

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DAYS OF PREGNANCY Fig. 3. Kinetics o f reactivity o f p a r a a o r t i c l y m p h n o d e cells of A O ( R T I " ) females to polyclonal m i t o g e n s in syngeneic ( o o ) or aUogeneic ( A O x D A - R T 1 ~') ( o - - - o ) pregnancy.

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at mid pregnancy (Fig. 3). The strongest reactivity was displayed by PALN cells in culture with Con-A, especially in allogeneic pregnancy (P < 0.001). However, at the end of pregnancy a depressed reactivity was observed. Effect o f pregnancy on the CD4+/CD8 + cell ratio in P A L N

The CD4+/CD8 ÷ cell ratio in the local draining lymph nodes of allogeneically pregnant female rats showed a constant increase of CD8 + cells in comparison to virgin controls (Fig. 4). Syngeneically pregnant females showed an increase in CD8 + cells during the 17th and 21st day of pregnancy. Discussion

Results of this study document pre- and peri-implantation immunosuppression of PALN cell reactivity to mitogens and alloantigens, and addressing local immunity and its expression in the mammalian uterus support previous studies (Chaouat et al., 1986; Clark et al., 1987; Chaouat et al., 1988). Maternal local immunological recognition and regulation appears very early even before implantation and stabilizes to near normal to nonpregnancy levels by the time of parturition. The present study, and those of others, highlight the importance of sequential analyses during all phases of pregnancy to understand the complex immune circuits operating. The mating and/or embryological antigenic message(s) to the mother requires periimplantational levels of estrogen and progesterone to initiate and express this suppressor activity (Beaman, 1990). Studies in progress indicate the antigenic message from the embryo is less critical because skin allografts as placebo embryos induce an identical antigen-specific suppression of the reactivities if peri-implantation levels of estrogen and progesterone are present. Analyses of the immunoreactivity of splenic lymphocytes during the period of local immunosuppression revealed no major changes to highlight the nature of local immunity in the cells of uterine lymphatics (Kapovic and Rukavina, 1991). If local immunoregulation and subsequent events leading to immunotrophism are relevant to feto-placental survival and well-being, measurement of their effects in allogeneic pregnancy should be possible during successful syngeneic pregnancies. The fact that monoclonal antibodies against suppressor factors produced locally and given to allogeneic mated females prior to implantation totally prevents pregnancy highlights the need to focus on the syngeneic pregnancy model and immunoregu!ation (Hoskin and Murgita, 1985a, 1985b). The systemic humoral immune alterations seen in the allogeneic pregnancy model may reflect systemic immune regulatory support needed in some but

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not all grafting encounters. This type of reasoning is supported by Croy and Chapeau (1990) who studied successful pregnancies in immunodeficient mouse models, using SCID and BEIGE mice. In these models there was evidence of normal local uterine immunoreactivity and no humoral" immunity to the feto-placental unit. Litter sizes and feto-placental units were small; however, pregnancy was successful to term. The marked two-fold increase in alloreactivity seen at mid-gestation in allogeneically pregnant females deserves comment. This may mirror the role of certain cytokines produced by T cells claimed to be "immunotrophic" for the sustained growth of the feto-placental unit. The increased response of these cells to Con-A stimulation points in the same direction. Studies are needed to test the effects of monoclonal antibodies to panels of relevant cytokines on pregnancy outcome or feto-placental growth rates. Normalization of immunoreactivity by the completion of gestation was unexpected. This study documents alloreactivities that are hypo-, hyper- or normo-responswe depending on the phase of pregnancy. This may cast doubts on prior claims of the presence or absence of evidence of immunoregulation in parous animals tested only in the postpartum period or even at the termination of pregnancy. The CD4+/CD8 + cell ratio in the para-aortic lymph nodes was decreased throughout all phases of allogeneic pregnancy. This altered ratio could be considered as specific because it was not seen in syngeneic pregnancy. It was due to a dramatic increase in the percentage of CD8 + cells. It seems acceptable to credit the increase in CD8 + suppressor cells for the decrease in alloreactivity seen in the pre- and peri-implantation period. Hyper-reactivity at mid pregnancy in spite of unchanged (low) levels of CD4+/CD8 + ratio could be explained by higher participation of anducer and growth promoting cells and decreased number of suppressor cells among CD8 + cell populations. In that situation, CD4 + cells could also be released of restraint and stimulate alloreactivity. It could be supposed that dynamic changes of cell subpopulations could occur during pregnancy, suppressing and/or enhancing alloreactivity and tissue growth. Studies addressing systemic immune responses to normally derived fetal alloantigens and to intrauterine skin allografts as placebo embryos are the subjects of our next reports.

Acknowledgments The authors wish to thank Dr. Alan E. Beer for helpful discussions and advice during the preparation of the manuscript, Mrs. Marija Kastela for secretarial help and the Research F u n d of the Repubhc of Croatia for financial support.

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