Allogeneic Pregnancy Research Articles

Murine peritoneal macrophages in syngeneic and allogeneic pregnancies

We have previously observed that some functional characteristics of peritoneal macrophages (MOp) are altered during syngeneic murine pregnancy. To determine if these alterations are related to the immunological stimulation that the embryo produces on the mother, we evaluated MOp activity in allogeneic pregnancy. We also compared expression of the la antigen, ability to phagocyte and reduce nitro blue tetrazolium (NBT) and to produce interleukin-1 (IL-1) in allogeneic and syngeneic pregnancies. We observed that at Day 7 of pregnancy the increment in MOpIa + percentages was more evident in allogeneic (P < 0.05) than syngeneic pregnancies, and that these values remained high during the second week of gestation. We also observed a significant decrease in the macrophages that reduced NAT during the first week both in allogeneic and syngeneic pregnancies. Yet, in the former, the percentages of MOpNBT + were still low in the last week of pregnancy (P < 0.05). No differences were found in IL-1 production or in estradiol and progesterone levels between the 2 types of pregnancies. Thus, it is possible to postulate that during the first week of pregnancy the strong antigenic challenge that the embryo represents may activate MOp and that this activation could be augmented when major antigenic differences between mother and embryo are present.

Anti-paternal cytotoxic lymphocyte (CTL) activity during allogeneic or xenogeneic pregnancy in equids

In situ hybridization employing a cRNA probe derived from a 428-bp fragment of equine relaxin was used to localize relaxin mRNA, and immunocytochemistry was used to localize relaxin itself, in tissues of the placenta-endometrium interface recovered between 33 and 153 days of gestation from mares carrying intraspecific horse, interspecific mule and extraspecific donkey conceptuses. Immunocytochemical staining was also used to localize trophoblast-specific and class I major histocompatibility complex (MHC) antigens on some specimens, Relaxin mRNA and relaxin were both present in the single-cell non-invasive trophoblast layer of the allantochorion between 45 and 153 days of gestation in all three types of equine pregnancy examined, Both, however, were absent from the invasive trophoblast cells of the progenitor chorionic girdle and the differentiated trophoblast cells of the endometrial cups throughout the latters' 60–80-day period of development and regression. Discrete and irregularly spaced clusters of elongated pseudostratified trophoblast cells on the allantochorion remained negative for relaxin mRNA and ligand, but stained strongly for equine trophoblast-specific antigens. These areolae-like structures of the mature horse placenta overlie the mouths of endometrial glands between adjacent microcotyledons and they are clearly involved with the uptake of uterine milk for fetal sustenance. It is speculated that their loose attachment to the endometrium and weak expression of class I MHC antigens may serve to tolerize the mother to the paternally-inherited histocompatibility antigens of the fetus.

Anit-paternal cytotoxic lymphocyte (CTL) activity during allogeneic or xenogeneic pregnancy in equids

Anit-paternal cytotoxic lymphocyte (CTL) activity during allogeneic or xenogeneic pregnancy in equids

Anticardiolipin, but not the [formula omitted] Id anti-DNA antibody induces pregnancy failure

The primary antiphospholipid syndrome or the antiphospholipid syndrome in systemic lupus erythematosus patients (defined as secondary antiphospholipid syndrome) are characterized by the presence of thrombosis, thrombocytopenia and recurrent fetal loss in association with anticardiolipin antibodies. To determine the causal role of these antibodies in the pathogenesis of pregnancy failure we studied the effects of immunization with monoclonal anti-DNA antibody (designated 16 6 Id; no cardiolipin reactivity) and anticardiolipin monoclonal antibody (designated 2C4C2; binds DNA as well) on the outcome of allogeneic pregnancies in BALB/c mice. Mating of BALB/c females 4 weeks after active immunization with the 16/6 Id, anti-DNA monoclon antibody resulted in normal pregnancy outcome, similar to control mouse groups. In contrast to that, immunization with the 2C4C2 anticardiolipin antibodies resulted in severe gestational failure with low pregnancy rate, low numbers of fetuses and high rates of resorptions. The fertility index of those mice was extremely low as compared to the 16 6 Id-immunized mice or the control groups. Furthermore, a correlation was shown between the presence of anticardiolipin antibody levels in the sera of the mice at the time of gestation and the pregnancy fate. The 2C4C2-immunized mice which produced high levels of anticardiolipin antibodies demonstrated severe pregnancy failure, whereas normal gestations were observed in the 16 6 Id primed or the control mouse groups that did not produce measurable amounts of the latter antibodies. Thus, our studies demonstrate that anticardiolipin but not the 16 6 Id anti-DNA antibodies can induce severe gestational impairment.

T cell awareness of paternal alloantigens during pregnancy.

During pregnancy a semiallogeneic fetus survives despite the presence of maternal T cells specific for paternally inherited histocompatibility antigens. A mouse transgenic for a T cell receptor recognizing the major histocompatibility (MHC) antigen H-2Kb was used to follow the fate of T cells reactive to paternal alloantigens. In contrast to syngeneic and third-party allogeneic pregnancies, mice bearing a Kb-positive conceptus had reduced numbers of Kb-reactive T cells and accepted Kb-positive tumor grafts. T cell phenotype and responsiveness were restored after delivery. Thus, during pregnancy maternal T cells acquire a transient state of tolerance specific for paternal alloantigens.

Presence of species-specific, monomorphic antigens on sheep and goat binucleate cells

Immunocytochemical assays for sheep and goat species-specific, monomorphic antigens were developed utilizing polyclonal antisera from sheep and goats immunized by interspecific pregnancy. The assays were applied to cell isolates from sheep and goat fetal cotyledons collected from allogeneic pregnancies at Days 35, 40 and 120 of gestation. The isolates contained 7 to 48% binucleate cells (BNC). Using these assays, the sheep-specific antigen was detected on sheep cotyledonary cell isolates on all days of gestation tested (P < 0.001); the assay also detected the antigen on the BNC subset of the cotyledonary cell isolate population (P < 0.001). The caprine-specific antigen was shown to be present on cotyledonary cell isolates (P < 0.05), although the presence of the antigen could not be demonstrated with statistical confidence on goat BNC due to insufficient numbers of discernible cells. Binucleate cells contribute to the formation of the syncytial layer of the placenta by fusing with maternal epithelial cells and with the syncytium. The species-specific antigen (or antigens) is present on BNC at the appropriate time of gestation at which it (they) could play a role in the humoral immune response to interspecific and hybrid pregnancies observed in ewes and does.

Seminal fluid and the expression of MHC class I antigens in the placenta of the rat.

To determine whether seminal fluid influences the expression of MHC class I antigens on the surface of basal trophoblast cells in the placenta of the rat. Transfer of DA x DA embryos into a WF (allogeneic) or DA (syngeneic) recipient made pseudopregnant by hormonal treatment followed by mating with a vasectomized male (seminal fluid) or by mechanical stimulation (no seminal fluid). Antigen expression was determined by electron microscopic immunocytochemistry using the appropriate gold-labeled monoclonal antibodies. Seminal fluid did not affect the expression of MHC class I antigens on the surface of the basal trophoblast in either allogeneic or syngeneic matings. The suppression of the expression of paternal class I antigens on the surface of the basal trophoblast cells in allogeneic pregnancies most likely occurs at the genome level shortly after fertilization.

Uterine beta-adrenergic receptors in allogeneic and syngeneic pregnancy.

Two different mechanisms of regulation in uterine beta-adrenergic receptors in allogeneic pregnancy (AP) and syngeneic pregnancy (SP) are described in this work. Firstly we noted changes in beta-adrenergic sensitivity to isoproterenol in AP, while in SP differences in uterine reactivity to isoproterenol were found. In binding assays with the beta-antagonist [3H]dihydroalprenolol, changes in beta-receptor affinity were seen in AP, while in SP the maximal number of binding sites is altered. In addition, there are differences in uterine concentrations of cyclic AMP in both types of pregnancies as well as in the nucleotide response to isoproterenol. The differences in the reactivity and expression of uterine beta-adrenoceptors in both types of pregnancies may be due to a distinctive immunological role of the semiallogeneic fetus in AP.

Amniotic fluid enhances allogeneic cytotoxic T cell responses, whereas it suppresses mitogen-stimulated lymphocyte proliferation.

Mouse amniotic fluid has been shown to suppress T lymphocyte proliferation and suggested to be important in regulating immunity during pregnancy. In allogeneic pregnancy, cytotoxic T cells in pregnant lymphocytes against paternal transplantation antigen are impaired. We examined the effect of amniotic fluid to the alloreactive CTL responses. Although the amniotic fluid suppressed Con A or LPS stimulated lymphocyte proliferation as previously reported, the amniotic fluid taken from syngeneic C57BL/6 pregnant mice or allogeneic C57BL/6 x BALB/c pregnant mice enhanced the anti-H-2d or anti-H-2k CTL responses dose-dependently. We speculate that amniotic fluid contains not only immunosuppressive factors but also immunoenhancing factors which upregulate the allogeneic CTL responses.

Lymphokine production by decidual cells in allogeneic and syngeneic murine pregnancy

The importance of T cells during pregnancy has been well established in the murine system. Depletion of CD4 and/or CD8 positive cells from the maternal circulation increases fetal abortion and inhibits placental and fetal growth. T cell mediated regulation depends on T cell-derived lymphokines such as IL-6, IL-10, GM-CSF and IL-3. Among these factors, GM-CSF and IL-3 have been shown not only to stimulate trophoblast growth but also to promote placental function. All these growth factors having a short half life in vivo must exert their effect proximally to the site of production. In the present study, biologically active GM-CSF and IL-3 produced by T cells in the maternal decidual cap, which is the closest to the feto-placental unit maternal component, was detected. Cytoplasmic staining of totla decidual cap cells in various strain combinations, usng monoclonal antibodies to lymphokines, showed that the numbers of cells producing these factors vary depending on the day of pregnancy and on the strain combination used. Using indicator cell lines and neutralizing antibodies, it was seen that GM-CSF and IL-3 are produced at the eleventh day of pregnancy and secreted in both allogeneic and syngeneic pregnancies by decidual cap cells. T cell depletion experiments in vivo showed that these factors are produced by T lymphocytes. The production and secretion of biologically active CSF-1 was also evaluated in ths study. Although this is not a T cell-derived lymphokine, it is shown to be produced in the uterus and affect trophoblast growth. The results indicate that on the eleventh day of pregnancy only syngeneically pregnant mice produce sufficient quantities of CSF-1 to stimulate biologic activity. It is therefore demonstrated that the maternal immune system stimulates the growth of the feto-placental unit, obeying different rules in allogeneic and syngeneic pregnancy.

Pregnancy-associated, lymphocyte-derived suppressor factor inhibits protein kinase C activity

For successful allogenic pregnancy to occur, suppression of maternal defense responses toward the fetus are vital. Suppressor factors elaborated by decidual cells or immune cells may facilitate this suppression. In order for appropriate cellular responses to occur an intact signal transduction/second messenger system must be present. The calcium/phospholipid-dependent protein kinase, Pk-C, plays an important role in regulating immune responses, and may also be important in regulating uterine cell responses and implantation events. Pk-C activation is necessary for IL-2 synthesis and IL-2 receptor synthesis through activation of the proto-oncogenes c- jun and c- fos. These proto-oncogene gene products combine to form the heterodimer AP-1 which then activates IL-2 gene transcription for both peptide and receptor. If Pk-C activity becomes abrogated then appropriate cell responsiveness is diminished. We have shown that Pk-C activity is decreased in the particulate fraction of 4–7 day pregnant spleen, thymus and draining lymph node (DLN) cells. Spleen cells did not exhibit any change in cytosolic Pk-C activity, the thymus was found to have a decrease in both cytosol and particulate fractions, and the DLN cells exhibited a translocation effect whereby particulate Pk-C decreased and cytosolic Pk-C activity increased. Supernatant from 3-day cultures of DLN cells from pregnant animals was shown to inhibit proliferation of spleen cells. In addition, the supernatant was able to directly lower Pk-C activity. We hypothesize that DLN cells secrete a factor(s) that is able to suppress immune response through abrogation of Pk-C activity, thereby decreasing AP-1 formation resulting in decreased IL-2 synthesis and IL-2 receptor synthesis.

Characterization of gamma delta T lymphocytes at the maternal-fetal interface.

A specific subset of gamma delta T lymphocytes bearing a V gamma 6V delta 1-encoded TCR is known to populate the normal nonpregnant murine uterine and vaginal epithelium. However, gamma delta T lymphocytes residing at the maternal-fetal interface during pregnancy have not yet been investigated. Using mAb and cytofluorographic analysis, we analyzed gamma delta TCR-bearing lymphocytes obtained from placental/decidual tissues of allogeneic (C57B1/10 X BALB/c and BALB/c X C57B1/10) pregnancies. Gestations were analyzed at several time points during the second half of pregnancy, with lymphocytes from both maternal spleen and nonpregnant C57B1/10 uteri analyzed as controls. We found that all gamma delta T lymphocytes at the maternal-fetal interface are maternally derived. Relative to the total T lymphocyte population, the percentage of gamma delta TCR-bearing T lymphocytes at the maternal-fetal interface is enriched three- to four-fold compared with maternal spleen, and twofold compared with nonpregnant uteri. Although one-third of gamma delta T lymphocytes from pregnant animals express a cell-surface marker associated with activation (IL-2R), gamma delta cells from uteri of nonpregnant mice fail to express IL-2R. In terms of absolute numbers, we estimate that reproductive-tract gamma delta T cells are increased nearly 100-fold in pregnant animals compared with nonpregnant animals. To characterize the TCR-gamma delta repertoire in the placenta/decidua, we generated 21 TCR-gamma delta-bearing hybridomas from lymphocytes in this tissue. Analysis of these hybridomas revealed at least six distinct gamma delta receptor types expressed at the maternal-fetal interface, with V gamma 6V delta 1 encoded TCR representing the predominant population. As specific resident constituents of the reproductive tract, gamma delta T lymphocytes may be involved in regulating a variety of physiologic and pathophysiologic events in reproductive biology.

Further evaluation of the pregnancy-linked down-regulation of the paternal antigen-specific splenic cytotoxic T lymphocyte activity in allogeneically pregnant mice.

Cytotoxic T lymphocyte (CTL) activity directed against paternal alloantigen was examined in allogeneically pregnant mice using various allogeneic combinations. The spleen cells from pregnant C57BL/6 (H-2b) mice mated with BALB/c (H-2d) male mice generated less anti-H-2d CTL after in vitro sensitization than those from unpregnant or syngeneically mated C57BL/6 mice. Different allogeneic combinations including the incompatibility at only D region of H-2 or minor histocompatibility loci were effective for downregulating the anti-paternal CTL activity in pregnancy. The downregulation of anti-paternal CTL activity induced by allogeneic pregnancy occurred at day 10 to day 18 of pregnancy, most extensively at day 14. The allogeneic pregnancy also downregulated the allogeneic CTL activities that had been amplified by injecting alloantigens before mating.

In vivo treatment with interferon-gamma during early pregnancy in mice induces strong expression of major histocompatibility complex class I and II molecules in uterus and decidua but not in extra-embryonic tissues.

Allopregnant (NFR/N [Swiss-derived] H-2q females x 57/Bl H-2b males) and syngeneically pregnant (NFR/N x NFR/N) mice were subjected to daily injections (10(5) U/mouse/day, from Day 5.5 of gestation) of recombinant rat or mouse interferon-gamma (IFNg) in order to investigate its ability to induce extra-embryonic major histocompatibility complex (MHC) expression and antipaternal immune reactions if administered during the first part of the gestation period. In addition, a limited number of IFNg-treated embryo-transferred NFR/N mice carrying C57/B1 embryos (representing a complete allogenic pregnancy) were investigated. Mouse and rat IFNg caused the same type of histological and physiological changes, and most of the experiments were performed by using rat IFNg. Several IFNg-treated mice (irrespective of type of mating) showed a drop in weight and a high rate of resorptions at Day 12.5 of gestation. This interference with pregnancy appeared not to be caused by immunological reactions against the feto-placental unit (no leukocyte infiltration and no significant effect on serum levels of antipaternal antibodies in preimmunized allopregnant IFNg-treated mice). Immunohistochemical stainings of cryosectioned tissues at Day 9.5 of pregnancy revealed that IFNg treatment caused a strong induction of MHC class I and class II expression on most cells in the uterus and on several cells in the maternal decidua, while there was a complete absence of detectable MHC class I and class II expression in the extra-embryonic tissues. Characteristic for a Day 12.5 placenta of an IFNg-treated mouse (including embryo-transferred mice) was a strongly MHC class II-induced maternal decidua and a completely MHC class II-negative fetal placenta. The pattern of IFN-induced MHC class I expression was similar to that of class II, with the exception of class I expression on scattered cells within the basal zone. Thus, the present study provides immunohistological evidence that IFNg administered in vivo during the first part of gestation is not capable of inducing MHC expression on murine extra-embryonic cells despite an extremely high expression of MHC molecules on decidual cells in intimate contact with extra-embryonic tissues. It is likely that the resistance to IFNg-mediated induction of MHC expression on extra-embryonic cells is of basic importance for the protection of mammalian semi-allogeneic fetuses.

Reactivity to alloantigens and polyclonal mitogens and CD4 +/CD8 + cell ratio shifts of cervical lymph node and spleen cells during pregnancy in rats

Cervical lymph node (CLN) cells and spleen cells were harvested from virgin and pregnant rats bearing syngeneic or allogeneic fetuses at all stages of pregnancy including the pre-implantation period. The specific and non-specific alloreactivity of these cells were analyzed in MLR against mitomycin-C treated paternal strain or unrelated cells. Mitogen stimulation of the cell cultures utilized PHA, Con-A and PWM. Cells bearing T cell markers were labeled in an indirect assay using the monoclonal antibodies W3/25 and MRC OX 8. Specific alloreactivity is enhanced at mid-pregnancy in both cell populations. Non-specific alloreactivity was suppressed in the cervical lymph node cells. Spleen cells demonstrated an increased non-specific alloreactivity and T polyclonal mitogen reactivity (PHA and Con-A) at mid-pregnancy. Reactivity to Con-A was depressed in the early phase and at the end of allogeneic pregnancy in the CLN. The CD4 +/CD8 + ratio was very low during all phases of pregnancy.

The effects of anti-CD4 and anti-CD8 antibody treatment on placental growth and function in allogeneic and syngeneic murine pregnancy

During pregnancy, maternal immune recognition of paternal alloantigens has been shown to result in an increased influx of maternal T cells into the spleen, draining lymph nodes and decidua. In previous studies we have shown that polyclonal or monoclonal anti-T cell antibody treatment of allogeneically pregnant mice results in decreased placental proliferation and phagocytosis in vivo. In the present study we compare the effect of such antibody treatment during allogeneic and syngeneic pregnancy. We show that monoclonal anti-CD8 treatment of both types of pregnant females reduces placental proliferation and phagocytosis. Anti-CD4 antibody treatment, on the other hand, only affects placental proliferation, indicating that there is a complex network of immune interactions affecting placental growth and function.

Induction of class II MHC antigen expression on the murine placenta by 5-azacytidine correlates with fetal abortion

During the gestational cycle the placental tissue does not express class II MHC antigens and whether this phenomenon is important to fetal survival has not yet been evoked. It has been reported that class II antigen expression precedes renal and cardiac graft rejection, which may also be the case in fetal abortion. In a recent report we showed that placental cells can be induced to express class II antigens in vitro and that these cells undergo different regulatory mechanisms depending on their anatomical position in the placenta. Thus, spongiotrophoblast-derived cells express these antigens after interferon-γ treatment, whereas labyrinthine trophoblast-derived cells are induced by 5-azacytidine. In the present study we examined the effect of 5-azacytidine on class II antigen expression in the placenta and fetal abortion in vivo. We report that 5-azacytidine, when given to pregnant females before the ectoplacental cone formation, dramatically increases fetal loss, which correlates with class II antigen expression in the labyrinthine trophoblast zone. No site effects of 5-azacytidine on placental cell proliferation, splenic T and B cell responses, or reproductive capability of treated females were observed. However, after treatment with 5-azacytidine placental cells can stimulate maternal spleen cells to proliferate in a mixed cell reaction, whereas untreated controls cannot. Furthermore, the abortive effect of 5-azacytidine can be rescued in allogeneic pregnancy by anti-paternal class II monoclonal antibody injection into the animals during the 5-azacytidine treatment. These results suggest that the maintenance of the class II antigen-negative expression on the placenta is indeed necessary to avoid maternal immune attack and ensure fetal survival.

An investigation of allogeneic pregnancy in multiparous mice subjected to in vivo depletion of CD8 (Ly2)-positive lymphocytes by monoclonal antibody treatment

Adult thymectomized C57/Bl (H-2 b) and DBA/1 (H-2 q) female mice were subjected to treatment with rat anti-mouse CD8 and mouse anti-rat Ig (kappa) prior to entering their third pregnancy with CBA/Ca (H-2 k) males. The treatment protocol drastically reduced the number of CD8 (Ly2)-carrying lymphocytes (T-cytotoxic/suppressor phenotype) in the spleen and para-aortic lymph nodes, as assessed by immuno-staining. All mice were investigated on day 18 of their third gestation. The following data were collected from experimental and control groups: (1) resorption frequency, (2) weight of the placenta, fetuses, spleen and para-aortic lymph nodes, (3) immunohistochemical analysis of maternal lymphoid tissues, (4) level of antipaternal IgG serum antibodies, (5) content of “background” IgM and IgG-secreting cells in spleen and para-aortic lymph nodes. Neither the resorption frequency nor placental/fetal weight was affected by anti-CD8 treatment. However, the formation of anti-paternal antibodies was enhanced in anti-CD8 treated C57/B1 mice.

Cyclosporin A prevents sensitization after blood transfusion in multiparous rats.

1. Humoral immune responses to allogeneic blood transfusions and semi-allogeneic pregnancies were monitored by the indirect haemagglutination assay in strains of inbred rats. 2. Similar titres and ranges of cross-reactive alloantibodies were induced by blood transfusions and pregnancy; the maternal alloantibody response to paternal antigens was not increased by an unrelated antigenic stimulus. 3. Immunosuppression with cyclosporin A was effective in preventing the primary humoral immune response produced by blood transfusion but not in abrogating the established immune response to previous pregnancy. 4. These studies have established an animal model of sensitization that both stimulates the clinical situation of many potential renal transplant recipients and permits analyses of the different antigenic stimuli involved.

Maintained allogeneic pregnancy in rats depleted of T cytotoxic/suppressor cells by OX8 monoclonal antibody treatment

It has been suggested that CD8 positive suppressor T-cells might be of importance in the non-rejection of the fetus. In the present investigation allogeneically pregnant (Lewix × DA) rats were subjected to in vivo treatment with monoclonal OX8 antibodies, reactive with the rat equivalent of CD8 receptor. This treatment protocol drastically reduced the numbers of OX8 positive cells in spleens and para-aortic lymph nodes. On the day of delivery these rats, together with normal IgG treated controls, were dissected and analysed for effects on: (1) fetal survival; (2) weight and immunohistology of spleens and para-aortic lymph nodes; (3) total numbers of IgM- and IgG-secreting cells within these organs. The OX8 treated rats passed through pregnancy as successfully as did the controls. Both groups showed the same type of pregnancy-induced changes in their lymphoid organs, including dramatic growth of the para-aortic lymph nodes and increase in Ig-secretors within both spleen and para-aortic lymph nodes. This pattern was the same in all pregnant rats investigated, including untreated syngeneically mated Lewis rats. It is concluded that OX8 positive T “suppressor” cells do not play an important role in the maintenance of the fetal-placental unit.