Lack of class I major histocompatibility antigens on trophoblast of periimplantation blastocysts and term placenta in the pig.

In this study, the pattern of expression of class I major histocompatibility (MHC) antigens and mRNA on periimplantation blastocysts and term placental tissue was determined for the pig. Class I MHC antigens could not be detected immunohistochemically either on extra-embryonic membranes or on the embryonic portion of Day 14, 16, 22, and 25 blastocysts. Nor could class I MHC antigens be detected on the outer trophoblast epithelium and inner endodermal surface of the chorioallantoic membrane or on the outer and inner surfaces of the amnion at term. However, MHC class I antigens were detected on the vascular mesoderm found in both the chorion and amnion at term, and in Day 25 extra-embryonic membranes. Uterine endometrial cells and tissues and maternal peripheral blood leukocytes stained strongly for class I MHC antigens. There was a large difference in the intensity of class I MHC mRNA signal, detected by Northern blot analysis, in embryo/fetus-derived tissues compared to that in maternal tissues. The embryos appeared to express even less class I MHC mRNA than did the extra-embryonic membranes. In addition, in situ hybridization of Day 16 blastocysts indicated class I MHC mRNA to be ubiquitously expressed at low levels in embryos and extra-embryonic tissues compared to uterine endometrial tissue controls. Taken together, these results indicate that class I MHC antigens are either not expressed on the surface of the extra-embryonic/fetal membranes during gestation in the pig or are expressed at very low levels, and that specific mRNA is expressed at correspondingly low levels.

A progesterone-dependent immunomodulatory protein alters the Th1/Th2 balance.

In the presence of progesterone, lymphocytes from pregnant females produce an immunomodulatory protein known as progesterone induced blocking factor (PIBF). We tested the effect of this protein on cytokine production by mitogen-activated lymphocytes. Spleen cells from Balb/c mice were incubated with Con A in the presence or absence of PIBF. Supernatants from the activated cells were collected and the concentrations of IL-3, IL-4, IL-10 and IFN gamma were determined by ELISA. In supernatants from spleen cells activated in the presence of PIBF the concentration of IFN gamma was not substantially different from controls however, the same spleen cells produced significantly more IL-10, IL-3 and IL-4 than those cultured without the progesterone-induced protein. When CD4+ and CD8+ enriched cell suspensions were used as producers of the cytokines it was found that both populations reacted with an equally increased production of IL-3, IL-4 and IL-10 in the presence of PIBF. Although cytokine-producing Th cells can be identified within the CD4+ population, the present findings suggest that involvement of CD8+ cells in altered cytokine production cannot be excluded. These data indicate that the PIBF affects the Th1/Th2 balance, and via altered cytokine ratios it contributes to decreased cell-mediated responses during pregnancy.

Pregnancy impairs resistance of C57BL/6 mice to Leishmania major infection and causes decreased antigen-specific IFN-gamma response and increased production of T helper 2 cytokines.

Resolution of cutaneous leishmaniasis in infected mice is associated with a polarized Th1 immune response by the host, whereas maternal immune responses during pregnancy appear to be biased toward humoral (Th2) and away from cell-mediated (Th1) responses. The objective of this study was to evaluate whether the putative Th2 bias in pregnant C57BL/6 mice would impair their normal ability to mount a curative Th1 response against Leishmania major infection. Pregnant C57BL/6 mice developed larger cutaneous lesions that showed no signs of resolution up to 70 days after infection. The infection appeared to be contained but not cured, as the footpad lesion remained stable, neither decreasing (as in normal C57BL/6 mice) nor showing uncontrolled expansion leading to death (as in susceptible mouse strains such as BALB/c). The number of parasites harvested from the footpads of pregnant mice was markedly higher than controls throughout the course of infection. The increased severity of infection in pregnant mice was accompanied by reduced IFN-gamma and increased IL-4, IL-5, and IL-10 production by spleen and popliteal lymph node cells stimulated in vitro with Leishmania Ags. Furthermore, IgG1 was elevated in the serum of pregnant mice as opposed to an increase of IgG2a in infected but nonpregnant controls. These observations support the existence of a bias toward Th2 cytokine expression during pregnancy and suggest that these cytokines effectively down-regulate the course of a normal Th1 response against a parasite infection in the periphery.

T helper 1 response against Leishmania major in pregnant C57BL/6 mice increases implantation failure and fetal resorptions. Correlation with increased IFN-gamma and TNF and reduced IL-10 production by placental cells.

Maternal immune responses can influence fetal survival and several cytokines have harmful or protective effects on pregnancy. The Th1 cytokines IFN-gamma and IL-2 can cause fetal loss, whereas the Th2 cytokine IL-10 is protective. However, infections such as leishmaniasis show the opposite pattern: resistance is associated with the preferential mounting of a Th1 response, whereas a Th2 response exacerbates the disease. We therefore asked whether the curative Th1 response against Leishmania major in genetically resistant C57BL/6 mice, would compromise concurrent pregnancy. The number of resorptions as assessed by uterine scars was significantly increased in infected C57BL/6 mice and this was associated with a decreased production by placental cells of the Th2 cytokines IL-4 and IL-10 and increased production of IFN-gamma and TNF. Interestingly, the frequency of pregnancy failure before implantation in C57BL/6 mice was also substantially increased. In contrast to C57BL/6 mice, early infection did not reduce implantations in BALB/c mice that mount a Th2 anti-L. major response and succumb to infection. For both resorptions and implantations, there appeared to be a short period early in infection that was detrimental to pregnancy, followed by a period with lesser effects, and a later period that again induced higher resorptions or pre-implantation losses. These results suggest that a beneficial anti-parasite Th1 response can adversely affect pregnancy outcome. Furthermore, Th1 cytokines may be deleterious for not only placental maintenance but also preimplantation events.

IL-10 prevents naturally occurring fetal loss in the CBA x DBA/2 mating combination, and local defect in IL-10 production in this abortion-prone combination is corrected by in vivo injection of IFN-tau.

CBA x DBA/2 placentae are quantitatively or qualitatively deficient in their production of the anti-inflammatory Th2-type cytokines IL-4 and IL-10 compared with the nonresorption-prone CBA x BALB/c mating combination. Wastage in this mating combination is accompanied by increased levels of local inflammatory cytokines. In addition, alloimmunization enhances the placental production of IL-4 and IL-10 in CBA x DBA/2 matings. Furthermore, rIL-10 by itself completely reverses the high incidence of fetal resorption after i.p. injection. Conversely, anti-IL-10 increases the resorption rate, but only in CBA x DBA/2 matings. On the other hand, injecting either anti-IFN-gamma or pentoxifillin (an anti-TNF agent) partially reduces the resorption. When given together, they produce a synergistic remission of fetal loss. Finally, we report that recombinant ovine trophoblast protein, an IFN-tau which is known to influence reproductive outcome in ruminants, can also counteract increased CBA x DBA/2 fetal resorption. It simultaneously induces increased placental IL-4 and IL-10 production in this mating combination. These results indicate that the placentally produced anti-inflammatory cytokines can play a vital role in the survival to term of the fetal allograft, by counteracting deleterious inflammatory cytokines.

Alloimmunization against well defined polymorphic major histocompatibility or class I MHC transfected L cells antigens can prevent poly IC induced fetal death in mice.

METHOD: It is possible to induce increased fetal resorption in a number of inbred murine matings by injecting Poly (I) Poly (C12U) 3.5 days postconception, a maneuver associated with natural killer-mediated damage to the feto placental unit such as occurs in spontaneous fetal resorptions. RESULTS: We show here that alloimmunization can block this effect. In addition, maternal immune responses induced by alloimmunization against isolated mutant class I or class II, as well as by immunization with class I MHC alloantigens (Kd) transfected L cells are sufficient to restore normal fetal viability. It is not necessary that the maternal immune response be specifically directed against paternal alloantigens fr the fetal protection to ensue, since the effect occurs in inbred matings when the mother is immunized against unrelated class I or class II alloantigens. As in previous studies conducted in the murine species, not all MHC alloimmunizations are protective. In addition, as control, immunization with a monomorphic class I MHC molecular (37), transfected L cells, sheep red blood cells or hen egg lysozyme is without effect. CONCLUSION: These results indicate that defined MHC antigens can mediate fetal protection from induced fetal resorption, and suggest that one driving force in promoting MHC antigen polymorphism in mammals is their capacity to confer protection from NK mediated fetal demise.

Differentiation of subsets of CD4+ and CD8+ T cells.

Our knowledge of the cytokine secretion patterns of T cells and other cells is clearly becoming more complex. The T helper 1 (Th1) and Th2 patterns may represent the extremes of a spectrum of cytokine regulatory patterns controlled by several cell types. CD8+ T cells can also secrete either Th1-like or Th2-like cytokine patterns, and they can contribute to bystander B cell activation. Interactions occur between immune cytokine regulatory networks and other systems, and pregnancy and responses against infection can profoundly influence each other.

Cytotoxicity of tumour necrosis factor-alpha and gamma-interferon against primary human placental trophoblasts.

Tumour necrosis factor-alpha (TNF-alpha) and gamma interferon (IFN-gamma) are expressed within human placental villi during normal pregnancy, yet their functions remain unknown. Since villous cytotrophoblasts are within the paracrine reach of this expression, the effects of TNF-alpha and IFN-gamma on a purified population of term placental cytotrophoblasts were examined. After 4 days of culture TNF-alpha alone induced a loss of trophoblast viability as measured by both metabolic capacity (MTT reduction) and DNA content. The combination of TNF-alpha and IFN-gamma enhanced the damaging effect. Neutralizing antibodies against TNF receptor p55, but not p75, partially reversed the TNF-alpha-induced cytotoxicity. After 24 h of culture, TNF-alpha and IFN-gamma increased the fraction trophoblasts containing nicked DNA, and after 60 h, increased the detachment of cells characterized by a distorted morphology, lower DNA content, and fragmented DNA. These results suggest that a physiological role of TNF-alpha and IFN-gamma expression in the placental villi may be to regulate the apoptotic death of villous cytotrophoblasts. The studies also predict potential harmful effects on placental development and function following aberrant inflammatory cytokine expression triggered by intravillous infections.

Demonstration of functional cytokine-placental interactions: CSF-1 and GM-CSF stimulate human cytotrophoblast differentiation and peptide hormone secretion.

The placental syncytiotrophoblast (ST) is a terminally differentiated epithelial cell monolayer that constitutes the outermost boundary between fetal and maternal tissues and performs a variety of synthetic, secretory, and transport functions essential for the maintenance of pregnancy. Although it is known that the ST arises from the underlying germinal layer of mononuclear cytotrophoblasts (Langhans' cells) by a process of cell fusion, the molecular mechanisms involved in this process are unclear. In order to address this question, we have investigated the effects of macrophage colony-stimulating factor (CSF-1) and granulocyte-macrophage colony-stimulating factor (GM-CSF), lymphohemopoietic cytokines implicated in mammalian placental development, on the in vitro morphological and functional differentiation of human trophoblast. Both CSF-1 and GM-CSF stimulated cytotrophoblast aggregation into large multinucleated structures composed of extensive patches of syncytium interspersed with mononuclear cells. Concomitant with this morphological differentiation was upregulation of the production of the placental hormones placental lactogen and chorionic gonadotrophin. Placental fibroblasts derived from the villous stroma that underlies the trophoblastic epithelium were found to produce both GM-CSF and CSF-1 under the control of the trophoblast-derived cytokines IL-1 and TNF alpha. These observations suggest that a network of interrelated cytokines operates within the basal (fetal) aspects of the villous stroma where they are situated to play a significant role in the morphological and functional development of the human placenta.

Maternal anti-placental reactivity in natural, immunologically-mediated fetal resorptions.

Observations on maternal recognition of the fetus and the demonstration of the effects of cytokines on reproductive events led to the "immunotrophism" model, which suggests that maternal immune recognition of fetally-derived Ags results in the release of cytokines that promote the growth of the placenta; any disturbance in this balance of cytokines could result in deleterious consequences for the placenta and, in turn, the fetus. We have focused our attention on the murine CBA/J x DBA/2 model of spontaneous abortions and compared them with normal CBA x BALB/c pregnancies. Our results indicate that the extent of stimulation of maternal strain lymphocytes in response to stimulator placental cells in mixed lymphocyte-placenta reactions (MLPR) was much higher in the normal mating combination compared with the abortion-prone mating combination. Cytokine analysis of the supernatants from MLPR indicates that there is significantly higher production of TNF-alpha, IFN-gamma, and IL-2 in supernatants from the abortion-prone combination than in supernatants from the normal combination. Furthermore, MLPR-stimulated cells induce resorptions in normal pregnant mice; maternal strain lymphocytes stimulated by placentas from the abortion-prone combination induce high rates of fetal resorptions, but lymphocytes stimulated with placentas from the normal combination do not. Together, these results suggest that immunologically mediated fetal resorptions probably result from improper or inappropriate maternal responses to placental Ags. Our observations also suggest that such effects are probably mediated by cytokines.

Functional, long-term cultures of human term trophoblasts purified by column-elimination of CD9 expressing cells.

We have extended previous observations of expression of the trypsin-resistant cell surface antigen CD9 on placental fibroblasts to virtually all cells in the villous stroma and developed a method for eliminating CD9 expressing cells from trypsinized placental preparations. Preparations incubated with the mouse anti-human CD9 monoclonal antibody 50H.19 were passed through a goat anti-mouse immunoglobulin column that captures CD9 expressing cells. Approximately 95 per cent of the eluted cells stained positive with the villous trophoblast specific antibody GB25 and could be cryopreserved and thawed with > 80 per cent recovery in culture. One week cultures contained fewer than 0.3 per cent vimentin positive (mesenchymal) cells and maintained secretion of hCG. Two week cultures remained free of fibroblasts and macrophages. Clusters of trophoblasts that formed spontaneously during the first week of culture were shown by microinjection of carboxyfluorescein and by staining with anti-desmoplakin antibody to be a patchwork of mononuclear cells and syncytial units. Although the DNA content of the culture decreased by 35 per cent during the 2 week culture, the metabolic capacity and protein content remained relatively constant. Thus, CD9 immuno-elimination gives a high yield of enriched and viable trophoblasts that can be cultured for at least 2 weeks with almost no contamination by stromal cells.

Prevention of spontaneous abortion in DBA/2-mated CBA/J mice by GM-CSF involves CD8+ T cell-dependent suppression of natural effector cell cytotoxicity against trophoblast target cells.

Spontaneous resorption (abortion) that occurs at a high rate in DBA/2-mated CBA/J female mice is dependent upon asialoGM1+ natural effector-type cells, can be ameliorated by alloimmunization or administration of GM-CSF, and is augmented by in vivo injection of anti-CD8 antibody. The abortion rate was similarly augmented by administration of monoclonal anti-GM-CSF neutralizing antibody, but the GM-CSF physiologically active in preventing abortion during normal pregnancy did not appear to be derived from maternal CB8+ T cells putatively responding to antigens on the fetoplacental unit. Rather, depletion of CD8+ cells in vivo prevented GM-CSF from reducing the rate of resorptions. GM-CSF administration rapidly downregulates natural effector cells able to kill a trophoblast target cell line in vitro, and anti-CD8+ antibody boosts total splenic cytotoxic cell activity. Further, anti-CD8 completely abrogated the ability of GM-CSF to suppress anti-trophoblast killer cell activity. These cells are known to be asialoGM1+ and injection of anti-asialoGM1 reduced the abortion rate appropriately in mice that had received anti-CD8. These data suggest that GM-CSF acts indirectly to prevent abortion in DBA/2-mated CBA/J mice through a mechanism that requires CD8+ maternal T cells, and systemic regulation of the level of anti-trophoblast killer cell activity may determine the success or failure of pregnancy in this system.

The role of cytokines in gestation.

Lymphohemopoietic cytokines are now recognized to be central participants in the cellular communication events underlying the complex and dynamic remodeling processes required to accommodate the semiallogeneic conceptus during mammalian reproduction. Cytokines are identified to be particular importance in mediating communications between the conceptus and maternal cells, particularly the uterine epithelium and infiltrating leukocytes, both prior to implantation and as the placenta develops. In this review we summarize recent experimental data concerning the synthesis of various cytokines in uterine and conceptus-derived tissues and highlight current hypotheses for their roles in establishing and maintaining successful pregnancy. It is concluded that complex and finely balanced cytokine networks underpin precise regulatory mechanisms controlling the rate and degree of conceptus development and invasion into maternal tissues.

Synthesis of T helper 2-type cytokines at the maternal-fetal interface.

Clinical and experimental evidence has indicated that the maternal immune response is biased toward antibody production and away from cell-mediated immunity during pregnancy, especially in the vicinity of the fetoplacental unit. Because antibody responses are often associated with the Th2 cytokine pattern, this suggests that Th2-type cytokines might predominate locally in the regulation of the maternal immune response. In order to test this hypothesis, we examined the local and distal release of cytokines during murine pregnancy using ELISA assays. We report here that the Th2-specific cytokines IL-4, IL-5, and IL-10 were readily detectable in cell supernatants derived from fetal-placental units in all three trimesters of gestation. IL-3 was also present. These cytokines were detected in lysates of freshly isolated, day 12 decidual and placental cells, and in supernatants as early as 15 min after the beginning of culture. The presence of functional IL-10 was confirmed by specific bioassay. IL-10 mRNA was localized to the decidua at day 6 of gestation by in situ hybridization. IFN-gamma was also found in the supernatants from the first trimester of pregnancy, but was barely detectable in the second, and undetectable in the third trimester. Cytokine expression was consistently detected in samples from individual mice. None of these cytokines was produced by unstimulated spleen or mesenteric lymph nodes from pregnant mice. IL-4, IL-10, and IFN-gamma were produced by Con A-stimulated spleen cells from virgin mice, but in ratios opposite to those found in the placenta. These observations indicate that Th2-specific cytokines are normally produced at the maternal-fetal interface. The continuous presence of IL-4, IL-5, and IL-10, with early and transient expression of IFN-gamma, can provide a molecular basis for the antibody/Th2-like bias of the maternal immune response during pregnancy.

A porcine trophoblast cell line that secretes growth factors which stimulate porcine macrophages.

We have previously described experiments in both the mouse and the human indicating that cytokines capable of activating macrophages (colony-stimulating factor-1 [CSF-1], granulocyte-macrophage colony-stimulating factor [GM-CSF], and interleukin-3 [IL-3]) are produced by, and/or stimulatory of, trophoblast cells in these species. In contrast to the complex hemochorial placenta of the mouse and humans, the pig has a simple diffuse type of placenta, designated as epitheliochorial. To determine whether similar phenomena might not apply to the porcine pregnancy, we have isolated a cell line, designated Jag-1, from the trophoblastic tips of Day 14 porcine embryos. We report here that this cell line is cytokeratin-positive, vimentin-negative, and therefore of epidermal origin. It also shares various morphological characteristics with porcine trophoblast as demonstrated at both the light and electron microscopic levels. In addition, Jag-1 cells and primary trophoblast tissue from Day 14 blastocyst do not express classical major histocompatibility (MHC) class I and class II antigens, a unique feature of trophoblast in many species. To determine the ability of this cell line to produce cytokines, we have developed an assay for porcine macrophage growth factors that utilizes uptake of tritiated thymidine. This assay responds positively to recombinant bovine GM-CSF and, more importantly, detects a similar activity in supernatants of the porcine trophoblast cell line and of Day 14 blastocysts. Thus porcine trophoblast cells, like their murine and human counterparts, produce and potentially interact with lymphohematopoietic cytokines that are traditionally associated with macrophages.

Bidirectional cytokine interactions in the maternal-fetal relationship: is successful pregnancy a TH2 phenomenon?

Pregnant females are susceptible to intracellular pathogens and are biased towards humoral rather than cell-mediated immunity. Since TH1 cytokines compromise pregnancy and TH2 cytokines are produced at the maternal-fetal interface, we hypothesize that these TH2 cytokines inhibit TH1 responses, improving fetal survival but impairing responses against some pathogens.

The trophoblast as an integral component of a macrophage-cytokine network.

The trophoblast, an epithelial cell of fetal origin that forms the physical barrier between the mother and developing conceptus, becomes a component of the host immune system during pregnancy. Of the classical immune cells, it most closely resembles the macrophage, also present in high numbers in the pregnant uterus. The macrophage and trophoblast, as cell classes, share characteristics such as phagocytosis, syncytialization, invasiveness, expression of the proteins CD4, CD14, IgG receptor (FcR), non-specific esterase, granulocyte macrophage-CSF (GM-CSF), colony stimulating factor 1 (CSF-1), interleukin-1 (IL-1), interleukin-6 (IL-6), tumour necrosis factor (TNF-alpha), transforming growth factors (TGF), platelet-alpha derived growth factor (PDGF) and receptors for these cytokines. In the uterus both cell types appear regulated by a common element, the uterine epithelium, that secretes cytokines such as CSF-1, GM-CSF, TNF alpha, TGF beta, IL-6, and leukaemia inhibitory factor (LIF) that target both macrophages and trophoblasts. The common characteristics and regulation that make teleological sense in terms of co-ordinating local uterine immunity during pregnancy may also be important in transmission of congenital diseases such as AIDS. The production by the uterine epithelium of a number of cytokines previously only associated with mononuclear phagocyte production and function predicts the existence of a similar, but broader, shared cytokine network encompassing trophoblast and the principal immune regulatory cell, the T lymphocyte.

Monoclonal antibodies directed against a widespread oncofetal antigen: the alpha-fetoprotein receptor.

Accumulating evidence based on alpha-fetoprotein (AFP) cell binding and uptake has shown the presence of a receptor for AFP on the surface of fetal and neoplastic cells. In order to further study this receptor, monoclonal antibodies (MAbs) made against pooled human mammary tumor membrane extracts were screened for their ability to inhibit the binding of radiolabeled AFP to the Ichikawa and TA3/Ha malignant cell lines. IgM-producing clones 167H.1 and 167H.4 were found to inhibit the binding of AFP to its receptor. Conversely, the MAb reaction was inhibited by an excess of AFP but not by serum albumin at an equal molar concentration. The possibility that these MAbs recognize AFP has been ruled out. In addition, we describe a method for the purification of the AFP receptor which yielded fractions reactive to both 167H.4 and AFP. The results obtained strongly suggest that 167H.1 and 167H.4 are directed against the binding site for AFP on the AFP receptor. Using 167H.4 we found positive immunohistochemical staining in 6/6 human mammary tumor specimens whereas 3/3 benign mammary adenomas were negative. Immunostaining of fetal muscle with either 167H.4 or an anti-AFP antiserum yielded a similar staining pattern. The staining of live Ichikawa cells with 167H.4 showed a surface receptor distribution (capping) similar to the one described in previous reports using labeled AFP. The widespread expression of the AFP receptor observed previously is consistent with the results obtained using the MAbs described herein. The potential use of these MAbs for basic and clinical studies is discussed.

Immune signalling at the maternal-fetal interface and trophoblast differentiation.

In this review the afferent and efferent signals involved in immune signalling at the maternal-fetal interface are highlighted in the light of recent information. MHC antigen expression is reviewed. Immunizing mothers against class I and II MHC antigens can prevent spontaneous fetal resorption in mice. In addition, the CSF family of cytokines is not only secreted in placental tissues, but also play a role in trophoblast proliferation and differentiation. GM-CSF in particular appears to promote trophoblast syncytialization and the synthesis of human chorionic gonadotropin and human placental lactogen. Recent knock-out experiments indicate that CSF-1 is essential for complete fertility. Finally, the fact that the CSF cytokines promote HIV release from macrophages indicates that the knowledge gained in this area could lead to a better understanding of the transmission of the HIV virus from the mother to the fetus through the trophoblast.