Immune aspects of embryo-maternal cross-talk in the bovine uterus.

This mini-review summarizes the results of recent transcriptome studies of bovine endometrium during the estrous cycle and during the pre-implantation phase, with a focus on immune response genes. Gene expression changes in the bovine endometrium during the estrous cycle were compared to a similar study in equine endometrium. The results indicate species-specific expression patterns, particularly for genes with immune functions. These are presumably the consequence of adaptations to differences in the physiology of reproduction in each species, including development of the conceptus, hormone profiles during the estrous cycle, and insemination. The results from a number of transcriptome studies during the pre-implantation phase, as well as comparison to the effects of human interferon alpha on bovine endometrial gene expression, suggest that during pregnancy there is no general suppression of the maternal immune system, but rather a fine-tuned regulation of immune cells. This presumably facilitates tolerance to the immunologically 'foreign' conceptus and at the same time activation of the immune system to defend against microbial and viral infections. Furthermore, comparison of differentially expressed genes in bovine endometrium to similar studies in human endometrial samples reveals a number of similar changes, indicating the existence of shared mechanisms in preparation for embryo implantation.

Immune balance at the foeto-maternal interface as the fulcrum of reproductive success.

Viviparity has many evolutionary advantages but brings with it the problem of the semi-allogeneic foetus having to coexist with the mother for the duration of pregnancy. In species with haemochorial placentation this problem is particularly evident as foetal trophoblast cells are extensively intermingled with maternal tissue and are directly exposed to maternal blood. Fascinating adaptations on both the foetal and maternal side have allowed for this interaction to be re-directed away from an immune rejection response not only towards immunotolerance, but in fact towards actively supporting reproductive success. Recent data have shown that some of these remarkable adaptations are conserved between mice and humans. Thus, a subset of trophoblast cells that is directly exposed to the maternal uterine environment shares the feature of expressing an unusual antigen repertoire on their surface. Paternal antigens can be recognized by maternal immune cells, in particular uterine natural killer cells that express cognate receptors, to regulate the extensive remodelling events that take place at the implantation site. Detailed genetic dissection experiments in the mouse have further demonstrated the direct impact of antigenic dissimilarity on foetal growth. With the availability of inbred strains, in vitro culture systems of trophoblast stem cells, and in-depth genetic, genomic and epigenomic data the mouse will be a valuable model system to study the intricate immune crosstalk at the foeto-maternal boundary. These insights will pave the way towards unravelling the mutual and synergistic interactions between trophoblast and its surrounding maternal environment, and in doing so help understand pregnancy pathologies.

Trophoblast debris modulates the expression of immune proteins in macrophages: a key to maternal tolerance of the fetal allograft?

Interactions between maternal immune cells and the placenta are of substantial interest since diseases of pregnancy, such as recurrent miscarriage, villitis of unknown etiology and preeclampsia may arise due to inadequate adaptation of the maternal immune system. During normal pregnancy trophoblast debris is shed from the placenta into the maternal blood in large quantities. This trophoblast debris is then rapidly cleared from the maternal circulation. In this study, we exposed trophoblast debris generated from an in vitro placental explant model to peripheral blood-derived macrophages and quantified a variety of molecules that are important in immune responses by ELISA or flow cytometry. Phagocytosis of trophoblast debris resulted in reduced cell-surface expression of MHC-II molecules, the costimulatory molecules (CD80, CD86, CD40 and B7H3), monocyte chemoattractant protein-1 (MCP-1), inter-cellular adhesion molecule 1 (ICAM-1) and IL-8 receptors in macrophages while the expression of programmed death-1 ligand 1 (PD-L1) was upregulated. In addition, phagocytosis of trophoblast debris induced the secretion of the anti-inflammatory cytokines IL-10, IL6 and IL1Ra and decreased the secretion of pro-inflammatory cytokines IL-1ß, IL12p70 and IL-8 by macrophages. Phagocytosis of trophoblast debris also increased macrophage expression of the immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO). We have shown that phagocytosis of trophoblast debris from normal placentae alters the phenotype of macrophages such that they are likely to deviate maternal immune responses towards tolerance and away from inflammation. This may be one of the mechanisms that allow the human fetal allograft to survive in direct contact with the maternal immune system.

Cytokine profile in the endometrium of normal fertile and women with repeated implantation failure.

BACKGROUND: Repeated Implantation Failure (RIF) is one of the most intricate obstacles in assisted reproduction. The cytokine and chemokine composition of uterine cavity seem to play important roles in the implantation process. OBJECTIVE: To compare the cytokine profile in the endometrium of normal fertile women and those with repeated implantation failure. METHODS: After enzymatic digestion of endometrial tissues, whole endometrial cells and endometrial stromal cells from RIF and normal fertile women were cultivated and stimulated for cytokine secretion. The levels of IL-10, TGF-ß, IFN-γ, IL-6, IL-8 and IL-17 in culture supernatants of the two groups were assayed by ELISA and compared together. RESULTS: Endometrial stromal cells and whole endometrial cells of normal fertile women produced higher levels of IL-6, IL-8 and TGF-ß compared to RIF group, although this difference was statistically significant only in endometrial stromal cells (p=0.005, 0.002 and 0.001, respectively). In addition, endometrial stromal cells of normal fertile women produced lower levels of IL-10 in comparison with RIF group (p=0.005). CONCLUSION: Disturbances in cytokine production at the feto-maternal interface could be a cause of implantation failure. A pro-inflammatory cytokine milieu seems to be pivotal for successful implantation.

CXCR7 and syndecan-4 are potential receptors for CXCL12 in human cytotrophoblasts.

The placenta forms the interface between the mother and the fetus. During placental development cytotrophoblasts differentiate to form the syncytium or to invade the decidual wall to breach maternal vessels and establish the blood flow in the intervillous space. This process is still not well understood but it is proposed that chemokines and their receptors are involved in guiding cytotrophoblasts to the decidua and maternal vessels as well as attracting immunocompetent cells to the implantation site. CXCL12 is a chemokine expressed by cytotrophoblasts and is involved in cytotrophoblast invasion, differentiation and survival. One of its receptors, CXCR4, has been detected on cytotrophoblasts. Recent data show that CXCR7 and syndecan-4 might partially mediate CXCL12 function in other cell types. In this study, we examined CXCR7 and syndecan-4 expression at the maternal-fetal interface via immmunolocalization in placental tissue sections and in isolated cytotrophoblasts. We further used immunoblot analyses to confirm the data. We were able to show that cytotrophoblasts express both receptors and that upregulation occurs during the differentiation process of cytotrophoblasts towards the invasive phenotype. On a functional level CXCR7 seems not to be involved in JAR cell chemotaxis, suggesting a different function of this receptor. In conclusion, we propose that CXCL12 binds to CXCR4, but also to CXCR7 and syndecan-4. These three receptors could mediate different functions of CXCL12, such as cell migration, directed invasion, proliferation and survival. The latter molecules might also be involved in the development of placental pathologies, such as preeclampsia or choriocarcinoma growth.

Magnesium sulfate normalizes placental interleukin-6 secretion in preeclampsia.

Interleukin-6 (IL-6) is one of the main proinflammatory mediators of hypertension and endothelial dysfunction in preeclampsia. In this study, we investigated the capacity of the preeclamptic placenta to secrete IL-6 and the effect of magnesium sulfate (MgSO(4)) on it. Placentas from normotensive (37-40 weeks) and preeclamptic (36-40 weeks) pregnancies were dually perfused for 6 h in the absence [normotensive (n = 3); preeclamptic (n = 4)] and presence [normotensive (n = 3); preeclamptic (n = 4)] of MgSO(4). Perfusate samples from the maternal and the fetal circulations were collected at each 30 min throughout the perfusion period and examined for IL-6 by enzyme-linked immunoassay. Statistical analysis was performed using the 2-way analysis of variance. In the absence of MgSO(4), IL-6 levels in the maternal and the fetal circulations of preeclamptic placentas (4.2 ± 1.3 and 0.9 ± 0.5 pg/mL/g cotyledon; respectively) were significantly higher, when compared with normotensive placentas (1.9 ± 0.5 and 0.2 ± 0.2 pg/mL/g cotyledon; respectively) (P < 0.05). Addition of MgSO(4) to the perfusate of normotensive placentas did not affect IL-6 secretion. However, exposure of preeclamptic placentas to MgSO(4) resulted in decreased IL-6 levels in the maternal circulations (1.7 ± 0.3 pg/mL/g cotyledon), when compared with the control group (P < 0.05). In the fetal circulation, the addition of MgSO(4) resulted only in a nonstatistical significant tendency toward decreased IL-6 levels, when compared with the control group. Our findings indicate that the perfused preeclamptic placenta secretes increased levels of IL-6 into the fetal and the maternal circulations and that MgSO(4) may normalize these increased secreted IL-6 levels.

Interaction between HLA-G and monocyte/macrophages in human pregnancy.

Several lines of evidence suggest that the human leukocyte antigen (HLA)-G play a key role in the regulation of human pregnancy. A sub-population of cells highly represented at the decidua belong to the myeloid-derived monocyte/macrophage lineage, which potentially interact with HLA-G expressing cells. It is proposed that HLA-G protects decidual trophoblasts from lysis by blocking the effector function of decidual monocyte/macrophages. The interaction between HLA-G and monocyte/macrophages may therefore contribute to a successful pregnancy. Here we examine existing knowledge on the convergent role of HLA-G and monocyte/macrophages in pregnancy and define the synergy that exists between these two elements in the decidua. Key features of the HLA-G gene product are discussed followed by the main characteristics of decidual monocyte/macrophages. A hypothetical model for the interaction between HLA-G and monocyte/macrophage cells at the fetal-maternal interface is proposed.

Is obstetric antiphospholipid syndrome a primary nonthrombotic, proinflammatory, complement-mediated disorder related to antiphospholipid antibodies?

UNLABELLED: Pregnancy loss is the main obstetrical complication of the obstetric antiphospholipid syndrome. Classically, such losses have been attributed to placental thrombosis and infarcts, although in many cases there is no evidence of decidual thrombosis or placental vasculopathy, and instead inflammatory signs are present. In addition, the prevalence of systemic thrombosis is low in obstetric antiphospholipid syndrome, suggesting an alternative pathogenesis. There is evidence that antiphospholipid antibodies, mainly beta2-glycoprotein-I/anti-beta2-glycoproteina-I complexes, activate both classical and alternative complement pathways. Complement proteins may injure trophoblast cells, recruiting and activating monocytes and neutrophils. Free radicals and proteolytic enzymes could also attack trophoblastic cells, and amplification of the causal loop between tissue factor, inflammatory cells, and complement proteins could also be a factor. Overall, these diverse mechanisms may explain both inflammatory and thrombotic placental alterations. The role played by certain pro-inflammatory cytokines, mainly tumor necrosis factor-alpha, and the altered balance between angiogenic and anti-angiogenic factors remains to be clarified. In the end, obstetric antiphospholipid syndrome seems to be a clinical subset of classical APS. In these women, systemic thrombotic risk seems to be low. Current knowledge about inflammatory pathway involvement in obstetric antiphospholipid syndrome will permit us to modify the time to start heparin treatment, currently recommended to begin it as soon as possible after pregnancy confirmation. TARGET AUDIENCE: Obstetricians & Gynecologists, Family Physicians. LEARNING OBJECTIVES: After completion of this article, the reader will be able to recall manifestations of obstetric antiphospholipid syndrome, describe nonthrombotic mechanisms that may affect obstetric outcomes in women with antiphospholipid syndrome, and predict changes in the evaluation and treatment of obstetric patients with antiphospholipid syndrome should inflammatory factors prove to be an important feature of antiphospholipid syndrome.

Decidual leucocyte populations in early to late gestation normal human pregnancy.

Most research on human decidual leucocytes to date has focused on the predominant CD56+ uterine natural killer (uNK) cell population in early pregnancy. Few reports have documented decidual leucocyte populations after 13 weeks gestation and in late pregnancy. Placental bed (decidua basalis) and non-placental bed (decidua parietalis) biopsies from normal pregnancies were taken from women undergoing termination of pregnancy in the 1st and 2nd trimesters and following Caesarean section in the 3rd trimester. Immunohistochemistry was used to quantify the numbers of decidual cells expressing CD56, CD3, CD8, CD94, NKG2A and CD14 and double labelled CD161+CD3+ NKT-like cells. Although a significant reduction in CD56+ uNK cells was found in 3rd trimester samples compared with 1st and 2nd trimester decidua, a substantial residual CD56+ leucocyte population was identified in 3rd trimester decidua. Expression of the KIR CD94/NKG2A mirrored that of CD56 at all gestational ages, providing an explanation for the absence of cytotoxic responses at the fetal-maternal interface. There was no difference in leucocyte populations between decidua basalis and decidua parietalis. Double immunohistochemical labelling revealed small numbers of decidual CD3+CD56+ and CD8+CD56+ cells, which decreased in number at term, and CD161+CD3+ cells, which increased in number at term. No differences in leucocyte populations were detected between decidua parietalis and decidua basalis. In contrast to previous reports, a substantial residual CD56+ cell population was demonstrated in 3rd trimester decidua. Decidual cytotoxic T-lymphocytes did not alter in number during gestation, while in contrast CD14+ macrophages decreased at term, representing the smallest decidual population assessed.

Preeclampsia--a placenta developmental biology perspective.

There are abundant theories in the scientific literature that propose a range of pathophysiological pathways for preeclampsia. In this review we discuss some of the contributions made to this field from the perspective of a placental developmental biology laboratory. We discuss an underlying immune component of preeclampsia associated with expression of HLA-G and also a beneficial function of decidual NK cells. We conclude by summarizing newer findings regarding the anti-angiogenic expression of soluble fms-like tyrosine kinase (sFlt-1) and its role in the development of preeclampsia.

Benefits and burden of the maternally-mediated immunological imprinting.

The ontogenetic development of both the immune and the nervous system entirely depend on external environmental signals that induce a lifelong learning process. The resulting collective immunological knowledge about the external world is transmitted in an epi-genetic fashion to the offspring, but only from the maternal and not the paternal side, with maternal IgG as the main transgenerational vector. As products of thymus-dependent responses, maternal IgG have undergone immune maturation by somatic hypermutations and are, therefore, acquired immunological phenotypes representing a great deal of the mother's immunological experience. During a limited neonatal imprinting period, maternal antibodies induce T cell-dependent idiotypic responses. These exert up to life-long determinative influences which may even be dominant over seemingly genetic predispositions. Such long-term immunological imprinting effects can be detected as (a) selection of the adult T and B cell repertoires, (b) anti-microbial protection by antigen-reactive antibodies (idiotypes) and anti-idiotypes, (c) allergen-specific suppression of IgE responsiveness by allergen-reactive IgG idiotype or corresponding anti-idiotype and (d) induction of autoimmune diseases by maternally-derived autoantibodies. Hence, immunological imprinting by maternal IgG antibodies will mostly be beneficial, but in case of autoantibodies can also be a burden for the initial development of the nascent immune system.

Pregnancy immunology: decidual immune cells.

Human pregnancy is a complex process. Placental development depends on the function of secretory molecules produced by placental trophoblast cells as well as by maternal uterine immune cells within the decidua. These decidual immune cells are T cells, natural killer cells, macrophages and dendritic cells. The interactions between the trophoblast cells and the maternal immune cells have an impact on the outcome of the pregnancy. Knowledge about the phenotypes and functions of the maternal immune cells in normal and pathological pregnancies including recurrent spontaneous abortions, preeclampsia and hydatidiform moles may improve our understanding of the immunobiology of the normal pregnancy as a whole and may provide approaches for improving the treatment of pathological pregnancies.

The role of macrophages in utero-placental interactions during normal and pathological pregnancy.

The intimate association between maternal and placental tissues elicits an interesting immunological paradox. Placental tissue contains paternal antigens, but under normal circumstances the semi-allogeneic fetus and placenta are not attacked by the maternal immune system. Interestingly, this tolerance to fetal antigens occurs in the presence of a large number of maternal leukocytes, almost all of which are members of the innate immune system. Macrophages are one of the most abundant leukocytes in the decidua and their numbers remain constant throughout gestation. They are recruited to the decidua by both stromal cells and trophoblast cells, where they adopt a specialized phenotype that may assist in various aspects of decidual homeostasis, placental development, and tolerance to the semi-allogeneic trophoblast. Aberrant behavior of these macrophages can affect trophoblast function and placental development, potentially leading to a spectrum of adverse pregnancy outcomes ranging from pre-eclampsia to fetal growth restriction or demise. This review will focus on the phenotype and putative functions of decidual macrophages in normal pregnancy, and how abnormal activation of these cells can affect various aspects of placental development.

Human NK cells in pregnant uterus: why there?

Human Natural Killer (NK) cells are present in great number in pregnant uterine mucosa. They must be there for specialized functions, but which ones? This review discusses important recent observations that further contribute to this fascinating debate. Firstly, an array of corroborating findings indicates that uterine NK cell proliferation is synchronized with the cyclic surge of progesterone. Secondly, uterine NK cells are unlikely to exert a direct control on the embryo implantation. Thirdly, these NK cells influence the uterine vascular remodeling in early pregnancy but might not be the single key element that control trophoblast invasion. Finally, uterine NK cells are likely to be an important component of the local maternal immune response to pathogen infections.

AT1-receptor autoantibodies and uteroplacental RAS in pregnancy and pre-eclampsia.

Pre-eclampsia is a common, pregnancy-induced disorder, consisting of hypertension and proteinuria. The condition is one of the leading causes for maternal and perinatal morbidity and mortality. Nonetheless, the underlying molecular mechanisms remain unclear. Immunological mechanisms and the renin-angiotensin system have been implicated in the development of pre-eclampsia. Agonistic autoantibodies to the angiotensin II type I receptor (AT1-AA) have been identified in pre-eclamptic patients, unifying the two hypothesis. Evidence has also accumulated for the existence and importance of a local, utroplacental renin-angiotensin system. We summarize recent data emphasizing the pathophysiological role for the autoantibodies and the uteroplacental renin-angiotensin system.

Alternatively activated macrophages regulate extracellular levels of the hormone placental lactogen via receptor-mediated uptake and transcytosis.

Alternatively activated (M2) macrophages regulate immune responses and tissue remodelling. In many tissues including placenta, M2 express stabilin-1, a multidomain protein that exerts a dual role as a scavenger receptor for acetylated low density lipoprotein (acLDL) and SPARC (secreted protein acidic and rich in cysteine) and as an intracellular cargo carrier for SI-CLP. Using yeast two-hybrid screening, we identified the developmental hormone placental lactogen (PL) as a novel ligand of stabilin-1. In Chinese hamster ovary-stabilin-1 cells and M2, FACS and confocal microscopy demonstrated that stabilin-1 mediates internalization and endosomal sorting of PL. In M2 macrophages, PL was partially degraded in lysosomes; part of PL escaped degradation and was delivered to novel PL+ storage vesicles lacking endosomal/lysosomal markers. During formation, PL+ vesicles underwent transient interaction with the trans-Golgi network (TGN). Upon placement of PL-loaded M2 into PL-free medium, PL was secreted into the supernatant. Leupeptin, an inhibitor of lysosomal hydrolases, reduced PL degradation, enhanced sorting of PL into the TGN/storage vesicle pathway and increased PL secretion. Thus, processing of PL in M2 macrophages occurs either by the classical lysosomal pathway or by a novel TGN-associated trans-secretory pathway. Macrophages isolated from human placental villi efficiently endocytosed PL-FITC and transported it to the storage vesicles. Our data show that extracellular PL levels are determined by uptake, degradation, storage, and release in M2. During pregnancy PL concentration reaches 10 microg/ml in maternal circulation and stays below 0.5 microg/ml in fetal circulation. We propose that stabilin-1-positive macrophages determine the difference in PL levels between maternal and fetal circulation.

Expression of CD28 and cytotoxic T lymphocyte antigen 4 at the maternal-fetal interface in women with unexplained pregnancy loss.

OBJECTIVE: To investigate the expression of CD28 and CTLA-4, two costimulatory molecules involved in T-cell activation at the maternal-fetal interface in women with unexplained pregnancy loss. METHODS: A total of 57 women, 39 with unexplained spontaneous abortion (SA) and 18 with unexplained recurrent spontaneous abortion (RSA), were enrolled in the study. A high-resolution spectratyping analysis of fluorescent bands was performed to detect CD28 and CTLA-4 expression in decidual tissues. RESULTS: The mean expression of CTLA-4 mRNA was significantly higher in women with SA than in controls (P<0.05). Moreover, the expression of CTLA-4 was higher in SA patients with genotype AA and phenotype A+ (AA+AG) than in controls (P<0.01). The expression of CTLA-4 was not significantly different in patients with RSA and in controls. The expression of CD28 was significantly decreased in patients with RSA (P<0.01) and SA (P<0.05) compared with controls. The mean ratios of CTLA-4/CD28 were significantly higher in patients with RSA (P<0.01) and SA (P<0.05) than in controls. CONCLUSIONS: This study suggests that an imbalance in CTLA-4/CD28 expression or suppressed T-cell activity at the maternal-fetal interface may confer susceptibility to unexplained pregnancy loss.

Nonthermal effects of mobile-phone frequency microwaves on uteroplacental functions in pregnant rats.

Exposure to high-density microwaves can cause detrimental effects on the testis, eye, and other tissues, and induce significant biologic changes through thermal actions. To examine nonthermal effect of continuous wave (CW) 915MHz microwaves used in cellular phones, we compared the effects of microwaves with those of heat. Thirty-six pregnant rats were assigned to six groups: rats exposed to microwaves at 0.6 or 3mW/cm(2) incident power density at 915MHz for 90min, rats immersed in water at 38 or 40 degrees C, which induces about the same increase in colonic temperature of 1.0 or 3.5 degrees C as 0.6 or 3mW/cm(2) microwaves, respectively; rats immersed in water at 34 degrees C, which is considered to be thermoneutral; and control rats. We identified significant differences in the uteroplacental circulation, and in placental endocrine and immune functions between pregnant rats immersed in water at 34 and 38 degrees C, but not between rats immersed at 38 degrees C and those exposed to microwaves at 0.6mW/cm(2). By contrast, we observed significant decreases in uteroplacental blood flow and estradiol in rats exposed to microwaves at 3mW/cm(2) as compared with those immersed in water at 40 degrees C. These results suggest microwaves at 0.6mW/cm(2) at 915MHz, equal to a specific absorption rate (SAR) of 0.4W/kg, which is the maximum permissible exposure level recommended by the American National Standards Institute (ANSI), do not exert nonthermal effects on blood estradiol and progesterone, on splenic natural killer cell activity, on the uteroplacental circulation.