Joan S. Hunt

Cytokine networks in the uteroplacental unit: macrophages as pivotal regulatory cells

A rich array of potent regulatory molecules has been identified in the uteroplacental unit. Most recently uncovered are the cytokines, families of polypeptides that establish intercellular communications, a paracrine effect, and often bind to synthetic cells in autocrine regulatory loops. Nearly all of the disparate maternal and fetal cell types in the uteroplacental unit are integrated into the cytokine network. The highly versatile macrophage, abundant in uteroplacental tissues, has emerged as a potentially pivotal cell type because of its unique ability to send and receive cytokine signals. Elevated levels of cytokines, possibly secreted when uteroplacental macrophages are activated by either bacterial endotoxins or receptor-bound cytokines, may compromise pregnancy. In particular, cytokines have been implicated in the induction of pre-term labor associated with infections. Intensive research is required to delineate the temporal patterns of cytokine synthesis that characterize pregnancy, to evaluate the events leading to normal and premature pregnancy termination and to establish protocols for therapeutic interventions in cases of infection.

Sex steroid hormones and macrophage function

Macrophages are versatile cells whose activities are programmed by environmental signals. In this review, we discuss the potential impact of sex steroid hormones on macrophage activation and production of various effector molecules. The evidence accumulated to date supports the postulate that estrogens, progesterone, androgens and testosterone profoundly influence host defense by controlling the ability of macrophages to participate in immune responses.

Transcriptional Regulation of VEGF-A by the Unfolded Protein Response Pathway

Background Angiogenesis is crucial to many physiological and pathological processes including development and cancer cell survival. Vascular endothelial growth factor-A (VEGFA) is the predominant mediator of angiogenesis in the VEGF family. During development, adverse environmental conditions like nutrient deprivation, hypoxia and increased protein secretion occur. IRE1α, PERK, and ATF6α, master regulators of the unfolded protein response (UPR), are activated under these conditions and are proposed to have a role in mediating angiogenesis. Principal Findings Here we show that IRE1α, PERK, and ATF6α powerfully regulate VEGFA mRNA expression under various stress conditions. In Ire1α−/− and Perk−/− mouse embryonic fibroblasts and ATF6α-knockdown HepG2 cells, induction of VEGFA mRNA by endoplasmic reticulum stress is attenuated as compared to control cells. Embryonic lethality of Ire1α−/− mice is due to the lack of VEGFA induction in labyrinthine trophoblast cells of the developing placenta. Rescue of IRE1α and PERK in Ire1α−/− and Perk−/− cells respectively, prevents VEGFA mRNA attenuation. We further report that the induction of VEGFA by IRE1α, PERK and ATF6 involves activation of transcription factors, spliced-XBP-1, ATF4 and cleaved ATF6 respectively. Conclusions/Significance Our results reveal that the IRE1α-XBP-1, PERK-ATF4, and ATF6α pathways constitute novel upstream regulatory pathways of angiogenesis by modulating VEGF transcription. Activation of these pathways helps the rapidly growing cells to obtain sufficient nutrients and growth factors for their survival under the prevailing hostile environmental conditions. These results establish an important role of the UPR in angiogenesis.

HLA and maternal-fetal recognition.

Despite genetic differences, mothers do not reject their semiallogeneic embryos. Regulated expression of the major histocompatibility antigens (HLA) by placental trophoblast cells, which intervene between the embryo and maternal blood and tissues, is now believed to play an important role in this surprising feature of pregnancy. Transcription and translation of the highly polymorphic class I HLA‐A, ‐B, ‐C genes whose products stimulate graft rejection are blocked in trophoblast cells. Instead, these cells express HLA‐G, a nonpolymorphic gene. Moreover, the cells do not express class II HLA‐D antigens, and factors such as interferons that usually enhance HLA expression have no effect on trophoblast cells in situ. Thus, multiple regulatory mechanisms prevent the cells that sequester the embryo from the mother from expressing the potentially deleterious paternal HLA antigens, immunological rejection is avoided and successful pregnancy ensues.—Hunt, J. S.; Orr, H. T. HLA and maternal‐fetal recognition. FASEB J. 6: 2344‐2348; 1992.

B7 Family Molecules Are Favorably Positioned at the Human Maternal-Fetal Interface

Abstract The human placenta utilizes both active and passive mechanisms to evade rejection by the maternal immune system. We investigated the pattern of expression of the B7 family of immunomodulatory molecules B7-H1 (PD-L1), B7-2 (CD86), and B7-1 (CD80) at the term maternal-fetal interface. Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses showed that B7-H1 mRNA is abundant in term placenta and that cytotrophoblasts are sources of this message. Immunohistochemistry demonstrated that B7-H1 is constitutively expressed by the syncytiotrophoblast and by extravillous cytotrophoblasts, both of which are juxtaposed to maternal blood and tissue. By contrast, placental stromal cells, including macrophages, lacked the protein. Expression of B7-H1 protein was low in first-trimester placenta compared to second- and third-trimester tissue (P < 0.05) and was enhanced in cultured cytotrophoblasts by treatment with either interferon-γ or epidermal growth factor (P < 0.05), suggesting that one or both of these mediators regulates B7-H1 expression in the placenta. RT-PCR and immunofluorescence analysis of term placental tissue revealed different patterns of expression of the immunostimulatory protein, B7-2. In contrast to B7-H1, B7-2 mRNA and protein were absent in cytotrophoblast cells but present in maternal macrophages and some fetal macrophages. The B7-1 mRNA and protein were absent at the maternal-fetal interface. These studies document expression of the B7 family proteins at the maternal-fetal interface and demonstrate that B7-H1 is positioned such that it could facilitate protection of fetal cells against activated maternal leukocytes. Conversely, B7-2 was absent on trophoblasts and was appropriately localized to fetal and maternal macrophages, which may participate in antigen presentation.

Progesterone inhibits inducible nitric oxide synthase gene expression and nitric oxide production in murine macrophages.

The purpose of this study was to determine whether the female hormones estradiol‐17β (E2) and progesterone (P4) influence inducible nitric oxide synthase (iNOS) and the production of nitric oxide (NO) by interferon‐γ(IFN‐γ)‐ and lipopolysaccharide (LPS)‐activated mouse macrophages. Treatment with P4 alone caused a time‐ and dose‐dependent inhibition of NO production by macrophage cell lines (RAW 264.7, J774) and mouse bone marrow culture‐derived macrophages as assessed by nitrite accumulation. RAW 264.7 cells transiently transfected with an iNOS gene promoter/luciferase reportergene construct that were stimulated with IFN‐γ/LPS in the presence of P4 displayed reduced luciferase activity and NO production. Analysis of RAW 264.7 cells by Northern blot hybridization revealed concurrent P4‐mediated reduction in iNOS mRNA. These observations suggest that P4‐mediated inhibition of NO may be an important gender‐based difference within females and males that relates to macrophage‐mediated host defense.

Placental Cell Expression of HLA-G2 Isoforms Is Limited to the Invasive Trophoblast Phenotype

The HLA-G message is alternatively spliced into multiple transcripts, two of which encode soluble isoforms. To initiate studies on the specific functions of the soluble isoforms, we produced soluble rHLA-G1 (rsG1) and rsG2 in human embryonic kidney 293 cells and characterized the proteins. Both isoforms were glycosylated and formed disulfide-bonded oligomers. Recombinant sG1 associated with β2-microglobulin, whereas rsG2 did not. Mouse mAb generated to rsG1 (1-2C3), which identified exclusively sG1, and mAb generated to rsG2 (26-2H11), which identified both soluble and membrane G2 (m/sG2), were used for immunohistochemical isoform mapping studies on placental tissue sections. Soluble G1 protein was abundant in many subpopulations of trophoblast cells, whereas m/sG2 protein was present exclusively in extravillous cytotrophoblast cells. Although both isolated placental villous cytotrophoblast cells and chorion membrane extravillous cytotrophoblast cells contained mRNAs encoding sG1 and sG2, protein expression was as predicted from the immunostains with m/sG2 present only in the invasive trophoblast subpopulation. Analysis of function by Northern and Western blotting demonstrated that both rsG1 and rsG2 inhibit CD8α expression on PBMC without changing CD3δ expression or causing apoptotic cell death. Collectively, the studies indicate that: 1) both sG1 and m/sG2 are produced in placentas; 2) transcription and translation are linked for sG1, but not G2; 3) expression of G2 is exclusively associated with the invasive phenotype; and 4) the two isoforms of sG may promote semiallogeneic pregnancy by reducing expression of CD8, a molecule required for functional activation of CTL.

Localization of transforming growth factor β and its natural inhibitor decorin in the human placenta and decidua throughout gestation

Transforming growth factor beta (TGF beta) produced at the human fetomaternal interface has been shown to play a crucial role in controlling trophoblast invasion of the uterus. Decorin, a naturally occurring chondroitin-dermatan sulphate proteoglycan which binds TGF beta can inhibit its activity. In this study, immunohistochemical techniques were used to determine the locations of TGF beta and decorin within the human placenta and decidua throughout normal gestation. In addition, sites of TGF beta 1 mRNA synthesis were identified in early and late placenta by in situ hybridization. Results revealed the presence of immunoreactive TGF beta in the cytoplasm of villous syncytiotrophoblast and extravillous trophoblast cells throughout gestation. TGF beta immunostaining was absent from villous cytotrophoblast at all gestational ages examined. The extracellular matrix (ECM) of the villous core at all stages of gestation and cells of the cytotrophoblastic shell of the term placenta were immunoreactive for TGF beta. Within decidual tissue, TGF beta was primarily localized in the ECM during the first trimester and only a small proportion of decidual cells exhibited intracellular labelling. At later gestational ages the majority of decidual cells showed intracellular labelling accompanied by a decrease in ECM staining. This switch may reflect increased TGF beta synthesis by the decidual cells, decreased release, or altered TGF beta binding to one or more ECM proteins. In situ hybridization indicated that TGF beta 1 mRNA was primarily localized to the syncytiotrophoblast cell layer with low intensity signals present in extravillous trophoblast cells, in trophoblast cell columns, and in large decidual cells. At term, TGF beta 1 mRNA was located in both the syncytiotrophoblast and villous mesenchymal cells. Decorin was immunolocalized to the ECM of the mesenchymal core of the chorionic villi throughout gestation and no immunoreactivity was observed in either villous or extravillous trophoblast. In the first trimester decidua, decorin was localized to the ECM whereas decidual cells, decidual leucocytes, and the uterine epithelium were negative. At later gestational ages, the ECM as well as a few decidual cells displayed weak immunoreactivity. A strong co-localization of TGF beta and decorin in the ECM of first trimester decidual tissue suggests that decorin may aid TGF beta storage or limit its activity in the ECM.

Uterine macrophages and environmental programming for pregnancy success

Macrophages are ubiquitous cells with an impressive range of functions. These include phagocytosis and coordination of the initiation and effector phases of immune responses, as well as production of bioactive proteins and lipids that profoundly influence cell growth, differentiation and function. Macrophages are highly individualized in tissues, where their activities are a reflection of targeting by systemic and local environmental signals. This review focuses on recent studies where uterine macrophage population densities and distribution have been mapped, chemotaxis, differentiation and activation have been investigated and production of potent effector molecules has been explored. Evidence supporting a major role for female sex steroid hormones and the uterine growth factors they control in governing these features of uterine macrophages is presented.

Localization and characterization of macrophages in murine uterus.

Macrophages are known to be present in the murine uterus and are known to be among those cells comprising the uterine decidual response to pregnancy. The extent of macrophage involvement in the decidual response has not been documented, and there are unresolved questions regarding expression of markers normally associated with macrophages on cells within the decidua.