Astrid Blaschitz

The functionality of HLA-G is emerging

Summary: In view of the recently published data, the HLA‐G class Ib gene appears to be a functional locus. This is based on the following observations: 1) HLA‐G is capable of presenting nonamer peptides and of exerting antigen‐presenting functions; 2) HLA‐G is a ligand for at least three natural killer (NK) and other cell inhibitory receptors of the immunoglobulin superfamily, namely leukocyte immunoglobulin‐like receptor‐1 /immunoglobulin‐like transcript (ILT)‐2, ILT‐4 and p49; 3) in addition to the extravillous cytotrophoblast cells, HLA‐G proteins have been detected in endothelial cells of placental chorionic villi, as well as in amniotic fluid and in some medullary thymic epithelial cells; 4) major histocompatibility complex (MHC) class Ib genes that share the unique characteristics of HLA‐G, including a high expression in placenta, have been reported in other mammalian species. In addition to the classical MHC class I roles (antigen presentation and ligarion to NK receptors inducing inhibitory and/or activatory signals), HLA‐G is likely to exert other, novel functions: first, HLA‐G was shown to be involved in the control of HLA‐E expression by furnishing the appropriate class I leader sequence nonamer peptide; second, we hypothesize that HLA‐G could be a regulator of placental angiogenesis; third, soluble HLA‐G isoforms may act as specific immunosuppressors during pregnancy. Such functional properties, although incompletely understood, are likely to be important in the outcome of human pregnancies but also in normal adult life.

Heterogeneity of microvascular endothelial cells isolated from human term placenta and macrovascular umbilical vein endothelial cells

The present study compares some phenotypic and physiologic characteristics of microvascular and macrovascular endothelial cells from within one human organ. To this end microvascular endothelial cells from human full-term placenta (PLEC) were isolated using a new method and compared with macrovascular human umbilical vein endothelial cells (HUVEC) and an SV40-transformed placental venous endothelial cell line (HPEC-A2). PLEC were isolated by enzymatic perfusion of small placental vessels, purified on a density gradient and cultured subsequently. Histological sections of the enzyme-treated vessels showed a selective removal of the endothelial lining in the perfused placental cotyledons. The endothelial identity of the cells was confirmed by staining with the endothelial markers anti-von Willebrand factor, Ulex europaeus lectin and anti-QBEND10. The cells internalized acetylated low-density lipoprotein and did not show immunoreactivity with markers for macrophages, smooth muscle cells and fibroblasts. The spindle-shaped PLEC grew in swirling patterns similar to that described for venous placental endothelial cells. However, scanning electron microscopic examination clearly showed that PLEC remained elongated at the confluent state, in contrast to the more polygonal phenotype of HPEC-A2 and HUVEC that were studied in parallel. The amount of vasoactive substances (endothelin-1,2, thromboxane, angiotensin II, prostacyclin) released into the culture medium and the proliferative response to cytokines was more similar to human dermal microvessels (MIEC) derived from non-fetal tissue than to HUVEC. Potent mitogens such as vascular endothelial growth factors (VEGF121, VEGF165) and basic fibroblast growth factor (FGF-2) induced proliferation of all endothelial cell types. Placental growth factors PIGF-1 and PIGF-2 effectively stimulated cell proliferation on PLEC (142 +/- 7% and 173 +/- 10%) and MIEC (160 +/- 20% and 143 +/- 28%) in contrast to HUVEC (9 +/- 8% and 15 +/- 20%) and HPEC-A2 (15 +/- 7% and 24 +/- 6%) after 48 h incubation time under serum-free conditions. These data support evidence for (1) the microvascular identity of the isolated PLEC described in this study, and (2) the phenotypic and physiologic heterogeneity of micro- and macrovascular endothelial cells within one human organ.

Fas and Fas-Ligand Are Expressed in the Uteroplacental Unit of First-Trimester Pregnancy

PROBLEM: Fas and Fas‐ligand (FasL) are thought to provide a strategy for reducing graft rejection in immunologically ‘privileged’ tissues by controlling injurious lymphocyte reactions. As the uteroplacental unit is often defined as an immune‐privileged site, we investigated the expression of Fas and FasL in this tissue in the first trimester of pregnancy.

Amnion Epithelial Cells, in Contrast to Trophoblast Cells, Express All Classical HLA Class I Molecules Together With HLA-G

PROBLEM: The expression of the non‐classical HLA‐G gene has been shown at the protein level on trophoblast‐derived embryonic tissue, like the extravillous cytotrophoblast. However, the presence of HLA‐G on embryoblast‐derived cells is currently controversial. The amnion epithelium is an embryoblast‐derived cell layer covering the amnion cavity and is the main source for the amnion fluid.

Expression of HLA class I molecules in human first trimester and term placenta trophoblast

Abstract.Expression of HLA class I molecules in trophoblast cells from various locations in normal human first trimester and term placenta was investigated by immunohistochemistry with a panel of monoclonal antibodies against the heavy chains or complete HLA class I molecules complexed with β2-microglobulin. These reagents were also employed to distinguish between the products of different HLA class I loci. In addition to previously characterized reagents, a novel monoclonal antibody against HLA-A molecules (TÜ155) was used. Various choriocarcinoma and transfected cell lines served as controls for the specificities of the monoclonal antibodies. Cells in close contact with maternal cells, such as invading trophoblast cells and cells of the basal plate, expressed β2-m micro globulin in association with HLA-G and HLA-C heavy chains. These class I heavy chains may also have been present as isolated molecules, although not in each of the cells. In contrast, cells of the chorion laeve exclusively expressed HLA-G, and not HLA-A, -B, or -C antigens. Our data support the often discussed immune protective function and the regulatory function of the HLA-G molecule, during invasion. In addition, by using monoclonal antibodies HCA2 (anti-HLA-A and -G), HC10 (anti-HLA-B and -C), TÜ149 (anti-HLA-B, -C, and some -A alleles), SFR8-B6 (anti-HLA-Bw6 and some -C), LA45 (some HLA-A and -B), TÜ48 (anti-HLA-Bw4 and some -A), and TÜ155 (anti-HLA-A), we show the presence of HLA-C molecules in all extravillous trophoblast cells of the cell columns and in the basal plate; the trophoblast cells of the chorion laeve lack this antigen. The function of this molecule is not clear, although a protective function against natural killer cell activity in the endometrium is postulated.

Insulin receptors in syncytitrophoblast and fetal endothelium of human placenta. Immunohistochemical evidence for developmental changes in distribution pattern

The localisation of insulin receptors (IR) was investigated on cryosections of human non-pathologic first trimester and full term placentae by indirect immunohistochemistry with three different monoclonal antibodies (MABS). In placentae from 6 to 10 weeks post-menstruation (p-m.), only syncytiotrophoblast was stained, predominantly that of mesenchymal villi and syncytial sprouts, which are areas of high proliferative activity. In placentae from 11 to 14 weeks p-m., endothelial cells commenced to react with the IR MABS and the syncytiotrophoblast was less intensely labelled than at weeks 6 to 10 p-m. In term placentae, the microvillous membrane of the syncytiotrophoblast showed only patches of weak immunoreactivity. In contrast, the endothelial cells in the placenta but not in the umbilical cord were strongly stained. The amniotic epithelium in the chorionic plate and fibroblasts in the stroma were conspicuously labelled. The data indicate: (1) the receptor density on villous syncytiotrophoblast decreases and that of fetal endothelium increases throughout gestation; (2) syncytiotrophoblast of human term placentae expresses a low level per unit area of surface IR; and (3) the majority of IR in human term placentae is located in fetal endothelium. Apart from yet unknown functional effects of maternal and fetal insulin at the placental barrier, the results suggest a growth promoting effect on the trophoblast of maternal insulin in first trimester as well as developmental effects of fetal insulin on the feto-placental vessels at term.

Paracrine regulation of distinct trophoblast functions in vitro by placental macrophages

Abstract In view of the accumulating evidence for paracrine mechanisms regulating trophoblast function, we tested the hypothesis that placental macrophages affect trophoblast activity in a paracrine fashion. Trophoblast was isolated from 17 term placentas (–IP). One aliquot of cells was further immunopurified (+IP) using an HLA class I antibody. This increased the proportion of trophoblast (+IP >97%; –IP ∼70%) as identified by rigorous immunocytochemistry. Most (∼70%) non-trophoblast cells in –IP were macrophages. The cells were cultured for 5 days with a daily medium change. In addition, +IP cells from seven placentas were cultured with lipopolysaccharide (LPS)-stimulated or -unstimulated macrophage-conditioned media. The concentrations of lactate, trophoblast-specific hormones, human chorionic gonadotropin-β (hCG-β) and human placental lactogen (hPL), of several prostanoids and of endothelin-1 and angiotensin II were determined in the culture media. The accumulated amounts of substances released into the culture media, corrected for the greater proportion of trophoblast in +IP cultures, were on average two- to threefold higher (hCG-β: 18-fold) in +IP than in –IP, with the exception of endothelin-1,2 (no change), angiotensin II (–70%) and 6-keto-prostaglandin-F1α (–40%). [3H]leucine incorporation into the trichloroacetic acid (TCA)-precipitable pool measured on day 5 was twofold higher in +IP than in –IP. Addition of conditioned media reverted these changes. The data demonstrate that placental macrophages in culture affect trophoblast biosynthetic activity in a paracrine fashion. We conclude that macrophages are important regulators of trophoblast activity.

Immunohistochemical evidence for the heterogeneity of maternal and fetal vascular endothelial cells in human full-term placenta

The heterogeneity of endothelial cell surface antigen expression was studied in 5 human full-term placentae by means of indirect immunohistochemistry using 9 monoclonal antibodies and by staining with fluorescent-conjugated Ulex europaeus lectin, both of which are widely used endothelial cell markers. (1) A highly specific, homogeneous staining of fetal and maternal placental vessels of all sizes and anatomical regions was observed by the monoclonal antibodies PAL-E, QBEND10 and 1F10. These antibodies were even more specific than Ulex europaeus lectin, factor VIII antibody and von Willebrand factor antibody, which cross-reacted with some non-endothelial cells and structures. The reactivity of PAL-E, QBEND10 and 1F10 with residual surface cells of the basal plate strongly suggests an endothelial origin of these cells. (2) In contrast to other organs, PAL-E, QBEND10 and HM 15/3 strongly stained endothelial cells of the macrovascular system in the human placenta. This might indicate an organ-associated heterogeneity of fetal endothelial cells. (3) Monoclonal antibodies against receptors for transferrin and IgG (FcγRII) labeled the endothelial cells of fetal placental vessels with increasing intensity distal to the insertion of the umbilical cord. The vessels of the umbilical cord itself were unreactive. This might suggest a heterogeneity of macro- and microvascular endothelial cells.

Localisation of the high affinity factilitative glucose transporter protein GLUT 1 in the placenta of human, marmoset monkey (Callithrix jacchus) and rat at different developmental stages

In the present study, the facilitative D-glucose transporter protein GLUT 1 was localised by immunohistochemistry in the placenta of human, marmoset (Callithrix jacchus) and rat at different developmental stages. A polyclonal antiserum agains a 13-amino-acid peptide of the GLUT 1 carboxy terminus was used. It identified a protein of around 50 kDa molecular weight in immunoblotting of the placental tissues. GLUT 1 was located in the syncytiotrophoblast, in cytotrophoblast cells and in fetal endothelium. Similar staining patterns, except in human extravillous cytotrophoblast cells, were observed at all differentiation stages, despite differences in the internal placental architecture of the species. In the marmoset placenta, GLUT 1 was undetectable in endothelial cells of maternal vessels. In rat placentae, trophoblastic giant cells, epithelial cells of both visceral and parietal yolk sac, yolk sac vessels and the stratum spongiosum were stained. Reicherts membrane did not immunoreact. Preadsorption of the antiserum with a 13-amino-acid peptide resulted in the loss of immunoreactivity. The results suggest that GLUT 1 is a prominent isoform of glucose transporters in mammalian placentae. It is generally abundant in placental cell populations bordering on the maternal and fetal circulations and may therefore facilitate an effective glucose supply to the fetus and placenta.

Hyperglycaemia-induced subcellular redistribution of GLUT1 glucose transporters in cultured human term placental trophoblast cells.

Aims/hypothesis. We have recently shown that hyperglycaemia down-regulates the GLUT1 glucose transport system of term placental trophoblast. The reduction in GLUT1 protein alone was, however, not sufficient to explain the decrease in net glucose uptake, suggesting additional mechanisms. Therefore, we hypothesised that hyperglycaemia in vitro leads to a GLUT1 translocation from the trophoblast surface to intracellular sites.¶Methods. This was tested in our study by determining the subcellular distribution of GLUT1 in human term placental trophoblast (n = 5 placentas) cultured for 48 h with 5 compared with 25 mmol/l d-glucose in vitro using immunogold labelling.¶Results. Electron microscopic examination of cell profiles showed that 73 % of total GLUT1 molecules reside in the trophoblast plasma membrane under basal conditions. The reduced GLUT1 expression (–20 %; p < 0.05) after culture of the cells with 25 mmol/l glucose was accompanied by an internalisation of plasma membrane GLUT1, resulting in a loss of 40 % (p < 0.05) in cell surface transporter labelling. Western blotting identified a characteristically broad band between 55–65 kDa, confirming the specificity of the GLUT1 antiserum.¶Conclusion/interpretation. We postulate that in addition to down-regulating human GLUT1 protein concentrations, glucose exerts its autoregulatory effect on hexose transport in term placental trophoblast by altering GLUT1 partitioning between the plasma membrane and intracellular sites in favour of the latter. [Diabetologia (2000) 43: 173–180]