Biomedical subjects

Y W Loke

Publications and source records attributed to Y W Loke.

At least 55 records · Page 3Linked to original sources

PubMed1996

Evidence for the expression of HLAA-C class I mRNA and protein by human first trimester trophoblast.

In this study, evidence is provided that normal human first trimester extravillous trophoblast expresses class I HLA-C molecules in addition to HLA-G. cDNA from highly purified trophoblast cells obtained by flow cytometric sorting was amplified by reverse-transcriptase PCR using HLA locus-specific primers. The identity of the product was confirmed by Southern blotting and hybridization by a second HLA-C-specific oligonucleotide. HLA-C mRNA was clearly demonstrated in all trophoblast samples as well as in JEG-3 and BeWo choriocarcinoma cells. JAR choriocarcinoma cells did not express HLA-C. The presence of HLA-C protein in extravillous trophoblast was investigated using a panel of Abs: L31 is specific for heavy chains of all HLA-C alleles; Q1/28 reacts with all HLA class I products except HLA-G; HC-10 has preferential reactivity with HLA-B and HLA-C heavy chains. We performed 35S metabolic and 125I surface labeling of normal first trimester trophoblast and found abundant HLA-C intracellularly together with low levels of expression of both the beta 2m-associated forms and free heavy chains on the surface. Flow cytometric analysis of normal trophoblast confirmed the expression of a class I HLA molecule distinct from HLA-G by positive reactivity with Q1/28. Immunohistologic studies of first trimester placenta and the implantation site clearly showed expression of HLA-C in all extravillous trophoblast populations. Our results demonstrate the presence of two HLA class I molecules, HLA-G and HLA-C, on the surface of extravillous trophoblast. These results have implications in understanding how maternal uterine lymphocytes, notably the abundant NK-like cells, might recognize the implanting placenta.

Base Sequence↗

PubMed1996

Nonclassical HLA-G molecules are classical peptide presenters.

BACKGROUND: The physiological functions of the classical HLA (human leukocyte antigen) molecules, HLA-A, HLA-B and HLA-C, are to present peptides to T cells and to inhibit the activity of natural killer cells. In contrast, the functions of nonclassical HLA-molecules, such as HLA-E, HLA-F and HLA-G, remain to be established. The expression of HLA-G is largely limited to the placental trophoblast, where it might mediate protection of the fetus from rejection by the mother. Achieving the aim of understanding the function of HLA-G should be facilitated by information on the biochemical properties of HLA-G molecules, especially on their potential ability to act as peptide receptors. RESULTS: To study peptide presentation by HLA-G, we used stably transfected LCL721.221 cells as a source of HLA-G molecules and analysed the spectrum of extracted peptides by individual and pool sequencing. Our results indicate that HLA-G molecules, like classical HLA molecules, are associated with a wide array of peptides derived from cellular proteins. Peptides presented by HLA-G usually consisted of 9 amino acids, and adhered to a specific sequence motif, with anchor residues at position 2 (isoleucine or leucine), position 3 (proline) and the carboxy-terminal position 9 (leucine). Thus, the HLA-G peptide ligand motif follows the principles of classical HLA motifs, although it displays its own unique features. Peptide-binding assays indicated that two of the three anchor residues were sufficient for binding, and that the three natural HLA-G ligands that we identified bound, not only to HLA-G, but also to HLA-A2. This was not surprising, because the binding pockets of HLA-A2 and HLA-G overlap in their ability to recognize anchor residues at positions 2 and 9. Likewise, some, but not all, HLA-A2 peptide ligands could also bind to HLA-G. CONCLUSIONS: Nonclassical HLA-G molecules present peptides essentially in the same way as classical HLA molecules do. We determined the peptide motif that is specifically recognized by HLA-G; its basic features are described by the sequence XI/LPXXXXXL: This information should help to elucidate the physiological role of HLA-G molecules at the fetal-maternal interface. Most likely, this role is to protect fetal cells from lysis by natural killer cells, and possibly to present foreign peptides to a class of T cells that has not yet been identified.

Amino Acid Sequence↗

PubMed1996

Human decidual leukocytes do not proliferate in response to either extravillous trophoblast or allogeneic peripheral blood lymphocytes.

The maternal leukocytes present in human decidua during the early weeks of pregnancy are composed of CD56+ NK cells (approximately 80%), CD3+ T cells (approximately 10%), and CD14+ macrophages (approximately 10%). These leukocytes are present in large numbers at the implantation site where trophoblast invades. The migrating extravillous trophoblast (EVT) cells express HLA class I antigens HLA-G and HLA-C. To examine whether EVT or other allogeneic cells are capable of inducing a local proliferative response in maternal decidual leukocytes, we have assayed [3H]thymidine incorporation by decidual leukocytes co-cultured with irradiated trophoblast or peripheral blood lymphocytes (PBL). No proliferative response was seen in decidual leukocytes either to trophoblast or to PBL as stimulators. In addition trophoblast did not stimulate a proliferative response in PBL. These findings indicate that extravillous trophoblast is not capable of initiating a classical immune response at the implantation site.

Cell Division↗

PubMed1996

Immune phenotype and cytotoxic activity of lymphocytes from human term decidua against trophoblast.

Flow cytometric data were used to compare the phenotype of term decidual lymphocytes and peripheral blood lymphocytes. Unlike peripheral blood lymphocytes, a significant percentage of CD3+, CD4+, CD8+ and CD16+ term decidual lymphocyte populations expressed the CD69 activation marker. The relative proportions of CD38 in CD3+, CD4+ and CD8+ populations were more than twice as large in term decidual lymphocytes as in peripheral blood lymphocytes. As reported for early decidual lymphocytes, the expression of CD38 and CD69 by term decidual lymphocytes suggests that these cells are also regionally activated. However, term decidual lymphocytes showed no spontaneous cytotoxicity against normal trophoblast or its tumoral counterpart, JEG cells. After stimulation with interleukin-2, these lymphocytes became cytotoxic, as did peripheral blood lymphocytes. The relevance of this latter result to the immune control of the physiological and pathological invasion of the decidua by the trophoblast is discussed.

ADP-ribosyl Cyclase↗

PubMed1996

Immunology of human implantation: an evolutionary perspective.

The concept of the fetus as an allograft, which has been much cherished by reproductive immunologists, is in need of re-appraisal. We speculate that the relationship between the allogeneic conceptus and mother is not governed by the laws of classical transplantation immunity. Instead, this relationship is more akin to the allorecognition system seen in invertebrates.

Animals↗

PubMed1996

Evaluation of oestrogen and progesterone receptor expression in uterine mucosal lymphocytes.

Expression of the oestrogen and progesterone receptors on uterine mucosal leukocytes has been examined by dual immunohistology. Neither the oestrogen receptor nor the progesterone receptor was expressed by lymphocytes, macrophages or the distinctive population of uterine natural killer (NK) cells. Although the accumulation and survival of these NK cells appears to be hormonally dependent, the effects must therefore be indirect.

Decidua↗

PubMed1996

Evolution of the fetal-maternal immunological relationship.

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Female↗

PubMed1996

Functions of human decidual NK cells.

The main population of lymphocytes found in the human decidua during early pregnancy are NK-like cells with a distinctive phenotype, CD56bright CD16- CD3-. These cells are in close association with invading trophoblast that may be their in vivo target. We have examined three aspects of decidual NK function in vitro: cytotoxicity, proliferation, and cytokine production. The functional assays indicate uterine lymphocytes differ fundamentally from both PBL and even from classical circulating NK cells. Their role in the establishment of normal pregnancy remains unknown.

Cytotoxicity, Immunologic↗

PubMed1995

The role of integrins in adhesion of decidual NK cells to extracellular matrix and decidual stromal cells.

At the time of implantation, the decidua is infiltrated by a unique population of NK cells with large granular lymphocyte morphology, which are thought to influence placental trophoblast invasion and differentiation. The mechanisms used by these cells to migrate within decidua are not known, but in other biological processes such as wound healing and tumor invasion cell-matrix interactions are important. These interactions are mediated by specific receptors, mostly belonging to the family of integrins. Decidual NK cells are observed to bind to type IV collagen and fibronectin, but not to laminin. Adhesion to collagen was inhibited with an anti-alpha 1 integrin subunit mAb, whereas adhesion to fibronectin was blocked with anti-alpha 4, -alpha 5, and -beta 1 integrin subunit mAbs. Binding of decidual NK cells to decidual stromal cells was partially blocked with mAbs to the alpha 4 and alpha 5 integrin subunits. These results provide insight into the possible mechanisms utilized by decidual NK cells for migration and retention within the pregnant uterine mucosa.

3T3 Cells↗

PubMed1995

Screening for cytokine mRNA in human villous and extravillous trophoblasts using the reverse-transcriptase polymerase chain reaction (RT-PCR).

During the process of placental implantation, sessile villous trophoblast cells migrate from the villi into the decidua as isolated motile extravillous trophoblast cells. There is differential expression of the epidermal growth factor-receptor (EGF-R) and c-erbB2 proteins on villous and extravillous trophoblast populations. Using monoclonal antibodies to EGF-R and c-erbB2, we have obtained highly purified populations of villous and extravillous trophoblast by fluorescence activated cell sorting. These cells were examined by the reverse transcriptase-polymerase chain reaction (RT-PCR) using nested internal primer pairs for the following cytokines: CSF-1, GM-CSF, TNF-alpha, TGF-beta1, IFN-gamma, IL-2, LIF and also for LIF-receptor. TNF-alpha and TGF-beta 1 were present in all trophoblast populations. GM-CSF and CSF-1 were only found in some samples, with preferential expression of CSF-1 in villous populations. IFN-gamma, IL-2 and LIF mRNA were not found, although all samples contained LIF-receptor mRNA. These cytokines (CSF-1, TGF-beta, TNF-alpha and GM-CSF) are likely to influence trophoblast growth and differentiation in an autocrine manner, since their receptors are also present on trophoblast. These results illustrate a quick and simple method to analyse for the presence of cytokine and other transcripts in trophoblast subpopulations during early pregnancy.

Base Sequence↗

PubMed1995

Decidua in human implantation.

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Decidua↗

PubMed1995

Secretion of colony stimulating factor-1 by human first trimester placental and decidual cell populations and the effect of this cytokine on trophoblast thymidine uptake in vitro.

The present in-vitro study using an enzyme-linked immunosorbent assay has identified the cell types responsible for colony stimulating factor-1 (CSF-1) production at the first trimester human placental uterine interface. The major sources were observed to be decidual stromal cells and decidual CD56+ natural killer (NK) cells, but decidual CD3+ T cells did not produce CSF-1, reflecting functional differences between these two decidual lymphoid populations. Of a variety of cytokines tested, only interleukin-2 (IL-2) was found to augment CSF-1 secretion by decidual NK cells. Trophoblast cells also secreted CSF-1, but the amounts were small relative to decidual stromal cells and NK cells. Therefore, most of the CSF-1 present at the implantation site appears to be maternally derived. Co-culture of decidual NK cells on a monolayer of irradiated trophoblast did not augment CSF-1 secretion by decidual NK cells, indicating that the production of this cytokine is not stimulated by contact with fetal trophoblast. CSF-1 was found to increase [3H]thymidine uptake by trophoblast cultured on laminin for 72 h, but no such response was seen in trophoblast cultured on fibronectin, indicating that these extracellular matrix proteins have differential effects on the response of trophoblast to this cytokine.

CD56 Antigen↗

PubMed1995

Human trophoblast adhesion to matrix proteins: inhibition and signal transduction.

At the time of implantation, the extracellular matrix proteins laminin and fibronectin are abundant in the decidua and are distributed pericellularly around each individual stromal cell. First trimester human trophoblast expresses both laminin and fibronectin receptors, specifically the alpha 1 beta 1, alpha 5 beta 1, alpha 6 beta 1 and alpha 6 beta 4 integrin heterodimers. In this study we have demonstrated that in-vitro adhesion of first trimester human trophoblast to purified extracellular matrix proteins and to purified decidual stromal cell monolayers can be inhibited by monoclonal antibodies directed against appropriate integrin subunits and by synthetic peptides containing an arginine-glycine-aspartic acid sequence. Monoclonal antibodies (mAbs) to the alpha 5 and beta 1 integrin subunits and a synthetic peptide significantly inhibited adhesion to fibronectin. Binding of trophoblast to laminin was blocked with mAbs to the alpha 6 and beta 1 but not alpha 1 and beta 4 integrin subunits. Similarly, integrin-mediated adhesion to monolayers of decidual stromal cells could be blocked with mAbs to the alpha 5, alpha 6, beta 1 and beta 4 integrin subunits. Integrin-mediated signal transduction in normal and malignant trophoblast was investigated by Western blotting. A 115 kDa protein was the major tyrosine phosphorylated protein detected in trophoblast after binding to laminin or fibronectin. The profile of tyrosine phosphorylated proteins differed for malignant trophoblast.

3T3 Cells↗

PubMed1995

[Adhesion molecules and trophoblastic invasion].

Cell Adhesion↗

PubMed1994

Screening for cytokine messenger ribonucleic acids in purified human decidual lymphocyte populations by the reverse-transcriptase polymerase chain reaction.

At the time of human embryo implantation, large numbers of maternal CD56brightCD16- NK cells appear in the uterus. These unusual lymphocytes are believed to control the migration and differentiation of highly invasive fetally derived trophoblast cells, which infiltrate into the maternal uterus to remodel the spiral arteries during the first trimester. One possible mechanism of control is by cytokine production. In this study, highly purified (> 99%) populations of first trimester decidual CD56brightCD16- NK cells and CD3+ T lymphocytes were obtained by using a FACS. These cells were examined by reverse transcriptase PCR for their expression of mRNAs for the following cytokines: granulocyte-macrophage (GM)-CSF, CSF-1, TNF-alpha, IFN-gamma, TGF-beta 1, leukemia-inhibitory factor (LIF), and IL-2. Then, the expression was compared with that of resting PBL. The identity of the PCR products was verified by Southern blotting and hybridization with cytokine-specific probes. Both decidual CD56brightCD16- NK cells and CD3+ T cells were found to express mRNA for CSF-1, TNF-alpha, IFN-gamma TGF-beta 1, and LIF, but GM-CSF mRNA was detected only in CD56bright NK cells. IL-2 mRNA was detected in only some decidual T cell samples, and then only after at least two rounds of amplification. In contrast, peripheral blood CD56brightCD16- NK cells, CD56dimCD16+ NK cells, and CD3+ T cells expressed mRNA only for TNF-alpha and TGF-beta 1, but not for GM-CSF, CSF-1, IFN-gamma, LIF, or IL-2. These results suggest that both decidual NK cells and decidual T cells produce a variety of cytokines that may be involved in the control of trophoblast migration and differentiation during pregnancy.

Antigens, CD↗

PubMed1994

Resistance of HLA-G and HLA-A2 transfectants to lysis by decidual NK cells.

Trophoblast cells from normal first trimester pregnancies have been shown to express the nonclassical Class I molecule, HLA-G, which is nonpolymorphic and has a heavy chain of 40 kDa. These HLA-G-expressing trophoblast cells infiltrate into maternal decidua, which contains abundant uterine-specific CD56bright natural killer (NK) cells. We believe HLA-G may act as a protective molecule against decidual NK lysis and thus allow trophoblast survival in the maternal tissues. To test this hypothesis, we have constructed HLA-G and HLA-A2 transfectants using LCL 721.221 HLA-null cells. We observed that both of these antigens protected target cells from NK effectors isolated from decidua or peripheral blood, although the effect of HLA-G is not as marked as that of HLA-A2. Our results, therefore, show that in this experimental system expression of a nonclassical Class 1 HLA molecule is also correlated with NK resistance in the same way as a classical Class I HLA molecule.

Blotting, Northern↗

PubMed1994

Generation of an antibody to HLA-G in transgenic mice and demonstration of the tissue reactivity of this antibody.

A method was devised to generate antibodies against the non-classical class I HLA-G antigen. This consisted of immunising HLA-A2/beta 2m double transgenic mice with HLA-G transfected into mouse Ltk- cells. A polyclonal antibody was obtained which appears to be specific for HLA-G. The staining pattern of this antibody was restricted solely to all populations of extravillous trophoblast. No fetal tissues reacted with this antibody, including those where HLA-G mRNA has been demonstrated, such as fetal eye, thymus and liver. This study confirms that HLA-G is a trophoblast-specific protein, although it remains a possibility that the technique of immunohistology is not sufficiently sensitive to detect low level HLA-G antigen expression in non-trophoblast tissues.

Animals↗

PubMed1994

Demonstration of the low affinity alpha subunit of the granulocyte-macrophage colony-stimulating factor receptor (GM-CSF-R alpha) on human trophoblast and uterine cells.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a classical haematopoietic cytokine which has also been implicated in placental growth and development. In this study we have performed a detailed immunohistological localization of the low affinity GM-CSF receptor (GM-CSF-R alpha) in human first trimester implantation site and non-pregnant endometrium. We have also investigated receptor expression and GM-CSF binding in vitro by normal first trimester trophoblast using flow cytometric analysis and compared this with JEG-3 and JAR choriocarcinoma cells. In the first trimester, the GM-CSF-R was found to be present on villous cytotrophoblast and all populations of extravillous trophoblast. Expression by villous syncytiotrophoblast was weak or absent, but this increased markedly by term. GM-CSF-R were also expressed by fetal Hofbauer cells within the mesenchyme of the chorionic villi and by uterine glandular epithelium and decidual macrophages within maternal decidua. GM-CSF-R was not expressed by glands in proliferative phase endometrium but began to appear during the secretory phase, suggesting hormonal regulation of the receptor on uterine glandular epithelium. Flow cytometric comparison of normal isolated first trimester trophoblast and JEG-3 and JAR choriocarcinoma cells revealed two- to threefold higher surface expression of GM-CSF-R by choriocarcinoma cells and higher binding capacity for rhGM-CSF than normal trophoblast. These results suggest that GM-CSF may regulate growth and development of human trophoblast. GM-CSF may also influence placental development and function by acting via decidual and fetal macrophages, and uterine glandular epithelium, which are the other cell populations to express the receptor.

Choriocarcinoma↗