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Joan S Hunt

Publications and source records attributed to Joan S Hunt.

At least 19 recordsLinked to original sources
PubMed2006

Differential cellular expression of LIGHT and its receptors in early gestation human placentas.

LIGHT (homologous to lymphotoxins, exhibits inducible expression, competes with herpes simplex virus glycoprotein D for HVEM, a receptor expressed by T lymphocytes) is an apoptosis-inducing member of the tumor necrosis factor family of ligands. Messenger RNAs encoding LIGHT and its receptors, lymphotoxin-beta receptor (LTbetaR), decoy receptor-3 (DcR3) and herpes virus entry mediator (HVEM), are present in first trimester and term placentas. Proteins have been localized to specific cells in term but not earlier gestation placentas. Here, we have studied LIGHT and its receptors in early (6-7 weeks) and early-to-middle (8-13 weeks) gestation using immunohistology. Notable cell-specific, gestation-related features were identified. LIGHT and two of its receptors, a membrane-bound receptor that mediates apoptosis (LTbetaR) and a soluble receptor that interferes with LIGHT signaling (DcR3), were present in syncytiotrophoblast and cytotrophoblast cells in all samples but were detected in placental stromal cells only at week 8 and thereafter. HVEM, a membrane-bound receptor that protects against apoptosis, was expressed only on syncytiotrophoblast. These observations suggest that the LIGHT system may regulate early to middle stages of placental development via cell-specific, temporally programmed expression of the ligand and its receptors, and may also assist in preserving placental immune privilege.

Apoptosis↗
PubMed2006

Differential expression of human leukocyte antigen-G (HLA-G) messenger RNAs and proteins in normal human prostate and prostatic adenocarcinoma.

Human leukocyte antigen-G (HLA-G) is a major histocompatibility complex class Ib gene expressed in normal organs and in some tumors. The glycoproteins encoded by this gene are best known for their immunosuppressive properties. Because isoform-specific expression of HLA-G in male reproductive organs has not been reported, we investigated HLA-G1, -G2, -G5, -G6 mRNAs and proteins in four-to-five samples of normal prostate glands, prostates with benign prostatic hyperplasia and prostate adenocarcinomas using RT-PCR and immunohistochemistry. All tissues contained HLA-G1, -G2, -G5 and -G6 specific mRNAs, but only HLA-G5 protein was detectable. In normal prostate glands, HLA-G5 protein was prominent in the cytoplasm of tubuloglandular epithelia and in glandular secretions. Staining was reduced in samples of benign prostatic hyperplasia but remained localized to the cytoplasm of glandular epithelia and secretions. In prostatic adenocarcinomas, HLA-G5 protein was detectable mainly in the secretions. Thus, HLA-G5 but not HLA-G1, -G2 or -G6 is produced in the normal prostate and is present in prostatic secretions. In addition, normal cellular localization is disturbed in benign and malignant prostatic adenocarcinomas. The results are consistent with this molecule may influencing female immune receptivity to sperm and suggest that such immunosuppression could be disturbed in men with prostatic adenocarcinomas.

Adenocarcinoma↗
PubMed2006

Stranger in a strange land.

Mammalian mothers and their embryos/fetuses are almost invariably genetically different, which raises the question of how the mother's immune system is diverted so as to permit cohabitation with the 'foreign' body. Several decades of research have shown that multiple cooperative systems sanction uteroplacental immune privilege. These systems include production of several varieties of soluble immunosuppressive molecules in the uterus and the placenta and strict regulation of the molecules expressed on or by placental trophoblast cells. Trophoblast, a unique lineage without counterpart in adult tissues, is in direct contact with maternal blood and tissue. The major graft rejection-promoting molecules, human leukocyte antigens (HLAs), are tightly regulated in these cells, with none of HLA-A, HLA-B, or HLA class II antigens expressed. The HLA class Ib antigens, HLA-E, HLA-F, and HLA-G, are detectable on some subpopulations. Our studies have focused on the expression, regulation, and functions of the soluble isoforms of HLA-G, which circulate in maternal blood and are present at high levels in the pregnant uterus. These isoforms are derived from the single HLA-G gene by alternative splicing and are now known to have immunosuppressive properties. Ours and other studies indicate that soluble HLA-G proteins may comprise a unique tolerogenic system for establishing local immune privilege during pregnancy.

Animals↗
PubMed2006

The role of HLA-G in human pregnancy.

Pregnancy in mammals featuring hemochorial placentation introduces a major conflict with the mother's immune system, which is dedicated to repelling invaders bearing foreign DNA and RNA. Numerous and highly sophisticated strategies for preventing mothers from rejecting their genetically different fetus(es) have now been identified. These involve production of novel soluble and membrane-bound molecules by uterine and placental cells. In humans, the placenta-derived molecules include glycoproteins derived from the HLA class Ib gene, HLA-G. Isoforms of HLA-G saturate the maternal-fetal interface and circulate in mothers throughout pregnancy. Uteroplacental immune privilege for the fetus and its associated tissues is believed to result when immune cells encounter HLA-G. Unequivocally demonstration of this concept requires experiments in animal models. Both the monkey and the baboon express molecules that are similar but not identical to HLA-G, and may comprise suitable animal models for establishing a central role for these proteins in pregnancy.

Animals↗
PubMed2006

Isolation and culture of term human trophoblast cells.

Experimentation with most human cell types is restricted to the use of cell lines, and this limits our ability to extrapolate interpretations to the in vivo condition. However, in studying human trophoblast cells, we have a unique opportunity to obtain large quantities of readily available human tissue. In this chapter, we outline the methodology for purification of human trophoblast cells from term placentas. The procedures are based on enzymatic dissociation of villous placental tissue, followed by gradient centrifugation and immunomagnetic bead purification. Purity may be assessed by immunocytochemistry or flow cytometry using a number of markers to identify both cytotrophoblast cells and cellular contaminants. The resulting cytotrophoblast cell populations have excellent viability and purity, and may be subjected to long-term culture.

Cell Culture Techniques↗
PubMed2006

In vitro models for studying human uterine and placental macrophages.

Human monocytes and macrophages, which are also called mononuclear phagocytes, represent a major arm of the innate immune system. These cells not only protect against infection but are also central to tissue remodeling and production of chemokines, cytokines, and growth factors. Tissue macrophages reside in the human placenta and uterine decidua throughout pregnancy, where they comprise part of the host defense network and facilitate placental and extraembryonic development. The purpose of this chapter is to describe methods for establishing useful models of human uteroplacental macrophages: (1) differentiated U937 myelomonocytic cells, (2) peripheral blood monocytes, (3) peripheral blood monocyte-derived macrophages, (4) decidual macrophages, and (5) placental macrophages.

Cell Culture Techniques↗
PubMed2006

Methods for evaluating histocompatibility antigen gene expression in the baboon.

A wide variety of techniques has been developed for qualitative and quantitative analysis of gene expression in human cells and tissues. Two commonly used methods are reverse-transcription (RT)-polymerase chain reaction (PCR) to analyze the transcribed messenger RNAs (mRNA) and immunohistochemistry to detect the translated proteins. These techniques can be modified and adapted for use in analyzing gene expression in animal models. In particular, as a result of the close phylogenetic relationship between humans and nonhuman primates, human reagents, especially antibodies, cross-react with nonhuman primate tissues. However, the results are not always satisfactory as some antibodies may cross-react with irrelevant antigens in these tissues. In this chapter, we describe the use of RT-PCR and immunohistochemical techniques to analyze expression of Paan-AG, a novel class lb major histocompatibility complex antigen in the olive baboon (Papio anubis) placenta. We used Paan-AG-specific primers to amplify Paan-AG transcripts from baboon placenta, and generated Paan-AG isoform-specific polyclonal antibodies for use in immunohistochemistry.

Animals↗
PubMed2006

Analysis of the soluble isoforms of HLA-G mRNAs and proteins.

The human major histocompatibility complex (MHC) contains genes encoding the Human Leukocyte Antigens (HLA). Of these antigens, placental immunologists need study only the HLA class I molecules, because HLA class II expression is repressed in the fetal placental cells that are in direct contact with maternal blood and tissues containing maternal immune cells. The class I antigens are subdivided into two general categories. The class Ia antigens are highly polymorphic and are typified by HLA-A, -B, and -C; these are expressed by nearly all somatic cells and stimulate graft rejection when foreign to the host. By contrast, the HLA class Ib antigens, HLA-E, -F, and -G, have restricted expression, few variants, and appear rarely to be immunostimulatory. One class Ia antigen, HLA-C, and the three class Ib antigens are differentially expressed by trophoblast cell subpopulations. In order to understand immune privilege in the pregnant uterus and placenta, it is essential to study the unique structural and functional features of these four genes and their glycoprotein products. In this chapter, we focus on the first class Ib gene identified in human placentas, HLA-G, with emphasis on its two soluble isoforms, HLA-G5 and HLA-G6. We describe methods developed in our laboratory to distinguish mRNAs encoding HLA-G5 and HLA-G6, and antibody-based protocols for identification of the soluble isoforms.

Enzyme-Linked Immunosorbent Assay↗
PubMed2005

Trophoblast CD274 (B7-H1) is differentially expressed across gestation: influence of oxygen concentration.

Modulation of the maternal immune system by the placenta is a mechanism by which the fetus ensures its own survival in a genetically foreign environment. The immunoinhibitor CD274 (also called B7-H1 or PD-L1) is highly expressed in the placenta, positioned to interact with maternal leukocytes. Further, immunoblot analysis of first- and second-trimester placental lysates showed that CD274 expression is low in the first trimester but dramatically rises around the onset of the second trimester. As this coincides with the expected onset of maternal blood flow to the placenta and a corresponding rise in local oxygen tension, we explored the possibility that oxygen regulates CD274 expression in trophoblast cells by culturing term trophoblast cells under oxygen concentrations similar to those found in vivo. Indeed, CD274 protein levels paralleled the in vivo situation: expression increased with rising oxygen concentrations. Furthermore, downregulation of CD274 mRNA by low oxygen was rapid, occurring within 4-12 h. We conclude that oxygen is a potential mediator of CD274 expression in vivo such that it is induced coincidentally on exposure of fetal tissues to maternal blood. Further, the regulation of this immunomodulator by oxygen may implicate its alteration during and involvement in the pathogenesis of complications of pregnancy such as preeclampsia.

Antigens, CD↗
PubMed2005

HLA-G and immune tolerance in pregnancy.

Multiple mechanisms underlie the surprising willingness of mothers to tolerate genetically different fetal tissues during pregnancy. Chief among these is the choice of HLA-G, a gene with few alleles, rather than the highly polymorphic HLA-A and -B genes, for expression by the placental cells that interface directly with maternal blood and tissues. Novel aspects of this major histocompatibility complex class Ib gene include alternative splicing to permit production of membrane and soluble isoforms, deletions that dampen responses to interferons, and a shortened cytoplasmic tail that affects expression at the cell surface. Placental cells migrating into the maternal uterus synthesize both membrane and soluble isoforms, which interact with inhibitory receptors on leukocytes such as ILT2 and ILT4. Cytotoxic T lymphocytes either die or reduce production of one of their major coreceptor/activator cell surface molecules, CD8; natural killer cells are immobilized and mononuclear phagocytes are programmed into suppressive modes characterized by high production of anti-inflammatory cytokines. The idea that placental HLA-G proteins facilitate semiallogeneic pregnancy by inhibiting maternal immune responses to foreign (paternal) antigens via these actions on immune cells is now well established, and the postulate that the recombinant counterparts of these proteins may be used as powerful tools for preventing immune rejection of transplanted organs is gaining in popularity.

Alleles↗
PubMed2004

Fine mapping and positional candidate studies identify HLA-G as an asthma susceptibility gene on chromosome 6p21.

Asthma affects nearly 14 million people worldwide and has been steadily increasing in frequency for the past 50 years. Although environmental factors clearly influence the onset, progression, and severity of this disease, family and twin studies indicate that genetic variation also influences susceptibility. Linkage of asthma and related phenotypes to chromosome 6p21 has been reported in seven genome screens, making it the most replicated region of the genome. However, because many genes with individually small effects are likely to contribute to risk, identification of asthma susceptibility loci has been challenging. In this study, we present evidence from four independent samples in support of HLA-G as a novel asthma and bronchial hyperresponsiveness susceptibility gene in the human leukocyte antigen region on chromosome 6p21, and we speculate that this gene might contribute to risk for other inflammatory diseases that show linkage to this region.

Adult↗
PubMed2004

Potential regulatory sequences in the untranslated regions of the baboon MHC class Ib gene, Paan-AG, more closely resemble those in the human MHC class Ia genes than those in the class Ib gene, HLA-G.

The baboon major histocompatibility complex (MHC) class Ib gene, Paan-AG, is structurally similar to the human MHC class Ia gene, HLA-A, but exhibits characteristics similar to those of the class Ib gene HLA-G. These include limited polymorphism, alternative splicing of a single message, and restricted tissue distribution, with high expression in the placenta. In order to determine whether regulatory elements controlling expression of Paan-AG resemble those of HLA-A or HLA-G, we cloned the 5' and 3' untranslated regions of Paan-AG. Unexpectedly, sequence comparisons showed that potential regulatory elements in Paan-AG strikingly resembled those in HLA-A and differed in major respects from those in HLA-G. Unlike HLA-G, Paan-AG contained an intact interferon-gamma stimulated response element (ISRE) in the promoter. Studies using luciferase reporter assays showed that the Paan-AG ISRE was functional. The basal activity of the Paan-AG ISRE and its response to interferon-gamma was similar to that of class Ia MHC genes. Further, we identified an ISRE in the 3' untranslated region of Paan-AG that is known to be functional in HLA-A2 but is deleted in HLA-G. These experiments predict that functional studies may demonstrate differences in regulation of expression of Paan-AG and HLA-G genes, which could restrict the use of the baboon as a primate model for studying HLA-G expression and function.

Animals↗
PubMed2004

Recombinant HLA-G5 and -G6 drive U937 myelomonocytic cell production of TGF-beta1.

Throughout human pregnancy, activated maternal macrophages producing anti-inflammatory cytokines comprise a stable cell population in the uterus. This organ is also massively infiltrated with semiallogeneic, placenta-derived, invasive cytotrophoblast cells, which produce membrane and soluble isoforms of human leukocyte antigen (HLA)-G. Here, we investigated the possibility that two soluble isoforms of HLA-G, HLA-G5 and -G6, program macrophage production of cytokines. The model system consisted of human U937 myelomonocytic cells treated with phorbol 12-myristate 13-acetate (PMA) and interferon-gamma (IFN-gamma), which induced differentiation and activation but did not affect their viability or decrease their expression of the two inhibitory immunoglobulin-like transcript (ILT) receptors for HLA-G, ILT2 and ILT4. Exposure of the PMA/IFN-gamma-treated U937 cells to increasing concentrations of recombinant HLA-G5 or -G6 (rG5 and rG6) stimulated effects common to the two isoforms. High doses of both significantly decreased interleukin (IL)-10 and dramatically increased transforming growth factor-beta1. Differential effectiveness between the isoforms was demonstrated in dose-response studies, as was differential binding to ILT2 and ILT4 in receptor-blocking studies. No effects on production of IL-4, IL-1 receptor antagonist, IL-15, tumor necrosis factor alpha, IL-1beta, or IL-6 were observed. Collectively, the results are consistent with the postulate that environmental programming of decidual macrophages may be dictated in part by their proximity to soluble HLA-G-producing fetal cytotrophoblast cells.

Antigens, CD↗
PubMed2004

Soluble receptor (DcR3) and cellular inhibitor of apoptosis-2 (cIAP-2) protect human cytotrophoblast cells against LIGHT-mediated apoptosis.

LIGHT (tumor necrosis factor superfamily 14) is among the powerful apoptosis-inducing cytokines synthesized in human placentas. Here, we investigated mechanisms protecting cytotrophoblast (CTB) cells from LIGHT-mediated apoptosis. Viability assays and caspase-3 immunoblots using recombinant LIGHT were done to establish that CTB cells purified from term placentas resist LIGHT-induced apoptosis. Although the cells were also resistant to killing by another placental cytokine, interferon-gamma (IFN-gamma), a combination of the two induced apoptosis. Killing was prevented by DcR3-Fc fragment but not control human-Fc fragment, showing that apoptosis occurs via the LIGHT pathway and that soluble receptors provide protection. Next, two cellular inhibitors of apoptosis expressed in CTB cells, cellular inhibitor of apoptosis (cIAP)-1 and cIAP-2, were investigated for protection. Cellular IAP-1 was unchanged after stimulation with LIGHT whereas cIAP-2 mRNA and protein were elevated. The increase was abrogated by treating CTB cells with LIGHT + IFN-gamma, implying a central role for cIAP-2 in preventing LIGHT-mediated apoptosis and an ability of IFN-gamma to overcome cIAP-2 protection. Definitive evidence was provided in experiments that showed that cIAP-2 anti-sense morpholinos permit LIGHT to induce apoptosis in HT-29 cells. In summary, the data are consistent with the postulate that placental CTB cells are protected from LIGHT-mediated apoptosis by both soluble receptor, DcR3, and cIAP-2.

Apoptosis↗
PubMed2003

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 beta(2)-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 CD8alpha expression on PBMC without changing CD3delta 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.

Animals↗
PubMed2003

Temporal and spatial patterns of expression of inhibitors of apoptosis in human placentas.

The apoptosis cascade that plays a central role in normal and pathological processes is strictly controlled, in part by newly described members of the inhibitor of apoptosis (IAP) family (HIAP-1, HIAP-2, XIAP, NAIP, Survivin, and Livin). Here, we report the expression of IAP mRNAs and proteins in early and late gestation human placentas, term cytotrophoblast cells, and two choriocarcinoma cell lines, JEG-3 and Jar. Reverse transcriptase-polymerase chain reaction identified mRNAs derived from all of the currently known IAP genes in all samples. Analysis by immunoblotting revealed that IAP proteins are present in early and late gestation human placentas and that levels of IAPs are not identical in normal and transformed trophoblast cells. Immunohistochemical experiments performed on paraformaldehyde-fixed tissue sections taken from early and late stages of pregnancy demonstrated that expression patterns differed according to cell lineage and stage of cell differentiation. The results of this study are consistent with the postulate that IAP proteins have critical roles in placental cell survival and suggest that specific apoptosis inhibitors may protect normal and transformed trophoblast cells from cell death.

Adaptor Proteins, Signal Transducing↗