Biomedical subjects

J S Hunt

Publications and source records attributed to J S Hunt.

At least 37 records · Page 2Linked to original sources

PubMed1999

TRAIL (Apo-2L) and TRAIL receptors in human placentas: implications for immune privilege.

Mechanisms accounting for protection of the fetal semiallograft from maternal immune cells remain incompletely understood. In other contexts, interactions between TRAIL (TNF-related apoptosis-inducing ligand/Apo-2L) and its receptors kill activated lymphocytes. The purpose of this study was therefore to investigate the potential of the TRAIL/TRAIL-R system to protect the placenta against immune cell attack. Analysis by Northern blotting demonstrated mRNAs encoding TRAIL as well as the four TRAIL receptors (DR4, DR5, DcR1/TRID, DcR2/TRUNDD) in human placentas. Immunohistochemical experiments demonstrated that TRAIL protein is prominent in syncytiotrophoblast, an uninterrupted placental cell layer that is continuously exposed to maternal blood, as well as in macrophage-like placental mesenchymal cells (Hofbauer cells). Studies on cell lines representing trophoblasts (Jar, JEG-3 cells) and macrophages (U937, THP-1 cells) showed that both lineages contained TRAIL mRNA and that steady state levels of transcripts were increased 2- to 11-fold by IFN-gamma. By contrast, cell lineage-specific differences were observed in expression of the TRAIL-R genes. Although all four lines contained mRNA encoding the apoptosis-inducing DR5 receptor, only trophoblast cells contained mRNA encoding the DcR1 decoy receptor and only macrophages contained DcR2 decoy receptor transcripts. DR4 mRNA was present only in THP-1 cells and was the only TRAIL-R transcript increased by IFN-gamma. Cytotoxicity assays revealed that the two trophoblast cell lines were resistant, whereas the two macrophage lines were partially susceptible to killing by rTRAIL. Collectively, the results are consistent with a role for the TRAIL/TRAIL-R system in the establishment of placental immune privilege.

Apoptosis Regulatory Proteins↗

PubMed1999

Unique epitopes of lactoferrin expressed in human cytotrophoblasts involved in immunologic reactions.

OBJECTIVE: Lactoferrin is an iron-binding protein that has been implicated in protection against infections and allogeneic recognition reactions and in the control of cell growth. We studied the biochemical characteristics and expression of the unique lactoferrin epitopes (LF(1)) in human placentas. STUDY DESIGN: Immunohistologic studies of normal human term placentas were done by using monoclonal antibodies to LF(1). Double-antibody experiments were done by using monoclonal antibodies to markers of inflammation (macrophages, human leukocyte antigen [HLA-DR]). LF(1) was studied immunochemically by using lactoferrin fragments produced by the reaction of lactoferrin with trypsin or N-glycanase. RESULTS: Anti-LF(1) monoclonal antibodies reacted with most interstitial cytotrophoblasts in the basal plate and with villous cytotrophoblasts of some but not all chorionic villi. Cytotrophoblasts expressing LF(1) were associated with large numbers of HLA-DR-reactive macrophages. Anti-LF(1) monoclonal antibodies reacted with 2 distinct tryptic fragments of lactoferrin, and these reactivities were not affected by treatment with N-glycanase. CONCLUSION: Placental cytotrophoblasts express unique epitopes of lactoferrin (LF(1)). Such expression is increased in the presence of activated macrophages. This expression could be an extraembryonic response to inflammation and maternal allogeneic recognition as an effort to protect trophoblastic cells. The LF(1) epitopes might represent conserved polypeptide epitopes on 2 homologous lobes of lactoferrin.

Amidohydrolases↗

PubMed1999

Interferons enhance HLA-G mRNA and protein in transfected mouse fibroblasts.

The HLA class Ib gene, HLA-G, has a 16-bp deletion in its Enhancer A/interferon response element (IRE). We used a model system consisting of mouse fibroblasts transfected with 6.0 kb of HLA-G DNA, the S14/8 cells, to test the postulate that this deletion prevents interferons (IFNs) from enhancing transcription. Northern blot hybridization experiments showed that after 48 h of treatment with IFN-alpha, IFN-beta or IFN-gamma, steady-state levels of HLA-G mRNA in the S14/8 cell line were doubled. Proteins were also increased by IFNs as demonstrated in flow cytometry and immunocytochemical experiments that used monoclonal antibodies to all HLA class I antigens (W6/32), HLA-G heavy chains (87G) and light chains (beta2m). Thus, interferons enhance expression of HLA-G and would be expected to improve host defense at the maternal-fetal interface by increasing the ability of maternal immune cells to recognize and destroy infected HLA-G+ cells.

Animals↗

PubMed1999

A candidate interferon-gamma activated site (GAS element) in the HLA-G promoter does not bind nuclear proteins.

The HLA-G gene is highly expressed at the maternal-fetal interface, where it is believed to participate in the generation and maintenance of maternal tolerance to the fetal semiallograft. This gene has two elements through which interferon-gamma (IFN-gamma) could act to enhance its rate of transcription, an Enhancer A/ICS region and a candidate IFN-gamma activated site (GAS). In this study we investigated functionality of this candidate HLA-G GAS. Two HLA-G-expressing cell lines were tested, the human myelomonocytic cell line, U937, and a mouse fibroblast cell line, S14/8, which is stably transfected with the full length HLA-G gene. Nuclear proteins from IFN-gamma-treated U937 and S14/8 cells bound the interferon regulatory factor-1 (IRF-1) gene GAS sequence (TTC CCCGAA) but not the HLA-G gene's candidate GAS sequence (TTTCGAGAA). Excess unlabeled HLA-G-GAS oligonucleotide failed to inhibit binding of the IRF-1-GAS using the same nuclear extracts. These data indicate that a sequence in the HLA-G gene which would normally permit cytokine enhancement of gene expression, the GAS element, is nonfunctional. This is also true of another regulatory sequence, the Enhancer A/ICS element, suggesting that defects in IFN-gamma response elements prevent inappropriate up-regulation of HLA-G gene expression at the maternal-fetal interface.

Animals↗

PubMed1999

The CTLA-4 gene is expressed in placental fibroblasts.

In order to elucidate the mechanisms that ensure survival of the allogeneic fetus, we are investigating the expression pattern of genes that are involved in peripheral self-tolerance in tissues at the maternal-fetal interface. Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is a negative regulator of T cell activation and may modulate peripheral self-tolerance. Previously, we reported the preferential transmission of maternally-inherited shorter alleles at a 3'-UTR microsatellite locus to liveborn children, but random transmission of paternally-inherited alleles, suggesting that CTLA-4 may be involved in the maintenance of tolerance at the maternal-fetal interface. In this report, we demonstrate that CTLA-4 mRNA and protein are indeed expressed in fetal tissues at the maternal-fetal interface throughout gestation.

Abatacept↗

PubMed1999

Expression of cell adhesion molecules in murine placentas and a placental cell line.

Integrins and vascular cell adhesion molecule-1 (VCAM-1) are required for normal placental development. In this study, integrin subunits alpha4, alphav, beta1, and beta3, and VCAM-1 were investigated for expression in uteroplacental units (gestation day [g.d.] 6 and 8) and placentas (g.d. 10, 12, 14, 16, and 18) of Swiss-Webster mice. All subunits and VCAM-1 mRNA (identified by reverse transcriptase polymerase chain reaction [RT-PCR]) and protein (detected by immunofluorescence) were present in all tissues throughout gestation. VCAM-1 was expressed strongly in the ectoplacental cone and trophoblast giant cells, alpha4 was expressed strongly by trophoblast giant cells and moderately by spongiotrophoblast and labyrinthine trophoblast, and alphav was expressed more strongly in the spongiotrophoblast than in the labyrinthine zone. The beta1 was more strongly expressed in the labyrinthine than the spongiotrophoblast zone, while beta3 and VCAM-1 were essentially equal in the two zones. Trophoblast-like SM9-1 cells were positive for all of the adhesion molecules when tested by RT-PCR and immunocytochemistry. Adhesion molecule expression in SM9-1 cells was consistent with expression in the labyrinthine zone. Collectively, the results of this study demonstrate that murine placentas contain mRNA and protein for alpha4, alphav, beta1, beta3, and VCAM-1, and that expression is cell-specific. These results and the identification of an adhesion molecule-expressing trophoblastic cell line should facilitate future studies on the function of adhesion molecules in placental development.

Animals↗

PubMed1999

Uterine natural killer cells are targets for a trophoblast cell-specific cytokine, prolactin-like protein A.

PRL-like protein A (PLP-A) is a member of the PRL family expressed in trophoblast cells coincident with establishment of the chorioallantoic placenta. The purpose of this investigation was to identify targets for PLP-A. Using an alkaline phosphatase-tagging strategy, we show that PLP-A specifically interacts with a population of natural killer (NK) lymphocytes within the mesometrial compartment of decidua from pregnant and pseudopregnant rats. These observations are supported by the codistribution of PLP-A targets with cells expressing the rat NK cell surface marker, gp42, the absence of PLP-A binding in conceptuses from NK cell-deficient tg epsilon26 mice, and the specific interaction of PLP-A with a rat NK cell line, RNK-16. We have further demonstrated that PLP-A effectively suppresses RNK-16 cell cytolytic activities. Our results provide evidence for a new paradigm of embryonic-maternal communication involving a PLP-A signaling pathway between trophoblast cells and uterine NK lymphocytes.

Alkaline Phosphatase↗

PubMed1998

Regulation of TNF-alpha production in activated mouse macrophages by progesterone.

The purpose of this study was to investigate the relationships between macrophage production of TNF-alpha and female hormones. Northern blot hybridization experiments showed that the female sex steroid hormone, progesterone, decreases steady state levels of TNF-alpha mRNA in LPS-activated mouse macrophages (RAW 264.7 and ANA-1 cells) in vitro. The production of intracellular and secreted TNF-alpha protein, as determined by ELISA, was decreased in both progesterone- and dexamethasone-treated, LPS-stimulated macrophages. Estrogen had no effect on expression of the TNF-alpha gene in mouse macrophages and did not alter progesterone-mediated suppression. Additional experiments conducted to investigate the mechanism of action of progesterone showed that this hormone, like dexamethasone, elevates steady state mRNA levels of IkappaB alpha and increases the levels of IkappaB alpha protein that are translocated from the cytoplasm to the nucleus. Thus, progesterone is a potent inhibitor of steady state levels TNF-alpha mRNA and TNF-alpha protein production in activated macrophages and may achieve this result through effects on an inhibitor of NF-kappaB.

Animals↗

PubMed1998

Interferon-gamma gene expression in cycling and pregnant mouse uterus: temporal aspects and cellular localization.

Interferon-gamma (IFN-gamma) is a potent pro-inflammatory cytokine that modulates hematopoietic cell maturation, differentiation, activation, and apoptosis. To evaluate the postulate that locally produced IFN-gamma could influence uterine hematopoietic cells, specific protein was detected by immunohistochemistry and messenger RNA (mRNA) was identified by in situ hybridization in cycling and pregnant mouse uteri. In cycling uteri, IFN-gamma was limited to luminal and glandular epithelial cells during the estrus phase of the cycle. In pregnant uteri, IFN-gamma was prominent at early (gestation day 6-10) and late (gestation day 18) stages. IFN-gamma-producing cells identified by in situ hybridization included uterine epithelial cells, natural killer cells, macrophages, placental trophoblast cells, and cells in the degenerating metrial gland. Collectively, the data indicate that programming of immune and other cells via autocrine and paracrine pathways could be achieved by locally produced IFN-gamma, and suggest that uteroplacental IFN-gamma may be most influential during early and late stages of pregnancy.

Animals↗

PubMed1998

Soluble HLA-G in human placentas: synthesis in trophoblasts and interferon-gamma-activated macrophages but not placental fibroblasts.

The HLA class Ib antigen, HLA-G, is highly expressed in early gestation placentas where it is believed to modulate maternal-fetal immunological interactions. In this study, soluble isoforms (sHLA-G) encoded by intron 4-retaining transcripts were identified in first trimester placentas by immunohistochemistry using a mAb specific for the C-terminus of sHLA-G. Immunoreactive sHLA-G protein was localized to trophoblast cells and to villous mesenchymal cells with the morphological features of macrophages. Reverse transcriptase polymerase chain reaction analysis which used primers specific for intron 4 and the 3' untranslated region of the HLA-G gene showed that transcripts encoding sHLA-G were present in the trophoblast-derived Jeg-3 cells as well as interferon-gamma-activated myelomonocytic U937 cells but were absent and uninducible in placental fibroblasts. These results indicate that placental sHLA-G is synthesized in trophoblast cells and activated placental macrophages and support the postulate that placenta-derived sHLA-G modulates maternal and fetal immune cell functions during pregnancy.

Amino Acid Sequence↗

PubMed1998

Differential expression and regulation of a human transgene, HLA-B27, in mouse placental and embryonic cell lines.

Unlike other somatic cells, human placental trophoblast cells do not express the highly polymorphic HLA-A and HLA-B human leukocyte major histocompatibility antigens that would stimulate maternal immunological rejection of the fetus. To investigate mechanisms underlying cell lineage-specific expression, cell lines were generated from homozygous matings of HLA-B27 transgenic mice. Trophoblast cell lines were generated from gestation day 10 placentas and fibroblasts were cultured from gestation day 13/14 embryos. Polymerase chain reaction (PCR) readily identified HLA-B DNA in transgenic trophoblastic cells but specific mRNA was of low abundance, being detectable by reverse transcriptase PCR but not by Northern blot hybridization. HLA-B-specific protein in/on the trophoblast cells was undetectable by cell enzyme-linked immunosorbent assay and the protein was not induced by exposing the trophoblastic cells to interferon-gamma (IFN-gamma). Restricted expression was specific for the HLA-B transgene and its antigen; IFN-gamma-inducible endogenous H-2Db class I antigens were detectable on the trophoblast cells. In contrast to the trophoblastic cells, HLA-B27 transgenic fibroblasts expressed IFN-gamma-inducible HLA class I antigens as well as H-2Db antigens. Thus, the mechanism(s) regulating expression of the polymorphic HLA-B antigen in trophoblastic cells is gene-specific, IFN-gamma-resistant and operative at the level of transcription or immediate post-transcription.

Animals↗

PubMed1998

HLA expression at the maternal-fetal interface.

Pregnancy in the human presents an "immunological paradox," because of the unexpected willingness of mothers to accept genetically disparate tissues. The fact that the fetus can develop unharmed for nine months shows that protective mechanisms must exist to permit its survival. The conditions that permit the genetically dissimilar human fetus to evade rejection by its mother's immune system have been the subject of intense interest for several decades. As the placental cells, which are in contact with maternal blood or tissue, are devoid of HLA class II antigens, interest has focused on the expression of HLA class I molecules. Recent developments in the constitutive, transcriptional, and translational expression of HLA class I molecules on anatomically and morphologically different subpopulations of trophoblast cells will form the basis of this short review.

Female↗

PubMed1998

Hormonal regulation of uterine macrophages.

Macrophages are major cellular inhabitants of cycling and pregnant mammalian uteri. Their densities and patterns of tissue distribution in this organ fluctuate in concert with levels of circulating female sex steroid hormones, estrogens and progesterone, and their production of various effector molecules also may be hormonally regulated. Hormonal control may be achieved by direct binding to receptors or by indirect pathways where hormones modulate production of various autocrine and paracrine cytokines and growth factors that then target to resident macrophages and influence their secretory profiles. In this paper, we marshall evidence supporting the concept that progesterone acts as a powerful negative regulator of these versatile cells, reducing their migration into the uterus and impairing their ability to produce potent effector molecules such as nitric oxide that could interfere with the success of pregnancy.

Animals↗

PubMed1997

Female steroid hormones regulate production of pro-inflammatory molecules in uterine leukocytes.

Estrogens and progesterone could be among the environmental signals that govern uterine immune cell synthesis of pro-inflammatory substances. In order to investigate this possibility, we first mapped expression of the inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-alpha) genes in the leukocytes of cycling and pregnant mouse uteri, then tested the ability of estradiol-17 beta (E2) and progesterone to influence gene expression. Immunohistochemistry, in situ hybridization, and other experimental approaches, revealed that the iNOS and TNF-alpha genes are expressed in mouse uterine mast cells, macrophages and natural killer cells (uNK). Gene expression in each cell type was noted to be dependent upon stage of the cycle or stage of gestation, implying potential relationships with levels of female hormones and state of cell differentiation or activation. Further in vivo and in vitro experiments showed that individual hormones have cell type-specific effects on synthesis of iNOS and TNF-alpha that are exerted at the level of transcription. In uterine mast cells, iNOS and TNF-alpha are promoted by E2 whereas preliminary studies in macrophages suggest that transcription and translation of the two genes are unaffected by E2 but are inhibited by progesterone. Uterine NK cell production of iNOS and TNF-alpha is strongly related to cell differentiation, which is initiated and sustained by progesterone. Collectively, the results indicate that regulation of synthesis of pro-inflammatory molecules by hematopoietic cells in cycling and pregnant uterus comprises a new and potentially critical role for female steroid hormones.

Animals↗

PubMed1997

Fas ligand is positioned in mouse uterus and placenta to prevent trafficking of activated leukocytes between the mother and the conceptus.

Despite intimate juxtaposition of maternal and fetal tissues during mammalian pregnancy, reciprocal migration of cells is limited. To evaluate the postulate that cell traffic is restricted by expression of Fas ligand (FasL) in the uterus and placenta, FasL mRNA was identified by using reverse transcription-PCR, and FasL protein was identified by Western blotting and immunohistology. FasL mRNA and protein were detected at all stages tested (gestation days (g.d.) 6-18). At g.d. 6 to 10, immunoreactive FasL was prominent in glandular epithelial cells and decidual cells. Between g.d. 12 and 14, expression shifted to placental trophoblast cells bordering maternal blood spaces and fetal placental endothelial cells. Thus, FasL is appropriately positioned, first in the uterus and then in the placenta, to deter trafficking of activated Fas+ immune cells between the mother and the fetus. To test whether the absence of functional FasL affects pregnancy, uteroplacental units from homozygous matings of gld mice, a mutant strain lacking functional FasL, were examined. Extensive leukocytic infiltrates and necrosis at the decidual-placental interface were observed from day 10 onward, resorption sites were common, and small litters were delivered by gld mice. These observations are consistent with the idea that FasL at the maternal-fetal interface protects the placenta against a maternal leukocytic influx that reduces fertility.

Animals↗

PubMed1997

Expression of the inducible nitric oxide synthase gene in mouse uterine leukocytes and potential relationships with uterine function during pregnancy.

Nitric oxide (NO), a potent and versatile free radical, is synthesized in leukocytes by the inducible form of NO synthase (iNOS). In this study, leukocytes in pregnant mouse uterus were investigated for expression of the iNOS gene. Inducible NOS mRNA, which was identified by reverse transcriptase polymerase chain reaction, was high relative to an invariant mRNA, glyceraldehyde-3-phosphate dehydrogenase, in midgestation uteri (gestation days [g.d.] 10, 12, and 14) but was low in late-gestation uteri (g.d. 16 and 18). Inducible NOS protein, identified immunohistochemically in paraformaldehyde-fixed uteri taken from g.d. 6 through 18 using rabbit antibodies generated to mouse carboxyl terminus iNOS peptides, was prominent in a few myometrial mast cells at early stages and was strongly expressed from g.d. 6 through g.d. 14 in myometrial macrophage-like cells. Inducible NOS protein was first detected in uterine (u) natural killer (NK) cells at g.d. 8. Signals peaked in this lineage at g.d. 10 and declined thereafter. Uterine leukocytes cultured in vitro expressed the iNOS gene; a hybridoma cell line derived from mouse uNK cells (GWM1-2) contained iNOS mRNA, and cells migrating from mouse metrial gland explants included iNOS/ leukocytes. Large, granular iNOS + uNK cells were absent from the uteri of homologously mated pregnant TgE26 mice, an NK cell-deficient transgenic mouse strain, but immunoreactive iNOS was detectable in trophoblast, a cell lineage that did not contain immunoreactive iNOS in NK cell-competent Swiss-Webster mice. In TgE26 mothers gestating normal embryos, the same pattern was observed. Collectively, the results of this study demonstrate that iNOS is present in mouse uterine leukocytes including mast cells, macrophage-like cells, and uNK cells, and suggest that in the absence of uNK cells, the placenta synthesizes iNOS. These findings are consistent with the postulate that leukocyte NO contributes importantly to events associated with successful pregnancy that are likely to include relaxation of vascular smooth muscle.

Animals↗

PubMed1996

Expression of HLA-G in human mononuclear phagocytes and selective induction by IFN-gamma.

In situ hybridization studies have shown that at early but not late stages of gestation, human placental stromal cells, many of which are macrophages (Hofbauer cells), contain HLA-G message. In this study, the HLA-G protein was identified in the macrophage-like stromal cells by immunohistochemistry using the anti-HLA-G mAb, 87G. Expression of the HLA-G gene was then analyzed in macrophage cell lines (U937, HL-60, THP-1) and blood monocytes. HLA-G mRNA identified by using reverse transcriptase PCR was consistent with production of a transcript containing intron 4, which codes for a soluble form of HLA-G. Low levels of HLA-G mRNA were identified in mononuclear phagocytes by Northern blot hybridization, and little if any HLA-G Ag was detectable. By contrast, essentially all of the cells displayed high levels of HLA-B/C H chains detected by the mAb, 4E, and B2m. Treatment of macrophage cell lines and monocytes with IFN-gamma increased steady-state levels of HLA-G mRNA, stimulated higher levels of cell surface and intracellular HLA-G Ag in a dose-dependent manner, and increased the proportions of HLA-G relative to HLA-B/C. INF-alpha and IFN-beta enhanced steady-state levels of HLA-G mRNA and in some lines modestly increased the numbers of weakly positive cells but were poor inducers of cell-surface and intracellular HLA-G and did not increase HLA-G relative to HLA-B/C. Thus, mononuclear phagocytes express low levels of HLA-G mRNA and protein, and IFN-gamma selectively enhances expression of this HLA class Ib gene relative to HLA class Ia, which could influence the repertoire of peptides presented during embryogenesis as well as during inflammatory situations in adults. Soluble HLA-G might influence both fetal and maternal immune responses.

Adult↗

PubMed1996

An IgG-transporting Fc receptor expressed in the syncytiotrophoblast of human placenta.

During normal human pregnancy, maternal IgG crosses the placenta and provides passive immunity for the fetus. In so doing, IgG passes through two cellular barriers: the syncytiotrophoblast and the fetal capillary endothelium. The Fc region of IgG is required for its transport across the placenta, but the Fc receptors responsible have not been identified definitively. We recently reported the isolation from a placental cDNA library of clones encoding the alpha chain of a human homologue of the major histocompatibility complex class I-related Fc receptor, the neonatal Fc receptor (FcRn). In mice, FcRn is essential for the transport of maternal IgG to the fetus and the neonate. We report here the localization of human FcRn mRNA within the placenta by in situ hybridization, and of human FcRn protein by immunohistochemistry. Both methods show that human FcRn is expressed in syncytiotrophoblast, and is, thus, appropriately located to transport maternal IgG across the first barrier. We confirm previous findings that specific binding of IgG to placental membranes is greater at pH 6.0 than pH 7.5. This corresponds with the pH dependence of IgG binding to FcRn and is consistent with the presence of FcRn in syncytiotrophoblast. We propose a transport model in which maternal IgG binds FcRn at low pH in endosomes within the syncytiotrophoblast. FcRn is not expressed in fetal capillary endothelia, and the mechanism of IgG transport across the second barrier remains unknown.

Amino Acid Sequence↗