Polarized Porcine Trophoblastic Cell Lines Spontaneously Secrete Interferon-Gamma (original) (raw)
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Experimental Cell Research, 2002
We report here original properties of a porcine trophectoderm cell line, TBA B4-3, that developed a polarized phenotype with high transepithelial electrical resistance (TER) values and functional tight junctions (TJs) when grown on a microporous membrane. We found that treatment of polarized TBA B4-3 cells with a strong protein kinase C (PKC) agonist, phorbol 12myristate-13-acetate (PMA), induced 3-4 days later a transient interferon-gamma (IFN-␥) mRNA expression and vectorial IFN-␥ protein secretion toward the apical side of the monolayer. Exposure of TBA B4-3 cells to PMA first resulted in a rapid and profound disorganization of the monolayer structure mainly characterized by the appearance of multilayered polyp-like foci structures, a strong decrease of the TER, and a increase of permeability correlated with changes in the organization and localization of the TJ-associated proteins (ZO-1 and occludin) and filamentous actin (f-actin). After PMA removal, spontaneous return to the initial polarized monolayer state occurred, characterized by TER rising to prestimulation values, TJ protein relocalization, and multilayered cell structures fading. This return was strictly correlated with transient IFN-␥ gene induction. Our report represents the first example of an inducible expression of IFN-␥ by a polarized epithelial cell. After PMA treatment, the close correlation between establishment of cell polarity and IFN-␥ gene expression suggests a link between these phenomena. This also suggests a novel biological mechanism by which transient and reversible disorganization of a polarized monolayer of epithelial cells could trigger regulated expression of a cytokine gene by these cells. © 2002 Elsevier Science (USA)
Veterinary Immunology and Immunopathology, 2003
Interferon-g (IFN-g) is a major effector cytokine of the immune system with an expression pattern strictly restricted to cells of the lymphoid lineage. Several years ago, we reported that, during early pregnancy, the trophectoderm of the pig blastocyst, which represents a monolayer of polarized epithelial cells secretes high amount of IFN-g in a transient and developmentally regulated manner. In an effort to study the molecular basis of this atypical IFN-g gene expression, a pig trophectoderm cell line, TBA B4-3, was established in our laboratory. These cells developed a polarized phenotype with high transepithelial electrical resistance (TER) when grown on a microporous membrane. We found that treatment of polarized TBA B4-3 cells with the strong PKC agonist PMA induced, 3-4 days later, a transient IFN-g mRNA expression and vectorial IFN-g protein secretion. In order to better understand IFN-g gene regulation in TBA B4-3 cells, we examined in this system the effect of several drugs and factors known to affect the inducibility of this cytokine in T lymphocytes, the main source of IFN-g in the immunocompetent animal. We found that cyclosporine A (CsA) treatment of TBA B4-3 cells induces a partial inhibition of IFN-g secretion, thus indicating a minor role for the calcineurin signaling pathway in IFN-g expression. In addition, we found that although PMA alone can induce IFN-g secretion, the calcium ionophore A23187 synergizes with PMA for induction. We also analyzed by Southern blot the methylation status of a CpG dinucleotide in the 5 0 flanking region of IFN-g promoter and found that it was unmethylated in TBA B4-3 cells and in several pig epithelial cell lines that do not express IFN-g thus indicating the absence of correlation between demethylation and the ability to express IFN-g. Taken together, these results indicate that the mechanisms involved in IFN-g induction in TBA B4-3 cells are atypical compared to those presently known to operate in the T cell lineage.
PLoS ONE, 2013
Type-I interferons (IFNs) form a large family of cytokines that primarily act to control the early development of viral infections. Typical type-I IFN genes, such as those encoding IFN-α or IFN-β are upregulated by viral infection in many cell types. In contrast, the gene encoding IFN-ε was reported to be constitutively expressed by cells of the female reproductive tract and to contribute to the protection against vaginal infections with herpes simplex virus 2 and Chlamydia muridarum. Our data confirm the lack of induction of IFN-ε expression after viral infection and the constitutive expression of IFN-ε by cells of the female but also of the male reproductive organs. Interestingly, when expressed from transfected expression plasmids in 293T, HeLa or Neuro2A cells, the mouse and human IFN-ε precursors were inefficiently processed and secretion of IFN-ε was minimal. Analysis of chimeric constructs produced between IFN-ε and limitin (IFN-ζ) showed that both the signal peptide and the mature moiety of IFN-ε contribute to poor processing of the precursor. Immunofluorescent detection of FLAG-tagged IFN-ε in transfected cells suggested that IFN-ε and chimeric proteins were defective for progression through the secretory pathway. IFN-ε did not, however, act intracellularly and impart an antiviral state to producing cells. Given the constitutive expression of IFN-ε in specialized cells and the poor processing of IFN-ε precursor in fibroblasts and cell lines, we hypothesize that IFN-ε secretion may require a co-factor specifically expressed in cells of the reproductive organs, that might secure the system against aberrant release of this IFN.
Paracrine activities of porcine trophoblastic interferons
Reproduction, 1994
Interferon-\g=g\IFN-\g=g\ and a type I IFN (spl IFN) are transiently coexpressed by trophoblastic cells of pig conceptuses at implantation between day 12 and day 20 of gestation. The local effects of these trophoblastic IFNs were examined on endometrial cells and on trophoblast by measuring antiviral activity and the induction of (2\m=' \,5\m=' \)-oligoadenylatesynthetase activity. Trophoblastic vesicles were shown to be susceptible to infection by vesicular stomatitis virus and transmissible gastroenteritis virus. Vesicular stomatitis virus multiplied by about 1000 times in trophoblastic vesicles, and endogenous trophoblastic IFNs or exogenous recombinant IFN-\g=g\ or spl IFN had no effect on virus production. No (2\m=' \,5\m=' \)-oligoadenylatesynthetase activity could be measured on the trophoblast, even after treatment with IFN-\g=g\ or spl IFN. These results clearly show that trophoblastic IFNs cannot induce antiviral resistance or (2\m=' \,5\m=' \)-oligoadenylatesynthetase activity in the trophoblast, suggesting that these IFNs have no autocrine function. Endometrial epithelial and stromal cells in primary cultures displayed distinct sensitivity to the antiviral effect of IFN-\g=g\ and spl IFN. Stromal fibroblasts were highly sensitive to spl IFN but weakly sensitive to IFN-\g=g\; epithelial cells were sensitive to both IFNs. The same sensitivity pattern was obtained when measuring the (2\m=' \,5\m=' \)-oligoadenylatesynthetase activity. Flushing fluid, containing IFN-\g=g\ and type I IFN, was a potent inducer of antiviral effect and (2\m=' \,5\m=' \)-oligoadenylatesynthetase activity. It is therefore postulated that the endometrial epithelium is the most likely target of trophoblastic IFNs. It is possible that these IFNs play a role in the viral protection of conceptuses.
Journal of Interferon & Cytokine Research, 2000
Interferon-c (IFN-c) is an abortion-inducing factor, yet its effects in such a reaction are subject to various levels of regulation. The trophoblast cell line TROPHO-1 can be induced by IFN-c to express mRNA and surface class II major histocompatibility complex (MHC) proteins after 8 and 48 h of stimulation, respectively. Untreated cells, however, show an intracellular accumulation of class II antigens earlier (6 h), indicating the existence of MHC pools in the cystosol independent of any induction. On addition of IFN-c , immunofluorescence, subcellular fractionation, and ELISA experiments showed that class II antigen activity detected in the endosomal compartments of the cells could be measured in the culture supernatants. These soluble class II proteins, when isolated and purified using magnetic bead isolation techniques and tested in SDS-PAGE gel and Western blot experiments, had a molecular weight of 70 kDa. Administration of these molecules to pregnant mice as culture supernatants increased the abortion rate and decreased maternal hematocrit levels, effects that could be immunoabsorbed by anti-I-A d monoclonal antibodies (mAb). These results indicate that although surface class II molecules are not expressed on trophoblast cells, they accumulate in endosomal compartments and can be released from the cells on addition of IFN-c. This new IFN-c property, to mobilize intracellular pools of class II MHC antigens in trophoblast cells independent of de novo protein synthesis and induce their release to the extracellular matrix, is a mechanism that appears to be involved in the fetal rejection process, facilitating priming of the maternal organism against the fetal allograft.
Reproduction in Domestic Animals, 2007
The interferon-tau (IFN-s) secretion levels after hatching by bovine blastocysts derived from in vitro maturated oocytes (Group A) and from in vivo (Group B) were investigated considering embryo quality. Only very homogeneous blastocysts of excellent or good quality were considered from day 7 of culture (Group A) and day 7 after artificial insemination with frozen-thawed from the same bull used for in vitro fertilization (Group B). All embryos were individually cultured into a 50 ll droplet of synthetic oviduct fluid medium with 10% fetal calf serum. After 24-h culture both Group A (n ¼ 44) and B (n ¼ 40) secreted <54 pM IFN-s. After 48-, 72-, 96and 120-h culture, Group A daily secreted 143 ± 24 pM IFN-s (n ¼ 19) vs 85 ± 12 pM IFN-s (n ¼ 21) for Group B (p < 0.01), 491 ± 128 pM IFN-s (n ¼ 29) vs 216 ± 37 pM IFN-s (n ¼ 23) (NS), 499 ± 135 pM IFN-s (n ¼ 26) vs 353 ± 93 pM IFN-s (n ¼ 21) (NS), 559 ± 136 pM IFN-s (n ¼ 22) vs 333 ± 75 pM IFN-s (n ¼ 20) (NS), respectively. Taken all together during 5 days, Group A produced per embryo 1690 ± 290 pM IFN-s (n ¼ 22) vs 982 ± 182 pM IFN-s (n ¼ 20) for Group B (p < 0.05). For all culture time there were sizable percentages of embryos that did not produce concentrations of IFN-s above a certain cut-off level, and as such were not used to compute the means. In respect of the embryo quality whatever the groups after days 7-12 of culture, IFN-s secretions were 1815 ± 453 pM (n ¼ 10) for the embryos of excellent quality vs 1356 ± 200 pM (n ¼ 28) for those of good quality (NS) and 360 ± 188 pM (n ¼ 4) (p < 0.05) for embryos of fair quality. A positive relationship between IFN-s production and in vitro development of quality I embryos was observed, whatever the embryos origins and, the embryos completely produced in vitro secreted more IFN-s than the embryos produced in vivo.
Secretion of biologically active interferon tau by in vitro-derived bovine trophoblastic tissue
Biology of Reproduction, 1995
Secretion of interferon tau (IFNt) by trophoblastic tissue has been shown to be the first embryonic signal for pregnancy recognition. Therefore we tried to derive biologically active trophoblastic tissue by in vitro techniques. Since conventional in vitro conditions for bovine embryo development were not sufficient for long-term culture, we tested more complex culture conditions, including M6nezo B2 or Buffalo rat liver (BRL) cell-conditioned medium, for their ability to support proliferation and IFNt secretion by in vitro-derived trophoblastic tissue. IFNt activity was determined by using a biological assay based on the inhibition of the cytopathic effect of vesicular stomatitis virus on Madin-Darby bovine kidney cells. When cultures of individual hatched blastocysts were started in 60-pl drops of BRL cell-conditioned medium, mean IFNt secretion (antiviral units/ml/48 h) corresponded to 1200 on Day 11 and to 5000 on Day 13 (p < 0.01). To characterize trophoblast cell-specific secretions, the inner cell mass was removed from all embryos by microsurgery on Day 13. IFNT secretion by trophoblastic tissue increased to mean levels of > 105 antiviral units/ml/48 h on Day 23, stayed high for about 1 wk, and then slowly declined to levels below 10 3 antiviral U/ml/48 h. The specificity of the cytoprotective effect of IFNc was tested by Western blot analysis and by immunoneutralization with use of a polyclonal antiserum specific to IFNt. Our results demonstrate that viable trophoblastic tissue can be maintained entirely in vitro and secretes high amounts of IFNt.
American Journal of Reproductive Immunology, 1997
IFN-receptors in human first trimester and term placental tissues and on isolated trophoblast c~c~l l s. AJRl 1997; 37:443-448 0 Munksgaard, Copenhagen PROBLEM: Type-I interferon (IFN) is the protein recognizing pregnancy in ruminants. Although IFN is secreted in early pregnancy, its role is not still clear in other species. Like other cytokines, IFN exerts its biological functions through specific membrane receptors. We have investigated the potential action of IFN in human pregnancy by studying the distribution of the receptors in the human placenta. METHOD: Reactivity to monoclonal antibodies (mAbs) to the type-I IFN-receptor (R) was analyzed by immunohistochemistry in human placental tissues and in cytospins of first trimester trophoblast cells. RESULTS: Type-I IFN-R immunoreactivity was observed mostly in first trimester villous cytotrophoblasts and in the cytotrophoblast cell columns. Trophoblast in the decidua, the epithelium of the uterine glands, and most of the isolated trophoblast cells were also immunoreactive. CONCLUSION: The expression of type-I IFN-R in the highly proliferating and migrating trophoblast suggests that this cytokine has a role in trophoblast growth and invasion. The production of IFN by the trophoblast cells, although detectable throughout ges
Secretion polarity of interferon-β in epithelial cell lines
Archives of Biochemistry and Biophysics, 2002
Epithelial cells are an attractive target for local gene delivery in gene therapy for which cytokine genes such as interferon (IFN) genes are promising. However, how the secretion of the gene products is regulated in epithelial cells has been insufficiently investigated. Here, we have studied the secretion polarity of IFN-b expressed via gene transfection in mouse epithelial Pam-T cells on a bicameral culture system. In transient expression, IFN-b was predominantly secreted from the cell membrane side on which the transfection was carried out. Meanwhile, the secretion of constitutive IFN-b from stable transformants was apparently unpolarized. Interestingly, the transformants displayed a polarized secretion of transiently expressed IFN-b in a transfection-side-dependent manner, their stable IFN-b secretion remaining unpolarized. These results suggest that epithelial cells have at least dual protein sorting-secretion pathways, transient and stable, for the same secretory proteins, such as IFNs.
Regulation of Gene Expression in Mouse Trophoblast Cells by Interferon-gamma
Placenta, 2007
We have previously shown that interferon-gamma (IFN-g) activates phagocytosis and induces nitric oxide production in cultured mouse trophoblast cells. In the present study we examined the effect of this cytokine on ectoplacental cone and gene expression in trophoblast cells. Ectoplacental cones were obtained during the postimplantation period on gestational day 7.5 from CD-1 mice and exposed to 100 U/mL IFN-g. Ectoplacental cone morphology, cell proliferation and death were also determined upon IFN-g treatment. Complementary DNA macroarray and semiquantitative RT-PCR were used to analyze gene expression. IFN-g treatment did not alter ectoplacental cone morphology, trophoblast cell proliferation or death. However, using gene array technology, we observed that IFN-g affected the developing trophoblast, altering the level of mRNA expression, which resulted in upregulation of 35 genes and downregulation of seven others. The upregulation of transcription factors and immune response-associated genes suggests that IFN-g is involved in processes beyond immunological homeostasis and plays an important role in placental development and function.