Susceptibility of MHC class I expressing extravillous trophoblast cell lines to killing by natural killer cells.

Purified human first trimester extravillous trophoblast (EVT) cell lines HTR-8 and HT-116 were examined for susceptibility to natural killer (NK) cell-mediated lysis. Based upon nucleic acid sequencing of an amplified fragment of cDNA, Western blot analysis and immunostaining of fixed and live cells, it was shown that both EVT cell lines expressed HLA-G mRNA and protein within the cytoplasm when cultured on laminin-coated plates. Very weak HLA-G expression was detectable on the cell surface under these conditions. However, strong cell surface expression of a classical MHC class I molecule (most likely HLA-C) was exhibited by these EVT cell lines when grown on laminin, as indicated by W6/32 FACS analysis (Ab recognizing pan MHC class I), and Western immunoblotting with HC10 (Ab recognizing HLA-B/C). When these EVT cells, cultured on laminin, were used as targets for peripheral blood natural killer (NK) cells in a standard chromium release assay, both HTR-8 and HT-116 cells were lysed by NK cells in a dose-dependent manner. The respective percentage specific lysis at an effector to target (E/T) ratio of 100 was 28+/-7, and 48+/-14. The choriocarcinoma cell lines JAR and JEG-3 which were respectively MHC class I negative and HLA-G positive were resistant to NK cell lysis. Thus, there was no clear relationship between the MHC class I expression and NK cell resistance or susceptibility among the EVT cell lines and choriocarcinoma cells. These findings raise the possibility that NK cells may take part in the surveillance of the invasive EVT cells during normal placentation.

Interferon gamma regulates a unique set of proteins in fresh human bladder transitional cell carcinomas.

Poly(A) mRNA was isolated from human placental trophoblast cells stimulated with 100 U/mL of interleukin-2 and 5 microg/mL of phytohemagglutinin and reverse-transcribed. The cDNA coding for the mature interferon-gamma (IFN-gamma) protein was amplified using specific primers, cloned into the pGEX-4T2 vector, and expressed in Escherichia coli. Treatment of four fresh bladder transitional cell carcinoma (TCC) biopsies (TCCs 845-1, grade II, Ta; TCC 925-1, grade II, Ta; TCC 919-1, grade III, T1; TCC 950-1, grade III, T1) with the purified recombinant trophoblast IFN-gamma (50 U/mL, 20 h), followed by proteome analysis using two-dimensional gel electrophoresis, revealed several major proteins whose level of expression were affected by this cytokine. Of these, five (tryptophanyl-tRNA synthetase, the interferon gamma-inducible protein gamma3, mangase superoxide dismutase, and two unknown proteins of apparent molecular masses of 35.8 and 11.2 kDa, respectively) were upregulated in at least 75% of the tumors analyzed while one was downregulated (aldose reductase). Proteins were identified using a combination of techniques that included microsequencing, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) immunoblotting and comparison with the transitional cell carcinoma isoelectric focusing (IEF) database (http://biobase.dk/cgi-bin/celis). Proteome profile analysis of primary cultures from a low-grade lesion (TCC 846-1, Grade II, Ta) labeled in the presence and absence of IFN-gamma showed that all of the proteins disregulated in vivo were also affected in the cultures. The cultured cells, on the other hand, exhibited additional changes that were not detected in vivo and that may reflect adaptation to the culturing conditions. Taken together, the results provide a first glance at the effect of IFN-gamma on the protein expression profiles of TCCs, and in due course may form the basis for more comprehensive studies aimed at evaluating the usefulness of this cytokine in bladder cancer management.

Effects of human trophoblast-induced interferons on the expression of proto-oncogenes c-fms/CSF-1R, EGF-R and c-erbB2 in invasive and non-invasive trophoblast.

The human cytotrophoblast is the first fetal cell type to arise during embryogenesis and differentiate along two pathways to the invasive (extravillous) and non-invasive (villous) populations. The non-invasive villous trophoblast differentiate morphologically and biochemically to form terminally differentiated multinucleated syncytial trophoblast. First trimester invasive and non-invasive trophoblast were isolated from human placentae (5-12 weeks) and were cultured in vitro. The villous trophoblast cells differentiated in vitro to form aggregated syncytial cells which was associated with increased expression of epidermal growth factor receptor (EGF-R). The invasive trophoblast cells expressed colony-stimulating factor receptor (c-fms/CSF-1R) and c-erbB2 proteins but low levels of EGF-R. We studied the effects of human trophoblast-induced interferon (IFN)-alpha/beta on the expression of c-fms/CSF-1R, EGF-R and c-erbB2 whose ligands are reported to be involved in the regulation of growth and differentiation of normal invasive and non-invasive trophoblast cells. Human trophoblast-induced IFN-alpha/beta (100 IU/ml) reduced the expression of EGF-R in both invasive and non-invasive trophoblast cells as determined by quantitative enzyme-linked immunosorbant assay ('ELISA') and western immunoblot methods. The same amount of IFN activity reduced the expression of c-fms/CSF-1R and c-erbB2 proto-oncogene products in invasive trophoblast cells. These results may suggest a possible role of trophoblast-induced IFNs in the regulation of normal trophoblast growth, differentiation and function.

Epstein-Barr virus permissively infects human syncytiotrophoblasts in vitro and induces replication of human T cell leukemia-lymphoma virus type I in dually infected cells.

Epstein-Barr virus (EBV) and human immunodeficiency virus type 1 (HIV-1), as well as human T-cell leukemia-lymphoma virus type I (HTLV-I), may interact in the pathogenesis of human retroviral infections. The placental syncytiotrophoblast layer represents a barrier protecting the fetal compartment from exposure to retroviruses. We studied the interactions of EBV with HIV-1 and HTLV-I in human term syncytiotrophoblast cells to investigate the significance of double infections in transplacental transmission of human retroviruses. We found that syncytiotrophoblast cells could be productively infected with EBV. Dual infection of the cells with EBV and HTLV-I resulted in full replication cycle of otherwise latent HTLV-I. In contrast, the restricted permissiveness of syncytiotrophoblasts for HIV-1 was not influenced by coinfection of the cells with EBV. Infection of syncytiotrophoblast cells with EBV, but not HTLV-I, induced interleukin-2 and interleukin-6 secretion, and augmented secretion occurred on coinfection with both viruses. Coinfection of syncytiotrophoblast cells with EBV and HTLV-I induced tumor necrosis factor-beta and transforming growth factor-beta 1 secretion, but infection with either virus alone did not lead to secretion of these cytokines. Permissive replication cycle of HTLV-I was induced by the EBV immediate-early gene product Zta. Pseudotype formation between EBV and HTLV-I in coinfected syncytiotrophoblast cells was not found. Our data suggest that activation of HTLV-I gene expression by EBV in coinfected syncytiotrophoblast cells may be a mechanism for transplacental transmission of HTLV-I.

High interferon alpha levels in placenta, maternal, and cord blood suggest a protective effect against intrauterine herpes simplex virus infection.

Interferons (IFN) are produced by the placenta during pregnancy, and they can be detected in the maternal and fetal blood. Although the antiviral potential of IFNs is well established, it remains unclear whether the IFNs associated with pregnancy can prevent transplacental spread of viral infection. The present study was undertaken in order to determine the possible protective effect of placentally produced IFN-alpha on fetal acquisition of herpes simplex virus (HSV). Nine mothers with a known history of genital HSV infection were studied. In five cases IFN-alpha was detected in the placenta, maternal, and fetal blood, whereas in three cases IFN-alpha could not be detected. in the remaining case, IFN-alpha was found only in the maternal blood. As corroborated by the serological evidence of early HSV infection in the cord blood, the single case of vertical HSV transmission was observed in the group of IFN nonproducers. Furthermore, virus transmission did not occur in cases where IFN-alpha was present in the placenta and simultaneously in the maternal and fetal circulations. Thus, the present data indicate that high levels of IFN during pregnancy may protect the fetus from acquiring a possibly fatal intrauterine HSV infection.

Altered expression of the tumor suppressor/oncoprotein p53 in SV40 Tag-transformed human placental trophoblast and malignant trophoblast cell lines.

The expression of the tumor suppressor/oncoprotein p53 has been investigated in normal human placental villous trophoblast, in vitro propagated invasive extravillous trophoblast, SV40 tumor antigen (Tag)-immortalized extravillous trophoblast, human cytomegalovirus (hCMV)-infected syncytiotrophoblast and malignant trophoblast (choriocarcinoma) cell lines (JAR, JEG-3 and BeWo) using quantitative enzyme-linked immunosorbent assay (ELISA) and Western immunoblot methods using monoclonal antibodies specific for wild-type and mutant p53. The normal villous and extravillous trophoblast cells expressed low levels of the wild-type p53 protein, whereas normal terminally differentiated multinucleated syncytiotrophoblast cells, as well as hCMV-infected syncytiotrophoblast, showed a higher expression of the wild-type p53 protein. SV40 Tag-immortalized invasive trophoblast cells also showed a high expression of the wild-type p53 protein which remained complexed with the Tag protein. All the choriocarcinoma cell lines over expressed the mutant form of the p53 protein. The increased expression of p53 protein in the SV40 Tag-immortalized invasive trophoblast and choriocarcinoma cells paralleled with increased expression of the mouse double minute 2 (mdm2) oncogenic protein. Transforming growth factor (TGF)-beta inhibited proliferation of normal extravillous trophoblast cells. The antiproliferative effects of TGF-beta were reduced in SV40 Tag-immortalized cells and non-detectable in choriocarcinoma cell lines JAR, BeWo and JEG-3. The inactivation of p53 owing to complexing with Tag in the immortalized premalignant trophoblast and p53 mutation in the malignant trophoblast may be responsible for their aberrant proliferation and refractoriness to antiproliferative effects of TGF-beta observed in these cells as compared to the normal trophoblast. These results may suggest the role of p53 protein in trophoblast differentiation, transformation and tumorigenesis.

Functional characteristics of human trophoblast interferons.

Human trophoblast populations from first-and third-trimester placentas produce interferons (IFNs) in the presence of growth factors (CSF and PDGF) or when infected with virus. The highly invasive extravillous trophoblast population produced a higher level of IFNs (three- to eightfold, P < 0.05) than the noninvasive villous trophoblast population when stimulated with growth factors and/or virus. The level of IFN produced was dependent on the type of trophoblast population, the type of inducer and the stage of differentiation of the trophoblasts. Tandem immunoaffinity chromatography of the virus-induced trophoblast IFNs resulted in the isolation of trophoblast IFN-alpha and -beta types. The purified trophoblast IFNs have antiviral, antiproliferative and immunoregulatory properties. Furthermore, the trophoblast IFNs inhibited the expression of proto-oncogenes such as EGF-R, c-erbB2 and c-fms reported to be involved in normal trophoblast growth and differentiation. These data suggest essential roles of interferons in normal human development during pregnancy.

Isolation and characterization of human placental trophoblast subpopulations from first-trimester chorionic villi.

A method for the simultaneous preparation of highly enriched human placental trophoblast populations (villous and extravillous) from first-trimester placental villi (5 to 12 weeks) by using sequential trypsinization, percoll gradient centrifugation, and negative selection with anti-CD9 immunomagnetic separation is described. The purification method resulted in the isolation of four distinct trophoblast populations identified on the basis of morphology and phenotyping: (i) mononuclear villous cytotrophoblast cells which, through differentiation, become committed to syncytium formation; (ii) an extravillous trophoblast population which appeared as a "crazy pavement" and, with subsequent subculturing, differentiated morphologically to mononuclear cells; (iii) an extravillous trophoblast fraction which fused to form multinucleated trophoblast giant cells; and (iv) floating intermediate extravillous trophoblast cells which fused together to form cell clumps and which further differentiated to a mononuclear anchoring intermediate extravillous trophoblast. Short-term cultures of the freshly isolated cell fractions consisted of heterogeneous trophoblasts at different differentiation stages as determined by their varied biochemical and morphological properties. All the isolated trophoblast populations expressed the cytokeratin intermediate filament and the epithelium-specific cell-cell adhesion molecule E-cadherin. The isolated villous trophoblasts in culture expressed integrins alpha 6 and beta 4 and reduced levels of beta 1 subunits, whereas the proliferating extravillous trophoblast cultures expressed alpha 1, alpha 3, and alpha 5 and high levels of beta 1 integrin subunits, vitronectin receptor (alpha V beta 3/beta 5), and major histocompatibility complex class 1 molecules. Furthermore, the isolated trophoblast populations secreted metalloproteases (such as type IV collagenases [mainly 72- and 92-kDa enzymes, i.e., gelatinases A and B]) and urokinase plasminogen activator, as evaluated by substrate gel zymography. This method of isolation should facilitate in vitro studies of trophoblast proliferation, differentiation, invasion, virus interactions, cytokenesis, and immunology.

Bidirectional enhancing activities between human T cell leukemia-lymphoma virus type I and human cytomegalovirus in human term syncytiotrophoblast cells cultured in vitro.

The syncytiotrophoblast layer of the human placenta has an important role in limiting transplacental viral spread from mother to fetus. Human cytomegalovirus (HCMV) is capable of establishing a latent infection in syncytiotrophoblast cells, with restriction of gene expression to immediate-early and early proteins. We analyzed the extent of replication of human T cell leukemia-lymphoma virus type I (HTLV-I) in human term syncytiotrophoblasts infected with HTLV-I alone or coinfected with HTLV-I and HCMV. Although syncytiotrophoblasts could be infected with cell-free HTLV-I, no viral protein expression was found in the singly infected cells. On the contrary, coinfection of the cells with HTLV-I and HCMV resulted in simultaneous replication of both viruses. Bidirectional enhancing activities between HTLV-I and HCMV were mediated primarily by the Tax and immediate-early proteins, respectively. The stimulatory effect of HTLV-I Tax on HCMV replication appeared to be mediated partly by tumor necrosis factor beta and transforming growth factor beta-1. We observed formation of pseudotypes with HTLV-I nucleocapsids within HCMV envelopes, whereas HCMV was not pseudotyped by HTLV-I envelopes in dually infected syncytiotrophoblast cells. Our data suggest that in vivo dual infection of syncytiotrophoblast cells with HTLV-I and HCMV may facilitate the transplacental transmission of both viruses.

Interactions between human immunodeficiency virus type 1 and human cytomegalovirus in human term syncytiotrophoblast cells coinfected with both viruses.

Human cytomegalovirus (HCMV) and human immunodeficiency virus type 1 (HIV-1) may interact in the pathogenesis of AIDS. The placental syncytiotrophoblast layer serves as the first line of defense of the fetus against viruses. We analyzed the patterns of replication of HIV-1 and HCMV in singly an dually infected human term syncytiotrophoblast cells cultured in vitro. Syncytiotrophoblast cells exhibited restricted permissiveness for HIV-1, while HCMV replication was restricted at the level of immediate-early and early gene products in the singly infected cells. We found that the syncytiotrophoblasts as an overlapping cell population could be coinfected with HIV-1 and HCMV. HIV-1 replication was markedly upregulated by previous or simultaneous infection of the cells with HCMV, whereas prior HIV-1 infection of the cells converted HCMV infection from a nonpermissive to a permissive one. No simultaneous enhancement of HCMV and HIV-1 expression was observed in the dually infected cell cultures. Major immediate-early proteins of HCMV were necessary for enhancement of HIV-1 replication, and interleukin-6 production induced by HCMV and further increased by replicating HIV-1 synergized with these proteins to produce this effect. Permissive replication cycle of HCMV was induced by the HIV-1 tat gene product. We were unable to detect HIV-1 (HCMV) or HCMV (HIV-1) pseudotypes in supernatant fluids from dually infected cell cultures. Our results suggest that interactions between HIV-1 and HCMV in coinfected syncytiotrophoblast cells may contribute to the transplacental transmission of both viruses.

Basal and interferon-induced 2',5'-oligoadenylate synthetase activity in human placental trophoblast and trophoblast-derived malignant cell lines.

Human placental trophoblasts produce interferon (tro-IFNs) when stimulated with viral inducers. Since the antiviral and cellular functions of IFNs are partly mediated by the 2',5'-oligoadenylate synthetase (2-5A synthetase) pathway, the aim of the present study was to determine the basal and IFN-induced levels of 2-5A synthetase in villous trophoblast cultures. A considerable basal level of 2-5A synthetase was observed in syncytiotrophoblast cultures from both first and third trimester. In contrast no basal activity was detectable in placental fibroblast- and trophoblast-derived malignant cell lines (Far, FEG-3, and BeWo). Stimulation with tro-IFN-beta, -alpha and leucocyte-IFN (leu-IFN)-alpha increased the enzyme activity in first and third trimester human syncytiotrophoblast cultures. Treatment with recombinant-IFN (rec-IFN)-gamma significantly enhanced 2-5A synthetase activity in first trimester syncytiotrophoblast, but had no effect on third trimester syncytiotrophoblast. Tro-IFN-beta, -alpha and leu-IFN-alpha induced high levels of 2-5A synthetase activity in placental fibroblast, BeWo and FEG-3 cell-lines, whereas rec-IFN-gamma treatment did not induce 2-5A synthetase activity in any of these cells. No detectable 2-5A synthetase activity was found in the Far cell line. The capability of cells deriving from the fetoplacental unit to raise an antiviral response by the induction of 2-5A synthetase may be important in defending the fetus against viral infection from the mother. Furthermore 2-5A synthetase in cells of the fetoplacental unit may play a role in their normal growth and development.

Human trophoblast interferons: production and possible roles in early pregnancy.

Human villous and extravillous trophoblast populations were isolated from first- and third-trimester placentae and were stimulated with viral and non-viral inducers to produce interferons (IFNs). Polyriboinosinic/polyribocytidylic acid [poly(I:C)] induced exclusively IFN-beta in trophoblast cultures, whereas viruses induced mixtures of IFN-alpha subtypes and -beta. The level of IFN production was dependent on the trophoblast population, type of inducer and the stage of differentiation of the trophoblast. First-trimester extravillous trophoblast cultures produced greater than five-fold more IFN than third-trimester villous trophoblast on a per cell basis, whereas term syncytiotrophoblast produced twice as much IFN as term mononuclear villous trophoblast when stimulated with the same inducer. Pretreatment of trophoblast cultures with platelet-derived growth factor and granulocyte/macrophage-colony stimulating factor (GM-CSF) increased the trophoblast IFN production. Tandem high-performance affinity chromatography of the virus-induced trophoblast IFNs resulted in the isolation of trophoblast IFN-alpha and -beta with specific antiviral activities of 0.75-2.73 x 10(8) IU/ml protein. The trophoblast-induced IFNs have antiproliferative and immunosuppressive properties, and, furthermore, activated natural killer cell activity. These data may suggest the possible roles of these IFNs during embryonic development with regard to protection of the fetus against viral infection and maternal immunity.

Concurrence of high levels of interferons alpha and beta in cord and maternal blood and simultaneous presence of interferon in trophoblast in an African population.

A high concentration of interferon-alpha (IFN-alpha) (> 5 U/ml) in cord blood was used as the criterion for establishing our study group. In a collection from deliveries by 269 Kenyan women, 16 such cord samples with matching maternal blood and placental biopsies were identified. These 16 were studied in detail together with 23 randomly selected among those with low cord IFN-alpha levels. The levels of IFN- in retal blood correlated with levels in their mothers for both IFN-alpha and beta but not for IFN-gamma. IFN-alpha was furthermore demonstrated in villous and decidual trophoblast from 15 (94%) placentae from donors with high IFN-alpha in the cord blood but not in the placenta of any low IFN level donors. In contrast, IFN-beta was not demonstrated in any placenta. These observations suggest simultaneous IFN induction in the three compartments, transplacental IFN transport, or trophoblast production of IFN to both circulations. Looking for IFN inducers, we did serologic tests for nonspecific indicators of inflammation and for specific virus and protozoan infections, but these showed no relation to elevated IFN levels. Immunohistology also revealed no evidence of a number of placental infections. The cause of the high levels of IFN-alpha could still be infectious but remains unexplained and may be noninfectious.

Cell-mediated transmission of human T cell lymphotropic virus type I to human malignant trophoblastic cells results in long-term persistent infection.

We investigated permissiveness of the malignantly transformed trophoblast (choriocarcinoma) cell lines JAR, BeWo and JEG-3 to the human T cell lymphotropic virus type I (HTLV-I). After co-culture with the productively infected cell line MT-2 the choriocarcinoma cell lines were analysed for infection over a period of three months. The presence of HTLV-I viral DNA was examined by PCR using primers targeting the gag, pol, env and pX specific sequences. All amplified segments were found consistently in the cell cultures throughout the period of study. Further analysis that aimed to characterize the size variation of the integrated proviral DNA by Southern blotting revealed the presence of integrated proviral sequences which consisted, for the most part, of incomplete genomes. The presence of the full-length HTLV-I genome, however, was unequivocally confirmed in clonally expanded cell cultures derived from the originally infected parental cells. In order to analyse virus expression at the transcriptional level, we used reverse transcriptase (RT)-mediated PCR that was targeted at intra-exon regions (gag, pol, env and pX), and the splicing sites of the env and pX-tax/rex mRNAs. When compared with MT-2 cells, substantially lower levels of all transcripts were found in all the cell lines analysed. We were unsuccessful in attempts to detect viral protein expression using polyvalent or Tax- and Gag-specific monoclonal antibodies by Western blot analysis or immunoprecipitation, and we could not detect any RT activity released into the supernatant of the infected cells either. Collectively, these data suggest that the trophoblastic cells may become persistently but essentially non-productively infected with HTLV-I.

Production of interferons in human placental trophoblast subpopulations and their possible roles in pregnancy.

The human cytotrophoblasts are the first fetal cells to arise during embryogenesis and are the progenitor cells to villous (noninvasive), syncytiotrophoblast (noninvasive), "intermediate" extravillous (invasive), and "anchoring" extravillous (invasive) trophoblast subpopulations. These trophoblast subpopulations were isolated from first- and third-trimester placentae and were stimulated with Sendai virus, granulocyte-macrophage colony-stimulating factors (GM-CSF), and platelet-derived growth factor (PDGF) to produce interferons (IFNs). GM-CSF and PDGF induced very low levels of IFN in first-trimester extravillous and villous trophoblast subpopulations. Highly proliferating and invasive intermediate extravillous trophoblast cultures produced five- to eightfold more IFNs than villous trophoblast cultures and two- to fivefold more IFN than the syncytiotrophoblast cultures when stimulated with Sendai virus. Syncytiotrophoblast cultures produced higher levels of IFNs (up to twofold) than villous trophoblast cultures when stimulated with the same virus. Pretreatment of first-trimester extravillous and villous trophoblast cultures with GM-CSF and PDGF followed by infection with Sendai virus resulted in greater IFN production than when the cultures were stimulated with virus alone. The levels of IFN produced were dependent on the type of trophoblast, the type of inducer, and the stage of differentiation of the trophoblasts. The purified trophoblast IFNs have potent antiviral activities when assayed on human amniotic WISH cells, and they inhibited proliferation of normal trophoblasts and trophoblast-derived malignant cells in vitro without any toxicity. Furthermore, the trophoblast IFNs activated NK cell activity and suppressed mitogen-stimulated lymphocyte proliferation at concentrations of between 10 and 1,000 IU/ml. The possible functions of the trophoblast IFNs during pregnancy are discussed with respect to human placental and fetal protection and development.

Human trophoblast interferons enhance major histocompatibility complex class I antigen expression on human term trophoblast cells in culture.

The expression and regulation of major histocompatibility complex class I (MHC class I) antigens by virus-induced human trophoblast interferons (tro-IFNs) were examined in term trophoblast cultures. Flow cytometry studies using fluorescence monoclonal antibodies against MHC class I antigens revealed that isolated cytotrophoblasts can express MHC class I antigens. The expression of these antigens increased with stimulation of trophoblast cultures with tro-IFN-alpha and -beta. One hundred IU tro-IFN-alpha and -beta/ml induced no significant higher levels of MHC class I antigens as compared with the control, whereas 1000 IU tro-IFN-alpha and -beta/ml did. The tro-IFN-enhanced expression of MHC class I antigens may be important as it increases the efficiency of local and viral antigen presentation, cytotoxicity by T cell response and local inflammatory processes, thereby preventing virus spread from mother to fetus.

Immunosuppressive effects of human placental trophoblast interferon-beta on lymphocytes in vitro.

Human placental trophoblast cells produce predominantly interferon-beta-type (IFN-beta) when stimulated with viral inducers. The aim of the present study was to determine the in vitro antiproliferative effect of the trophoblast interferon-beta (tro-IFN-beta) on mitogen-stimulated and resting lymphocytes. The antiproliferative effect of the tro-IFN-beta was compared to human recombinant IFN-beta. All activities of tro-IFN-beta and human recombinant IFN-beta ranging between 10-1000 IU/ml showed suppression of proliferative responses on mitogen-stimulated and resting lymphocytes compared to cultures without IFN treatment. The inhibitory level of both tro-IFN-beta and recombinant IFN-beta was significantly higher on the stimulated than on the resting lymphocytes. Although there was a variation in the inhibition of lymphocyte proliferation by both IFNs with respect to time, there was no statistically significant difference in the antiproliferative effect of the IFNs on both resting and mitogen-stimulated lymphocytes. Since IFNs are produced locally by the placenta during pregnancy, our data suggest that in addition to the antiviral activity, the human tro-IFN-beta may participate in the local control of the maternal immune response during pregnancy at the fetomaternal interface.

Antibody-dependent enhancement of HIV-1 infection in human term syncytiotrophoblast cells cultured in vitro.

We examined if Fc receptor-mediated antibody-dependent enhancement (FcR-ADE) or complement-mediated antibody-dependent enhancement (C'-ADE) of virus infection can contribute to increasing replication of HIV-1 in human syncytiotrophoblast (ST) cells. Here we report that both FcR-ADE and C'-ADE may result in enhanced virus release from HIV-1-infected ST cells. We show that FcR-ADE of HIV-1 infection in ST cells is mediated by FcRIII and other FcR(s) belonging to undetermined Fc classes and does not require CD4 receptors, whereas C'-ADE uses both CD4 and CR2-like receptors. FcR-ADE seems to be more efficient in enhancing HIV-1 replication than C'-ADE. While FcR-ADE leads to increased internalization of HIV-1, C'-ADE does not result in enhanced endocytosis of the virus. In addition, antibodies mediating FcR-ADE are reactive with the gp120 viral envelope antigen, whereas antibodies involved in C'-ADE react with the viral transmembrane glycoprotein gp41. Data suggest that both FcR-ADE and C'-ADE may contribute to the spread of HIV-1 from mother to the fetus.

In vitro cytotoxic activity of cord blood NK cells against herpes simplex virus type-1 infected purified human term villous cytotrophoblast.

Transplacental infection of the fetus with herpes simplex virus (HSV) is associated with high morbidity. The present study was undertaken to shed light on the possible participation of the fetal immune system in the elimination of HSV from placental unit. In a chromium release assay cultured term villous trophoblast cells, regardless of infection with HSV-1, were found resistant to lysis by cord blood natural killer (CBNK) cells. In contrast to this, CBNK cells exhibited a basal level of cytotoxic activity against placental fibroblasts, which was significantly increased by preceding infection of the target cells with HSV-1. Stimulation of CBNK cells with interferon-beta purified from trophoblast (tro-IFN-beta) increased the killing of both HSV-1 infected and uninfected fibroblast, while HSV-1-infected and uninfected term villous trophoblast cells remained resistant to lysis. IL-2-stimulated CBNK cells were able to lyse villous trophoblast cells at a low level, but no significant difference in the susceptibility of the HSV-1-infected and uninfected trophoblast cell was detected.

Physiological oxygen tension is relevant to MHC-1 expression, spontaneous transformation, and interferon response of in vitro aging murine fibroblasts.

Working from the hypothesis that modest deviations from physiological oxygen tension will influence cell characteristics important for infections/immunity and tumor development, cells were studied at three oxygen tensions during in vitro aging. Primary mouse embryo fibroblasts were established and subsequently passaged at 3, 6, and 18 kPa oxygen tension (6 representing normal tissue tension and 18 being the conventionally tension at in vitro cultures). The growth rate was slightly higher at 6 than 3 and 18 kPa. All cultures eventually stopped growing and subsequently transformed to nonmalignant cells with unlimited growth capacity. Cells kept at 3 kPa reached the highest number of cell doublings before crisis. Stimulation with PolyI:C resulted in detectable interferon response only at the high oxygen tension, and after transformation none of the cultures responded with interferon production. Expression of the major histocompatibility complex H-2K was elevated above and below physiological oxygen tension, indicating regulatory processes optimizing MHC expression at about physiological oxygen tension.