Toshio Bessho

Effect of antioxidants on apoptosis induced by influenza virus infection: inhibition of viral gene replication and transcription with pyrrolidine dithiocarbamate

Influenza virus (IV) infection induced apoptotic DNA fragmentation and the moderate overproduction of reactive oxygen species (ROS) in primary cultured chorion cells prepared from human fetal membranes, and IV particles were released from the infected cells. The antioxidant pyrrolidine dithiocarbamate (PDTC) inhibited the induced DNA fragmentation, ROS overproduction and IV particle release. Although Trolox inhibited ROS overproduction, it did not inhibit DNA fragmentation or IV production. The inhibitory effect of PDTC on DNA fragmentation was manifested when added up to 3 h after infection or by exposing the infected cells to it for only 1 h after infection. PDTC inhibited IV hemagglutinin (HA) viral (vRNA) and complementary (cRNA and mRNA) RNAs synthesis until 6 h after infection and delayed and decreased HA protein synthesis. However, HA RNA synthesis resumed after 12 h even in the presence of PDTC. These results suggested that PDTC inhibited apoptosis by inhibiting viral macromolecule synthesis rather than through its antioxidant effect, because Trolox did not inhibit apoptosis or IV production, although ROS overproduction was inhibited. The synthesis of specific viral macromolecules at the early stage of infection may play a critical role in the mechanism of apoptosis induction and moderate ROS overproduction may not be involved in the mechanism.

Possible Roles of Proinflammatory and Chemoattractive Cytokines Produced by Human Fetal Membrane Cells in the Pathology of Adverse Pregnancy Outcomes Associated with Influenza Virus Infection

Pregnant women are at an increased risk of influenza-associated adverse outcomes, such as premature delivery, based on data from the latest pandemic with a novel influenza A (H1N1) virus in 2009-2010. It has been suggested that the transplacental transmission of influenza viruses is rarely detected in humans. A series of our study has demonstrated that influenza virus infection induced apoptosis in primary cultured human fetal membrane chorion cells, from which a factor with monocyte differentiation-inducing (MDI) activity was secreted. Proinflammatory cytokines, such as interleukin (IL)-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-β, were identified as a member of the MDI factor. Influenza virus infection induced the mRNA expression of not only the proinflammatory cytokines but also chemoattractive cytokines, such as monocyte chemoattractant protein (MCP)-1, regulated on activation, normal T-cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1β, IL-8, growth-regulated oncogene (GRO)-α, GRO-β, epithelial cell-derived neutrophil-activating protein (ENA)-78, and interferon inducible protein (IP)-10 in cultured chorion cells. These cytokines are postulated to associate with human parturition. This paper, therefore, reviews (1) lessons from pandemic H1N1 2009 in pregnancy, (2) production of proinflammatory and chemoattractive cytokines by human fetal membranes and their functions in gestational tissues, and (3) possible roles of cytokines produced by human fetal membranes in the pathology of adverse pregnancy outcomes associated with influenza virus infection.

Imbalance between ROS production and elimination results in apoptosis induction in primary smooth chorion trophoblast cells prepared from human fetal membrane tissues.

We have previously demonstrated that induction of apoptosis was observed in the smooth chorion trophoblast cells of human fetal membranes prepared at term, and that apoptosis progressed rapidly during in vitro incubation of the tissues. Furthermore, we identified the contribution of ROS production system (e.g., oxidant enzymes, such as iNOS and Cox-2) to the apoptosis induction in the chorion cells, suggesting an important role of the two inducible enzymes in the induction process. In this study, we examined the role of ROS elimination system (e.g., antioxidant enzymes, such as glutathione peroxidase (GPx) and catalase) in the apoptosis induction of the chorion cells, since the apoptosis induction by oxidative stress is a result of imbalance between production and elimination of ROS. Treatment of chorion and amnion cells with mercaptosuccinic acid (MS, GPx inhibitor) and 3-amino-1,2,4-triazole (ATZ, catalase inhibitor) resulted in an inhibition of GPx and catalase activity, respectively. Furthermore, incubation with MS alone induced apoptosis in the chorion cells and apoptosis level was enhanced by the addition of ATZ, while ATZ alone hardly induced apoptosis in the chorion cells. However, none of these reagents induced apoptosis in the amnion cells. Moreover, an increase of the level of hemeoxygenase-1 gene expression was observed only in the amnion cells when both antioxidant enzyme activities were suppressed. Therefore, we concluded that GPx played a more critical role than catalase in the control of the apoptosis induction of the chorion cells, suggesting that the threshold levels of stress tolerance in the chorion cells are much lower than those in the amnion cells.

Inhibition of influenza-virus-induced apoptosis in chorion cells of human fetal membranes by nordihydroguaiaretic Acid.

Objectives: It has been postulated that the pathogenesis of influenza virus infection involves not only the virus-proliferation-mediated apoptotic cell death in infected cells, but also a direct reactive oxygen species (ROS)-induced cellular injury in the infected organs. We examined effects of an antioxidant, nordihydroguaiaretic acid (NDGA), on apoptosis induction and viral proliferation. Subsequently, the results were compared with those of pyrrolidine dithiocarbamate (PDTC), another antioxidant. Methods: The levels of ROS production were measured with 2′,7′-dichlorofluorescein diacetate; apoptosis induction and viral proliferation were analyzed by DNA fragmentation and plaque-forming assays, respectively. Results: The treatment of infected cells with NDGA inhibited ROS overproduction, apoptotic DNA fragmentation and virus proliferation. The maximum inhibition against DNA fragmentation (76%) was observed with 500 µM NDGA. The antiviral activity of NDGA against influenza virus was more potent than that of PDTC. Conclusions: The present study, therefore, suggests for the first time that NDGA, a known antioxidant reagent, inhibits the induction of apoptosis in human fetal membrane chorion cells infected with influenza virus through the more potent antiviral activity than that of PDTC.

Differentiation of monocytes to macrophages induced by influenza virus-infected apoptotic cells.

The effect of the culture supernatant of influenza virus (IV)-infected apoptotic and non-apoptotic cells on the differentiation of monocytes to macrophages was investigated. IV infection induced apoptotic DNA fragmentation in cultured chorion cells but not in amnion cells prepared from human foetal membrane tissue. To examine the differentiation of monocytes to macrophages, an adhesion assay was employed using the human monocytic leukaemia THP-1 cell line. THP-1 cells became adherent to a substrate by incubation with the culture supernatant of IV-infected chorion cells, but not with that of amnion cells. The spreading THP-1 cells were morphologically characteristic of macrophages and they phagocytosed latex particles. RT-PCR analysis revealed that the expression of class A scavenger receptor mRNA was induced in THP-1 cells by incubation with the culture supernatant of IV-infected chorion cells. These results suggested that monocytic THP-1 cells were morphologically and functionally differentiated to macrophages by IV-infected apoptotic cells due to a soluble factor released from the apoptotic cells.

Semi-quantitative RT-PCR-based assay, improved by Southern hybridization technique, for polarity-specific influenza virus RNAs in cultured cells

Complementary (c) DNAs against viral (v) RNA of negative polarity and complementary and/or messenger (c/m) RNA of positive polarity for influenza virus hemagglutinin (HA) were synthesized from total cellular RNA extracted from influenza virus- and mock-infected cells using polarity-specific primers, respectively. HA vRNA and c/mRNA were amplified readily by polymerase chain reaction (PCR) from influenza virus-infected cells during a virus productive period; however, non-specific PCR product was prone to amplification from mock-infected cells and cells at once after virus infection. Southern blots of the PCR products were hybridized with biotinylated DNA probe, which enabled the generation of specific signals to HA vRNA and c/mRNA. Mock-infected cells produced no signals. Furthermore, titration analyses revealed linear relationships between amount of target RNAs and generated signals. Accordingly, Southern hybridization made possible the quantitation of specific PCR products for HA vRNA and c/mRNA in cell culture and proved the lack of HA RNAs in mock-infected cells in the absence of virus. The RT-PCR based assay combined with Southern hybridization methodology was useful with respect for investigating the processes of replication and transcription of viral genes in cell culture before and during the virus productive period.

Effects of Mitogen-Activated Protein Kinase Inhibitors on Tumor Necrosis Factor-α Gene Expression and Apoptosis Induction in Cultured Human Fetal Membrane Chorion Cells Infected with Influenza Virus

Objectives: We investigated the involvement of p38 mitogen-activated protein (MAP) kinase in tumor necrosis factor (TNF)-α gene expression, apoptosis induction and virus replication in cultured human fetal membrane chorion cells infected with influenza virus. Methods: Influenza virus-infected chorion cells were incubated in the absence or presence of inhibitors of p38 MAP kinase, SB203580 and SB202190. TNF-α mRNA and hemagglutinin viral RNA (HA vRNA) were amplified with reverse transcriptase-polymerase chain reaction techniques. TNF-α protein concentrations were determined by enzyme-liked immunosorbent assay. The extent of apoptosis induction was estimated by DNA agarose gel electrophoresis. Pyrrolidine dithiocarbamate (PDTC) and ribavirin, which have been shown to inhibit apoptosis induction via the inhibition of viral gene replication, were used as positive control reagents. Results: PDTC and ribavirin inhibited the accumulation of TNF-α mRNA and HA vRNA in the virus-infected chorion cells, resulting in the suppression of TNF-α protein secretion. Both SB203580 and SB202190 suppressed TNF-α protein secretion, but not the accumulation of TNF-α mRNA as well as HA vRNA and the induction of apoptosis. Conclusions: These results suggest that p38 MAP kinase pathway is critical in TNF-α gene expression at a post-transcriptional level but not in the apoptosis induction and influenza virus replication in cultured chorion cells.

Presence of Vicia graminea lectin- and Vicia unijuga lectin-binding (Vgu) glycoproteins in human fetal membranes and some of their biochemical properties.

We have previously reported that Vicia graminea lectin (VGA)- and Vicia unijuga lectin (VUA)-binding glycoproteins (Vgu glycoproteins), malignant tumor-associated antigens, exist in human meconium and amniotic fluid. To examine the origin of Vgu glycoprotein, their presence, some of their chemical and serological properties and their biosynthesis in the human fetal membrane, amnion and chorion laeve and accompanying membrane cells were examined. Perchloric acid-soluble fractions were prepared from human amnion and chorion laeve, after which VUA-binding components (Vgu glycoproteins) were separated by HPLC and affinity chromatography using immobilized VUA. Biosynthesis of the antigens in primary cultured cells prepared from the amnion and chorion laeve were examined by pulse-labeling and immunoprecipitation using immobilized VUA and compared with those in cultured human cancer cells. The results indicated that the serological properties of VUA-binding components in fetal membranes were similar to those of meconium and amniotic fluid, that many molecular species of VUA-binding components were synthesized in amnion and chorion laeve cells and that about 40-50% of antigens synthesized are secreted from cells while antigens synthesized in cultured cancer cells human were hardly secreted with more than 95% of the antigens remaining in the cells. From these results, we concluded that a large part of Vgu glycoproteins found in amniotic fluid is synthesized in cells of the amnion and chorion laeve and secreted into the fluid, and that Vgu glycoproteins synthesized in cancer cells were not secreted, rather they were retained in the cells.