Differential mRNA Expression of Inflammatory Cytokines in Cultured Human Fetal Membrane Cells Responding to Influenza Virus Infection.

Abstract

When a virus encounters a susceptible cell, the virus enters and initiates cytocidal, persistent, latent or abortive infection. We recently reported that influenza virus (IV) induces persistent infection accompanied by cell survival and virus production in cultured amnion cells. In addition, cytocidal infection accompanied by apoptotic cell death and virus production occurred in cultured chorion cells. Both cell cultures were prepared from human fetal membranes. 1)IV induces cytocidal infection in a variety of cultures, such as HeLa and Madin-Darby canine kidney (MDCK) cell lines and human peripheral blood monocytes in vitro, all of which die through the mechanism of apoptosis.2-5) Furthermore, IV infection induces gene expression of inflammatory cytokines such as interleukin (IL)-1, IL-6, tumor necrosis factor (TNF)-a and interferon (IFN)-a/b, and C-C and C-X-C chemokines such as monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1a, RANTES (regulated on activation, normal T cell expressed and secreted) and IFN-inducible protein (IP)-10, 6,7) and proapoptotic factors such as Fas and Fas ligand,8) which occur in infected host cells undergoing apoptosis. Apoptosis and inducible gene expression of inflammatory mediators therefore seem to be correlated. However, whether IV induces gene expression of inflammatory mediators during persistent infection remains unknown.We therefore extensively examined expression of mRNAs for inflammatory cytokines such as IL-1b, IL-6, TNF-a, IFN-a, IFN-b, IFN-g, macrophage colony-stimulating factor (M-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF), and Fas during cytocidal and persistent infections by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Of note is the fact that IV infection induced the gene expression of a set of inflammatory cytokines during cytocidal infection accompanied by apoptotic cell death, but this induction was not present during persistent infection in the absence of apoptosis. In this paper we report a noticeable difference in gene expression of a set of inflammatory cytokines between cytocidal and persistent infections. MATERIALS AND METHODSMaterials PCR primers for IFN-a, IFN-b, IFN-g, M-CSF and GM-CSF were originally designed according to published cDNA sequences. 9-13) PCR primers for Fas were based on the published DNA sequences for the primers. 14)PCR primers for IL-1b, IL-6, TNF-a and glycerol-3-phosphate dehydrogenase (G3PDH) were purchased from CLON-TECH Labs. Inc. (CA, U.S.A.). DNA sequences for the PCR primers are shown in Table 1. Neutralizing antibodies against Fas (Medical & Biological Labs. Co., Ltd., Nagoya, Japan), IFN-b (Yamasa Shoyu Co., Ltd., Tokyo, Japan), TNF-a and IFN-g (Genzyme Diagnostic, MA, U.S.A.) were purchased from the listed suppliers.Cell Culture and Virus Infection Primary cultured chorion and amnion cells were prepared from human fetal membranes obtained by cesarean section during the month of normal parturition as described.15) HeLa cell line was obtained from...

Regulation of Matrix Metalloproteinases-2 and -9 Gene Expression in Cultured Human Fetal Membrane Cells by Influenza Virus Infection

“…Interestingly, we have reported that influenza virus infection induced apoptosis and the gene expression of interleukin (IL)-6, tumor necrosis factor (TNF)-α and interferon (IFN)-β in cultured human fetal membrane chorion trophoblast cells. [12][13][14] However, even though the virus proliferated in cultured amnion epithelial cells, it should be noted that we did not observe the above phenomena in these cells. [12][13][14] Overall, these findings suggest that the induction of apoptosis and gene expression of pro-inflammatory cytokines in chorion trophoblast cells may have a possible role in the etiology of adverse pregnancy outcomes that are associated with an intrauterine infection caused by an influenza virus.…”

“…[12][13][14] However, even though the virus proliferated in cultured amnion epithelial cells, it should be noted that we did not observe the above phenomena in these cells. [12][13][14] Overall, these findings suggest that the induction of apoptosis and gene expression of pro-inflammatory cytokines in chorion trophoblast cells may have a possible role in the etiology of adverse pregnancy outcomes that are associated with an intrauterine infection caused by an influenza virus. 5,15) The main etiological factor responsible for the development of both premature rupture of the membranes (PROM) and preterm labor has been reported to be an intrauterine bacterial infection.…”

“…RT-PCR was carried out as described previously. 13) PCR primers for MMP-2, MMP-9, MT1-MMP, TIMP-1, TIMP-2, β-actin, glycerol-3-phosphate dehydrogenase (G3PDH) were referred to the published DNA sequences as listed in Table 1. 13,34-36) They were custom-made by SigmaGenosis (Tokyo, Japan). PCR amplification was performed with optimal numbers of PCR cycle as listed in Table 1.…”

Apoptosis Induced by Influenza Virus-Hemagglutinin Stimulation may be Related to Fluctuation of Cellular Oxidative Condition.

“…7,8) While the relationship of viral replication in host cells to host cell apoptosis has been definitely shown, the role of virus contact with host cell surface as the stimulus to induce apoptosis has not been examined previously. We know that the viral surface components which have direct contact with the host cell surface are neuraminidase, hemagglutinin and membrane protein-2, however, the participation of these viral components in the induction of host cell apoptosis has not been elucidated yet.…”

Predominant Contribution of IFN-.BETA. Expression to Apoptosis Induction in Human Uterine Cervical Fibroblast Cells by Influenza-Virus Infection

“…1) Chorion trophoblast cells expressed significantly amounts of inflammatory cytokine gene mRNAs in response to IV infection, such as interleukin (IL)-1b, IL-6, tumor necrosis factor (TNF)-a, interferon (IFN)-b, IFN-g, granulocyte macrophage colony-stimulating factor (GM-CSF) and Fas antigen, however, these cytokines production was not directly related to the apoptosis induction. 2) Thus, we speculate that the expression of these inflammatory cytokines may be related to set up the antiviral response system in the maternal body.…”