engleski

Lung fibroblasts activation and apoptosis in infection with Sendai virus

Sendai virus (SeV) belongs to the family Paramyxoviridae subfamily Paramyxovirinae, genera Respirovirus. This RNA virus is indigenous to mice but it is not a human pathogen. Usually, it is propagated in the alantoic cavity of the chicken embryo, but also in some cell cultures, like Vero cells, CV-1 etc. MRC-5 cells (human fetal lung fibroblasts) are suitable host cells for numerous viruses, including Paramyxoviridae. However, we did not find information about propagation of SeV in MRC-5 cells. Previous studies showed that infection and replication of SeV in cell cultures lead to a cytopathic effect (CPE), but the mechanisms behind these phenomena are poorly understood. It has also been reported that lung fibroblasts have the potential to release IL-8/CXCL8, GM-CSF, and MCP-1/CCL2 in response to some cytokines, kininogens or viruses. The aim of our study was to investigate the interaction between SeV and MRC-5 cells. For that purpose we measured the levels of some cytokines (IL-6, IL-10, IL-12-p70, TNF-alpha, IFN-alpha, IFN-beta, GM-CSF) and chemokines (IL-8/CXCL8, MCP-1/CCL2) by ELISA test in the supernatants of infected cells. Our recent results showed that SeV also cause CPE in MRC-5 cells. To better understand the mechanisms of cytopathology caused by SeV, we used Focused Gene Expression cDNA Array Analysis (GEArrays, SuperArray Bioscience, Frederick, MD, USA) to compare expression profiles of genes in RNA samples from MRC-5 cells one and three days after infection with SeV. Two different GEArrayTM were used: GEArray Q series Human Apoptosis Gene Array and GEArray Q series Human Extracellular Matrix & Adhesion Molecules Genes Array. Selected genes identified by GEArray analysis will be also analyzed by quantitative real-time PCR to verify transcriptional responses. SeV induced the production of GM-CSF and some IL-8/CXCL8 and IL-6 in infected MRC-5 cells in comparison to non-infected cells. Non-infected MRC-5 cells produced constitutively IL-8/CXCL8, IL-6 and MCP-1/CCL2 and no differences in MCP-1/CCL2 we found between infected and non-infected MRC-5 cells. Additionally, low levels of IFN-beta, and no IFN-alpha, TNF-alpha, IL-10 and IL-12p70 were detected. Our first results indicated that increase in gene expression of growth arrest and DNA-damage-inducible, alpha (GADD45A) may be involved in process of apoptosis induced by SeV in MRC-5 cells. Additionally, alpha integrins and some matrix metalloproteinases (MMPs) like MMP1 and MMP24 may be involved in process of cytopathology caused by SeV. MMPs are thought to be responsible for the turnover and degradation of the extracellular matrix. However, recent findings indicate that metalloproteinases act on pro-inflammatory cytokines and chemokines to regulate varied aspects of inflammation and immunity. It is recently shown that MMP1 cleave the N-terminus of CCL2/MCP-1 to produce antagonist factors. Whether MMP-1 was responsible for the regulation of MCP-1/CCL2 production in MRC-5 cells infected with SeV should be analyzed in further experiments.

Sendai virus; MRC-5 cells; lung fibroblasts; apoptosis; cytopathology

Rad je kao poster prezenturan i na skupu Annual meeting of the Croatian Immunological Society, održanom od 08-10.10.2004., Opatija, Hrvatska.