title: Rat hepatocyte invasion by Listeria monocytogenes and analysis of TNF-alpha role in apoptosis.
abstract: Listeria monocytogenes, etiological agent of severe human foodborne infection, uses sophisticated mechanisms of entry into host cytoplasm and manipulation of the cellular cytoskeleton, resulting in cell death. The host cells and bacteria interaction may result in cytokine production as Tumor Necrosis Factor (TNF) alpha. Hepatocytes have potential to produce pro-inflammatory cytokines as TNF-alpha when invaded by bacteria. In the present work we showed the behavior of hepatocytes invaded by L. monocytogenes by microscopic analysis, determination of TNF-alpha production by bioassay and analysis of the apoptosis through TUNEL technique. The presence of bacterium, in ratios that ranged from 5 to 50,000 bacteria per cell, induced the rupture of cellular monolayers. We observed the presence of internalized bacteria in the first hour of incubation by electronic microscopy. The levels of TNF-alpha increased from first hour of incubation to sixth hour, ranging from 0 to 3749 pg/mL. After seven and eight hours of incubation non-significant TNF-alpha levels decrease occurred, indicating possible saturation of cellular receptors. Thus, the quantity of TNF-alpha produced by hepatocytes was dependent of the incubation time, as well as of the proportion between bacteria and cells. The apoptosis rate increased in direct form with the incubation time (1 h to 8 + 24 h), ranging from 0 to 43%, as well as with the bacteria : cells ratio. These results show the ability of hepatocyte invasion by non-hemolytic L. monocytogenes, and the main consequences of this phenomenon were the release of TNF-alpha by hepatocytes and the induction of apoptosis. We speculate that hepatocytes use apoptosis induced by TNF-alpha for release bacteria to extracellular medium. This phenomenon may facilitate the bacteria destruction by the immune system.
authors.affiliation: Laboratory of Cellular Microbiology, LIM 54, Department of Infectious Diseases, School of Medicine, University of São Paulo, 05403-000 São Paulo, SP, Brazil. saniasan@usp.br
authors.forename: Sânia Alves dos
authors.initials: SA
authors.lastname: Santos
authors.forename: Dahir Ramos de
authors.initials: DR
authors.lastname: Andrade
authors.forename: Dahir Ramos de
authors.initials: DR
authors.lastname: Andrade Júnior
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000502:physiology*; D005260:Female; D022781:Hepatocytes / Q000276:immunology / Q000382:microbiology*; D008089:Listeria monocytogenes / Q000472:pathogenicity / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*
chemical_list: D014409:Tumor Necrosis Factor-alpha