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Characterization of influenza virus-induced death of J774.1 macrophages
1Radiation Pathophysiology and Toxicology Department, Armed Forces Radiobiology Research Institute, Bethesda, Maryland 20889-5603, USA. lowy@mx.afrri.usuhs.mil
Abstract:
The mechanism and role of influenza virus (IV)-induced pathogenesis of macrophages during respiratory infection are ill defined. Reported here are findings on IV-induced cytopathic effects (CPEs) for an in vitro experimental system using the murine macrophage cell line J774.1. CPE was elicited by 0.2 or greater multiplicity of infection (m.o.i.). CPEs showed a lag of 6-8 h postinfection and occurred most rapidly between 6 and 12 h. J774.1 cells did not support productive IV replication, but immunofluorescence demonstrated that IV protein synthesis occurred. Light microscopy and DNA staining showed that after death cells had very condensed cytoplasm and nuclei. Cell remnants were surrounded by intact plasma membrane (PM) as demonstrated by exclusion of a membrane-impermeant dye. Time-lapse video microscopy recordings between 6 and 10 h postinfection showed sequential structural changes, including previously undescribed events. Notable changes were a rapid cytokinesis (zeiosis; "cell boiling"), followed by nuclear shrinkage, and an unusual transient blebbing of the PM. DNA fragmentation occurred after 12 h, producing a wide size range. UV-inactivated virus failed to induce CPEs, and CPE was blocked by amantadine. N-Acetylcysteine and pyrrolidine dithiocarbamate, but not other inhibitors of reactive oxygen intermediates, reduced or blocked the CPE. Most changes observed are those attributed to apoptotic processes rather than necrotic cell death. The kinetics and inhibitor effects suggest that IV infection and replication must be initiated to activate CPEs.
Insights
Influenza virus (IV) infection causes cytopathic effects (CPEs) in macrophages, characterized by apoptosis-like cell death. These IV-induced changes require viral replication and protein synthesis, offering insights into respiratory infection pathogenesis.
Area of Science:
- Virology
- Immunology
- Cell Biology
Background:
- The pathogenesis of influenza virus (IV)-induced macrophage damage during respiratory infections is not fully understood.
- Investigating IV's impact on macrophages is crucial for understanding host-pathogen interactions in respiratory diseases.
Purpose of the Study:
- To elucidate the mechanism and role of IV-induced cytopathic effects (CPEs) in macrophages.
- To characterize the cellular changes and kinetics of IV-induced cell death in a murine macrophage model.
Main Methods:
- Utilized an in vitro experimental system with the murine macrophage cell line J774.1 infected with IV.
- Employed light microscopy, DNA staining, immunofluorescence, and time-lapse video microscopy to observe cellular changes.
- Assessed the role of viral replication, protein synthesis, and reactive oxygen intermediates using inhibitors and UV-inactivated virus.
Main Results:
- IV infection elicited CPEs in J774.1 cells at multiplicities of infection (m.o.i.) of 0.2 or greater, with a lag of 6-8 hours and peak activity between 6-12 hours.
- While productive replication was not supported, IV protein synthesis occurred, preceding characteristic apoptotic-like changes including zeiosis, nuclear shrinkage, and plasma membrane blebbing.
- DNA fragmentation was observed after 12 hours; CPE induction was dependent on viable virus and partially inhibited by N-Acetylcysteine and pyrrolidine dithiocarbamate.
Conclusions:
- IV infection induces apoptosis-like CPEs in macrophages, dependent on viral replication and protein synthesis.
- The observed cellular events, including zeiosis and DNA fragmentation, provide new insights into IV-induced macrophage death pathways.
- These findings contribute to understanding the complex interplay between influenza viruses and the innate immune system during respiratory infections.