Use of back-scatter electron signals to visualise cell/nanowires interactions in vitro and in vivo; frustrated phagocytosis of long fibres in macrophages and compartmentalisation in mesothelial cells in vivo

Our plain-English summary of Use of back-scatter electron signals to visualise cell/nanowires interactions in vitro and in vivo; frustrated phagocytosis of long fibres in macrophages and compartmentalisation in mesothelial cells in vivo, by A. Schinwald, K. Donaldson (Particle and Fibre Toxicology, 2012).

The study

• Authors: A. Schinwald, K. Donaldson
• Journal: Particle and Fibre Toxicology
• Published: 2012;9:34
• Cited by: 79 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Background: Frustrated phagocytosis has been stated as an important factor in the initiation of an inflammatory response after fibre exposure. The length of fibrous structures has been linked to the potential of fibres to induce adverse health effects for at least 40 years. However, we only recently reported for the first time the threshold length for fibre-induced inflammation in the pleural space and we implicated frustrated phagocytosis in the pro-inflammatory effects of long fibres. This study extends the examination of the threshold value for frustrated phagocytosis using well-defined length classes of silver nanowires (AgNW) ranging from 3-28 μm and describes in detail the morphology of frustrated phagocytosis using a novel technique and also describes compartmentalisation of fibres in the pleural space.

Methods: A novel technique, backscatter scanning electron microscopy (BSE) was used to study frustrated phagocytosis since it provides high-contrast detection of nanowires, allowing clear discrimination between the nanofibres and other cellular features. A human monocyte-derived macrophage cell line THP-1 was used to investigate cell-nanowire interaction in vitro and the parietal pleura, the site of fibre retention after inhalation exposure was chosen to visualise the cell- fibre interaction in vivo after direct pleural installation of AgNWs.

Results: The length cut-off value for frustrated phagocytosis differs in vitro and in vivo. While in vitro frustrated phagocytosis could be observed with fibres≥14 μm, in vivo studies showed incomplete uptake at a fibre length of ≥10 μm. Recently we showed that inflammation in the pleural space after intrapleural injection of the same nanofibre panel occurs at a length of ≥5 μm. This onset of inflammation does not correlate with the onset of frustrated phagocytosis as shown in this study, leading to the conclusion that intermediate length fibres fully enclosed within macrophages as well as frustrated phagocytosis are associated with a pro-inflammatory state in the pleural space. We further showed that fibres compartmentalise in the mesothelial cells at the parietal pleura as well as in inflammatory cells in the pleural space.

Conclusion: BSE is a useful way to clearly distinguish between fibres that are, or are not, membrane-bounded. Using this method we were able to show differences in the threshold length at which frustrated phagocytosis occurred between in vitro and in vivo models. Visualising nanowires in the pleura demonstrated at least 2 compartments--in leukocyte aggregations and in the mesothelium--which may have consequences for long term pathology in the pleural space including mesothelioma.

In plain English

This methodology paper tested a microscope technique, backscatter scanning electron microscopy, for seeing how cells handle long, thin fibres. The fibres were silver nanowires in five average lengths (3, 5, 10, 14 and 28 μm). No asbestos was tested. In laboratory dishes, a human macrophage cell line (THP-1) fully took up the 3 and 5 μm wires and mostly took up the 10 μm wires. The 14 and 28 μm wires caused 'frustrated phagocytosis', where a cell cannot fully enclose a fibre. In female mice injected with wires into the chest cavity, macrophages on the lining of the chest wall (the parietal pleura) only partly enclosed 10 μm wires. No 3 μm wires were seen on the pleura after 24 hours. After one week, 5 and 10 μm wires were seen on and inside mesothelial cells and looked intact, while wires inside macrophages were starting to break down. The authors concluded that this placement in the mesothelium may matter for long-term pleural disease, including mesothelioma. They called the mesothelial findings descriptive and said they need confirmation by more quantitative studies.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Methodology study using laboratory cell-culture experiments (in vitro) and a mouse experiment (in vivo)
• Who was studied: The human monocytic cell line THP-1 in culture, and female C57BI/6 mice aged 8 weeks, with 4 mice killed at 24 hours and 4 at 7 days after injection
• Exposure: Silver nanowires in five length classes (average lengths 3, 5, 10, 14 and 28 μm). In culture, doses ranged from 0.4 to 4.0 μg/cm2, adjusted to give roughly equal fibre numbers. Mice received 5 μg per mouse injected into the pleural cavity. No asbestos was tested.
• Main results:
  • In culture, THP-1 cells completely took up the 3 μm and 5 μm nanowires.
  • In culture, the 10 μm nanowires were mostly taken up, with a small percentage of fibre ends sticking out of cells.
  • In culture, obvious frustrated phagocytosis was seen with the 14 μm and 28 μm nanowires.
  • In culture, only the 28 μm nanowires caused a significant decrease in THP-1 membrane integrity and a loss of proliferation and metabolic activity.
  • In mice, no 3 μm nanowires were seen on the parietal pleura surface 24 hours after injection.
  • In mice at 24 hours, most 5 μm nanowires were fully taken up by pleural macrophages. Some 10 μm nanowires were only partly taken up, which the authors described as frustrated phagocytosis.
  • The 10 μm length was fully taken up in culture but only incompletely taken up by cells on the mouse parietal pleura.
  • The 5 μm fibres could be fully enclosed by macrophages but were associated with inflammation. The inflammation finding comes from the authors' earlier study (reference 14), which used the same animals.
  • In mice after 1 week, 5 μm and 10 μm nanowires were seen on the surface of mesothelial cells of the parietal pleura or taken up by them.
  • After 1 week, nanowires in the mesothelial layer looked intact, while those inside macrophages were starting to degrade.
• The authors' conclusion: The authors concluded that backscatter imaging showed nanowires in at least two places in the pleura: in clusters of white blood cells and in the mesothelium. They wrote that this may have consequences for long-term pleural disease, including mesothelioma. They also concluded that frustrated phagocytosis is not the only factor in the onset of pleural inflammation.
• Limitations the authors state:
  • The authors stated that the mesothelial compartment finding was merely descriptive and needs confirmation by more quantitative studies.
  • The authors stated that THP-1 macrophages are not a good model for the length-dependent events seen in the mouse pleural space.
  • The authors noted that the cultured cells were a human cell line kept in a very abnormal environment compared with the mouse pleura.
  • The authors noted that silver ion measurements may not help clarify how long the nanowires persist.

Topics

Medical terms in this study, explained in our Lexicon: Frustrated Phagocytosis, Macrophage, Mesothelial Cells, Inflammation, Scanning Electron Microscopy (SEM), Parietal Pleura, Pleura, Mesothelium.

Related studies

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• In vitro biodegradation of chrysotile fibres by alveolar macrophages and mesothelial cells in culture: comparison with a pH effect — M.C. Jaurand, A. Gaudichet, et al. (1984)
• Role of fibre characteristics on cytotoxicity and induction of anaphase/telophase aberrations in rat pleural mesothelial cells in vitro: correlations with in vivo animal findings — M. Yegles, X. Janson, et al. (1995)
• Interactions between magnetic nanowires and living cells: uptake, toxicity, and degradation — M. Safi, M. Yan, et al. (2011)
• Phagocytosis of asbestos fibers by human pulmonary alveolar macrophages — T. McLemore, M. Corson, et al. (1979)
• Phagocytosis of chrysotile fibers by pleural mesothelial cells in culture — M.C. Jaurand, H. Kaplan, et al. (1979)

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