The mechanism of pleural inflammation by long carbon nanotubes: interaction of long fibres with macrophages stimulates them to amplify pro-inflammatory responses in mesothelial cells

Our plain-English summary of The mechanism of pleural inflammation by long carbon nanotubes: interaction of long fibres with macrophages stimulates them to amplify pro-inflammatory responses in mesothelial cells, by F.A. Murphy, A. Schinwald, C.A. Poland, et al (Particle and Fibre Toxicology, 2012).

The study

• Authors: F.A. Murphy, A. Schinwald, C.A. Poland, K. Donaldson
• Journal: Particle and Fibre Toxicology
• Published: 2012;9:8
• Cited by: 159 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Carbon nanotubes (CNT) are high aspect ratio nanoparticles with diameters in the nanometre range but lengths extending up to hundreds of microns. The structural similarities between CNT and asbestos have raised concern that they may pose a similar inhalation hazard. Recently CNT have been shown to elicit a length-dependent, asbestos-like inflammatory response in the pleural cavity of mice, where long fibres caused inflammation but short fibres did not. However the cellular mechanisms governing this response have yet to be elucidated. This study examined the in vitro effects of a range of CNT for their ability to stimulate the release of the acute phase cytokines; IL-1β, TNFα, IL-6 and the chemokine, IL-8 from both Met5a mesothelial cells and THP-1 macrophages. Results showed that direct exposure to CNT resulted in significant cytokine release from the macrophages but not mesothelial cells. This pro-inflammatory response was length dependent but modest and was shown to be a result of frustrated phagocytosis. Furthermore the indirect actions of the CNT were examined by treating the mesothelial cells with conditioned media from CNT-treated macrophages. This resulted in a dramatic amplification of the cytokine release from the mesothelial cells, a response which could be attenuated by inhibition of phagocytosis during the initial macrophage CNT treatments. We therefore hypothesise that long fibres elicit an inflammatory response in the pleural cavity via frustrated phagocytosis in pleural macrophages. The activated macrophages then stimulate an amplified pro-inflammatory cytokine response from the adjacent pleural mesothelial cells. This mechanism for producing a pro-inflammatory environment in the pleural space exposed to long CNT has implications for the general understanding of fibre-related pleural disease and design of safe nanofibres.

In plain English

This laboratory study tested carbon nanotubes, not asbestos, on human cell lines. The authors exposed a human mesothelial cell line (Met5A) and macrophages from a human monocyte cell line (THP-1, a stand-in for pleural macrophages) to five multiwalled carbon nanotube (CNT) samples of different lengths and to carbon black, mostly at 5 μg/cm2. Directly exposed mesothelial cells showed no change in four inflammatory signalling proteins (IL-1b, IL-6, IL-8, TNFa). Macrophages released significantly more IL-1b and IL-6 only with the two long-fibre samples. The macrophages could not fully engulf these fibres (frustrated phagocytosis). Blocking uptake with cytochalasin D reduced IL-1b release. Fluid from macrophages exposed to the longest sample (NTlong2) made mesothelial cells release 20-fold, 30-fold and 6-fold more IL-6, IL-8 and TNFa than the combined total from both cell types exposed directly. Fluid from macrophages given short or tangled samples had no effect. The authors concluded this describes a mechanism for long-fibre pleural inflammation. They could not rule out trace nanotubes in the fluid but judged any such amounts well below the threshold of effect.

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: In vitro (cell culture) laboratory experiment
• Who was studied: Human cell lines: the Met5A mesothelial cell line, and macrophages derived from the THP-1 human monocyte cell line, used as a surrogate for pleural macrophages. No humans or animals were studied. Experiments were in triplicate (n = 3).
• Exposure: Five multiwalled carbon nanotube samples and nano-particle carbon black. The samples were: short (NTshort, 1-2 μm), two tangled (NTtang1, 1-5 μm; NTtang2, 5-20 μm) and two long (NTlong1, mean 13 μm, 24.04% of fibres longer than 15 μm; NTlong2, mean 36 μm, 84.26% longer than 15 μm). Doses for viability testing were 5 to 50 μg/cm2. A sub-lethal dose of 5 μg/cm2 was used for the activation studies. Asbestos was not tested.
• Main results:
  • In mesothelial (Met5A) cells exposed directly, no CNT sample changed IL-1b, IL-6, IL-8 or TNFa levels.
  • In macrophages (THP-1) exposed directly, only the two long-fibre samples significantly increased IL-1b and IL-6. NTlong1 also increased IL-8. NTlong2 appeared to raise TNFa, but this was not statistically significant.
  • Macrophages could not fully take up the long fibres (frustrated phagocytosis). The phagocytosis inhibitor cytochalasin D reduced IL-1b release from macrophages treated with the long samples in a dose-dependent way, but it did not reduce the response to LPS.
  • Mesothelial cells treated with fluid (conditioned media) from NTlong2-exposed macrophages released 20-fold more IL-6, 30-fold more IL-8 and 6-fold more TNFa than the combined total from each cell type exposed directly. For IL-1b, conditioned media from LPS-, NTlong1- or NTlong2-exposed macrophages gave about a two-fold increase.
  • In Table 1, IL-8 from mesothelial cells given NTlong2 conditioned media was 12130 ± 2254 pg/ml. This compares with 121 ± 29 pg/ml with NTshort conditioned media and 458 ± 16 pg/ml for mesothelial cells exposed directly to NTlong2.
  • Conditioned media from macrophages treated with carbon black, short or tangled CNT had no effect on mesothelial cytokine production.
  • Mesothelial IL-1b and IL-6 release was reduced when the macrophages had been co-exposed to NTlong2 and cytochalasin D to prevent phagocytosis.
• The authors' conclusion: The authors concluded that their data describe a mechanism for starting a long-fibre inflammatory response in the pleural space through frustrated phagocytosis. In this mechanism, macrophages engaging long CNT amplify the inflammatory response of mesothelial cells.
• Limitations the authors state:
  • Very small amounts of CNT remaining in the conditioned media could not be excluded, though the authors considered any such amounts well below the threshold of effect.
  • Macrophage viability could not be measured by trypan blue because the cells clumped together.
  • Further research is needed on what pleural or alveolar macrophages containing CNT actually secrete in the pleural space.
  • The study addressed only inflammation. It did not address other long-fibre effects on mesothelial cells, such as genotoxic ones.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Mesothelioma.

Medical terms in this study, explained in our Lexicon: Inflammation, Macrophage, Mesothelial Cells, Aspect Ratio (Length-to-diameter ratio), Pleural Cavity, Cytokine, Frustrated Phagocytosis, Phagocytosis.

Related studies

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• Asbestos, carbon nanotubes and the pleural mesothelium: a review of the hypothesis regarding the role of long fibre retention in the parietal pleura, inflammation and mesothelioma — K. Donaldson, F.A. Murphy, et al. (2010)
• Effects of sustained stimulation with multi-wall carbon nanotubes on immune and inflammatory responses in mice — A. Yamaguchi, T. Fujitani, et al. (2012)
• Different Cellular Response of Human Mesothelial Cell MeT-5A to Short-Term and Long-Term Multiwalled Carbon Nanotubes Exposure — L. Ju, W. Wu, et al. (2017)
• Multi-walled carbon nanotubes induce stronger migration of inflammatory cells in vitro than asbestos or granular particles but a similar pattern of inflammatory mediators — G.A. Westphal, N. Rosenkranz, et al. (2019)
• SWCNT suppress inflammatory mediator responses in human lung epithelium in vitro — E. Herzog, H.J. Byrne, et al. (2009)

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