Multi-walled carbon nanotubes induce stronger migration of inflammatory cells in vitro than asbestos or granular particles but a similar pattern of inflammatory mediators

Our plain-English summary of Multi-walled carbon nanotubes induce stronger migration of inflammatory cells in vitro than asbestos or granular particles but a similar pattern of inflammatory mediators, by G.A. Westphal, N. Rosenkranz, A. Brik, et al (Toxicology in Vitro, 2019).

The study

• Authors: G.A. Westphal, N. Rosenkranz, A. Brik, D. Weber, I. Föhring, C. Monsé, N. Kaiser, B. Hellack, M. Mattenklott, T. Brüning, G. Johnen, J. Bünger
• Journal: Toxicology in Vitro
• Published: 2019;58:215-223
• Cited by: 15 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

Biopersistent pro-inflammatory fibers are suspected human carcinogens. Cytotoxicity and transcription of pro- and anti-inflammatory mediators of different fibers were investigated in functional relationship to chemotaxis in vitro as a model for fiber-induced inflammation of the lung. We challenged NR8383 rat macrophages with multi-walled carbon nanotubes (MWCNT) and various asbestos fibers. The resulting cell supernatants were than studied using the Particle-induced Cell Migration Assay (PICMA) and cytotoxicity was determined using the LDH test. Expression of inflammatory mediators was analyzed with qPCR and verified by ELISA. Chrysotile A and the rigid, needle-shaped NM-401 caused the strongest cytotoxic effects and the largest number of migrated cells. In contrast, the MWCNT NM-400, NM-402, and NM403 were apparently non-cytotoxic but induced pronounced cell migration showing a very steep dose response. However, the strength of cell migration and cytotoxicity of the asbestos fibers were correlated. The expression profile of inflammatory mediators was comparable, although cytotoxicity of the MWCNT NM-401 and NM-403 differed strongly. Induction of the corresponding proteins was confirmed for CCL2, CCL3, CXCL1, CXCL3, IL1RA (IL1RN), CSF1, GDF15 and TNFa. Chrysotile A and NM-401 induced much stronger chemotaxis than the non-fibrous particles reported in our previous study. Cytotoxic and chemotactic effects correspond to the induction of inflammatory mediators.

Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.

In plain English

This was a laboratory study on cells, not a study of people. The authors exposed rat immune cells (the NR8383 macrophage cell line) to four types of multi-walled carbon nanotubes (NM-400, NM-401, NM-402, NM-403) and four UICC reference asbestos samples (chrysotile A, chrysotile B, amosite, crocidolite). They then tested whether fluid from the exposed cells made other cells (dHL-60) migrate, as a model of fiber-induced lung inflammation. They also measured cell damage and inflammatory signaling molecules. All fibers caused dose-dependent cell migration. The doses giving half the maximum effect were lower for the nanotubes (0.8 to 4.9 μg/cm2) than for the asbestos samples (8.8 μg/cm2 for chrysotile A to 40.9 μg/cm2 for amosite). The rigid, needle-like NM-401 and chrysotile A produced the most migrated cells. In a cell-damage test (LDH), NM-401 was the only nanotube that was toxic, while all four asbestos samples were. The gene expression pattern was similar after nanotube and asbestos exposure. The authors concluded that fibers caused stronger migration than the non-fibrous particles from their earlier study, with a similar pattern of mediators. They noted that cell tests model only short-term effects, and that the ground asbestos reference samples may underestimate asbestos toxicity.

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 laboratory experiment using cell lines (rat macrophages and differentiated human leukemia cells); no human subjects or live animals
• Who was studied: NR8383 rat alveolar macrophage cell line. Fluid from these cells was tested on HL-60 human leukemia cells that had been differentiated in the lab (dHL-60). Each migration sample was tested in at least three independent experiments.
• Exposure: Four multi-walled carbon nanotubes (NM-400, NM-401, NM-402, NM-403) and four UICC standard reference asbestos samples (chrysotile A, chrysotile B, amosite, crocidolite). Concentrations for the migration test were 0 to 64 μg/cm2, except NM-402, which declined at 32 μg/cm2. Nanosized silica was the positive control, and results were also compared with granular particles from the authors' previous study.
• Main results:
  • Doses giving half the maximum cell migration (EC50) for the nanotubes: 0.83 μg/cm2 (NM-402), 1.76 μg/cm2 (NM-403), 3.39 μg/cm2 (NM-400), and 4.88 μg/cm2 (NM-401)
  • EC50 values for cell migration for the asbestos samples: 8.77 μg/cm2 (chrysotile A), 10.01 μg/cm2 (chrysotile B), 21.49 μg/cm2 (crocidolite), and 40.87 μg/cm2 (amosite). The silica positive control had an EC50 of 39.92 μg/cm2.
  • The largest number of migrated cells came from the straight, needle-like nanotube NM-401, about 8 times the spontaneous migration.
  • Most nanotubes caused considerably stronger migration than the asbestos samples, but only NM-401 exceeded chrysotile A, which was the strongest of the asbestos samples.
  • Statistically significant differences from the silica positive control were reported for NM-401 and chrysotile A. The chrysotile A p-value is printed differently in the text (0.00212) and in the figure legend (0.0212).
  • In the LDH cell-damage test, nanotubes NM-400, NM-402 and NM-403 caused no measurable cytotoxicity.
  • NM-401 was strongly toxic in both cytotoxicity tests (LDH IC50 3.26 μg/cm2), considerably more than the asbestos samples.
  • LDH IC50 values for the asbestos samples: 9.8 μg/cm2 (chrysotile A), 11.9 μg/cm2 (chrysotile B), 35.1 μg/cm2 (crocidolite), and 46.8 μg/cm2 (amosite)
  • The gene expression profile of inflammatory mediators was similar after exposure to nanotubes and to asbestos. For this reason, follow-up testing was done only with NM-401 and NM-403.
  • In cells exposed to NM-401 and NM-403, induction of several mediators was reported at both the RNA and protein level. The article is inconsistent about CSF1, which is elsewhere described as not detectable at the protein level.
  • Compared with granular particles from the authors' earlier study (quartz, silica nanoparticles, barium sulfate), the test distinguished inert particles from those with medium and strong inflammatory effects.
• The authors' conclusion: The authors concluded that fibers caused clearly stronger cell migration than non-fibrous particles, with a similar pattern of inflammatory mediators. They also concluded that migration can occur independently of cell damage for the non-rigid, non-needle-like nanotubes.
• Limitations the authors state:
  • Cell tests model short-term (acute) inflammation, while the long-term fiber toxicity seen in living animals depends on fiber dimensions and how long fibers persist in the body.
  • The UICC asbestos reference samples were ground, so their fibers are much shorter than naturally occurring asbestos.
  • Because the asbestos samples were ground while the nanotubes were left more or less unchanged, asbestos toxicity may be underestimated.
  • Different cell densities were used in the different tests, so per cell the LDH test received about 10 times less dose.

Topics

Medical terms in this study, explained in our Lexicon: Inflammation, Macrophage, Chrysotile.

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