A 3-dimensional in vitro model of epithelioid granulomas induced by high aspect ratio nanomaterials

Our plain-English summary of A 3-dimensional in vitro model of epithelioid granulomas induced by high aspect ratio nanomaterials, by V.C. Sanchez, P. Weston, A. Yan, et al (Particle and Fibre Toxicology, 2011).

The study

• Authors: V.C. Sanchez, P. Weston, A. Yan, R.H. Hurt, A.B. Kane
• Journal: Particle and Fibre Toxicology
• Published: 2011;8:17
• Cited by: 37 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Background: The most common causes of granulomatous inflammation are persistent pathogens and poorly-degradable irritating materials. A characteristic pathological reaction to intratracheal instillation, pharyngeal aspiration, or inhalation of carbon nanotubes is formation of epithelioid granulomas accompanied by interstitial fibrosis in the lungs. In the mesothelium, a similar response is induced by high aspect ratio nanomaterials, including asbestos fibers, following intraperitoneal injection. This asbestos-like behaviour of some engineered nanomaterials is a concern for their potential adverse health effects in the lungs and mesothelium. We hypothesize that high aspect ratio nanomaterials will induce epithelioid granulomas in nonadherent macrophages in 3D cultures.

Results: Carbon black particles (Printex 90) and crocidolite asbestos fibers were used as well-characterized reference materials and compared with three commercial samples of multiwalled carbon nanotubes (MWCNTs). Doses were identified in 2D and 3D cultures in order to minimize acute toxicity and to reflect realistic occupational exposures in humans and in previous inhalation studies in rodents. Under serum-free conditions, exposure of nonadherent primary murine bone marrow-derived macrophages to 0.5 μg/ml (0.38 μg/cm2) of crocidolite asbestos fibers or MWCNTs, but not carbon black, induced macrophage differentiation into epithelioid cells and formation of stable aggregates with the characteristic morphology of granulomas. Formation of multinucleated giant cells was also induced by asbestos fibers or MWCNTs in this 3D in vitro model. After 7-14 days, macrophages exposed to high aspect ratio nanomaterials co-expressed proinflammatory (M1) as well as profibrotic (M2) phenotypic markers.

Conclusions: Induction of epithelioid granulomas appears to correlate with high aspect ratio and complex 3D structure of carbon nanotubes, not with their iron content or surface area. This model offers a time- and cost-effective platform to evaluate the potential of engineered high aspect ratio nanomaterials, including carbon nanotubes, nanofibers, nanorods and metallic nanowires, to induce granulomas following inhalation.

In plain English

This laboratory study developed a three-dimensional in vitro model in which primary murine bone marrow-derived macrophages from C57BL/6J mice were cultured without serum in agarose-coated wells and exposed to carbon black (Printex 90), UICC crocidolite asbestos fibers, and three commercial multiwalled carbon nanotube (MWCNT) samples. Doses of 0.05, 0.5, 5 and 10 micrograms per ml were tested; significant cell loss occurred at the two highest doses, so 0.05 and 0.5 micrograms per ml were used. At 0.5 micrograms per ml, crocidolite asbestos and MWCNT 3 produced stable aggregates after 3 days and MWCNT 1 and 2 after 7 days, while carbon black produced none at any dose or time; at 14 days asbestos and MWCNTs, but not carbon black, formed compact aggregates with epithelioid features. At 1.25 micrograms per ml for 6 hours, macrophages showed incomplete phagocytosis of asbestos fibers and MWCNTs. TNF-alpha and iNOS rose early, mannose receptor and arginase-1 later. The authors concluded that granuloma induction appeared to correlate with high aspect ratio and complex three-dimensional structure rather than iron content or surface area.

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 experimental study using a newly developed 3-dimensional serum-free macrophage culture model, with reference particles and three commercial MWCNT samples
• Who was studied: Primary murine bone marrow-derived macrophages (BMDM) from 6- to 8-week-old C57BL/6J male mice, cultured in 2D and 3D for up to 14 days (Brown University)
• Exposure: Carbon black particles (Printex 90, negative reference), UICC crocidolite asbestos fibers (positive reference), and three commercial MWCNT samples, at 0.05, 0.5, 5 and 10 micrograms per ml (0.038, 0.38, 3.8 and 7.6 micrograms per cm2); 1.25 micrograms per ml for phagocytosis studies
• Main results:
  • Cell loss (measured by total DNA content) was significant at the two highest doses, so lower doses were used for the granuloma experiments
  • In 3D cultures at 0.5 micrograms per ml, crocidolite asbestos produced stable cellular aggregates by 3 days and MWCNT 1 and 2 by 7 days
  • MWCNT 3 induced aggregation after 3 days at either 0.05 or 0.5 micrograms per ml
  • Carbon black produced no cellular aggregation at any dose or time point tested
  • After 14 days, asbestos fibers and MWCNTs, but not carbon black, produced compact aggregates with ultrastructural features of epithelioid macrophages
  • Macrophages in 3D culture showed incomplete (frustrated) phagocytosis of both asbestos fibers and MWCNTs after 6 hours at 1.25 micrograms per ml
  • Macrophages exposed to asbestos or MWCNTs showed early TNF-alpha increase with reduced mannose receptor at 1 to 3 days, and increased mannose receptor after day 7
  • Asbestos or MWCNT exposure induced early iNOS (3 to 7 days) followed by progressively increasing arginase-1 expression
  • Surface area did not explain granuloma formation in this data set: MWCNT 2 (200 m2/g) induced granulomas while carbon black (reported in the discussion as 300 m2/g) did not
  • No significant differences in IL-1b release were detected between exposed and untreated cells at the sub-lethal doses used
  • The commercial MWCNT samples had iron contents of less than 0.1 to 1.8 wt-% compared with 22% for crocidolite asbestos, yet induced granulomas to the same extent
• The authors' conclusion: The authors concluded that epithelioid granuloma induction appeared to correlate with high aspect ratio and complex 3D structure of the carbon nanotubes rather than iron content or surface area, and that the model offers a platform for screening high aspect ratio nanomaterials
• Limitations the authors state:
  • The authors noted that in vitro nanotoxicology assays typically deliver high doses to target cells that may not reflect realistic human exposure
  • The authors could not detect significant differences in inflammatory mediator levels by ELISA or Western blot between exposed and untreated cells
  • The authors stated that the mechanisms of stable macrophage aggregate formation in non-immune granulomas are unknown and the activated macrophage phenotype in such granulomas has not been characterized

Topics

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

Medical terms in this study, explained in our Lexicon: Epithelioid, Granuloma, Aspect Ratio (Length-to-diameter ratio), Inflammation, Interstitial Fibrosis, Mesothelium, Macrophage, Crocidolite.

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