No abstract

Summary Carbon nanotubes have fibre-like shape1 and stimulate inflammation at the surface of the peritoneum when injected into the abdominal cavity of mice2, raising concerns that inhaled nanotubes3 may cause pleural fibrosis and/or mesothelioma4. Here we show that multi-walled carbon nanotubes reach the sub-pleura in mice after a single inhalation exposure of 30 mg/m3 for 6 hours. Nanotubes were embedded in the sub-pleural wall and within sub-pleural macrophages. Mononuclear cell aggregates on the pleural surface increased in number and size after 1 day and nanotube-containing macrophages were observed within these foci. Sub-pleural fibrosis increased after 2 and 6 weeks following inhalation. None of these effects were seen in mice that inhaled carbon black nanoparticles or a lower dose of nanotubes (1 mg/m3). This work advances a growing literature on pulmonary toxicology of nanotubes5 and suggests that minimizing inhalation of nanotubes during handling is prudent until further long term assessments are conducted.

We developed a dispersal method for multi-walled carbon nanotubes (MWCNTs) that allows quantitative assessment of dispersion on pro-fibrogenic responses in tissue culture cells as well as in mouse lung. Here we demonstrate that the dispersal of as-prepared (AP), purified (PD), and carboxylated (COOH) MWCNTs by bovine serum albumin (BSA) and dipalmitoylphosphatidylcholine (DPPC) influences TGF-β1, PDGF-AA and IL-1β production in vitro and in vivo. These biomarkers were chosen based on their synergy in promoting fibrogenesis and cellular communication in the epithelial-mesenchymal cell trophic unit in the lung. The effect of dispersal was most noticeable in AP- and PD-MWCNTs, which are more hydrophobic and unstable in aqueous buffers than hydrophilic COOH-MWCNTs. Well-dispersed AP- and PD-MWCNTs were readily taken up by BEAS-2B, THP-1 cells and alveolar macrophages (AM), and induced more prominent TGF-β1 and IL-1β production in vitro as well as TGF-β1, IL-1β and PDGF-AA production in vivo than non-dispersed tubes. Moreover, there was good agreement between the pro-fibrogenic responses in vitro and in vivo as well as the ability of dispersed tubes to generate granulomatous inflammation and fibrosis in airways. Tube dispersal also elicited more robust IL-1β production in THP-1 cells. While COOH-MWCNTs were poorly taken up in BEAS-2B and induced little TGF-β1 production, they were bio-processed by AM and induced less prominent collagen deposition at sites of non-granulomatous inflammation in the alveolar region. Taken together, these results indicate that the dispersal state of MWCNTs affects pro-fibrogenic cellular responses that correlate with the extent of pulmonary fibrosis and are of potential use to predict pulmonary toxicity.

Carbon nanotubes are gaining increasing attention due to possible health risks from occupational or environmental exposures. This study tested the hypothesis that inhaled multiwalled carbon nanotubes (MWCNT) would increase airway fibrosis in mice with allergic asthma. Normal and ovalbumin-sensitized mice were exposed to a MWCNT aerosol (100 mg/m(3)) or saline aerosol for 6 hours. Lung injury, inflammation, and fibrosis were examined by histopathology, clinical chemistry, ELISA, or RT-PCR for cytokines/chemokines, growth factors, and collagen at 1 and 14 days after inhalation. Inhaled MWCNT were distributed throughout the lung and found in macrophages by light microscopy, but were also evident in epithelial cells by electron microscopy. Quantitative morphometry showed significant airway fibrosis at 14 days in mice that received a combination of ovalbumin and MWCNT, but not in mice that received ovalbumin or MWCNT only. Ovalbumin-sensitized mice that did not inhale MWCNT had elevated levels IL-13 and transforming growth factor (TGF)-beta1 in lung lavage fluid, but not platelet-derived growth factor (PDGF)-AA. In contrast, unsensitized mice that inhaled MWCNT had elevated PDGF-AA, but not increased levels of TGF-beta1 and IL-13. This suggested that airway fibrosis resulting from combined ovalbumin sensitization and MWCNT inhalation requires PDGF, a potent fibroblast mitogen, and TGF-beta1, which stimulates collagen production. Combined ovalbumin sensitization and MWCNT inhalation also synergistically increased IL-5 mRNA levels, which could further contribute to airway fibrosis. These data indicate that inhaled MWCNT require pre-existing inflammation to cause airway fibrosis. Our findings suggest that individuals with pre-existing allergic inflammation may be susceptible to airway fibrosis from inhaled MWCNT.