Inhaled multiwalled carbon nanotubes potentiate airway fibrosis in murine allergic asthma

Abstract

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.

Figure 1.

Photomicrographs of multiwalled carbon nanotubes (MWCNT) before and after aerosolization. (A) SEM of bulk nanotubes. (B) Transmission electron microscopy (TEM) of bulk nanotubes. (C and D) TEM photomicrographs of nanotubes collected from filters after 6 hours of inhalation. Scale bars: A, B, and D, 100 nm; C, 2,000 nm.

Figure 2.

MWCNT distribution in lung tissue 1 day after inhalation exposure. (A) Low magnification (×20) of lung from a mouse exposed to MWCNT after 1 day. MWCNT aggregates deposited at alveolar duct bifurcation (ADB), terminal bronchioles (TB), and on alveolar surfaces as indicated by arrows (hematoxylin stain). (B) Light micrograph showing MWCNT in macrophages in a terminal bronchiole (TB) indicated by open arrows (hematoxylin stain). (C) Light micrograph showing MWCNT-positive alveolar macrophage (open arrow) on the bronchiolar epithelium in the lung of an ovalbumin-challenged mouse (Masson's trichrome stain). (D) Agglomerated MWCNT (solid arrow) and MWCNT-laden alveolar macrophages migrating through an alveolar septa (open arrows) (Masson's trichrome stain). (E) TEM of MWCNT within an alveolar macrophage. (F) Higher magnification of MWCNT in E showing nanotube structure. (G) TEM of unstained lung section showing aggregrate of MWCNT beneath the cell membrane of a type I epithelial cell. (H) Higher magnification of MWCNT shown in G showing distinct nanotube structure.

Figure 3.

Persistence of MWCNT in macrophages at 1 and 14 day after inhalation exposure. Bronchoalveolar lavage (BAL) cells from cytospin were counted and the data expressed as percentage MWCNT-postive or MWCNT-negative macrophages relative to the number of total macrophages. Shaded bars, sal/MWCNT; solid bars, OVA/MWCNT.

Figure 4.

Masson's trichrome stain for collagen (blue) around airways 14 days after inhalation (magnification: ×20). (A) Unsensitized, saline inhalation. (B) Ovalbumin-sensitized, saline inhalation. (C) Unsensitized, MWCNT inhalation. (D) Ovalbumin-sensitized, MWCNT inhalation. (E) Quantitative morphometry for collagen deposition around airways at 14 days. ^aP < 0.01 compared with sal/MWCNT; ^bP < 0.01 compared with OVA/sal. Data are the mean ± SEM of five (sal/sal) or seven animals (all others).

Figure 5.

IL-13 mRNA and protein levels at 1 day after inhalation of MWCNT. (A) IL-13 in BAL fluid (BALF) was measured by enzyme-linked immunosorbent assay (ELISA). No IL-13 was detected in mice that did not receive ovalbumin challenge (ND, not detectable), nor was IL-13 detected in any groups at 14 days (not shown). (B) IL-13 mRNA levels measured by Taqman quantitative real-time RT-PCR. *P < 0.05 compared with sal/sal control group.

Figure 6.

Levels of platelet-derived growth factor (PDGF) and transforming growth factor (TGF)-β1 protein in BALF 1 day after MWCNT inhalation. (A) PDGF-AA measured by ELISA. *P < 0.05 compared with sal/sal control group. (B) Total TGF-β1 measured by ELISA. *P < 0.05 compared with sal/sal or sal MWCNT. Data are the mean ± SEM of 9 (sal/sal) or 10 animals (all others) from samples assayed in duplicate.

Figure 7.

mRNA levels of IL-5 and chemokines in lung tissue at 1 day after inhalation exposure. Taqman quantitative real-time RT-PCR was used to measure changes in mRNA levels. (A) IL-5. *P < 0.05 compared with sal/sal. (B) CCL2/MCP-1. *P < 0.05 compared with sal/sal control group. (C) CCL11/eotaxin. *P < 0.05 compared with sal/sal. (D) CXCL9. *P < 0.05 compared with sal/sal. Data are the mean ± SEM of 9 (sal/sal) or 10 animals (all others) from samples assayed in duplicate.