Role of p53 in the chronic pulmonary immune response to tangled or rod-like multi-walled carbon nanotubes

Abstract

The fiber-like shape of multi-walled carbon nanotubes (MWCNTs) is reminiscent of asbestos, suggesting they pose similar health hazards when inhaled, including pulmonary fibrosis and mesothelioma. Mice deficient in the tumor suppressor p53 are susceptible to carcinogenesis. However, the chronic pathologic effect of MWCNTs delivered to the lungs of p53 heterozygous (p53^+/-) mice has not been investigated. We hypothesized that p53^+/- mice would be susceptible to lung tumor development after exposure to either tangled (t-) or rod-like (r-) MWCNTs. Wild-type (p53^+/+) or p53^+/- mice were exposed to MWCNTs (1 mg/kg) via oropharyngeal aspiration weekly over four consecutive weeks and evaluated for cellular and pathologic outcomes 11-months post-initial exposure. No lung or pleural tumors were observed in p53^+/+ or p53^+/- mice exposed to either t- or rMWCNTs. In comparison to tMWCNTs, the rMWCNTs induced the formation of larger granulomas, a greater number of lymphoid aggregates and greater epithelial cell hyperplasia in terminal bronchioles in both p53^+/- and p53^+/+ mice. A constitutively larger area of CD45R⁺/CD3⁺ lymphoid tissue was observed in p53^+/- mice compared to p53^+/+ mice. Importantly, p53^+/- mice had larger granulomas induced by rMWCNTs as compared to p53^+/+ mice. These findings indicate that a combination of p53 deficiency and physicochemical characteristics including nanotube geometry are factors in susceptibility to MWCNT-induced lymphoid infiltration and granuloma formation.

Keywords: Caron nanotubes; cancer; granuloma; immunotoxicology; p53.

Figure 1.

TEM images of nanomaterials used in the study A) tMWCNTs and D) rMWCNTs. B) TEM image of tMWCNTs in lung granuloma from a p53^+/− mouse at 11 months post-exposure. Cy indicates cytoplasm, and Nu indicates the nucleus. C) Inset showing a representative tMWCNT (arrow) near the nuclear membrane of a phagocytic cell. E) TEM image of rMWCNTs in granulomatous connective tissue from a p53^+/− mouse at the end of the 11-month study period. F) Inset showing a representative rMWCNT (arrow).

Figure 2.

Pulmonary alveolar macrophages containing tMWCNTs or rMWCNTs in the lungs of mice at the end of the 11-month study period. A) Hematoxylin-stained mouse lung tissue from both wild type and p53^+/− with arrows indicating the enclosed foreign material within the alveolar macrophages in the lungs (scale bar equals 20 μm). B) Hematoxylin & Eosin-stained (H&E) lung tissue from wild type and p53^+/− mice with arrows indicating macrophages containing tMWCNTs or rMWCNTs. Arrowheads indicate crystalline bodies within the cytosol of alveolar macrophages (scale bars equal 20 μm).

Figure 3.

rMWCNT exposure results in significantly larger lung granuloma area and a higher association with lymphoid tissue compared to tMWCNT, with a trend of larger formations in the lungs of p53^+/− mice. A) H&E stained lung sections from wild type and p53^+/− mouse lungs exposed to t- or r- MWCNTs near the terminal bronchioles (TB) of the lungs. Insets depict matured granuloma formations as indicated by arrows. (scale bar equal 200 μm, inset scale bars equal 50 μm). B) Average area of lung occupied by granuloma measured by quantitative morphometry (***p<0.001, *p<0.05 between tMWCNTs and rMWCNTs). C) Average number of granulomas per three lung sections per mouse. D) Average number of granulomas directly adjacent to lymphoid tissue per three lung sections per mouse. Number in parentheses above graph bar indicates number of animals per group. (***p<0.001, *p<0.05 between tMWCNTs and rMWCNTs).

Figure 4.

Formation of inducible lymphoid tissue in response to tMWCNT or rMWCNT exposure. A) Average number of lymphoid aggregates as a function of square mm lung area (**p<0.01 between tMWCNT and rMWCNT, **p<0.01, *p<0.05 compared to control). B) Average percent of lung area occupied by lymphoid tissue measured by quantitative morphometry (*p<0.05 between genotypes, *p<0.05 compared to control). C) Inducible bronchus associated lymphoid tissue (iBALT) along the bronchial (Br) airway and D) ectopic lymphoid tissue (ELT) and surrounding alveolar (Alv) space of the lung of a wild type and p53^+/− mouse lung exposed to rMWCNTs (scale bars equal 50 μm). Lymphoid tissue area broken up into average area of E) iBALT and F) ELT (*p<0.05 between genotypes). Number in parentheses above graph bar indicates number of animals per group.

Figure 5.

Structure of pulmonary inducible lymphoid tissue. Lung tissue from p53^+/− mice exposed to rMWCNTs for 11 months was immunohistochemically stained for the T cell marker CD3 and the B cell marker CD45R (B220) depicting a B cell germinal center with peripheral T cells in both the iBALT and ELT formations. For reference a granuloma (G) is indicated to show negative staining of cellular aggregates (scale bars equal 100 μm).

Figure 6.

BrdU IHC staining of mouse lungs from control and MWCNT-resultant granulomas show increased proliferation with rMWCNT exposure. A) Tissue from wild type and p53^+/− mouse lungs exposed to t- or r- MWCNTs for 11 months was stained with BrdU where a dark brown stain indicates DNA synthesis and is also indicated by arrows. Arrowheads indicate the nanomaterial within the granuloma (scale bars equal 50 μm). B) Average number of granulomas with BrdU positively stained nuclei (**p<0.01 between genotypes). Number in parentheses above graph bar indicates number of animals per group.

Figure 7.

Bronchiolar hyperplasia. A) BrdU stained lung sections from control, tMWCNT and rMWCNT wild type and p53^+/− mice were imaged to show the bronchial epithelium near a terminal bronchiole (TB) as indicated by arrows. Arrowheads indicate BrdU positive epithelial cells in the hyperplastic epithelium (scale bars equal 50 μm). B) Quantitation of BrdU positive epithelial cells at the terminal bronchial bifurcation reported as %BrdU positive cells with standard error. Numbers in parenthesis indicate the number of animals analyzed per group. (*p<0.05 compared to control)

Figure 8.

Papillary hyperplasia of the bronchiolar epithelium at alveolar duct bifurcation. Serial sections of papillary hyperplasia at the terminal bronchus in a p53^+/− mouse 11 months post-initial exposure to rMWCNTs. This rare pathology was captured in the IHC T cell marker CD3 and B cell marker CD45R sections. Arrows indicate location of papillary attachment to the bronchiole wall (scale bars equal 200 μm, inset scale bars equal 50 μm).

Figure 9.

Collagen deposition 11 months following initial tMWCNT or rMWCNT pulmonary exposure. A) Trichrome-stained lung sections are imaged here depicting terminal bronchioles (TB). Arrows indicate fibrotic foci (scale bars equal 200 μm). B) Relative average μg of soluble collagen per mouse lung as determined using the Sircol assay (*p<0.05 between genotypes). Number in parentheses above graph bar indicates number of animals per group.