Chronic exposure of diesel exhaust particles induces alveolar enlargement in mice

Abstract

Background: Diesel exhaust particles (DEPs) are deposited into the respiratory tract and are thought to be a risk factor for the development of diseases of the respiratory system. In healthy individuals, the timing and mechanisms of respiratory tract injuries caused by chronic exposure to air pollution remain to be clarified.

Methods: We evaluated the effects of chronic exposure to DEP at doses below those found in a typical bus corridor in Sao Paulo (150 μg/m3). Male BALB/c mice were divided into mice receiving a nasal instillation: saline (saline; n = 30) and 30 μg/10 μL of DEP (DEP; n = 30). Nasal instillations were performed five days a week, over a period of 90 days. Bronchoalveolar lavage (BAL) was performed, and the concentrations of interleukin (IL)-4, IL-10, IL-13 and interferon-gamma (INF-γ) were determined by ELISA-immunoassay. Assessment of respiratory mechanics was performed. The gene expression of Muc5ac in lung was evaluated by RT-PCR. The presence of IL-13, MAC2+ macrophages, CD3+, CD4+, CD8+ T cells and CD20+ B cells in tissues was analysed by immunohistochemistry. Bronchial thickness and the collagen/elastic fibers density were evaluated by morphometry. We measured the mean linear intercept (Lm), a measure of alveolar distension, and the mean airspace diameter (D0) and statistical distribution (D2).

Results: DEP decreased IFN-γ levels in BAL (p = 0.03), but did not significantly alter IL-4, IL-10 and IL-13 levels. MAC2+ macrophage, CD4+ T cell and CD20+ B cell numbers were not altered; however, numbers of CD3+ T cells (p ≤ 0.001) and CD8+ T cells (p ≤ 0.001) increased in the parenchyma. Although IL-13 (p = 0.008) expression decreased in the bronchiolar epithelium, Muc5ac gene expression was not altered in the lung of DEP-exposed animals. Although respiratory mechanics, elastic and collagen density were not modified, the mean linear intercept (Lm) was increased in the DEP-exposed animals (p ≤ 0.001), and the index D2 was statistically different (p = 0.038) from the control animals.

Conclusion: Our data suggest that nasal instillation of low doses of DEP over a period of 90 days results in alveolar enlargement in the pulmonary parenchyma of healthy mice.

Figure 1

Respiratory mechanics parameters in saline or DEP exposed mice during 90 days. (A) tissue elastance (Htis), (B) tissue damping (Gtis) and (C) airway resistance (Raw). The central line defines the median, the borders above and below the shaded areas represent the 25% and 75% percentiles, respectively, and the error bars represent the 5% and 95% percentiles. The grey box represents saline group and stripped box represent DEP group. (D) Respiratory mechanics after methacholine challenge in saline (white circle) or DEP (black circle) exposed mice, where Raw means airway resistance. There were not statistically difference significant between groups.

Figure 2

Airspace enlargement in the lungs in saline or DEP exposed mice. (A) Mean linear intercept (Lm) (*p ≤ 0.001) and (B) parameter D2 (*p = 0.038). Lung parenchyma morphology. Photomicrography of mice lung parenchyma (HE), 90 days after exposure with saline (C) or a concentration of DEP of 30 μg/10 μL (D). Scale bar = 50 μm.

Figure 3

Elastic fibers (resorcin-fuchsin) and collagen fibers (sirius red) in the parenchyma region. Photomicrography of mice lung parenchyma showing elastic fibers (A, B) and collagen fibers (C, D) in saline (A, C) and DEP group (B, D). There were no statistically significant differences among groups in the proportion of elastic (p = 0.08) or collagen fibers (p = 0.208) occupied in the lung parenchyma. Scale bar = 20 μm.

Figure 4

CD3+ and CD8+ T cells density in lung parenchyma in saline or DEP exposed mice. Photomicrography of mice lung parenchyma showing CD3+ (A,B) and CD8+ T cells (C, D) in saline (A, C) and DEP group (B, D). Observe the increased density of these cells in the DEP group. Scale bar = 20 μm. The graphs show expression of CD3+ T lymphocytes and CD8+ T lymphocytes in saline and DEP group. The median is represented as horizontal bars. CD3+ T lymphocytes: *p ≤ 0.001 compared with saline group. CD8+ T lymphocytes: *p ≤ 0.001 compared with saline group.