Granuloma formation induced by low-dose chronic silica inhalation is associated with an anti-apoptotic response in Lewis rats

Abstract

Chronic human silicosis results primarily from continued occupational exposure to silica and exhibits a long asymptomatic latency. Similarly, continued exposure of Lewis rats to low doses of silica is known to cause delayed granuloma formation with limited lung inflammation and injury. On the other hand, intratracheal exposure to large doses of silica induces acute silicosis characterized by granuloma-like formations in the lung associated with apoptosis, severe alveolitis, and alveolar lipoproteinosis. To ascertain similarities/differences between acute and chronic silicosis, in this communication, we compared cellular and molecular changes in established rat models of acute and chronic silicosis. In Lewis rats, acute silicosis was induced by intratracheal instillation of 35 mg silica, and chronic silicosis through inhalation of aerosolized silica (6.2 mg/m(3), 5 d/wk for 6 wk). Animals exposed to acute high-dose silica were sacrificed at 14 d after silica instillation while chronically silica-treated animals were sacrificed between 4 d and 28 wk after silica exposure. The lung granulomas formation in acute silicosis was associated with strong inflammation, presence of TUNEL-positive cells, and increases in caspase-3 activity and other molecular markers of apoptosis. On the other hand, lungs from chronically silica-exposed animals exhibited limited inflammation and increased expression of anti-apoptotic markers, including dramatic increases in Bcl-2 and procaspase-3, and lower caspase-3 activity. Moreover, chronic silicotic lungs were TUNEL-negative and overexpressed Bcl-3 and NF-kappaB-p50 but not NF-kappaB-p65 subunits. These results suggest that, unlike acute silicosis, chronic exposures to occupationally relevant doses of silica cause significantly lower lung inflammation and elevated expression of anti-apoptotic rather than proapoptotic markers in the lung that might result from interaction between NF-kappaB-p50 and Bcl-3.

Figure 1. Acute but not chronic silicosis is associated with apoptosis

(A) Tissue sections from control (left), chronic (center), and acute (right) silicotic lungs were examined by TUNEL assay. Animals chronically and acutely exposed to silica were sacrificed at week 28 and day 14 after silica exposure, respectively. TUNEL-positive cells (nuclear condensates) present in the lung sections from acutely exposed animals are indicated by arrows and magnified in the inset. The figure is a representative of 5 animals per group. (B) Quantitative analysis of TUNEL activity for apoptotic cells at week 28 chronic silica exposure and day 14 acute silica exposure is represented as the number of nuclei that have apoptotic bodies (nuclear condensates) found within a field of granulomatous inflammatory sites (3 microscopic fields per animal/5 animals per group). No apoptotic bodies were found at day 4 or week 7 and 14 (data not shown). * Significant from p < 0.05.

Figure 2. BAL cells from animals chronically exposed to silica show anti-apoptotic markers

BAL cell lysates examined by Western blot analysis show the following: (A) procaspase 3 content at 14 week post chronic silica inhalation; (B) procaspase 3 content at day 14 after acute silica instillation. Loading controls were determined by GAPDH. Significantly more protein was loaded in the acute samples (100 μg) as was loaded in the chronic samples (10 μg) – thus the marked group differences in the amount of control procaspase 3 as well as total GAPDH. Data is representative of 5 animals per group (C, D) densitometry readings were normalized with GAPDH and were calculated from 5 animals per group and is represented as a change in % of control (* p <0.05).

Figure 3. Acute silicosis increases capase-3 activity in the lung

Caspase-3 activity of lung lysates was determined by ELISA as described in the Methods section. Each bar graph represents the enzyme activity ± SE of 3 different lung samples and the assay was run in duplicates at two different dilutions. Statistical significance was analyzed by GraphPad Prism.

Figure 4. Bcl-2 but not Bax is upregulated in chronic silicosis

(A) Changes in Bcl-2 expression by Western blots in BAL cell lysates at the indicated times after chronic silica exposure. Each lane is from an individual animal at the time point. Loading controls determined by GAPDH expression. (B) Densitometry of Figure 3A at day 4 or week 7, 14, and 28 after silica exposure. The readings were normalized with GAPDH and presented as a change in % of control. (C) Western blot analysis of lung cell lysates for the presence of Bcl-2 and Bax at 28 weeks, each lane represents an individual animal (* p < 0.05).

Figure 5. Bcl-2 expression is upregulated in lung granulomas from chronically silica-exposed animals

Lung sections from control (left), chronic 28 week (center), and acute 14 day (right) silicotic lungs were examined by IHC staining for Bcl-2 expression as described in Methods. The majority of stain was found within the epitheloid macrophages. Data represent 5 animals per group.

Figure 6. Chronic silicosis increases p50 but not p65 immunoreactivity in the lung

Lung sections from control, chronic 28 week, and acute 14 day silicotic lungs were analyzed by IHC as described in Methods. Upper panels (A–C) show p50, and lower panels (D–F) show p65 reactivity. The figures are representative of 5 animals per group.

Figure 7. Chronic silicosis increases p50 immunoreactivity in the lung

(A) Cell extracts made from control, chronic week 28, and acute day 14 silicotic lungs were examined by Western blot analysis as described in Methods. Data is representative of 5 animals per group and GAPDH was performed to verify proper loading. (B) Densitometry for p105, p50, and p65 was normalized with GAPDH and is presented as change % of control. * p < 0.05.

Figure 8. Bcl-3 is mainly localized to the nucleus in chronic silicotic granulomas

(A) Tissue sections from control, chronic week 28, and acute day 14 silicotic lungs were examined for Bcl-3 expression by IHC as described in Methods. The figure represents data from 5 animals per group. (B) Bcl-3 staining. Cells were enumerated by counting the number of nuclei that stained positive for Bcl-3 (suggesting nuclear localization) within a microscopic field of granulomatous inflammation. Three fields per animal, 5 animals per group. * p < 0.05. In control animals. other than occasional alveolar macrophages that demonstrated nuclear localization the vast majority of Bcl-3 staining did not appear localized to the nucleus (data not shown).