The role of surfactant protein A in bleomycin-induced acute lung injury

Abstract

Rationale: Surfactant protein A (SP-A) is a collectin family member that has multiple immunomodulatory roles in lung host defense. SP-A levels are altered in the bronchoalveolar lavage (BAL) fluid and serum of patients with acute lung injury and acute respiratory distress syndrome, suggesting the importance of SP-A in the pathogenesis of acute lung injury.

Objectives: Investigate the role of SP-A in the murine model of noninfectious lung injury induced by bleomycin treatment.

Methods: Wild-type (WT) or SP-A deficient (SP-A(-/-)) mice were challenged with bleomycin, and various indices of lung injury were analyzed.

Measurements and main results: On challenge with bleomycin, SP-A(-/-) mice had a decreased survival rate as compared with WT mice. SP-A(-/-) mice had a higher degree of neutrophil-dominant cell recruitment and the expression of the inflammatory cytokines in BAL fluid than did WT mice. In addition, SP-A(-/-) mice had increased lung edema as assessed by the increased levels of intravenously injected Evans blue dye leaking into the lungs. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling and active caspase-3 staining suggested the increased apoptosis in the lung sections from SP-A(-/-) mice challenged with bleomycin. SP-A also specifically reduced bleomycin-induced apoptosis in mouse lung epithelial 12 cells in vitro. Moreover, intratracheal administration of exogenous SP-A rescued the phenotype of SP-A(-/-) mice in vivo.

Conclusions: These data suggest that SP-A plays important roles in modulating inflammation, apoptosis, and epithelial integrity in the lung in response to acute noninfectious challenges.

Figure 1.

Surfactant protein A–deficient (SP-A^−/−) mice have significantly higher mortalities compared with wild-type (WT) mice in response to bleomycin challenge. WT and SP-A^−/− mice received intratracheal instillations of either (A) 2.5 U/kg, or (B) 5 U/kg bleomycin, and the percentages of surviving mice were plotted over a 21-day period. All saline-challenged mice (as a control) survived during the experimental period (data not shown). (A) P < 0.05; (B) P < 0.01; n = 20 in each group. (C) Representative histologic sections of lung tissue stained with hematoxylin and eosin. Increased cellularity, consolidation, and hemorrhage were seen in SP-A^−/− mice on Day 7 after bleomycin treatment (2.5 U/kg). Magnification ×100.

Figure 2.

Increased inflammatory phenotype in surfactant protein A–deficient (SP-A^−/−) mice after bleomycin (bleo) challenge. After saline or bleomycin treatment, bronchoalveolar lavage (BAL) fluid was collected from wild-type (WT) and SP-A^−/− mice on Day 7. (A) Total cell and (B) differential cell counts were performed. SP-A^−/− mice had a higher degree of neutrophil-dominant cell recruitment. MNC = mononuclear cells; PMN = polymorphonuclear neutrophils. n = 9–12 in each group. **P < 0.05. (C) The levels of KC, tumor necrosis factor (TNF)-α, and IL-1β protein in BAL fluid from WT and SP-A^−/− mice were measured by ELISA at the indicated time points after bleomycin challenge. n = 5 in each group. *P < 0.01; **P < 0.05.

Figure 3.

Increased high mobility group box (HMGB)1 protein expression in surfactant protein A–deficient (SP-A^−/−) mice after bleomycin challenge. After bleomycin challenge, bronchoalveolar lavage (BAL) was performed in wild-type (WT) and SP-A^−/− mice at the indicated time points. The levels of HMGB1 protein in BAL fluid were measured by ELISA. n = 5–14 in each group. **P < 0.05.

Figure 4.

Increased Evans blue dye leakage in the lungs of bleomycin-challenged surfactant protein A–deficient (SP-A^−/−) mice. Wild-type (WT) or SP-A^−/− mice were challenged with saline or bleomycin (bleo) intratracheally. On Day 7, Evans blue dye (50 mg/kg) was injected via the tail vein. Three hours after the injection, bronchoalveolar lavage (BAL) fluid and lung tissues were collected and the levels of Evans blue dye were assessed by measuring absorbance at 620 nm. (A) Representative pictures of lungs from the four treatment groups. The levels of Evans blue dye recovered in (B) BAL fluid and (C) lung tissue were increased in SP-A^−/− mice. n = 5 in each group. *P < 0.01; **P < 0.05.

Figure 5.

Analysis of cell injury/death after bleomycin challenge by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. TUNEL staining was performed with lung tissue sections from wild-type (WT) and surfactant protein A–deficient (SP-A^−/−) mice on Day 7 after bleomycin treatment. (A) Representative pictures are shown. Nuclei were counterstained with 4′-6-diamidino-2-phenylindole (DAPI). Magnification ×400. (B) TUNEL-positive cells were counted from 20 random fields per section, and TUNEL-positive index was calculated as indicated in Methods. Significant increase of TUNEL-positive cells was seen in SP-A^−/− mice in response to bleomycin. n = 5 in each group. *P < 0.01.

Figure 6.

Double staining of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and active caspase-3. TUNEL staining was performed in conjunction with intracellular staining for cleaved (active) caspase-3 using lung tissue sections from surfactant protein A–deficient (SP-A^−/−) mice on Day 7 after bleomycin treatment. Some active caspase-3 positive (apoptotic) cells are also TUNEL-positive. Nuclei were counterstained with 4′-6-diamidino-2-phenylindole (DAPI). Magnification ×400.

Figure 7.

Surfactant protein A (SP-A) prevents bleomycin-induced apoptosis in MLE12 cells. MLE12 cells were treated with bleomycin (bleo) with or without SP-A. The apoptotic cells were assessed by flow cytometric analysis with annexin V and propidium iodide (PI). Annexin V–positive, PI-negative (A+/P−) population was considered as indicative of early apoptotic cells in the dot plots, and annexin V-positive, PI-positive (A+/P+) population was considered as mid to late apoptotic cells. (A) Representative flow cytometric analysis for the treatment groups. (B) Percentages of A+/P− and A+/P+ population are shown. Data are average of three independent experiments. **P < 0.05.

Figure 8.

Exogenous surfactant protein A (SP-A) rescued bleomycin-induced lung injury in SP-A–deficient (SP-A^−/−) mice. (A) Wild-type (WT) (shaded bars) and SP-A^−/− (solid bars) mice were challenged with intratracheal saline or bleomycin (bleo), and exogenous SP-A (or saline as a control) was administrated intratracheally as indicated in Methods. On Day 7 after bleomycin challenge, total cell count in bronchoalveolar lavage (BAL) was counted. SP-A administration to SP-A^−/− mice reduced the number of BAL total cell count to the level of WT mice. SP-A administration did not affect saline-challenged mice. n = 8–9. **P < 0.05. (B) On Day 7 after bleomycin treatment, Evans blue dye leakage in BAL fluid was determined. SP-A treatment significantly reduced the Evans blue dye leakage in bleomycin-challenged SP-A^−/− mice, but not in WT mice. n = 8–10. *P < 0.01. (C) The mortality rate of SP-A–rescued SP-A^−/− mice (SP-A^−/− SP-A) was significantly lower than that of saline-rescued mice (SP-A^−/− Saline) in response to bleomycin challenge (P < 0.05). No significant difference in survival was seen in WT mice with SP-A rescue. n = 18–23 in each group.