Malondialdehyde-acetaldehyde-adducted protein inhalation causes lung injury

Abstract

In addition to cigarette smoking, alcohol exposure is also associated with increased lung infections and decreased mucociliary clearance. However, little research has been conducted on the combination effects of alcohol and cigarette smoke on lungs. Previously, we have demonstrated in a mouse model that the combination of cigarette smoke and alcohol exposure results in the formation of a very stable hybrid malondialdehyde-acetaldehyde (MAA)-adducted protein in the lung. In in vitro studies, MAA-adducted protein stimulates bronchial epithelial cell interleukin-8 (IL-8) via the activation of protein kinase C epsilon (PKCɛ). We hypothesized that direct MAA-adducted protein exposure in the lungs would mimic such a combination of smoke and alcohol exposure leading to airway inflammation. To test this hypothesis, C57BL/6J female mice were intranasally instilled with either saline, 30μL of 50μg/mL bovine serum albumin (BSA)-MAA, or unadducted BSA for up to 3 weeks. Likewise, human lung surfactant proteins A and D (SPA and SPD) were purified from human pulmonary proteinosis lung lavage fluid and successfully MAA-adducted in vitro. Similar to BSA-MAA, SPD-MAA was instilled into mouse lungs. Lungs were necropsied and assayed for histopathology, PKCɛ activation, and lung lavage chemokines. In control mice instilled with saline, normal lungs had few inflammatory cells. No significant effects were observed in unadducted BSA- or SPD-instilled mice. However, when mice were instilled with BSA-MAA or SPD-MAA for 3 weeks, a significant peribronchiolar localization of inflammatory cells was observed. Both BSA-MAA and SPD-MAA stimulated increased lung lavage neutrophils and caused a significant elevation in the chemokine, keratinocyte chemokine, which is a functional homologue to human IL-8. Likewise, MAA-adducted protein stimulated the activation of airway and lung slice PKCɛ. These data support that the MAA-adducted protein induces a proinflammatory response in the lungs and that the lung surfactant protein is a biologically relevant target for malondialdehyde and acetaldehyde adduction. These data further implicate MAA-adduct formation as a potential mechanism for smoke- and alcohol-induced lung injury.

Figure 1

Formation of MAA-adducted proteins. Two moles of malondialdehyde react with 1 mole of acetaldehyde to form a stable hybrid malondialdehyde-acetaldehyde protein adduct. The linear adduct is unstable and is rapidly degraded.

Figure 2

MAA adduction of human lung surfactant protein in vitro. Human surfactant protein D (SPD) and surfactant protein A (SPA) were purified from whole lung lavage fluid and chemically adducted with malondialdehyde and acetaldehyde as described in Methods. Purified SPD and SPA form hybrid aldehyde adducts similar to MAA-adducted bovine serum albumin (BSA).

Figure 3

In vitro stimulation of mouse lung by SPD-MAA adducted protein. Lung slices were treated with SPD or SPD-MAA in the presence or absence of Rö 31-8220 for 24 hr and media supernatants assayed for keratinocyte chemokine (KC). The significant (P<0.01) increase in KC release in response to SPD-MAA was inhibited by a 1 hr pretreatment with Rö 31-8220.

Figure 4

Nasal instillation of MAA-adducted protein causes lung inflammation in mice. Mice instilled with BSA-MAA (50 µg/mL) exhibited enhanced bronchoalveolar lavage (BAL) inflammatory cells consisting primarily of neutrophils. The total number of lung lavage cells significantly increased (P<0.01) with BSA-MAA instillation. No inflammation was observed in mice instilled with the sterile PBS vehicle or with non-adducted BSA.

Figure 5

Bronchoalveolar lavage (BAL) of mice instilled with SPD-MAA. Mice instilled with SPD-MAA (50 µg/mL) exhibited enhanced lung lavage inflammatory cells consisting primarily of neutrophils. The total number of lung lavage cells significantly increased (P<0.001) with SPD-MAA instillation. No inflammation was observed in mice instilled with the sterile PBS vehicle (control) or with non-adducted SPD.

Figure 6

MAA adducts activate tracheal epithelial cell PKC epsilon in vivo. Mice were nasally instilled with 50 µg/mL BSA-MAA or surfactant protein D- (SDP) MAA and tracheal epithelial PKCε activity assayed. Both BSA-MAA and SPD-MAA stimulated PKCε activity compared to control PBS or BSA (50 µg/ml), which had no effect (*P<0.05 vs. no instillation control).

Figure 7

Nasal instillation of SPD-MAA causes lung inflammation in mice. A. Mice instilled with SPD-MAA (50 µg/mL) demonstrated a peribronchiolar inflammation consisting primarily of neutrophils (10× magnification) on H&E stained lung sections. B. Inflammatory score of lungs and BAL KC levels after intranasal inhalation of sterile PBS, SPD, and SPD-MAA (50 µg/mL; 50 µL) after 3 weeks of daily intranasal inhalation exposure in mice (N=6 mice per group). Error bars are SE. *P<0.001 is statistically significant of SPD-MAA versus PBS-treated mice.

Figure 7

Nasal instillation of SPD-MAA causes lung inflammation in mice. A. Mice instilled with SPD-MAA (50 µg/mL) demonstrated a peribronchiolar inflammation consisting primarily of neutrophils (10× magnification) on H&E stained lung sections. B. Inflammatory score of lungs and BAL KC levels after intranasal inhalation of sterile PBS, SPD, and SPD-MAA (50 µg/mL; 50 µL) after 3 weeks of daily intranasal inhalation exposure in mice (N=6 mice per group). Error bars are SE. *P<0.001 is statistically significant of SPD-MAA versus PBS-treated mice.