In this study, we demonstrate that exposure to MAA-adducted proteins increases lung inflammation in an in vivo
mouse exposure model using direct nasal instillation. As evidenced by histology and bronchoalveolar lavage cell differentiation, this elevated inflammation primarily consisted of a peribronchiolar infiltration of neutrophils in response to nasal instillation with MAA-adducted protein. Mechanistically, this neutrophil recruitment was preceded by MAA-adducted protein stimulation of both epithelial cell PKCε activity and chemokine release into the lung. Ex vivo
examination of mouse lung slices exposed to MAA-adducted protein demonstrated that PKCε activation was required for this chemokine release. Similar results were observed irrespective of whether the nasally instilled protein was a native lung surfactant protein or a non-native lung protein such as BSA. These results suggested that the biological response is to the MAA moiety and not to the protein that is adducted. Furthermore, we demonstrate that lung surfactant proteins such as SPA and SPD, which are native to the lung airspaces, can be as effectively MAA-adducted as the previously established and well-characterized BSA-MAA (Wyatt et al., 2001
; Xu et al., 1997
). Because no significant difference in MAA adduction was detected between BSA and SPD, we chose to focus on using SPD, and not SPA, for our mouse instillation studies.
Equivalent adduction of a lung surfactant to that of BSA has important ramifications for the lung inflammation observed in response to SPD-MAA instillation. Previously, we demonstrated in submerged monolayers of bronchial epithelial cells that BSA-MAA rapidly activates PKC and results in the stimulated release of the neutrophil chemokine, IL-8 (Wyatt et al., 2001
). MAA-stimulated chemokine release was blocked by inhibition of PKCα and by pan-isoform inhibitors of PKC. Since those early studies, we have identified a sequential regulation of cytokine release in airway epithelial cells whereby PKCα activation precedes and is required for subsequent PKCε activation (Wyatt et al., 2010
). This pathway was confirmed using dominant-negative PKC isoform cell lines, thus avoiding pharmacologic inhibitor specificity concerns. In our current studies using both in vitro
mouse lung slices and in vivo
nasal instillation, both BSA-MAA and SPD-MAA significantly elevated PKCε activity and led to release of the mouse neutrophil chemokines, KC and MIP-2, functional homologues to human IL-8. Likewise, a significant increase in neutrophils was observed in the BALF and the peribronchiolar areas of the lung. Surfactant protein adduction may also impact subsequent lung susceptibility to infection as the availability of surfactant protein for innate anti-microbial defense could be impacted by MAA adduction. Indeed, chronic alcohol exposure in animal models has already demonstrated decreased levels of surfactant (Lazic et al., 2007
; Sozo et al., 2009
). These data strongly indicate that the identity of the adducted protein may be less important than the formation of the stable hybrid aldehyde adduct regardless of the protein it is covalently bound to in terms of chemokine regulation and inflammatory cell recruitment.
The in vivo
formation of lung MAA-adducted proteins represents an important consideration for lung pathophysiology beyond the establishment that surfactant proteins can be targets for MAA adduction and that lung exposure to SPD-MAA results in lung inflammation. In our early cellular studies, we observed that very high concentrations (1 mM) of both acetaldehyde and malondialdehyde are required for 2–5 days to produce PKC activation (Wyatt et al., 2001
). These conditions parallel the in vitro
conditions for MAA-adducting lysine-rich proteins (Tuma et al., 2001
). Thus, to achieve MAA adduction of native proteins in the lung, the airways would have to be exposed in vivo
to chronically high levels of both acetaldehyde and malondialdehyde. To model this, we co-exposed mice to both alcohol and cigarette smoke for up to 6 wk and demonstrated that the levels of acetaldehyde and malondialdehyde required for the formation of MAA-adducted protein were only reached under co-exposure conditions (McCaskill et al., 2011
). Importantly, MAA-adducted proteins were only detected in the lungs of the smoke and alcohol co-exposed mice. A dominant MAA-adducted protein identified by smoke and alcohol co-exposure in this study was SPD (McCaskill et al., 2011
). While we demonstrated that airway PKCε activity was elevated under conditions of SPD-MAA formation in the cigarette smoke and alcohol co-exposure model, the functional effects of such MAA formation were not examined. Here, we provide evidence for the first time that directly instilling SPD-MAA into a mouse lung can mimic the type of lung neutrophilia observed in previously reported studies of chronic cigarette smoke and alcohol exposed mice (Elliott et al., 2007
The intracellular signaling pathway that links the formed SPD-MAA in the lung to PKC-stimulated chemokine release and subsequent neutrophil recruitment remains to be defined. MAA-adducted proteins bind to cells through membrane scavenger receptors (Terpstra et al., 2000
; Duryee et al., 2005
). Previously, we have shown that airway epithelial cells express several classes of scavenger receptors (Wyatt et al., 2005
) including scavenger receptor A (SRA) (Bowdish and Gordon, 2009
), also known as macrophage scavenger receptor I (MSR-I). A specific ligand for SRA, fucoidan, was capable of competing with and blocking BSA-MAA from binding to SRA. Similar to MAA-adducted protein, fucoidan treatment also resulted in the activation of PKC, suggesting that SRA ligand binding is one pathway to PKC activation in airway epithelium. Importantly, this study showed that migration of epithelial cells into a wound was delayed under conditions of exposure to BSA-MAA or fucoidan (Wyatt et al., 2005
). Thus, the impact of MAA-adducted proteins on airway wound repair may represent another important pathway for chronic inflammatory lung disease/remodeling in addition to the effect of MAA adducts on neutrophil recruitment.
Chronic inflammatory lung diseases now represent the third leading cause of death in the United States affecting over 25 million adults (Mathews et al., 2011
). While the vast majority of chronic lung disease is due to cigarette smoking, little has been studied with regard to alcohol and chronic inflammatory lung disease. Because clinical observations have established that alcohol use disorders (AUD) are associated with the co-morbidity of cigarette smoking, the lung of a smoker who drinks would represent the target organ ideally capable of creating an environment of high malondialdehyde and acetaldehyde levels required for the formation of MAA-adducted protein. However, individual differences in the expression of phase I metabolizing enzymes such as aldehyde dehydrogenases could impact the chronic formation of MAA-adducted lung protein. Likewise, potential differences in the expression of lung epithelial cell scavenger receptors could alter responses to MAA adduct-induced lung injury. Indeed, genetic polymorphisms in SR-A expression have been linked to chronic obstructive pulmonary disease (Hersh et al., 2006
; Ohar et al., 2010
). Future studies translating preclinical mouse data on MAA adduct-induced lung injury to individuals with AUD will be necessary to define a role for MAA adducts in alcohol and cigarette smoke-induced chronic lung disease.