Acute Lung Injury Edema Fluid Decreases Net Fluid Transport across Human Alveolar Epithelial Type II Cells

Abstract

Most patients with acute lung injury (ALI) have reduced alveolar fluid clearance that has been associated with higher mortality. Several mechanisms may contribute to the decrease in alveolar fluid clearance. In this study, we tested the hypothesis that pulmonary edema fluid from patients with ALI might reduce the expression of ion transport genes responsible for vectorial fluid transport in primary cultures of human alveolar epithelial type II cells. Following exposure to ALI pulmonary edema fluid, the gene copy number for the major sodium and chloride transport genes decreased. By Western blot analyses, protein levels of ␣ENaC, ␣1Na,K-ATPase, and cystic fibrosis transmembrane conductance regulator decreased as well. In contrast, the gene copy number for several inflammatory cytokines increased markedly. Functional studies demonstrated that net vectorial fluid transport was reduced for human alveolar type II cells exposed to ALI pulmonary edema fluid compared with plasma (0.02 ؎ 0.05 versus 1.31 ؎ 0.56 l/cm 2 /h, p < 0.02). An inhibitor of p38 MAPK phosphorylation (SB202190) partially reversed the effects of the edema fluid on net fluid transport as well as gene and protein expression of the main ion transporters. In summary, alveolar edema fluid from patients with ALI induced a significant reduction in sodium and chloride transport genes and proteins in human alveolar epithelial type II cells, effects that were associated with a decrease in net vectorial fluid transport across human alveolar type II cell monolayers. Impaired alveolar fluid clearance (AFC;2 i.e. the resolution of alveolar edema) is a common characteristic among patients with acute lung injury (ALI) and acute respiratory distress syndrome. The level of AFC impairment has significant prognostic value in determining morbidity and mortality (1, 2). Multiple clinically relevant experimental studies have tried to uncover the underlying mechanisms that reduce AFC in ALI, and several pathways have been implicated (3, 4).In the alveolar environment, basal AFC is determined predominately by amiloride-sensitive (epithelial sodium channel (ENaC)) and -insensitive sodium channels and the activity of the Na,K-ATPase (3, 5-8). Several stimuli can up-regulate AFC including ␤-adrenergic agonists via cAMP-dependent mechanisms (3, 4). In the mouse and human lung, cAMP-dependent alveolar epithelial fluid transport is dependent on CFTR activity, especially in mediating ␤-adrenergic receptor-driven alveolar epithelial fluid transport (9 -11).We and others have reported that, in the early phase of ALI, pulmonary edema fluid contains high levels of several proinflammatory cytokines, including IL-1␤, TNF␣,. Several of these proinflammatory cytokines have been studied in experimental fluid transport experiments. For example, during short in vitro exposures, TNF␣ increases AFC, which is mediated predominantly by both TNF␣ receptor-dependent and -independent effects (15, 16). In contrast, for exposures up to 24 h, TNF␣ decreases the expression of ENaC (␣, ␤, and ␥ subunit...

Allogeneic human mesenchymal stem cells for treatment of E. coli endotoxin-induced acute lung injury in the ex vivo perfused human lung

“…Cytomix reduced vectorial fluid transport across epithelial type II cells by 70% over 24 h, an effect that was not associated with an increase in apoptosis or necrosis of the type II cells, as previously measured by flow cytometry (33). The addition of human MSCs to the lower chamber restored the cytomix-induced decrease in net fluid transport through a cell contact independent mechanism.…”

“…To further test potential mechanisms of benefit of MSCs on alveolar fluid transport, we exposed primary cultures of human alveolar epithelial type II cells grown on a Transwell plate with an air-liquid interface to cytomix (33), a mixture of the most biologically active cytokines in ALI pulmonary edema fluid (34). Cytomix reduced vectorial fluid transport across epithelial type II cells by 70% over 24 h, an effect that was not associated with an increase in apoptosis or necrosis of the type II cells, as previously measured by flow cytometry (33).…”

“…To test one mechanism that could account for the therapeutic effect of KGF in these experiments, we used an in vitro model of alveolar epithelial human type II cells, as in our prior studies (33). The addition of cytomix, a mixture of the most biologically active proinflammatory cytokines present in ALI pulmonary edema fluid (IL-␤, TNF␣, and IFN␥) (34), reduced net fluid transport by 70% in primary cultures of human alveolar epithelial type II cells.…”

Human Mesenchymal Stem (Stromal) Cells Promote the Resolution of Acute Lung Injury in Part through Lipoxin A4

“…To injure the cells, we exposed the AT II cells and MSCs to a mixture of three different cytokines referred to as cytomix (IL-1b, TNF-a, and IFN-g, 50 ng/ml each; R&D Systems). We previously reported that these were the major proinflammatory cytokines in pulmonary edema fluid from patients with ARDS (20). Experiments were carried out in eight different groups …”

“…Freshly isolated human AT II cells were plated on collagen I-coated Transwells for 96 h in culture medium containing 10% FBS and an additional 24 h in serum-free medium (19,20). Then, 250,000 MSCs were added to the lower compartment.…”