TGF-beta is a critical mediator of acute lung injury

Abstract

We have shown that the integrin alphavbeta6 activates latent TGF-beta in the lungs and skin. We show here that mice lacking this integrin are completely protected from pulmonary edema in a model of bleomycin-induced acute lung injury (ALI). Pharmacologic inhibition of TGF-beta also protected wild-type mice from pulmonary edema induced by bleomycin or Escherichia coli endotoxin. TGF-beta directly increased alveolar epithelial permeability in vitro by a mechanism that involved depletion of intracellular glutathione. These data suggest that integrin-mediated local activation of TGF-beta is critical to the development of pulmonary edema in ALI and that blocking TGF-beta or its activation could be effective treatments for this currently untreatable disorder.

Figure 1

β6^–/– mice are protected against bleomycin-induced pulmonary edema. Lung permeability to protein (L/P, lung/plasma ratio) (a) and extravascular water content of the lung (W/D, wet/dry ratio) (b) in wild-type (β6^+/+) and β6^–/– mice 5 days after treatment with intratracheal saline or bleomycin. Data (means ± SEM) are representative of at least two comparable experiments with five mice per group; ^AP < 0.05 relative to saline-treated mice and bleomycin treated β6^–/– mice.

Figure 2

Effect of airspace instillation of bleomycin on differential cell count and macrophage expression of MHC II in BAL fluid in β6^+/+ and β6^–/– mice. Means (±SEM) of differential cell count obtained from BAL of each of six mice in both experimental groups. Values for MHC II expression on macrophages were obtained by flow cytometry.

Figure 3

Soluble chimeric TGF-β type II receptor inhibits TGF-β1 activity in β6-expressing cells and protects β6^+/+ mice against bleomycin-induced pulmonary edema. TGF-β activity is measured as relative luminescence of TMLC cells stably expressing a portion of the plasminogen activator inhibitor-1 promoter (12) in response to exogenous rhTGF-β1 (a) or β6-transfected SW480 cells (b) in the presence of increasing concentrations of TGF-β type II receptor–IgG-Fc chimera. Means (±SEM) of three experiments done in duplicate are shown. (c) Lung protein permeability in wild-type mice treated with saline or bleomycin and given either TGF-β type II receptor chimera or saline every 48 hours beginning either just before (pretreatment) or 24 hours after (treatment) intratracheal instillation. Data (means ± SEM) are representative of at least two comparable experiments with five mice per group; ^AP < 0.05 relative to saline-treated mice; ^BP < 0.05 relative to bleomycin-treated mice that received vehicle.

Figure 4

Lung histology. Low-power photomicrographs of lung sections stained with hematoxylin and eosin. (a) β6^–/– mouse instilled with PBS shows a normal lung morphology. (b) β6^–/– mouse instilled with bleomycin does not show any red blood cell leak or accumulation of proteinaceous material into its airspaces. (c and d) β6^+/+ mice instilled with PBS and injected either with the soluble chimeric TGF-β type II receptor (c) or its vehicle (d) have a normal lung morphology. (e) Accumulation of red blood cells and proteinaceous material in the alveolar spaces of a β6^+/+ mouse instilled with bleomycin and injected with the vehicle for the soluble chimeric TGF-β type II receptor. (f) Absence of red blood cells in the airspaces of a β6^+/+ mouse instilled with bleomycin and pretreated with the soluble chimeric TGF-β type II receptor.

Figure 5

Soluble chimeric TGF-β type II receptor protects β6^+/+ mice against E. coli endotoxin-induced pulmonary edema. Lung protein permeability in wild-type mice treated with saline or E. coli endotoxin and given either TGF-β type II receptor chimera or saline just before intratracheal instillation. Data (means ± SEM) are representative of two comparable experiments with five mice per group; ^AP < 0.05 relative to saline-treated mice; ^BP < 0.05 relative to bleomycin-treated mice that received vehicle.

Figure 6

rhTGF-β1 decreases the TER across primary cultures of rat ATII cell monolayers by decreasing their intracellular content of reduced GSH. Transepithelial resistance of rat ATII monolayers treated with rhTGF-β1 alone (a) or in the presence of either anti–TGF-β Ab (b) or exogenous GSH (c). (d) Effects of rhTGF-β1 on ATII cell content of GSH and GSSG. (e) Effects of inhibitors of the myosin light chain kinase on the TGF-β–induced decrease in TER. Means (±SEM) of three experiments done in duplicate are shown for each panel. For all experiments, ^AP < 0.05 relative to vehicle-treated ATII cell monolayers.