Constitutive activation of prosurvival signaling in alveolar mesenchymal cells isolated from patients with nonresolving acute respiratory distress syndrome

Abstract

Acute respiratory distress syndrome (ARDS) is a clinical syndrome characterized by stereotypic host inflammatory and repair cellular responses; however, mechanisms regulating the resolution of ARDS are poorly understood. Here, we report the isolation and characterization of a novel population of mesenchymal cells from the alveolar space of ARDS patients via fiber-optic bronchoscopy with bronchoalveolar lavage (BAL). BAL was performed on 17 patients during the course of ARDS. Immunofluorescence staining and multiparameter flow cytometric analysis defined a population of alveolar mesenchymal cells (AMCs) that are CD45-/prolyl-4-hydroxylase+/alpha-smooth muscle actin+/-. AMCs proliferated in ex vivo cell culture for multiple passages; early passage (3-5) cells were subsequently analyzed in 13 patients. AMCs isolated from patients with persistent or nonresolving ARDS (ARDS-NR, n = 4) demonstrate enhanced constitutive activation of prosurvival signaling pathways involving PKB/Akt, FKHR, and BCL-2 family proteins compared with AMCs from patients with resolving ARDS (ARDS-R, n = 9). Exogenous transforming growth factor-beta1 markedly induces PKB/Akt activation in AMCs from ARDS-R. ARDS-NR cells are more resistant to serum deprivation-induced apoptosis compared with ARDS-R. This study identifies a novel population of mesenchymal cells that can be isolated from the alveolar spaces of ARDS patients. AMCs in patients with ARDS-NR acquire an activational profile characterized by enhanced prosurvival signaling and an antiapoptotic phenotype. These findings support the concept that apoptosis of mesenchymal cells may be an essential component of normal repair and resolution of ARDS and suggest that dysregulation of this process may contribute to persistent ARDS.

Fig. 1

Morphological and phenotypic characterization of cultured alveolar mesenchymal cells (AMCs) from patients with acute respiratory distress syndrome (ARDS). A: morphological appearance (×20 magnification) of adherent cells recovered by bronchoalveolar lavage (BAL) and plated on tissue culture plastic within 24 h from a normal volunteer (NV, left) and from a representative patient with ARDS on day 7 postdiagnosis (middle). Subsequent trypsinization and passage in culture of the cells from ARDS patients produced homogeneous cultures of spindle-shaped cells (right). B: CD45 staining of BAL cells that were processed as described in A. C: AMC cultures at passage 3 were stained for the collagen cross-linking enzyme, prolyl-4-hydroxylase (PH, left) and for α-smooth muscle actin (α-SMA, right). D: AMC cultures at passage 3 were growth-arrested for 24 h. Whole cell lysates were obtained and assessed by Western immunoblotting for vimentin and α-tubulin. E: the Western blot in D was stripped and probed sequentially for cellular fibronectin and α-tubulin. The bottom panels demonstrate the band densitometry, relative to controls, for all AMC cell lines analyzed. ARDS-R, resolving ARDS; ARDS-NR, nonresolving ARDS; C, control; NS, not significant.

Fig. 2

Serial morphological and flow cytometric analyses of freshly isolated BAL cells from patients during early and late ARDS. Freshly isolated BAL cells from 2 representative ARDS patients were analyzed immediately (without plating) by multiparameter flow cytometry with similar results. BAL cells were stained for expression of CD45 and PH on day 1 postdiagnosis (left), repeat bronchoscopy was performed, and BAL cells were reanalyzed on the same patient 4 days later (day 5, right). Top: forward (FSC) vs. side scatter (SSC) gates used to determine expression of CD45 and PH. Middle: CD45 expression vs. FSC. Essentially all BAL cells expressed the common leukocyte antigen, CD45, on day 1 (left). BAL cells from the same patient 4 days later contained a significant number of CD45⁻ cells (right). Bottom: PH expression on CD45⁻ gated BAL cells. Immunophenotyping of BAL cells are described in materials and methods.

Fig. 3

Constitutive activation of the antiapoptotic protein kinase, PKB/Akt, in AMCs from patients with ARDS-NR. A: AMCs isolated from 13 patients with ARDS were grown to 80% confluence in media containing 10% FBS. Whole cell lysates were collected and analyzed with SDS-polyacrylamide gel electrophoresis and immunoblotting with an antibody against phosphorylated (Ser⁴⁷³) PKB/Akt. The blot was then stripped and probed for total Akt. Representative samples from the first 9 patients studied are shown. Lysates from untreated normal human lung fibroblasts that were cultured under identical conditions were used for normalization (N: normal). B: densitometric analysis was done of the Western immunoblots from all 13 ARDS patients. The density of each band was measured and compared with that from untreated normal (control) human lung fibroblasts. Fold increases over control for each patient with ARDS-R and ARDS-NR are shown. P = 0.003 for ARDS-R vs. ARDS-NR.

Fig. 4

Exogenous transforming growth factor (TGF)-α1 markedly upregulates PKB/Akt activation in AMCs from patients with ARDS-R. A: AMCs isolated from patients with ARDS-R (n = 5) and ARDS-NR (n = 3) were isolated and grown to 80% confluence as described in materials and methods. Cells were then growth-arrested in media containing 0.01% FBS for 24 h and treated with or without TGF-β1 (2 ng/ml) for 16 h. Whole cell lysates were subjected to Western immunoblotting with a phospho-specific antibody to phosphorylated (Ser⁴⁷³) PKB/Akt. The blot was then stripped and probed for total Akt. A representative blot is shown. B: band densities from the Western blots in A were measured using NIH imaging software, and the background density was subtracted. The band densities for the induction of phospho-Akt by TGF-β1 in ARDS-R and ARDS-NR are shown. Statistical analysis was done by a 1-way ANOVA with Bonferroni posttest. *P < 0.001 for TGF-β1 induction compared with the respective baseline/constitutive levels of phosphorylated (active) Akt. **P < 0.001 for baseline/constitutive level of phosphorylated (active) Akt in ARDS-NR vs. ARDS-NR. C: for each set of AMCs from ARDS-R and ARDS-NR, band densities of TGF-β1-induced Akt phos-phorylation were divided by the band densities of the respective untreated/basal values to determine the fold induction with TGF-β1. P = 0.035 for ARDS-R vs. ARDS-NR.

Fig. 5

Activation of PKB/Akt-dependent antiapoptotic signaling pathways in AMCs from patients with ARDS-NR. A: mesenchymal cells were isolated from 13 patients with ARDS and cultured as described in materials and methods. Whole cell lysates were obtained and analyzed by Western immunoblotting for phosphorylation (S²⁵⁶) of the forkhead family transcription factor FKHR. The blot was then stripped and probed for α-tubulin. Representative samples from normal, unstimulated fibroblasts and AMCs from patients with ARDS-R and ARDS-NR are shown. B: densitometric analysis of the Western blots described in A on all 13 AMC cell lines were compared with normal controls (on the same blot). The percent change in density over controls was plotted for each of the patients with ARDS-R and ARDS-NR. P = 0.01 for ARDS-R vs. ARDS-NR. C: relative fold-increase in phospho-Akt for each AMC cell line (from Fig. 3B) was plotted against the change in phospho-FKHR (from Fig. 4A) and assessed for correlation by regression analysis (r² = 0.88; P < 0.05). D: AMCs from ARDS-R and ARDS-NR patients and control fibroblasts were analyzed by Western immunoblotting for BCL-2 and then stripped and probed for BAX. A representative blot is shown. E: densitometric ratios for BCL-2/BAX were determined for ARDS-R fibroblasts (n = 9), for ARDS-NR (n = 4), and for normal control fibroblasts (n = 3). P < 0.01 for ARDS-NR vs. ARDS-R using ANOVA by Bonferroni’s posttest analysis.

Fig. 6

Susceptibility to serum deprivation-induced apoptosis in AMCs from ARDS-R vs. ARDS-NR. AMCs isolated from all 4 patients with ARDS-NR and 4 patients with ARDS-R were plated at an equal density in a 96-well plate. Cells were cultured in DMEM with 10% FBS for 4 days, and the medium was changed to DMEM with 0.1% FBS. Cells were cultured under serum-deprivation conditions for 6 days, and apoptosis was assessed with an ELISA for single-stranded (ss) DNA as described in materials and methods. P = 0.028 by nonparametric Mann-Whitney test; n = 4 for each AMC cell line. The experiment was repeated 3 times with similar results.