Background and Overview
Article Title/Citation: Alveolar fibroblasts in acute lung injury: biological behaviour and clinical relevance.
To determine the biological behavior of fibroblasts found in the alveolus during acute lung injury
To evaluate the clinical relevance of positive alveolar fibroblast cultured from patients with acute lung injury (ALI)/acute respiratory distress syndrome (ARDS)
Classically, the pathologic features of acute lung injury have been described as occurring in 3 interrelated phases: an exudative phase, characterized by pulmonary edema and hemorrhage, a proliferative phase of organization and attempts at repair, and, in some patients, a fibrotic phase in which normal lung architecture is not restored and the risk of death is increased.1 Normal wound healing after ALI/ARDS is believed to require the participation of fibroblasts and myofibroblasts to initiate repair. Resolution of ALI/ARDS requires replacement of the fibroblasts and myofibroblasts by normal components of the lung alveolar/capillary unit. Why normal repair occurs after ALI/ARDS in some patients, yet in others, fibrogenesis progresses without restoration of normal lung tissue, is as yet unknown. Since it is difficult to obtain cells that participate in the process of injury and repair in ALI/ARDS patients directly from lung tissue, the authors attempted to isolate fibroblasts obtained by bronchoalveolar lavage (BAL). It is hoped that isolation and characterization of fibroblasts from the alveolar space could shed light as to their biologic activity and role in normal or fibrotic repair in ALI/ARDS.
Funding source of study
This work was supported by the European Commission (Framework Programme 7, European IPF Network) and by the Agence Nationale dela Recherche (ANR-06-Physio, Fibroblastes et fibrogenese pulmonaire). C. Quesnel, B. Crestani and M. Dehoux were supported, in part, by grants from Contrat d’initiation a` la Recherche Clinique de l’Assistnace Publique-Hopitaux de Paris CRC05105. C. Quesnel also received funding from the Societe´ de Reanimation de Langue Franc¸aise and theSociete´ de Pneumologie de Langue Francaise. P. Soler was supported by a contrat d’interface INSERM-AP-HP.
Study design and methodology:
Bronchoscopy with bronchoalveolar lavage (BAL) was performed and resultant cells and fluid was processed:
Alveolar permeability was estimated by calculating the ratio of BAL fluid protein to serum protein.
Cells were cultured for at least 28 days.
Cells grown from BAL were used for immunocytochemical characterization 24 hours and 21 days after initial plating and then at passage 1 and 3.
BAL cell lines were randomly chosen and their proliferation, migration and collagen1 production capacities were compared to fibroblasts derived from normal explanted lung tissue.
C-terminal propeptide of type I procollagen (PICP) was measured by enzyme immunoassay. Transforming growth factor (TGF-β1), monocyte chemoattractant protein-1 (MCP-1) and IL-8 concentrations were measured by ELISA.
Alveolar fibroblasts cultured on Lab-Tek slides were fixed with acetone. Anti-human antibodies (Ab) directed against collagen 1, vimentin, prolyl-4-hydroxylase (α-4H), desmin, pancytokeratin, CD31, alpha-smooth muscle actin (α-SMA), CD45, smooth muscle myosin 1 CD14, CD34 or isotype-matched control Ab were used.
Cells cultured on slides were fixed after 21 days of culture and were analyzed by immunofluorescence for collagen 1 and CD45 by confocal microscopy.
After 72h of incubation with 0.1% FCS alone (basal condition) or with PGE, proliferation was measured as bromodeoxyuridine (BrdU) incorporation.
Patient selection and enrollment: Mechanically ventilated patients in three intensive care units (ICU) were prospectively enrolled if BAL was to be performed for a diagnosis of ventilator-associated pneumonia (VAP). The patients were then classified into one of three groups: acute lung injury (ALI), acute respiratory distress syndrome (ARDS) or ventilated without ALI/ARDS, which acted as the control group.
Outcomes/endpoints being measured: Patients were followed for 28 days and clinical data were recorded, including duration of ventilation after bronchoscopy, length of ICU stay and survival. Alveolar fibroblasts were identified from BAL fluid and studied for proliferation, migration, and collagen-1 synthesis. The relationship between clinical outcomes and alveolar fibroblast characteristics was explored.
Demographic data were expressed as mean +SD and biological data as median (range).
All proportional values were compared with the Fisher’s exact test or the Chi-squared test for multiple comparisons.
The continuous variables were compared by the Mann–Whitney test or Wilcoxon paired test when appropriate.
Multiple comparisons were tested by the Kruskal–Wallis or Friedman test followed by Dunn’s multiple comparison post hoc analysis.
Durations of ventilation after the BAL procedure were analyzed by Kaplan–Meier survival curves and compared by log-rank analysis.
Correlations were assessed with the Spearman rank-order test.
Statistical significance was accepted as p<0.05.
Enrollment & baseline characteristics:
Subjects enrolled included 17 ALI patients, 31 ARDS patients and 20 ventilated patients that did not have ALI or ARDS who served as controls.
The groups were not significantly different with regard to age, percent with pulmonary infection, length of ICU stay, time on mechanical ventilation before BAL, total time on mechanical ventilation.
There was a significant difference in survival, with 35% of ALI patients, and 39% of ARDS patients dead by 28 days after onset, compared with 10% of control subjects.
Summary of primary & secondary outcomes
In the BAL fluid of ALI/ARDS patients, a population of cells expressing CD45+, prolyl-4-hydroxylase and weakly CD34+ (consistent with fibrocytes) were observed in the first 24 hours after plating, though they formed less than 1% of the total adherent cell population.
Alveolar fibroblasts, defined as cells that expressed mesenchymal markers (vimentin, collagen 1, and propyl-4-hydroxylase) were cultured in 12 of 68 BAL cell pellets (6 from ALI patients, 6 from ARDS patients and none from control patients) and were usually detected after the 1st week in culture
The migration of alveolar fibroblasts was heightened at basal conditions when compared to control fibroblasts but were hyporesponsive to the inhibitory effects of PGE2.
Collagen 1 mRNA and protein expression was higher in alveolar fibroblasts compared with control fibroblasts.
Patients were divided into 2 groups according to whether or not alveolar epithelial cells were identified in BAL fluid
- No difference in PaO2/FIO2 ratio, etiology of ALI/ARDS, length of ICU stay and 28 day mortality
- However, there was a significant decrease in total days on mechanical ventilation in patients with a positive culture for alveolar fibroblasts
Author’s Discussion and Conclusions
Brief summary of author’s main points:
A small number of fibrocytes (cells co-expressing collagen 1 and the leukocyte marker CD45) were detectable in the BAL cell pellet in the first few days in culture
Alveolar fibroblasts were cultured from 25% of BAL fluid samples from ALI/ARDS patients
Alveolar fibroblasts from ALI/ARDS patients exhibited a persistently active phenotype with enhanced migratory activity and collagen 1 expression
The presence of alveolar fibroblasts in BAL fluid was associated with a shorter duration of mechanical ventilatory support
Author’s conclusions: Activated alveolar fibroblasts can be found in some patients with ALI/ARDS and may reflect the organizing phase of ALI.
Reviewer’s Discussion and Conclusions
The authors offer a method for future study of alveolar fibroblasts, which may be important in the resolution of ALI/ARDS.
They describe an activated fibroblast phenotype and found it to be associated with less time on mechanical ventilation, suggesting accelerated lung repair
Formerly, the presence of mesenchymal cells in ALI/ARDS patients was felt to be worrisome for progressive fibrogenesis, not necessarily indicative of normal repair
Study limits and weakness:
The authors studied patients who were already planned to undergo BAL for suspected ventilator-associated pneumonia, suggesting some recent deterioration in their clinical status. It is possible that such patient s do not reflect the same pathophysiology of patients who are not suspected as having pneumonia.
Some patients did have pneumonia diagnosed by the results of the BAL procedure. Data from these patients was not shown separately. Results from patients with pneumonia may have reflected a response to an acute infection rather ALI/ARDS alone.
It is unclear how well fibroblasts cultured from the alveolar space reflect fibroblast activity within the lung parenchyma. Although difficult to obtain, it would be useful to have BAL data from at least a few ALI/ARDS patients who also had a lung tissue available for analysis to confirm this is an appropriate model.
Another study of patients with non-resolving ALI/ARDS, found that mesenchymal cells in the BAL fluid had alterations in pro-survival/anti-apoptotic signaling,2 suggesting that the characteristics of the cells may be important in whether repair proceeds normally or progresses to fibrosis.Applicability & impact on healthcare providers: It would highly advantageous to have a treatment available for ALI/ARDS patients that would accelerate repair. If alveolar fibroblasts are found to be important components of the lung tissue recovery process following ALI/ARDS, a better understanding of their role could lead to treatments that enhance recovery.
Conclusions and recommendations: Normal repair of lung injury may involve the participation of alveolar fibroblasts. A better understanding of the role these cells play in resolution of ALI/ARDS may be useful in tailoring treatment for patients with this devastating disorder.
- Ware, L.B., Matthay, M.A. The Acute Respiratory Distress Syndrome. N Engl J Med: 342, 1334-1349, 2000.
- Horowitz, J.C., Cui, Z., Moore,T.A. et al. Constitutive activation of prosurvival signaling in alveolar mesenchymal cells isolated from patients with nonresolving acute respiratory distress syndrome. Am J Physiol Lung Cell Mol Physiol: 290, L415-L425, 2006
Ngozika A. Orjioke, M.D.
Janice M. Liebler, M.D.
Pulmonary and Critical Medicine Division
University of Southern California
Los Angeles, CA