Mesenchymal stromal cells in bronchoalveolar lavage as predictors of bronchiolitis obliterans syndrome

Abstract

Rationale: Bronchoalveolar lavage fluid (BAL) from human lung allografts demonstrates the presence of a multipotent mesenchymal stromal cell population. However, the clinical relevance of this novel cellular component of BAL and its association with bronchiolitis obliterans syndrome (BOS), a disease marked by progressive airflow limitation secondary to fibrotic obliteration of the small airways, remains to be determined.

Objectives: In this study we investigate the association of number of mesenchymal stromal cells in BAL with development of BOS in human lung transplant recipients.

Methods: Mesenchymal colony-forming units (CFUs) were quantitated in a cohort of 405 BAL samples obtained from 162 lung transplant recipients. Poisson generalized estimating equations were used to determine the predictors of BAL mesenchymal CFU count.

Measurements and main results: Higher CFU counts were noted early post-transplantation; time from transplant to BAL of greater than 3 months predicted 0.4-fold lower CFU counts (P = 0.0001). BOS diagnosis less than or equal to 365 days before BAL was associated with a 2.11-fold higher CFU count (P = 0.02). There were 2.62- and 2.70-fold higher CFU counts noted in the presence of histologic diagnosis of bronchiolitis obliterans (P = 0.05) and organizing pneumonia (0.0003), respectively. In BAL samples obtained from BOS-free patients greater than 6 months post-transplantation (n = 173), higher mesenchymal CFU counts (≥10) significantly predicted BOS onset in both univariate (hazard ratio, 5.61; 95% CI, 3.03-10.38; P < 0.0001) and multivariate (hazard ratio, 5.02; 95% CI, 2.40-10.51; P < 0.0001) Cox regression analysis.

Conclusions: Measurement of mesenchymal CFUs in the BAL provides predictive information regarding future BOS onset.

Figure 1.

CONSORT diagram illustrating the selection of patients included in the data analysis. BAL = bronchoalveolar lavage; GEE = generalized estimating equation; BOS = bronchiolitis obliterans syndrome.

Figure 2.

Flow cytometric analysis of cell surface markers on culture-expanded mesenchymal CFUs. Bronchoalveolar lavage (BAL) CFUs from lung transplant recipients (control, acute rejection [AR], and bronchiolitis obliterans syndrome [BOS]) were expanded in culture and immunostained for CD44, CD90, CD73, and CD105 surface markers with specific mAbs. Cells from the three groups were found to be predominantly positive for the surface markers studied. All histograms demonstrate specific mAbs in black and control isotype-specific IgGs in gray. The percentage of positive cells relative to the total number of cells analyzed (mean ± SD) is shown above the respective histograms. n = 5 individual patients in each group.

Figure 3.

Multilineage differentiation potential of culture-expanded bronchoalveolar lavage (BAL) mesenchymal CFUs. (A) Cell lines generated from BAL CFUs of control, acute rejection (AR), or bronchiolitis obliterans syndrome (BOS) patients (n = 5 in each group) were investigated for the in vitro multilineage differentiation capacity. Accumulation of lipid droplets (indicating adipocytic differentiation) was demonstrated by staining with oil red O in treated cells. Osteocytic differentiation was indicated by calcium deposition as demonstrated by alizarin red staining (red color) in treated cells. No staining was observed in control untreated cells. (B) Real-time polymerase chain reaction was performed to analyze the expression of specific adipogenic and osteogenic related mRNAs under inductive culture conditions. Relative expression of PPAR-α mRNA (indicative of adipogenic activity) and osteopontin mRNA (indicative of osteogenic activity) are shown in control and treated conditions.

Figure 4.

Distribution of mesenchymal CFU counts in bronchoalveolar lavage (BAL) samples obtained from lung transplant recipients. Numbers of mesenchymal CFUs in the BAL was quantitated by culturing BAL cells in plastic adherent condition. CFU counts per 2 × 10⁶ cells plated in a 100-mm dish were reported (n = 405).

Figure 5.

Relationship of mesenchymal CFU count bronchoalveolar lavage (BAL) samples and time post-lung transplantation. Mean CFU count over various time periods post-lung transplantation are demonstrated in all BAL samples (blue diamonds; n = 405). A bimodal distribution with higher mean mesenchymal CFUs in BALs obtained at time post-transplant of less than or equal to 3 months and greater than or equal to 24 months was seen. The late increase was not seen when BAL samples obtained in presence of BOS (n = 46) or within 6 months of BOS onset (n = 36) were excluded (red squares).

Figure 6.

Mesenchymal CFUs in the bronchoalveolar lavage (BAL) as predictors of bronchiolitis obliterans syndrome (BOS) onset in lung transplant recipients. (A) Kaplan-Meier curve demonstrating time to BOS in lung transplant recipients grouped by number of mesenchymal CFUs in BAL (CFU ≥10, dashed line; CFU <10, solid line). (B) CFU-group specific adjusted time to BOS plots based on survival estimates obtained using hazards estimated with Cox models adjusted for average patient profile. Average covariate profile is as follows: time from transplant to BAL = 1.74 year; probability of being male = 52%; probability of histologic diagnosis of acute rejection = 11.9%, lymphocytic bronchitis = 10%; probability of pretransplant diagnosis of idiopathic pulmonary fibrosis = 32.4%, chronic obstructive pulmonary disease = 39.9%, cystic fibrosis = 13.9%; probability of single lung transplantation = 57.8%.