Chronic aspiration of gastric fluid induces the development of obliterative bronchiolitis in rat lung transplants

Abstract

Long-term survival of a pulmonary allograft is currently hampered by obliterative bronchiolitis (OB), a form of chronic rejection that is unique to lung transplantation. While tracheobronchial aspiration from gastroesophageal reflux disease (GERD) has clinically been associated with OB, no experimental model exists to investigate this problem. Using a WKY-to-F344 rat orthotopic left lung transplant model, the effects of chronic aspiration on pulmonary allograft were evaluated. Recipients received cyclosporine with or without 8 weekly aspirations of gastric fluid into the allograft. Six (66.7%) of 9 allografts with aspiration demonstrated bronchioles with surrounding monocytic infiltrates, fibrosis and loss of normal lumen anatomy, consistent with the development of OB. In contrast, none of the allografts without aspiration (n = 10) demonstrated these findings (p = 0.002). Of the grafts examined grossly, 83% of the allografts with chronic aspiration but only 20% without aspiration appeared consolidated (p = 0.013). Aspiration was associated with increased levels of IL-1 alpha, IL-1 beta, IL-6, IL-10, TNF-alpha and TGF-beta in BAL and of IL-1 alpha, IL-4 and GM-CSF in serum. This study provides experimental evidence linking chronic aspiration to the development of OB and suggests that strategies aimed at preventing aspiration-related injuries might improve outcomes in clinical lung transplantation.

Figure 1. Histological evidence consistent with the development of obliterative bronchiolitis associated with gastric fluid (GF) aspiration

In lung allografts with gastric fluid aspiration (A, B, E, F), fibroproliferative tissue significantly reduced the size of the bronchiolar lumen, whereas the lumen remained normal in allografts (C, G) and isografts (D, H) not receiving aspiration. A mild degree of peribronchiolar cell infiltration was observed in allografts receiving gastric fluid aspiration (A, B, E, F). Trichrome staining (lower panels) demonstrated a substantially greater extent of fibroproliferation surrounding the bronchioles of allotransplants with gastric fluid aspiration (E, F) compared to those observed in allotransplants (G) and in isotransplants (H) without gastric fluid aspiration. (Bar represents 250 μm.)

Figure 2. The number of OB-like lesions per 100× (10× ocular and 10× objective lenses) microscopic field

The mean is indicated by the bar, and is 0.87 lesions per 100× field for all animals that received gastric fluid aspiration. The mean for animals that were positive for OB-like lesions was 0.97 lesions per 100× field.

Figure 3. Positive immunohistochemical staining for T cells in peribronchiolar infiltrates

In allografts subjected to chronic gastric fluid aspiration, cells staining positive for CD3 surrounded small bronchioles 9–10 weeks after transplantation (left). CD3-positive T cells appeared to infiltrate through the lamina propria to the basement membrane and epithelium. Narrowed bronchiolar lumen were also noted. [The bar in the left panel represents 500 μm. The panel on the right is a magnified view of the area circled in the left panel.]

Figure 4. Gross and histological analyses of lung grafts (arrow) 9–10 weeks after transplantation

In isotransplants (A,D) and allotransplants without gastric fluid aspiration (B,E), the majority of the lung grafts (arrow) were functional and grossly normal, as seen in the native right lung. After repetitive gastric fluid aspirations (150 μL once a week), the pulmonary allografts developed adhesions to the surrounding chest wall and became dusky and consolidated (C). Marked fibrosis and inflammation throughout the lung was observed, and the lumen of the bronchiole was significantly narrowed (F). (H&E; bar represents 500 μm.)

Figure 5. Cytokines in the BAL from allografts with and without gastric fluid aspiration

Levels of IL-1α, TNF-α and TGF-β were markedly elevated (p < 0.05) in the BAL of allografts receiving repeated gastric fluid aspiration (n = 4) compared to that of allografts without aspiration (n = 9). Furthermore, levels of IL-1β, IL-6 and IL-10 were elevated in the BAL of allografts receiving gastric fluid aspiration compared with that of the native right (nontransplanted) lungs (p < 0.05). There was no difference observed in cytokine levels between the allografts (left lung) and native right lungs in allotransplant recipients that did not receive gastric fluid aspiration.

Figure 6. Serum cytokine levels 8-weeks after transplantation with and without gastric fluid aspiration

Levels of IL-4, IL-1α and GM-CSF were elevated in the serum of animals receiving allografts with repeated gastric fluid aspiration (n = 4) compared to that of animals with allografts and no aspiration (n = 4). Cytokine levels in animals receiving no allograft and no aspiration (n = 4) are shown for comparison. [*p < 0.05; **p < 0.01.]