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. Author manuscript; available in PMC: 2017 Jun 27.
Published in final edited form as: Am J Transplant. 2008 Jun 28;8(8):1614–1621. doi: 10.1111/j.1600-6143.2008.02298.x

Chronic Aspiration of Gastric Fluid Induces the Development of Obliterative Bronchiolitis in Rat Lung Transplants

B Li a, M G Hartwig a, J Z Appel a, E L Bush a, K R Balsara a, Z E Holzknecht a, B H Collins a, D N Howell b, W Parker a, S S Lin a,c, R D Davis a,*
PMCID: PMC5485647  NIHMSID: NIHMS662505  PMID: 18557728

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-1α, IL-1β, IL-6, IL-10, TNF-α and TGF-β in BAL and of IL-1α, 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.

Keywords: Bronchiolitis obliterans, chronic aspiration, gastroesophageal reflux, pulmonary allografts

Introduction

Over the last 20 years, lung transplantation has become a therapeutic option for appropriately selected patients with end-stage lung disease. However, successful approaches for managing chronic rejection of pulmonary allografts have not yet been developed. Reflecting this need, the survival rate of pulmonary allografts at 5 years after transplantation is a disappointing 49%, lagging behind the survival rates of other solid organ transplants (1). Long-term survival of lung transplant recipients is limited by the development of bronchiolitis obliterans syndrome (BOS), a clinically measured decrease in allograft function. In light of BOS being the predominant cause of death in adult lung transplant recipients greater than 1 year posttransplantation (1), elucidating the mechanism leading to its development has become a clinical priority.

Obliterative bronchiolitis (OB), first described in 1984 by Burke and colleagues, is considered to be the histological hallmark of BOS (2). It is a fibroproliferative process that is characterized histologically by peribronchiolar lymphocytic infiltrates and fibrous scarring and luminal narrowing of the small airways. Clinical studies suggest that both immune- and non-immune-mediated factors contribute to the development of BOS and thus OB. Non-alloimmune injury, in particular, is increasingly recognized as having an important impact on the development of OB (3,4). For example, the association between chronic aspiration and OB after heart–lung transplantation has been repeatedly observed since 1990 (5), and high levels of gastroesophageal reflux with resultant aspiration are known to be very common following pulmonary transplantation (5–10). Other than leading to an increase in acute rejection by exacerbating the alloimmune response, aspiration of gastric contents might act independently of the alloimmune response to promote allograft injury and the development of OB.

Previous work from our group showed that chronic aspiration of gastric fluid significantly accelerates acute rejection and subsequent pulmonary allograft dysfunction in a mildly histoincompatible, nonimmunosuppressed rat lung transplant model (11). In that study, no evidence of OB was observed in the face of severe acute rejection. In this study, we sought to develop a model that would evaluate the effects of chronic aspiration in pulmonary allografts without a significant degree of acute rejection. By utilizing a controlled amount of gastric fluid aspirate in the setting of lung transplantation with immunosuppression, we sought to elucidate the mechanisms underlying aspiration-induced injury in pulmonary allografts free of acute rejection.

Materials and Methods

Animals

Male Fisher 344 (F344) (RT1lvl) and Wistar Kyoto (WKY) (RT1l) rats, inbred strains which are MHC class I (RT-1)-incompatible with one another, were utilized as recipient and donor, respectively, for orthotopic left lung allograft transplantation. Five isografts using F344 rats as donors were performed and served as controls. The rats weighed between 250 to 300 g at the time of transplantation. F344 rats were purchased from Harlan Sprague Dawley (Indianapolis, IN), and WKY rats were purchased from Charles River Laboratories, Inc. (Wilmington, MA). All rats were housed in specific pathogen-free (SPF) conditions in the animal care facilities in the Medical Science Research Building at Duke University Medical Center in accordance with the institutional guidelines. All animal care and procedures were approved by the Duke Institutional Animal Care and Use Committee.

Orthotopic lung transplantation

Left lungs from WKY rats were orthotopically transplanted into F344 rats based on the nonsuturing external cuff technique reported previously (12). Briefly, the donor was anesthetized with inhaled isoflurane (2–3%) and then administered pentobarbital (100 mg/kg) intraperitoneally. The donor was then ventilated via a tracheotomy (60 breaths per minute; tidal volume of 5 mL/kg), and a midline sternolaparotomy was performed. The animal was treated with i.v. heparin (2 U/g) before flushing of the pulmonary artery (PA) with 25 mL of preservation solution at 4°C, with the pH of the preservation solution adjusted to 7.4 using Tris base. The heart–lung block was then removed, and the left lung was isolated and separated. After procurement, the left pulmonary vein (PV), pulmonary artery (PA) and main bronchus were passed through polytetrafluoroethylene cuffs, everted over the cuffs and then secured with 7-0 silk sutures. A 16-gauge cuff was used for the PA, whereas the PV and main bronchus required 14-gauge cuffs. The procedure was performed using a surgical fibermatic microscope (JKH 1402, USA). After placement of the anastomotic cuffs, the donor graft was wrapped in preservation solution-soaked gauze and placed on ice.

The recipient animal was anesthetized with ketamine (60 mg/kg) and atropine (0.25 mg/kg) and then orotracheally intubated with a 14-gauge intravenous catheter. The recipient was anesthetized with isoflurane (2–3%) during the procedure and ventilated at a rate of 60 breaths per minute and a tidal volume of 5 mL/kg. A left thoracotomy was made in the third intercostal space, followed by dissection of the left lung hilum. After the hilar structures were dissected, the left PV, PA and main bronchus were then isolated and cross-clamped with microaneurysm clamps (Mizuho America, Beverly, MA) at the most proximal end, and small incisions were made anteriorly for subsequent insertion of each respective cuff. After insertion, the cuffs were fixed by using 7-0 silk sutures, and ventilation and perfusion were reestablished by removing the hilar clamps. Clamps were sequentially removed, first from the PV, then from the bronchus and finally from the PA. For most transplants, the warm ischemic time was less than 30 min, and the cold ischemic time was less than 3 h. After ensuring that all anastomosis were secure, the native, left lung of the recipient was removed, the chest was closed in three layers and a pleural drainage tube was maintained until the recipient recovered from anesthesia.

The first dose of cyclosporine (10 mg/kg) was administered subcutaneously immediately following recovery of the animal from anesthesia after the transplant surgery. The subsequent administrations were given to 10 rats at 5 mg/kg three times a week and 14 rats at 10 mg/kg twice a week until the animals were sacrificed at 9–10 weeks after the transplant.

Gastric fluid collection and aspiration

Gastric fluid was collected from rodents as previously described (11) using the following procedure: A small midline incision was made over the upper abdomen and the peritoneal cavity was entered. The stomach and proximal duodenum were dissected free. The proximal duodenum was then ligated with 2-0 silk suture and a small gastrostomy created via which a gastrostomy tube was inserted. The gastrostomy tube was placed to straight drain and the gastric fluid collected in a 50-mL sterile conical tube. The gastric fluid was then filtered through a 70 μm strainer (BD Biosciences, Bed-ford, MA) to remove particulate matter, pooled and frozen for future aspiration experiments. The gastric fluid pH was 2–2.5 just prior to intratracheal installation.

Those animals subjected to gastric fluid aspiration (n = 9) received once weekly gastric fluid instillation into the left lung starting 1 week posttransplant as previously described (11). In order to accomplish this, each recipient was sedated and orotracheally intubated with a 16-gauge intravenous catheter. The subject was then placed with the head directed upward at a 35–40 degree angle in the left lateral decubitus position. A small silastic catheter was inserted into the distal trachea, through which 0.5 mL/kg of gastric fluid was injected. The small silastic catheter, as well as the endotracheal tube, was then flushed clear with air. One group (n = 10) of rats receiving pulmonary allografts received no gastric fluid aspiration. All experiments were terminated 9 to 10 weeks posttransplant, 1 week after the final aspiration event.

Procurement of serum and BAL samples

Blood samples were collected from the tail vein 8 weeks postlung transplant. Serum was obtained by allowing whole blood samples to clot for 1–2 h at 37°C, followed by centrifugation at 1000 × g for 10 min. The BAL was obtained by flushing twice with 1.5 mL 1% BSA in PBS. Serum and BAL samples were collected and stored frozen at −80°C until needed. Serum samples were prepared for cytokine analysis by diluting 1 volume of the serum sample with three volumes of the Bio-Plex diluent (Bio-Rad Laboratories, Inc., Hercules, CA) provided for use with serum samples. Cytokine standards for serum cytokine determination were diluted in the same diluent as was the serum. BAL samples were not diluted prior to assessment of cytokine levels, and standards for cytokine determination in BAL samples were diluted in the same wash buffer that was used to collect the BAL samples.

Assessment of cytokine concentrations in serum and BAL samples

Levels of IL-1α, IL-1b, IL-2, IL-4, IL-6, IL-10, TNF-α, GM-CSF and IFN-γ in serum and BAL samples were assayed using the Bio-Plex cytokine assay (Bio-Rad) according to the manufacturer’s instructions. The assay was run using a Luminex 100 instrument (Bio-Rad), and the Bio-Plex Rat Cytokine 9-plex Panel and the Bio-Plex reagent kit were used. The instrument was calibrated and the 0.2–3200 pg/mL cytokine standard curve for sample cytokine concentration determination was used. Assays were run in duplicate. Bio-Plex Manager Software 4.1 (Bio-Rad) was used to analyze the data.

The levels of TGF-β1 in serum and BAL samples were tested using a TGF-β1 ELISA assay (R&D Systems, Inc., Minneapolis, MN) according to the manufacturer’s instructions. A brief description of the procedure is as follows: 100 μL BAL samples were activated with 20 μL of 1 N HCl and neutralized by adding 13 μL of 1.2 N NaOH/0.5 M HEPES. Forty microliters of serum samples were activated with 10 μL of 1 N HCl and neutralized by adding 8 μL of 1.2 N NaOH/0.5 M HEPES. Neutralized serum samples were appropriately diluted with Assay Diluent RD1-21 and neutralized BAL samples were appropriately diluted with Assay Diluent RD1-73 prior to addition of the samples to the wells of the assay plate. Standards, positive controls and negative controls were also added prior to 2-h incubation at room temperature. Following this incubation, an enzyme-antibody conjugate specific for TGF-β1 was added to each well and incubated for 2 h at room temperature. Finally, a substrate solution was added and incubated for 30 min before quenching the reaction with a stop solution. Absorbance at 405 nm (A405) was determined using an EL 340 Bio Kinetics Reader (Bio-Tek Instruments, Winooski, VT). The absorbance at 540 nm was taken to be background and was subtracted from the absorbance at 405 nm to obtain the absorbance value corresponding to the level of TGF-β1. All samples, standards and controls were assayed in duplicate.

Histology and immunohistochemistry

Tissue specimens were fixed in 10% Neutral Buffered Formalin for at least 24 h, then processed into paraffin blocks for microtomy. Tissues were then sectioned at 5 lm thickness and mounted onto positively charged microslides (Erie Scientific Company, Portsmouth, NH). Sections for histological analysis were then stained with either a hematoxylin and eosin stain or Mason’s trichrome stain. For immunohistochemical staining of tissues, a standard immunoperoxidase protocol was followed using DAB as the chromogen and hematoxylin as a counterstain.

Statistical analysis

Statistical analysis was performed using Prism 3.0 software (GraphPad Software, Inc., San Diego, CA). An unpaired t-test was used for comparison of differences between animals receiving gastric fluid aspiration and those not receiving gastric fluid aspiration. A paired t-test was used for comparison of differences between left lung allografts and right (native) lungs from transplant recipients.

Results

OB after chronic gastric fluid aspiration

Histology slides were reviewed by two pathologists (BL and DNH [blinded]). Sixty-seven percent (6 out of 9) of lung allografts receiving gastric fluid aspiration for 2 months showed evidence consistent with the development of OB. These findings include cellular fibroproliferative tissue, significant reduction in the size of bronchiolar lumena, and mild degrees of peribronchiole inflammatory cell infiltrates (Figure 1A, B, E, F). Based on a lack of profound fibrosis and the presence of infiltrative cells, the OB pathology appears to be active, rather than inactive OB. Substantial, widespread infection was found in one of the allografts that received gastric fluid aspiration, and lesions consistent with OB were also observed in that allograft. At least two sections of each lung, one from the upper region of the lung and one from the lower region of the lung, were examined. All sections were examined in their entirety, with an average of 13 100× microscopic fields (10× ocular and 10× objective lenses) being examined per lung. As shown in Figure 2, the number of OB-like lesions in the lungs that were positive for OB-like lesions varied over slightly more than a 2-fold range, with between 0.83 and 2.0 lesions per 100× microscopic field. In contrast to allografts receiving chronic gastric fluid aspiration, all of the allografts receiving no aspiration did not show any evidence of OB (p = 0.002).

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

Figure 1

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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.

Figure 2

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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.

As shown in Figure 1E, and F, trichrome staining revealed cellular fibroproliferative tissue surrounding the bronchiolar lumen and reducing the size of that lumen. In addition to fibrosis, mononuclear infiltrates were observed. As shown in Figure 3, these cellular infiltrates stained heavily for CD3, indicating that the cells were predominantly T cells. Attempts at staining with markers for CD4 proved to be difficult for technical reasons, predominantly because of the lack of availability of appropriate reagents to detect the rat antigen in fixed tissues. In general, foreign bodies as evidenced by visualization under a polarized light microscope were not observed near OB lesions. This is consistent with the idea that the observed lesions were not simply granulomatous reactions around foreign bodies. However, slide sections are necessarily thin, and it is possible that foreign bodies may be associated with the lesions in a plane outside that of the tissue section.

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

Figure 3

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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.]

Of note was a general lack of evidence of acute rejection in the grafts, based on a paucity of perivascular lymphocytes. This contrasts with our previous study in which immunosuppression was not utilized and in which extensive acute rejection was observed (11). However, it is possible that acute rejection was taking place and not observed due to timing or perhaps to changes due to OB, which may have overshadowed any evidence of acute rejection.

Effect of chronic gastric fluid aspiration on lung function

When lung volume expansion was examined grossly utilizing a constant ventilator pressure of 20 mm H2O, 83.3% (5 out of 6 rats evaluated: 3 were not evaluated.) of animals receiving gastric fluid aspiration had lung allografts that could not be inflated to more than 50% of their normal volume. The allografts were relatively consolidated and dark in color (Figure 4, arrows), with five of the grafts showing essentially no expansion (<5% of lung function) at a pressure of 20 mm H2O. In contrast, only 20% (2 out of 10) of lung allografts in animals not receiving gastric fluid aspiration could not be inflated more than 50% of their normal volume (p = .013 for the difference between groups). Grossly, a majority of these allografts were similar to isograft controls and to native, nontransplanted right lungs (Figure 4A, B).

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

Figure 4

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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.)

Effect of chronic gastric fluid aspiration on cytokine production

To probe the mechanism underlying the development of OB induced by gastric fluid aspiration, we evaluated the concentrations of a panel of rat cytokines in BAL and serum samples obtained from transplant recipients receiving or not receiving chronic gastric fluid aspiration. IL-1α, IL-1β, IL-6, IL-10, TNF-α and TGF-β were detectable in BAL samples, while IFN-γ, IL-2, IL-4 and GM-CSF were not detectable. After repeated gastric fluid aspiration, the average levels of IL-1α, IL-1β, IL-6, IL-10, TNF-α and TGF-β1 were markedly elevated compared with those average levels in animals not receiving gastric fluid aspiration, although only the differences in IL-1α, TNF-α and TGF-β1 were statistically significant due to large variations in the sample population (Figure 5). Average levels of IL-1α, IL-1β, IL-6, IL-10, TNF-α and TGF-β1 were also elevated in the BAL fluid taken from the left (transplanted) lung compared to levels in the BAL fluid taken from the right (native) lung (Figure 5), although only the differences in IL-1β, IL-6 and IL-10 were statistically significant. As might be expected, in transplant recipients that did not receive chronic gastric fluid aspiration, there were no significant differences between cytokine levels in the BAL fluid taken from the right, native lung and those from the left, transplanted lung.

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

Figure 5

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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.

On the other hand, the serum concentrations of IL-1α, IL-4 and GM-CSF were increased in transplant recipients receiving chronic gastric fluid aspiration compared to the serum concentrations of these cytokines in animals not receiving gastric fluid aspiration (Figure 6). However, the TGF-β1 level in the serum of was not associated with gastric fluid aspiration (data not shown).

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

Figure 6

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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.]

Discussion

The specific etiology and pathogenesis of BOS are not well understood. Many factors have been described as probable risk factors or as potential risk factors. The current premise is that OB, the histological hallmark of BOS, represents a common lesion in which different inflammatory insults such as ischemia-reperfusion, acute rejection, infection and chemical injury can lead to a similar pathologic and clinical outcome (13).

In the setting of lung transplantation, it seems likely that an additional insult beyond an antiallogeneic immune response is necessary for the development of OB. This two-hit hypothesis has been demonstrated in a series of animal models looking at the relative contributions of viral injury and alloimmune injury following bone marrow transplantation (BMT). Winter et al. demonstrated that the endotracheal introduction of a parainfluenza virus into an animal following lung transplantation resulted in a histologic picture consistent with OB. This was not seen in control or syngeic transplanted animals (14,15). Additionally, in a fully mismatched BMT rodent model, the introduction of LPS, with its activation of the innate immune response through the toll-like receptor-4 pathway, resulted in a robust immune response with an injury pattern indicative of OB (16).

Among the chemical injuries that could potentially create an inflammatory environment in the lung, which might eventually lead to OB, are those related to gastroesophageal reflux disease (GERD). GERD is very common in lung transplant recipients (5,6,8,9), perhaps in part due to intraoperative manipulation of the vagal nerve and in part due to medication-induced gastroparesis. A possible role of tracheobronchial aspiration after GERD in the early development of BOS has been implicitly documented in our recent work published by Cantu et al. (17). Cantu showed that, in lung transplant patients with known GERD, early fundoplication led to significant improvements in freedom from BOS. Recently, elevated levels of bile acids in the BAL fluid were associated with early BOS development (18,19). A prolonged contact time of aspirated gastric contents may trigger epithelial lung injuries, leading to a host of reactions resulting in obliterative bronchiolitis.

In our previously published study (11), we evaluated the effects of chronic aspiration on pulmonary isografts and pulmonary allografts (strain WKY to strain F344) under conditions in which the lung grafts succumb to acute rejection. The results indicate that chronic aspiration of gastric contents promotes accelerated allograft failure and may promote a profibrotic environment. Further, this prior study demonstrated that chronic gastric fluid aspiration-induced injury to a transplanted lung was dependent on the immunologic differences between host and recipient, since chronic gastric fluid aspiration in pulmonary isografts was not associated with substantial graft injury.

In this study, we observed histologic evidence consistent with the development of OB in lung allografts, after providing chronic gastric fluid aspiration in a model that utilizes immunosuppression to avert acute rejection. The observation that chronic aspiration of gastric fluid, as it might occur in GERD, can lead to findings consistent with the development of OB in this model provides the first direct experimental evidence establishing a causal relationship between these two pathologies. Furthermore, the loss of >95% of lung function in many of the allografts receiving chronic gastric fluid aspiration but not in grafts without such aspiration suggests that GERD can lead to severe BOS in lung transplant recipients. The fact that OB was observed only in the face of immunosuppression may simply be due to the fact that severe acute rejection in the absence of immunosuppression led to complete graft failure before OB could be observed. However, it is possible that cyclosporine modulated the immune response in a manner that triggered the development of OB, and this possibility remains to be evaluated.

Evidence from the clinical arena has suggested that alloimmune-mediated injury to airway epithelial cells is an important factor contributing to the development of OB (20). This evidence includes increased airway epithelial cell expression of MHC class II proteins in patients with acute and chronic rejection. Further, cytotoxic T lymphocytes directed against donor airway epithelial cell HLA class I antigens have been identified in patients with OB. In this study, despite the use of cyclosporine to prevent acute rejection, some CD3+ T cells were still found in the allografts receiving chronic gastric fluid aspiration. This observation indicates that the immunosuppressive regimen used in this study for rats after transplant cannot completely block the alloimmune-dependent cellular response in the face of chronic gastric fluid aspiration. It therefore seems likely that the enhanced alloimmune response contributes to the development of OB in this model.

Another possibility is that autoimmune processes at least, in part, contribute to the development of OB. In the pulmonary allograft setting, it is speculated that alloreactive T and B cells may undermine recipient immunomodulatory mechanisms. Such loss of immunomodulation might result in activation of host T and B cells against collagen, type-V. Experimental support of this idea is compelling (21,22). For example, Burlingham et al. recently demonstrated a role for collagen type-V cell-mediated immunity in the progression and development of OB (21) through a series of experiments involving adoptive transfer of cells from immunized donors and by detecting collagen type-5 antigen in recipients. Further, CD4+CD25+ regulatory T cells have been demonstrated to modulate cellular autoimmunity to collagen type-V (23). Interestingly, collagen type-V is normally cross-linked and not immunogenic in its natural form. It seems possible that alteration of collagen type-V, possibly by acid from GERD, may potentiate the autoimmune response.

The chronic gastric fluid aspiration-induced upregulation of proinflammatory cytokines that we observed could play a major role in propagating the process or processes responsible for tissue damage and eventual airway fibrosis and obliteration (24). In particular, the proinflammatory cytokine IL-1a significantly increased in both the BAL fluid and the serum of allograft recipients receiving chronic gastric fluid-aspiration could be a mitigator. IL-1a is cell-associated and is produced continuously in alveolar macrophages from rats with bleomycin-induced pulmonary fibrosis (25). The systemic increase in the levels of this potent inflammatory cytokine may reflect a profound change in the microenvironment of the lung that could account for the fibrosis and poor outcomes of the grafts receiving chronic gastric fluid aspiration.

The observation that chronic gastric fluid aspiration caused increased levels of cytokines associated with inflammatory injury and repair (e.g. IL-1α, IL-1β, IL-6, IL-10, TGF-β and TNF-α) is of interest. Alveolar macrophages can be involved in the production of cytokines that mediate both injury (e.g. IL-6) and repair (e.g. TGF-β). However, it seems likely that a variety of perhaps newly recruited cells, including the CD3+ cells observed in this study, play a role in mediating the pathogenesis. Possible interplay between the T cells observed in this study and the macrophage and dendritic cell populations is suggested by the increase in GM-CSF that was observed as a result of aspiration. This cytokine, produced by T-helper cells, stimulates the growth and differentiation of monocytes and dendritic cells, and may thus play a key role in communication between the CD3+ cells observed in this study and the macrophage population.

The chronic gastric fluid aspiration-induced increases in TGF-β levels in the BAL fluid we observed in this study are not unexpected given the pathogenesis of OB. TGF-β is involved in normal tissue repair following lung injury, as well as in modulation of proliferation, differentiation and apoptosis (26). Further, TGF-β is chemotactic for fibroblasts and induces expression of extracellular matrix components (27). The cytokine is also markedly increased in patients with lung transplant-related BOS (27–29), and has been found to be upregulated in other experimental models of BOS as well (30). These data provide important insights for future investigations into defining the mechanistic basis of BOS.

In conclusion, this study validates in an experimental model the growing body of literature suggesting a role of gastroesophageal reflux in the development of OB after lung transplantation. The findings in this study also support the idea that OB is an immunologically mediated phenomenon involving the activation of T-lymphocytes in the face of immunosuppression. It is hoped that this model for chronic gastric fluid aspiration-induced OB will provide the basis for further studies aimed at intervention, treatment and prevention of OB after lung transplantation.

Acknowledgments

This work was supported in part by the American College of Surgeons Faculty Research Grant, the Parks Protocol Memorial Fund, the Duke Heart Center Career Development Award, the Fannie E. Rippel Foundation, and National Institutes of Health Grant 5T32GM069331-04.

Abbreviations

BAL

bronchoalveolar lavage

BOS

bronchiolitis obliterans syndrome

CDx

cluster of differentiation

DAB

3,3-diaminobenzidine

F344

Fisher 344

GERD

gastroesophageal reflux disease

HCl

hydrochloric acid

H&E

hematoxylin and eosin

NaOH

sodium hydroxide

OB

obliterative bronchiolitis

PA

pulmonary artery

PV

pulmonary vein

SPF

specific pathogen-free

WKY

Wistar Kyoto

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