Obliterative bronchiolitis (OB) is a frequent complication after lung transplantation and represents the most significant limitation to long-term survival in lung transplant recipients. It is a fibroproliferative disease that causes a progressive decline in airflow with eventual hypoxemia. Histologically, OB is characterized by subepithelial and luminal deposition of mature collagen and inflammatory cell infiltration, resulting in narrowing or complete blockage of airways (1). Evidence is accumulating that neutrophil products, including oxidants, play a critical role in the development of OB (2,3).
Induction of heme oxygenase (HO)-1 is a cellular response to oxidative stress (4) that may serve a cytoprotective function (5–7). Using a variety of models, our laboratory and others have shown a role for HO-1 in the pathophysiology of oxidative and inflammatory injury (5–9). HO-1 is the inducible form of HO, which is the initial and rate-limiting enzyme in heme catabolism that catalyzes the oxidative cleavage of heme molecules to biliverdin, free iron, and carbon monoxide. Biliverdin is subsequently converted to bilirubin by biliverdin reductase (10). HO-1 was identified as one of several antiapoptotic and antioxidant genes associated with prolongation of mouse cardiac allograft and xenograft survival (11,12). Recent studies have suggested that the cytoprotective activity of HO-1 against inflammatory disorders is at least in part mediated by its interactions with interleukin (IL)-10 (13–15), which also has graft protective effects (16).
In our recent study of human lung transplants with acute cellular rejection and OB, we documented increased HO-1 protein expression in acute cellular rejection and active OB lesions, whereas inactive OB lesions showed minimal HO-1 expression (17). To further define the role of HO-1 expression in lung transplant rejection, we studied the effects of genetic and pharmacologic HO-1 modulation on airway rejection in a murine heterotopic tracheal transplant model of OB.
MATERIALS AND METHODS
Pharmacologic and Genetic Alteration of HO-1 Expression
HO activity was inhibited using the metalloporphyrin, tin protoporphyrin (SnPP) or using mice containing the null mutation for HO-1 (HO-1−/−), whereas HO activity was increased using the metalloporphyrin, cobalt protoporphyrin (CoPP). Chemical alteration was achieved by subcutaneously injecting donor and recipient mice with 20 μmol/kg of SnPP or CoPP (Porphyrin Products, Logan, UT), 18 hr before transplantation. In addition, recipients were treated with SnPP or CoPP at 4 and 9 days after transplantation. HO-1−/− mice with a background of C57BL/6 were selected from the offspring of heterozygous/homozygous mating by Southern blotting of tail DNA. These mice were derived from an HO-1+/− breeding pair that were initially obtained from Drs. Poss and Tonegawa, Massachusetts Institute of Technology, Cambridge, MA (18). IL-10 null (IL-10 −/−) mice were purchased from The Jackson Laboratory, Bar Harbor, ME.
Murine Heterotopic Tracheal Transplantation
Subcutaneous tracheal transplantation was performed under general anesthesia in accordance with guidelines of the University of Florida Institutional Animal Care and Use Committee. BALB/c and C57BL/6 mice were used as donors and recipients. Tracheal transplantation was performed as described by Hertz et al. (19), with slight modifications. The trachea was isolated through a neck incision and separated from the esophagus and lung. It was excised, stripped of any attached soft tissues, and maintained in a sterile Petri dish containing cell culture medium (RPMI) (Sigma, St. Louis, MO) until the recipient was ready. The recipient mouse was anesthetized, and the posterior half of the mouse was shaved and prepared with 70% ethanol. A 1.0-cm incision was made in the right flank of the mouse. A narrow tunnel in the subcutaneous tissue was created by blunt end dissection, the donor trachea was inserted into the distal end of the tunnel, and the wound was closed with surgical clips.
Tracheal transplants were performed using mice of the same strain, BALB/c-to-BALB/c or C57BL/6-to-C57BL/6 (isografts), or of different strains, allografts, with each strain being used as a donor or recipient. The following are the number of transplantations performed for each time point and treatment condition: (1) BALB/c isografts day 9, n=3; day 16, n=4; day 21, n=8; (2) C57BL/6 isografts day 21, n=7; (3) C57BL/6-to-BALB/c allografts day 9, n=8; day 16, n=6; day 21, n=12; (4) BALB/c-to-C57BL/6 allografts day 21, n=6; (5) SnPP-treated C57BL/6-to-BALB/c allografts day 9, n=6; day 16, n=6; day 21, n=8; (6) CoPP-treated C57BL/6-to-BALB/c allografts day 9, n=6; day 16, n=6; day 21, n=8. HO-1−/− mice with a background of C57BL/6 were also used as donors or recipients for the tracheal transplantations. In these experiments, tracheal allotransplantations were performed using either HO-1−/− mice as donors and BALB/c mice as recipients (n=6) or HO-1−/− mice as recipients of trachea harvested from BALB/c mice (n=4). In addition, allotransplantations were performed using IL-10-deficient mice as donors (n=6) or recipients (n=6) and BALB/c mice as recipients or donors, respectively. The tracheal transplantations using HO-1−/− and IL-10-deficient mice were evaluated at 21 days after transplantation.
On days 9, 16, or 21 after transplantation, recipient mice were killed and the graft and surrounding soft tissues removed en bloc, fixed in 10% neutral buffered formalin, and then transversely sectioned to produce full cross-sections of the grafts. Tissues were processed routinely and embedded in paraffin, and 5-micron thick sections were cut for staining.
A slide from each animal sample was stained with a standard hematoxylin-eosin stain and semiquantitatively evaluated for findings of airway rejection including lymphocytic tracheitis, respiratory epithelial cell loss, and luminal or subepithelial granulation tissue. The degree of epithelial loss was graded as none (0), focal (1+), diffuse (2+), or total (3+). The amount of luminal obstruction by granulation tissue was graded as none (0), less than 25% (1+), 25% to 75% (2+), or more than 75% (3+). To better identify luminal or subepithelial collagen deposition, a Masson trichrome stain was performed. Collagen deposition was graded as none (0), mild (1+), moderate (2+), or marked (3+).
Immunohistochemical staining was performed on a Ventana immunostainer (Ventana Medical Systems, Inc., Tucson, AZ). After deparaffinization, hydration, and treatment with 3% hydrogen peroxide, tissue sections were placed in citrate buffer (Antigen Retrieval Citra; Biogenex, San Ramon, CA) at pH 6.0 and incubated in an 80°C water bath for 2 hr. A polyclonal rabbit anti-HO-1 antibody (SPA-895; StressGen Biotechnologies Corporation, Victoria, BC, Canada) with specificity for rat, human, and mouse HO-1 (20) was used to stain the mouse tracheal transplant samples. A 1:100 dilution of the antibody was selected by titration to optimize staining. IL-10 expression was evaluated using polyclonal rabbit anti-mouse IL-10 antiserum (Biosource, Camarillo, CA) at a dilution of 1:80. Slides were successively treated with biotinylated goat anti-rabbit IgG as secondary antibody (DAKO, Carpinteria, CA) at 1:400 dilution, peroxidase-conjugated avidin-biotin complex solution, and 3,3′-diaminobenzidine (DAB), before being counterstained with hematoxylin (Ventana Medical Systems). A positive control slide was included with each staining run. Negative control slides were subjected to the same staining protocol except that normal rabbit antiserum was used in place of the primary antibody. Cell types were assessed morphologically, and staining was graded as none (0), focal (1+), patchy (2+), or diffuse (3+).
Terminal Deoxynucleotide Transferase-Mediated dUTP Nick-End Labeling (TUNEL)
Apoptotic cells were evaluated by TUNEL assay, using the In Situ Cell Death Detection Kit (Roche Diagnostic, Chicago, IL). After deparaffinization and hydration, the slides were treated with 20 μg/ml proteinase K (Sigma) in 10 mM Tris/HCL, pH 7.4 to 8.0, for 30 min at 37°C. The slides were incubated in permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) for 2 min on ice, and DNA nick-end labeling was performed with terminal transferase and fluorescein isothiocyanate-labeled nucleotide in a humidified chamber for 60 min at 37°C. The slides were counterstained with 0.25 μg/ml propidium iodide for 15 min at 4°C, mounted with aqueous mounting medium, and viewed under fluorescence microscopy. Samples treated with 1 mg/ml DNase I for 10 min at room temperature served as positive control with each run, and slides treated with labeling solution without terminal transferase served as negative controls. Apoptotic epithelial cells in each tracheal cross-section were counted.
Data are expressed as mean ± SEM. Nonparametric analysis of variance (Kruskal-Wallis Test with Dunn’s Multiple Comparison Test) sum tests were used to compare the extensiveness of staining between the different tracheal transplant groups. An unpaired t test was used to evaluate the numbers of apoptotic epithelial cells. A value of P less than 0.05 was considered to be statistically significant.
Murine heterotopic tracheal allografts demonstrated features associated with human chronic airway rejection including lymphocytic airway inflammation, respiratory epithelial cell loss, and granulation tissue with fibrosis in the tracheal mucosa and/or lumen (Fig. 1A) (19,21). In contrast, BALB/c isografts were virtually indistinguishable from normal tracheas (Fig. 1B) and were surrounded by variable amounts of granulation tissue representing a local wound healing response. Interestingly, C57BL/6 isografts consistently showed a pronounced lymphocytic tracheitis (Fig. 1C), but in contrast to allografts, the respiratory epithelium was intact and the tracheas lacked luminal or mucosal granulation tissue. The presence of lymphocytic tracheitis in C57BL/6 isografts is probably a strain variation, and represents a form of lymphoid hyperplasia in these mice. The absence of the other features of rejection distinguishes the lymphoid hyperplasia from rejection and indicates a lack of the cytotoxic T-cell activity and fibroproliferation that are characteristic of cellular rejection.
Similar to our observations in humans (17), HO-1 protein expression in the mouse tracheal allografts significantly exceeded that of isografts (Fig. 2). HO-1 expression in isografts was minimal (weak staining of a few luminal or intramural macrophages with a score of 0.3±0.15;Fig. 2A), whereas allografts demonstrated more numerous stained macrophages and mesenchymal cells in the tracheal granulation tissue with a HO-1 staining score of 1.75±0.11 (Fig. 2B). Allografts from mice treated with the chemical inducer of HO, CoPP, resulted in a staining score for HO-1 of 2.36±0.15, whereas treatment with the HO activity inhibitor, SnPP, demonstrated a similar staining score (1.7±0.15) as untreated allografts. SnPP inhibits the enzyme activity of HO rather than inhibiting its protein or mRNA expression, and it is not surprising that HO-1 staining in the SnPP-exposed allografts was not reduced (22).
Modulation of HO-1 activity seemed to alter the progression of airway rejection. By day 21, tracheal transplants from HO-1−/− mice or mice receiving SnPP treatment resulted in a total loss of respiratory epithelium (score of 3) and complete granulation tissue occlusion of the lumen (score of 3) for a combined score of 6 in all HO-1–deficient allografts (Fig. 3). Allografts from untreated or CoPP-treated mice showed more variability. The loss of respiratory epithelium and the amount of intraluminal granulation tissue for the untreated and CoPP-treated allografts ranged from partial to complete for a combined score of 5.3±0.22 for untreated and 4.9±0.33 for CoPP treated (Fig. 3). These differences did not reach statistical significance.
Masson stains highlighted more collagen in the grafts for which HO-1−/− mice served as either donor or recipient (fibrosis score of 2.4±0.4) as compared with untreated allografts (0.7±0.4;Fig. 4). Comparisons at days 9 and 16 after transplantation revealed earlier onset of severe injury in the SnPP-treated animals, as determined by the extent of epithelial loss and the amount of intraluminal granulation tissue (Figs. 3 and 5). The injury scores for untreated, SnPP-treated, or CoPP-treated allografts, respectively, for day 9 are 1.0±0.4, 3.0±0.6, and 0.6±0.6 and for day 16 are 1.6±0.4, 3.5±0.9, and 1.2±0.4 (Fig. 3). Respiratory epithelial cell apoptosis also seemed to be more prevalent in allografts than isografts at day 9, particularly in animals treated with SnPP. TUNEL staining confirmed this impression, revealing significantly greater numbers of apoptotic epithelial cells in the SnPP-treated allografts (40.4±9.5 cells/graft) than the CoPP-treated allografts (10.7±1.3) or isografts (0.4±0.2). Untreated allografts demonstrated 17.2±4.5 TUNEL-positive epithelial cells per graft; however, this was not significantly different from the SnPP-treated allografts.
Lastly, use of IL-10−/− mice as either donors or recipients resulted in earlier development of severe airway rejection lesions similar to those observed in the grafts in which HO-1 was inhibited (Fig. 6). IL-10–deficient grafts showed complete loss of respiratory epithelium and complete filling of the lumen with granulation tissue, with a fibrosis score based on Masson staining of 2.0±0.3, as compared with HO-1−/− grafts with a score of 2.4±0.4. The IL-10–deficient grafts showed reduced HO-1 protein expression compared with grafts in which both donor and recipient were wild-type (Fig. 6A,B). Reduction in HO-1 expression, however, was only statistically significant when the IL-10−/− knockout mice were the tracheal donors (0.5±0.3 HO-1 staining in IL-10−/− donors as compared with 1.9±0.2 in wild-type allografts;Fig. 6C). Conversely, IL-10 staining was reduced in tracheal transplants from HO-1−/− donors (1.0±0.4 compared with 3.3±0.2 in wild type;Fig. 6D–F).
The relationship between HO-1 and allograft survival has become a topic of recent interest because of information derived from several recent animal and human studies. HO-1 was one of several genes linked to prolonged graft survival in a mouse cardiac allograft model (11). HO-1 has also been shown to be beneficial in a setting of heart and liver transplant ischemia/reperfusion injury (23–25). We previously noted increased HO-1 expression in human lung allografts with rejection (17), but its relationship to lung allograft survival is unknown. The current studies demonstrate rejection-associated changes in HO-1 expression in the heterotopic mouse tracheal transplant model and support a role for HO-1 as a contributor to the rejection phenotype.
HO-1 is induced by a variety of stimuli, including oxidants, and is believed to be a sensitive marker of oxidant stress (4). Under most circumstances, this induction is believed to be a cytoprotective response. The current studies add support to this contention. These experiments show that genetically or chemically induced deficiency of HO-1 translates into more rapid onset of a severe airway rejection lesion in the murine tracheal transplant model of chronic airway rejection. Grafts demonstrated features of rejection identical to those observed in humans (1).
Although previous studies in other organ systems have shown that increasing HO-1 resulted in additional protective effects for graft survival (11,25,26), the current studies revealed no clear histologic benefits with the use of CoPP. One explanation for this may be that HO-1 expression is increased in response to allotransplantation and providing more confers little added benefit. A second possibility is that HO-1’s protective effects are enhanced by concurrent immunosuppression. Support for this hypothesis comes from previous studies of cardiac rejection, in which CoPP by itself had little effect on the development of chronic cardiac rejection (transplant arteriosclerosis) (11), whereas the use of immunosuppressive agents (cobra venom F or CD4 monoclonal antibodies) that increase HO-1 produced amelioration of rejection (11,12,26–28).
IL-10 is an anti-inflammatory cytokine that inhibits the production of proinflammatory mediators such as tumor necrosis factor-α and interferon-γ, and increasing IL-10 was shown to reduce acute and chronic lung transplant rejection (16). Treatment with recombinant IL-10 or increasing IL-10 through viral vectors inhibited OB in a tracheal transplant model (29). Recently, IL-10–induced enhancement of HO-1 was shown to attenuate lipopolysaccharide-induced acute lung injury in mice (14). In addition, increasing HO-1 with an intratracheal injection of a recombinant AdV-HO-1 vector caused an increase in IL-10 and protected the lung from lipopolysaccharide-induced injury (15). Our results also support a relationship between IL-10 and HO-1, which may be important for the accelerated development of OB-like lesions in both IL-10– and HO-1–deficient mice.
These studies indicate that enhanced expression of HO-1 accompanies murine heterotopic airway rejection. Reduction of HO-1 activity by either genetic or chemical means results in accelerated development of a tracheal lesion that resembles human chronic airway rejection. Likewise, IL-10 deficiency is also associated with more rapid progression of airway rejection. It is not clear from these studies, however, whether enhancement of HO-1 activity will translate into improved graft survival. Further studies using other strategies for enhancing HO-1 expression, alone or through the use of agents with other immunosuppressive activities, may offer further insight into this question.
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