Introduction

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Lung transplantation has become a successful clinical therapy for end-stage pulmonary disease as surgical techniques and immunosuppressive regimens have improved (1). However, obliterative bronchiolitis (OB), a form of chronic graft rejection, affects 50% of all lung transplant recipients and is the leading cause of death after lung transplantation (2). This disorder most commonly occurs six or more months after transplantation, and may present with cough, recurrent episodes of bronchitis, a decrease of more than 15% in FEV₁, and hyperinflation on chest X-ray (CXR) (3). Histologically, epithelial cell desquamation with luminal fibrosis (4), as well as graft arteriosclerosis affecting both large elastic and small muscular arterioles of the pulmonary circulation (7), characterize chronic rejection.

Clinical studies have linked the development of OB and acute, severe graft rejection (8). However, human studies of the pathogenesis of OB have been difficult to interpret because of its varied clinical course (4), the heterogeneous distribution of the pathologic lesion and low diagnostic yield of transbronchial biopsy (< 20%) (11), and the presence of concurrent infection. Despite a large number of human studies of lung lymphocyte subtypes, donor-specific alloreactivity, major histocompatibility complex (MHC) class I and II expression, inflammatory cytokine messenger RNA (mRNA) expression, and histology of airway injury, the immunopathogenesis of OB remains unclear (12).

Animal models of orthotopic lung transplantation have been used largely to study early postoperative problems, such as ischemia-reperfusion, airway dehiscence, and acute rejection. Recently, these models have been applied to the study of OB. One orthotopic rat model develops the histopathologic lesion of airway obliteration relatively early, in association with lung necrosis (13), whereas another model does not appear to develop the characteristic lesion of OB (14). In 1993, Hertz and colleagues (15) developed a mouse heterotopic airway transplant model to study the pathogenesis of OB, and found that allografts demonstrated subepithelial inflammation, epithelial necrosis, and early fibroproliferation reproducing human OB after 21 d as opposed to isografts, which did not show these effects. Cyclosporin treatment reduced the development of OB in allografts at 30 d, and this effect was dose-dependent (16); however, epithelial injury and cellular inflammation were still present. Since mouse airway heterografts reproduced the characteristic histopathology of human OB, we used this model to study the inflammatory cell populations in airway allografts over time. We specifically analyzed CD4⁺, CD8⁺, and B lymphocytes, and macrophages and myofibroblasts, contrasting traditional allografts with isograft controls, as well as with severe combined immunodeficiency (SCID) mouse recipients lacking B and T lymphocytes to test the hypothesis that: (1) airway damage was predominantly immune-mediated; and (2) lymphocytes and macrophages were important in the pathogenesis of this process.

	Materials and Methods

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Mice

BALB/c (H2-d) and C57BL/6 (H2-b) (Charles River, Raleigh, NC), and SCID C57BL/6 (H2-b) mice (Jackson Laboratory, Bar Harbor, ME) were obtained from pathogen-free inbred colonies and housed in accordance with the rules and regulations of the Institutional Animal Care and Use Committee of the University of North Carolina.

Tracheal Transplantation and Immunosuppression

Allografts and isografts were obtained by transplanting 28 BALB/c tracheas into 12 C57BL/6, eight BALB/c, and eight SCID recipient hosts. The allograft and SCID experiments used fully mismatched (MHC I and II) animals. First, donor BALB/c mice were euthanized with 100% CO₂. Donor hearts and lungs were exposed via a midline incision through the skin and peritoneum extending through the rib cage and sternal notch. Thymus tissue was dissected away. The esophagus was separated from the trachea by blunt dissection, and the trachea was tied to a hollow, 2-mm-diameter polyethylene stent (Becton Dickinson and Company, Parsippany, NJ) matched for size, and then excised cephalad near the larynx and caudad at the hilum. The trachea was placed in ice-cold Dulbecco's modified Eagle's medium (DMEM) (Lineberger Center Core Facility, University of North Carolina, Chapel Hill, NC). Recipient mice were anesthetized via intramuscular injection with 20 µl of xylazine (American Animal Labs, Wisner, NE) and ketamine (Fort Dodge Labs, Fort Dodge, IA) in a 3:2 mix. After shaving a 1 cm × 1 cm area behind the head, a 3-mm transverse incision was made through the dermis and a 1.5 cm × 1.5 cm subcutaneous pouch was created via blunt dissection over the posterior upper back area. One airway graft, sutured to polyethylene tubing (Becton-Dickinson) was placed into each animal. The skin pocket was closed with size 4.0 silk suture and covered with bacitracin ointment (Fougera, Melville, NY). Animals were injected intraperitoneally Monday through Friday with cyclosporin 25 mg/kg (Sandoz, East Hanover, NJ), a dose previously shown to result in immunosuppression (16). Three allografts, two SCID allografts, and two isografts were harvested at each 2-, 6-, 10-, and 15-wk time point, and airway stents were removed.

Histologic Studies

Grafts were placed in ethanol/acetic acid fixative solution (Omnifix; An-Con Genetics, Melville, NY) at room temperature for 24 h, embedded in low-melting-point paraffin, cut into 6-µm-thick cross-sections (model 230 Rotary Microtome; Leica, Deerfield, IL) and stained with hematoxylin and eosin (H&E) (17). Allografts, SCID allografts, and isografts were examined for the presence or absence of fibroproliferation.

Antibodies

Rat monoclonal antimouse antibodies were used in optimal concentrations as primary antibodies, with the exception of the -smooth-muscle-actin antibody, which was an alkaline phosphatase-conjugated mouse antibody (Table 1).

Immunohistochemistry

Tracheas were frozen in polyvinyl alcohol/polyethylene glycol compound (Miles Inc., Elkhart, IN) and stored at 70°C. Twelve-micron-thick cross-sections of trachea were prepared with a cryostat (model 1800 Cryostat; Leica). Frozen sections were air dried for 5 min and then fixed for 5 min in acetone chilled to 4°C. All subsequent incubations were done at room temperature. After fixation, sections were blocked successively with a 10% milk solution (Sigma, St. Louis, MO), 5% normal goat serum (Vector Labs, Burlingame, CA), and avidin and biotin blocking solution (Vector Labs). An optimal concentration of each primary antibody was applied to serial sections for 60 min. The sections were then incubated with secondary, biotin-conjugated goat antirat antibody for 30 min. Subsequently, avidin-biotin complex (ABC) alkaline phosphatase-avidin reagent (Vector Labs, Burlingame, CA), diluted 1:5 in phosphate-buffered saline (PBS)/0.1% bovine serum albumin (BSA) was added for 30 min. Color was developed with Vector red or blue substrate solution (Vector Labs) for 30 min, and slides were then washed in distilled water. No counterstain was used, in order to avoid a color interaction between hematoxylin and the Vector substrate solutions. Slides were then mounted with Advantage mounting medium (Innovex Biosciences, Richmond, CA) and coverslipped. For each primary antibody, a negative control was prepared with normal serum alone, to ensure that effects of nonspecific binding were eliminated.

Morphometry

CD4⁺ cells, CD8⁺ cells, B cells, macrophages, and myofibroblasts were quantitated as the sum of cells in 10 separate, high-power (×40) fields of view evaluated per graft, including areas of tracheal submucosa and lumen. Neutrophils were counted in paraffin sections, using the same method. All cells were counted by two independent, blinded reviewers. Allografts (n = 6), SCID allografts (n = 2), and isografts (n = 4) were analyzed at each time point.

Statistical Analysis

Analyses were performed with commercially available software (18). The two-sample t test with unequal variances was used to compare the cell counts in allografts with those in isografts at each time point. A two-sided alpha level of P < 0.05 was considered to indicate significance.

	Results

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Histopathology

Low-power views of isografts and SCID hosts revealed a widely patent lumen from Week 2 through Week 15, without evidence of fibroobliteration, whereas allografts demonstrated luminal occlusion by granulation tissue at 6 wk and at all subsequent time points. High-power examination of cross-sections revealed differences in epithelial-cell integrity, inflammatory-cell infiltration, and growth of granulation tissue. At 2 wk, allografts exhibited disruption of the epithelial layer, with loss of cilia, squamous metaplasia, and swelling of the submucosa with mononuclear cell infiltrates (Figure 1a). As with the allografts, grafts to SCID hosts exhibited a mononuclear-cell infiltrate; however, the epithelium was generally intact, with a few areas of metaplasia (Figure 1g). Isografts showed neither epithelial abnormalities nor cellular infiltrate (Figure 1d). At 6 wk, allografts demonstrated epithelial denudation, scarring of the basement membrane (Figure 1b), and luminal obliteration by loose connective tissue, with scattered luminal and submucosal infiltrate as compared with isografts (Figure 1e) and grafts to SCID hosts (Figure 1h), which exhibited no abnormalities. By 10 wk, mononuclear cells were present but diminished in the allografts (Figure 1c), and by 15 wk, the allograft airway was scarred and contracted, with dense, fibrous tissue circumferentially and minimal luminal cellularity. Isografts (Figure 1f) and SCID allografts (Figure 1i) at 10 wk retained normal airway architecture. Few neutrophils or eosinophils were present in allografts, SCID allografts, or isografts at any of the time points of examination.

View larger version (122K): [in this window] [in a new window]   Figure 1.   H&E-stained mouse tracheal grafts. (a-c) Allograft at 2, 6, and 10 wk, respectively. (d-f ) Isograft at 2, 6, and 10 wk, respectively. (g-i) SCID C57BL/ 6 allograft at 2, 6, and 10 wk, respectively. L = lumen; C = cartilage. Paraffin sections 6 µm thick. Magnification: ×40 all images.

Immunohistochemistry

At 2 wk, allograft sections showed an abundance of CD4⁺(Figure 2a) and CD8⁺ (Figure 2d) lymphocytes, with a CD4⁺/CD8⁺ ratio of 0.75 (Figure 3, top). The mean cell count of CD4⁺ lymphocytes per allograft section was 220 ± 32.6 (mean ± SD) (Figure 3, top), compared with 21 ± 6.2 CD4⁺ lymphocytes per isograft (P < 0.001) (Figure 4, top), and the mean cell count of CD8⁺ lymphocytes per allograft section was 295 ± 22.0 (Figure 3, top), compared with 15 ± 7.7 CD8⁺ lymphocytes per isograft (P < 0.001) (Figure 4, top). SCID allografts revealed rare numbers of CD4⁺ lymphocytes but no CD8⁺ lymphocytes (data not shown). Allograft sections also exhibited moderate numbers of B lymphocytes in a peritracheal distribution (Figure 2g), with a mean of 87 ± 31.7 cells (Figure 3, top), compared with isograft sections, with a mean of 20.8 ± 3.0 cells (P < 0.05) (Figure 4, top), which stained positive for B cells in serosal areas of preserved bronchial-associated lymphoid tissue (BALT) (data not shown). SCID allografts did not contain B lymphocytes (data not shown). Allografts were infiltrated with large numbers of macrophages (Figure 5a), with a mean of 102.8 ± 10.7 (Figure 3, bottom), as compared with isografts, which had a mean of 19.2 ± 2.2 macrophages (P < 0.001) (Figure 4, bottom). SCID allografts also stained for large numbers of macrophages (data not shown). As expected, 2-wk allografts (Figure 5d), SCID allografts, and isografts demonstrated staining for myofibroblasts along the posterior membrane of the trachea and small-vessel walls.

View larger version (104K): [in this window] [in a new window]   Figure 2.   Allograft immunohistology, CD4+, CD8+, and B lymphocytes. (a-c) CD4+ (red) lymphocyte at 2, 6, and 10 wk, respectively; magnification: ×40. (d-f ) CD8+ (blue) lymphocyte at 2, 6, and 10 wk, respectively; magnification: ×40. (g and h) B (red) lymphocyte at 2 and 6 wk, respectively; magnification: ×20. Sections are frozen, 12 µm thick, and without counterstain. L = lumen; C = cartilage.

View larger version (22K): [in this window] [in a new window]   Figure 3.   Allograft cellular recruitment at 2, 6, 10, and 15 wk. (Top panel ) CD4+, CD8+, and B lymphocytes. (Bottom panel ) PMN, macrophages, and myofibroblasts. Note difference in ordinance scale compared with Figure 4.

View larger version (21K): [in this window] [in a new window]   Figure 4.   Isograft cellular recruitment at 2, 6, 10, and 15 wk. (Top panel ) CD4+, CD8+, and B lymphocytes. (Bottom panel ) PMN, macrophages, and myofibroblasts.

View larger version (140K): [in this window] [in a new window]   Figure 5.   Allograft immunohistology, macrophages, and myofibroblasts. (a-c) Macrophage (red) at 2, 6, and 10 wk, respectively; magnification: ×40. (d-f ) Myofibroblast (red) at 2 wk, magnification: ×20; 6 wk, magnification: ×20; and 6 wk, magnification: ×40. Sections are frozen, 12 µm thick, and without counterstain. L = lumen; C = cartilage; S = submucosa.

At 6 and 10 wk, allograft sections revealed a predominance of macrophages in a maturing, luminal, connective-tissue matrix (Figures 5b and 5c), with means of 117.8 ± 22.3 and 104.7 ± 14.1, respectively (Figure 3, bottom), as compared with CD4⁺ lymphocytes (Figures 2b and 2c), with means of 96.5 ± 10.1 and 40.6 ± 7.3 (Figure 3, top), and CD8⁺ lymphocytes (Figures 2e and 2f), with means of 49.7 ± 8.6 and 28.5 ± 4.9 (Figure 3, top), respectively, at 6 and 10 wk. The CD4⁺/CD8⁺ ratio in allograft sections was approximately 2:1 at 6 wk and 1.5:1 at 10 wk. B lymphocytes were scant at 6 wk (Figure 2h) and not evident at 10 wk in allografts. Myofibroblasts peaked at 6 wk in allograft sections and were dispersed within the lumen, as with the macrophage distribution (Figures 5e and 5f). Compared with isografts at 6 and 10 wk, allograft sections had significantly greater numbers of macrophages (P < 0.05), CD4⁺ and CD8⁺ lymphocytes (P < 0.001, P < 0.001), and myofibroblasts (P < 0.05) (Figures 3 and 4). SCID allografts did not stain positively for macrophages, T lymphocytes, or B lymphocytes at 6 or 10 wk.

By 15 wk the allografts had diminished cellularity. The mean number of CD4⁺ lymphocytes was 37.8 ± 3.0 (Figure 3, top), that of CD8⁺ lymphocytes was 25.5 ± 3.7 (Figure 3, top), and that of macrophages was 35.8 ± 4.1 (Figure 3, bottom). Allografts had significantly greater numbers of macrophages (P < 0.001) and of CD4⁺ and CD8⁺ lymphocytes (P < 0.001, P < 0.05, respectively) than did isografts. No CD4⁺ lymphocytes, CD8⁺ lymphocytes, macrophages, or B cells were present in SCID allografts at 15 wk. Companion sections were not available for myofibroblast staining at this time point, as the 15-wk grafts were severely scarred, with minimal tissue.

	Discussion

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In this study we characterized the immune cells infiltrating tracheal grafts during the development of airway injury and fibrosis as a first step to investigating the pathogenesis of OB in a mouse model. Our results reproduced the work of Hertz and colleagues (15), who found subepithelial inflammation, epithelial necrosis, denudation, squamous metaplasia, and fibroproliferative changes in allografts 10 d after transplantation, with 100% fibroobliteration at 21 d, whereas isografts exhibited no fibroproliferation. Our time course to fibroobliteration was delayed by the effect of cyclosporin treatment, as with recent findings by King (16).

The early and abundant lymphocyte infiltrate into trachael allografts, with a CD8⁺ predominance, is consistent with acute cellular rejection. At later time points the histopathology was more consistent with chronic rejection, with a macrophage and CD4⁺ predominance. These results suggest that acute and chronic airway rejection probably constitute a continuum of the host immune response. They support the clinical observations of a strong association between acute rejection and the development of chronic rejection (9, 19, 20), and an increased prevalence of OB in patients with histopathologic evidence of lymphocytic bronchitis/bronchiolitis (21), which may precede submucosal scarring and airway fibrosis (22). Acute cellular airway rejection may therefore be an underappreciated predecessor of OB. Second, these results confirm the immune nature of OB, since SCID allografts failed to develop epithelial injury and airway fibroobliteration. Although other factors (e.g., ischemia, cytomegalovirus (CMV), and infection) may influence a variety of immune pathways and thus play a role in allograft rejection, the failure of the SCID allografts to develop OB provides strong evidence that immune injury is the dominant mechanism of OB.

Our findings of airway obliteration and increased numbers of lymphocytes and macrophages at 2 wk, with macrophage and myofibroblast predominance at later time points, were similar to preliminary observations in the heterotopic rat-airway model of OB (23). By Day 28, heterotopically placed rat allografts demonstrated nearly complete airway occlusion and increased numbers of macrophages, CD4⁺ and CD8⁺ lymphocytes, and -smooth-muscle actin as compared with isografts. The finding that immunosuppressive drugs suppress airway inflammation in the rat implicated a role for immune injury (in particular, cell-mediated injury) in OB.

Using the rat orthotopic lung-transplant model to study chronic rejection, Uyama and coworkers (24) found epithelial ulceration, lymphocyte infiltration, and granulation tissue protruding into the bronchial lumen after 100 d, with a CD4⁺ lymphocyte predominance, increased local expression of MHC class II antigens and B lymphocytes, and decreased numbers of macrophages, but no unequivocal evidence of OB (25). Our finding of CD4⁺ lymphocyte persistence at 6 wk, during the fibroproliferative phase of rejection, was similar to that of Uyama and coworkers. However, our results differed because increased numbers of macrophages were present at many time points, suggesting that delayed-type hypersensitivity and cell-mediated direct cytotoxic responses may be present in this mouse model. Compared with the heterotopic mouse model, which developed epithelial injury and exhibited histopathologic effects consistent with OB, the orthotopic rat-lung model demonstrated more intense vascular and parenchymal inflammation injury, and only modest airway injury (13, 14).

Immune injury has been demonstrated in non-lung (heart, liver, kidney) animal models of chronic rejection, and is manifested by a fibroproliferative endarteritis or fibroobliterative lesions of organ tissue, such as hepatic bile ductules and renal tubules. Rat cardiac heterografts developed focal graft arteriosclerosis with a macrophage-predominant infiltrate as well as T cells, natural killer (NK) cells, and small numbers of B cells within arterial endothelium at 4 wk after transplantation, followed by a smooth muscle cell and macrophage predominance after 50 d (26- 28). Rat hepatic allografts demonstrated an infiltrate with CD4⁺ lymphocyte and macrophage predominance from Day 14 to Day 150 after transplantation (29). The rat orthotopic renal transplant model of chronic rejection developed endothelitis, interstitial cellular infiltration and fibrosis, thickening of glomerular basement membrane, mesangial expansion, and glomerulosclerosis, with peritubular capillary and glomerular-basement-membrane (GBM) thickening indicative of chronic rejection after 12 wk (30). Activated macrophages and predominantly CD4⁺ cells were increased in allografts compared with isografts at 24 wk after transplantation (31, 32). Hancock and coworkers (33) found a mononuclear cell infiltrate in both allografts and isografts; however, activated macrophages were detected in allografts at Week 12 and Week 24 versus isografts. Our findings of a mixed infiltrate of lymphocytes and macrophages at later time points are similar to the findings made in non-lung models of chronic rejection.

Human studies of chronic airway rejection have also focused on putative mechanisms of cell-mediated immune airway injury. They have demonstrated a spectrum of CD4⁺/CD8⁺ ratios in OB as opposed to acute rejection, infection, and clinically quiescent states. Some clinical studies have found an increased CD4⁺/CD8⁺ ratio in bronchoalveolar lavage fluid (BALF) of patients with OB (34), or a CD8⁺ predominance in BALF (35) and lung tissue 6-12 mo after transplantation (36, 37), but others have found no difference between T-lymphocyte subsets in acute rejection and chronic rejection (38). As in studies of the animal kidney, Winter and colleagues have identified B lymphocytes and immunoglobulin deposition around vessels in transbronchial biopsies from heart-lung transplant patients with OB (39), as have also Hasegawa and associates (40). The results of these clinical studies, taken together, seem to implicate a variety of immune mechanisms (both cell-mediated and humoral) in the pathogenesis of OB. The most consistent finding in clinical studies of OB has been increased numbers of bronchoalveolar lavage neutrophils (41, 42), a result suggesting that coexisting infection may have confounded the analysis of immune-cell typing.

Although the heterotopic mouse airway model appears to be useful in the study of OB, several potential limitations of this model were evident in this study. The tracheal graft is a large airway located subcutaneously, thereby eliminating the air-epithelium interface, and is not primarily vascularized, which may alter the immunopathogenesis of OB in this model. However, since the model reproduces the histopathogy of human OB, these shortcomings do not preclude the use of this model in this study. Although the tracheal graft is a large-airway graft, epithelial injury, submucosal fibrosis, and bronchiectasis have been found in the large airways of patients with known OB (43). In addition, the immunopathogenesis of OB in mice may differ from that in humans, with variable interstrain immune responses (44). However, the mouse immune system is closely homologous to the human genome in terms of MHC loci (45). A further point is that the present study did not evaluate all potential immune responses in OB, such as the presence of NK-like cells in chronic rejection (32), or the role of humoral immunity and immunoglobulin-mediated injury, which have been implicated in other models of chronic rejection (33, 46, 47).

In summary, we have characterized the cellular infiltrate in the mouse airway graft model of OB. Our results suggest an important role for CD4⁺ cells and macrophages in the evolution of airway injury to fibrosis. The finding that the heterotopic mouse airway graft may be a model of mixed acute and chronic rejection has important implications for the understanding of human OB. The rapid destruction of the epithelium in the allografts in the present study, and the lack of it in the SCID grafts strongly suggest that T (and maybe B) cells are critical to allorecognition of the respiratory epithelium, which is an important precursor to airway fibrosis. Having identified important cell types during the evolution of OB, we have directed our attention toward the assessment of T-cell cytokine-gene expression and growth factors, hoping that information derived from these experiments will lead to such therapeutic interventions as an anti-CD4⁺ antibody (48) or anticytokine antibodies against mixed Th1 and Th2 subtypes (49). In the future, identification of specific mechanisms of epithelial-cell injury may provide additional insight into the pathogenesis of OB.