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The Kinetics and Pattern of Tracheal Allograft Re-Epithelialization

Departments of Otolaryngology-Head and Neck Surgery, Surgery and The Institute for Gene Therapy and Molecular Medicine, and The Immunobiology Center, The Mount Sinai School of Medicine, New York, New York

Address correspondence to: Eric M. Genden, M.D., Mount Sinai School of Medicine, Department of Otolaryngology-Head and Neck Surgery, One Gustave Levy Place, New York, NY 10029. E-mail: eric.genden{at}mssm.edu

	Abstract

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Extensive tracheal defects may pose a life-threatening dilemma. Although tracheal transplantation may represent a reconstructive solution, very little is known regarding the immunobiology and behavior of tracheal allografts. The objective of this study was to assess the pattern and kinetics of re-epithelialization of orthotopic tracheal allografts in immunosuppressed recipients. Thirty-eight age-matched mice were randomly assigned to five experimental groups. BALB/c donor tracheal segments were orthotopically transplanted into either syngeneic BALB/c or MHC mismatched allogeneic C57BL/6 recipients with and without immunosuppression. On post-transplant days 7, 14, 28, 48, and 62, animals from each group were evaluated by serial histology, electron microscopy, and serial immunohistochemical analysis for mucosal phenotype, re-epithelialization pattern, and lymphocyte subpopulations. Nonimmunosuppressed recipients underwent recipient-derived basal cell re-epithelialization by Day 48, with differentiation into a sparse population of ciliated columnar epithelium by Day 62, whereas immunosuppressed recipients underwent basal cell re-epithelialization 28 d after transplantation and differentiation into a dense population of ciliated columnar epithelium by Day 48. The re-epithelialization process occurred in a definable pattern that was significantly enhanced with the addition of immunosuppression. Orthotopic tracheal transplants undergo progressive re-epithelialization with recipient-derived basal cells that differentiate into ciliated columnar epithelium in a definable pattern that is enhanced with the addition of immunosuppression.

Abbreviations: cyclosporine A, CsA • ketamine and xylazine anesthesia, KA • ratio of the lamina propria to the tracheal cartilage, LCR • phosphate-buffered saline, PBS

	Introduction

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A significant body of experimental work has been focused on the repair of airway epithelium in an effort to elucidate how these mechanisms may be implicated in inhalation injury, asthma, bronchopulmonary dysplasia, and post–lung transplant bronchiolitis obliterans. A variety of in vitro model systems have been introduced for the study of epithelial regeneration, including primary airway epithelial cells of human and bovine origin (1, 2), and guinea-pig bronchial epithelial cultures (3). Using these systems, it has been determined that epithelial repair is the result of a cascade of events beginning with migration of tracheal basal cells from the margin of the injury to the damaged region, followed by proliferation and differentiation into pseudostratified ciliated epithelium (4–6). This process can be stimulated by several fibroblast-derived growth factors or inhibited by variety of inflammatory mediators (7, 8). Although the process of autologous airway repair has been well defined using these in vitro models, very little is known about the effects of allotransplantation on the airway epithelia, or the role that the epithelium plays in graft survival. The lack of knowledge in this area has a significant impact on our ability to proceed safely with clinical tracheal transplantation.

The expression of epithelial MHC Class I antigen renders the airway epithelium the primary target of allograft rejection in both tracheal and lung allografts. Class II expression is limited to the dendritic cells within the lamina propria. Re-epithelialization with host-derived epithelium could have a significant impact on allograft rejection. In an effort to elucidate the effects of transplantation on the airway epithelium, Hertz and coworkers described a murine heterotopic tracheal transplant model in which donor tracheal segments were transplanted into a subcutaneous pouch in the recipient's neck (9). This work and subsequent work has demonstrated that when donor tracheal segments reject, fibroblasts and macrophages mediate a fibroproliferative response that obliterates the tracheal lumen, a response that can be avoided in the immunosuppressed recipient (10). There are, however, inherent shortcomings to a heterotopic model for the investigation of tracheal transplantation. Transplantation of the graft into a heterotopic recipient site discounts the influence of lymphocyte trafficking and the adjacent syngeneic mucosa on the allograft segment, and therefore does not represent an accurate depiction of the clinical airway system. The immune cells associated with heterotopic tracheal allograft rejection have been characterized (10); however, placement of the graft within the omentum undoubtedly changes the nature of the cellular infiltrate as well as discounts the role of lymphocyte trafficking. Medoff and coworkers (11) and others (12–14) have demonstrated the importance of lymphocyte trafficking and dendritic cell migration in allergic and allograft rejection response within the airway. The orthotopic transplant model preserves the airway continuity in an effort to maintain the immunobiology and physiology of the system. The effect of orthotopic transplantation has been highlighted in work performed by Ikonen and colleagues, who demonstrated in the rat model that re-epithelialization of the tracheal allograft occurred in nonimmunosuppressed recipients, and that this process prevents airway obliteration (15).

Recent work by our group using the murine orthotopic tracheal transplant model supports the earlier work of Ikonen and coworkers in the rat, and we have further demonstrated that rejecting orthotopic tracheal allografts do not obliterate, but rather manifest acute rejection characterized by a T cell infiltrate within the lamina propria, edema of the tracheal lumen, and a loss of the pseudostratified ciliated columnar epithelium (16). The murine recipients clinically manifest rejection with an audible stridor as a result of the infiltrate within the lamina propria; however, the tracheal airway remains patent.

In this study, we hypothesized that the re-epithelialization of the donor tracheal allograft with recipient-derived mucosa occurs in a definable pattern, and that the kinetics of this process can be positively influenced with the addition of immunosuppression.

	Materials and Methods

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Experimental Design
Thirty-eight age-matched mice were randomly assigned to five experimental groups (Table 1). BALB/c donor tracheal segments (five tracheal rings), were orthotopically transplanted into either syngeneic BALB/c or Class I and Class II MHC mismatched allogeneic C57BL/6 recipients. BALB/c and C57BL/6 mice (20 g; Taconic Farms, Germantown, NY) were used at 20 wk of age. In Group I, BALB/c donor tracheal segments were orthotopically transplanted as previously described (16) into syngeneic BALB/c recipients without immunosuppression. In Groups II and III, BALB/c donor tracheal segments were orthotopically transplanted into allogeneic C57BL/6 recipients; Group II was not immunosuppressed and Group III was treated with 7 mg/kg/d of intraperitoneal cyclosporine A (CsA; Sigma, St. Louis, MO). Groups IV and V were composed of C57BL/6 recipients which were heterotopically transplanted with a 10-ring tracheal segment composed of 5 rings of syngeneic trachea and 5 rings of allogeneic trachea, microsurgically sutured together as a single graft. Group IV was not immunosuppressed, whereas Group V was treated with CsA as described above.

Tracheal Grafting Procedure
Subcutaneous ketamine (50 mg/kg) and xylazine (10 mg/kg) (KA) anesthesia was administered preoperatively. Using an operating microscope (Wild M651; Wild Leitz, Willodale, ON, Canada), under KA anesthesia, the donor mouse tracheal segment was exposed through an anterior midline neck incision. Division of the strap muscles enabled identification of the entire laryngotracheal complex. The cervical esophagus and vascular structures were separated from the trachea with careful attention to preserve the recurrent laryngeal nerves. A five-ring circumferential tracheal segment was excised and placed into a glass dish with cooled physiologic saline. The recipient mouse was prepared using an operating microscope (Wild M651; Wild Leitz), under KA anesthesia. Heterotopic recipients underwent a midline laparotomy incision followed by the isolation and implantation of the donor trachea segment into the omentum. The tracheal segment was wrapped in omentum, and the abdomen was closed using 7–0 nylon sutures.

Groups IV and V were composed of allogeneic–syngeneic composite grafts that were created by suturing together ex vivo, a five-ring segment of syngeneic trachea and a five-ring segment of allogeneic trachea. The 10-ring allogeneic–syngeneic composite grafts were then implanted into the recipient omentum using the technique previously described.

The tracheal segment of the orthotopic recipient mouse was exposed through an anterior midline neck incision. Division of the strap muscles enabled identification of the entire laryngotracheal complex. An incision was made in the recipient trachea which resulted in a tracheal gap that easily accommodated the donor tracheal graft. The donor tracheal graft was orthotopically placed in the recipient tracheal defect and oriented such that the proximal end of the native trachea opposed the proximal end of the graft segment, and secured with 10–0 nylon interrupted transtracheal sutures. The strap muscles were approximated and the skin was closed with 7–0 nylon sutures. No oxygenation was administered to either the donor or recipient during the course of the procedure, and postoperatively the animals were placed under a warming lamp and monitored for 3 h.

Clinical Evaluation of Tracheal Airway
Recipient animals were evaluated once daily for airway compromise as manifested by the development of stridor, labored breathing, and decreased activity. If airway compromise, leading to decreased activity was detected, the animal was killed.

Histologic Evaluation
Seven, 14, 28, 48, and 62 d after tracheal transplantation, two mice from each group were killed and the tracheal graft segments were surgically removed. Tracheal segments were initially fixed in cold 10% neutral buffered formalin solution for hematoxylin and eosin (H&E) staining. The entire graft was sectioned at 3-µ intervals and stained with H&E. Six random sections from each graft were assessed for cartilage architecture, cilia ultrastructure, and the presence of lymphocytic infiltrate.

In addition, measurements of the cartilage thickness and the thickness of the lamina propria were determined from three separate predetermined points using computer-assisted morphometry (Zeiss Axioskop microscope, SAMBA 4000 Interactive Imaging Process; Zeiss, Thornwood, NY). The ratio of the lamina propria to the tracheal cartilage (LCR) was calculated and averaged for the three points. This assessment was performed on six random histologic sections from each graft.

Electron Microscopy
Transmission electron microscopy. Sixty-two days after tracheal transplantation, tracheal graft segments were harvested and fixed with 3% glutaraldehyde in phosphate-buffered saline (PBS) for 3 h, washed in PBS buffer, and treated for 1 h with 1% osmium tetroxide, dehydrated in graded steps of ethanol through propylene oxide, and embedded in Embed 812 (Electron Microscopy Sciences, Philadelphia, PA). Representative areas for ultrathin sections were chosen by light microscopy from 1-µm plastic sections stained with methylene blue and azure II. Ultrathin sections were stained with uranyl acetate and lead citrate.

Scanning electron microscopic preparation description. The tracheal specimens were removed after dehydration in 100% ethanol. They were then placed in the critical point drier, where the alcohol was exchanged for liquid CO₂. The CO₂ was removed at the critical temperature and pressure and coated with a light coat of gold palladium by sputter coating method. The specimen was then mounted on an aluminum stub and viewed with an S-530 Hitachi scanning electron microscope (Hitachi, Ontario, Canada).

Transmission electron microscopy and scanning electron microscopy were performed on all specimens. Quantification of ciliated cells per high-powered field (x40) (HPF) was calculated from three randomly chosen sections for each experimental group and reported as a mean. Scanning electron microscopy was performed and objectively assessed by a pathologist for morphology including cilia density, length, and architecture.

Immunohistochemical Assessment
Ten-micron-thick serial sections were cut from tracheal grafts 7, 14, 28, 48, and 62 d after tracheal transplantation for assessment of mucosal phenotype. BALB/c mouse epithelium was assessed using biotin-conjugated mouse anti-mouse H-2K^d monoclonal antibody (SF1–1.1), and C57BL/6 mouse epithelium was detected using biotin-conjugated mouse anti-mouse H-2K^b monoclonal antibody (AF6–88.5) (BD PharMingen, Boston, MA). Spleen and trachea were used as controls to assess for cross reactivity of the antibodies. Endogenous peroxidase activity is blocked by incubating slides in 0.3% H₂O₂ solution in PBS for 10 min. Slides were then rinsed and the primary antibody (1:200) was applied to the tissue sections and left to incubate at room temperature for 2 h. Slides were then washed with PBS three times, and streptavidin–horseradish peroxidase (Cat # 550,946; PharMingen, Boston, MA) was applied and incubated for 30 min at room temperature. The slides were then washed in PBS three times, and diaminobenzidine was applied and allowed to incubate for 5 min. After rinsing the slides in water, counterstaining with hematoxylin solution was performed for 30 s. The slides were then dehydrated with four changes of alcohol (95–100%), and a coverslip was applied. Serial sections were evaluated individually and in sequence from proximal to distal. The sections were then submitted for three-dimensional analysis.

Three-Dimensional Imaging
The graft segment is serially sectioned into 10-µm sections, every third of which was chosen to render the model. The sections are converted into digitized images and the ciliated cells were enhanced. The images are combined into three-dimensional models with computer image analysis software. The image acquisition required transferring the image from the section to the computer screen, followed by image processing where the objects are manually outlined into closed contours. Two-dimensional contours are formed to create a surface reconstruction. The computer program Surfdriver (www.surfdriver.com) was used to automatically combine 100 sections, each with 10 regions of interest, and allow linear and volume measurements of the reconstructed image to perform the three-dimensional reconstruction.

Immunohistochemical Assessment of Lymphocyte Subpopulation
Seven, 14, 28, 48, and 62 d after tracheal transplantation, two mice from each group were killed and the tracheal graft segments were surgically removed. Routine frozen sections were cut at 5 µ, and picked up on a glass slide, and allowed to dry over night at room temperature. The sections were then fixed in cold acetone (-20°C) for 2 min. The fixed slides were dried for 1 h at room temperature and then rinsed 2–3 times in PBS. Endogenous block of peroxidase with 0.03% H₂O₂ solution in PBS was performed for 10 min. The slides were rinsed in PBS and blocked with 5% normal rat serum. One of two diluted primary antibodies (RM2–4, CD4[L3T4] or 53–6.7, CD8[Ly-2]; BD PharMingen, San Diego, CA) (1:20) was applied and secondary antibody (biotin-conjugated rabbit, anti-rat; BD PharMingen, San Diego, CA), was applied at room temperature for 30 min. The slides were then rinsed in three changes of PBS at 2 min each. Strepavidin–horseradish peroxidase was applied to each slide and allowed to incubate at room temperature for 30 min. The slides were again rinsed in PBS, and diaminobenzidine solution was added to the slides and allowed to incubate for 5 min. The slides were then rinsed in water and counterstained in Hematoxylin, bluing reagent, and ammonia. The slides were then dehydrated, and coverslips were applied.

CD4 and CD8 cells were individually counted using computer-assisted morphometry (Zeiss Axioskop microscope, SAMBA 4000 Interactive Imaging Process; Zeiss). For each group, five slides were examined, and three single high-powered fields were randomly identified for each histologic section and computer enhanced to better identify the stained lymphocytes. The stained lymphocytes were manually counted and recorded.

Statistical Methods
Data in this study are presented as the mean ± SD. For all nonparametric data, a one-way ANOVA was performed as an initial test, followed by a post hoc test. The Dunnett's test was performed to compare control groups with multiple treatment groups (P = 0.05). Statistica v5.0 (Statsoft INC., Tulsa, OK), was used for all data analysis.

	Results

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Group I, Orthotopic Isografts
After transplantation, the orthotopic isografts did not demonstrate stridor or labored breathing at any time during the experimental period. Serial sections of the tracheal isografts assessed at 7, 14, 28, 48, and 62 d after tracheal transplantation demonstrated normal tracheal architecture with pseudostratified ciliated columnar epithelium (Figure 1A). The LCR remained consistent throughout the 62-d experimental period (0.79 ± 0.2) (Figure 2). Immunohistochemical assessment of the tissue origin demonstrated that the orthotopic isografts were epithelialized with BALB/c mucosa at each assessment point (Table 2). Scanning electron microscopy performed on Day 62 demonstrated morphologically normal ciliated epithelium (Figure 3A). Transmission electron microscopy demonstrated that the cilia density (11.13 ± 1.3) was not significantly different from the control epithelium (11.8 ± 2.1; (P < 0.05) (Figure 1B). Immunohistochemical assessment of lymphocyte subpopulations (Figures 4A and 4B) demonstrated the mean values that ranged from 0.7/HPF (CD4) and 0.2/HPF (CD8).

View larger version (518K): [in this window] [in a new window]   Figure 1. (A) Orthotopic tracheal isograft at 48 d after transplantation. Normal tracheal architecture with ciliated pseudostratified columnar epithelium (hematoxylin–eosin; original magnification: x25). (B) Cilia density as calculated by transmission electron microscopy, demonstrating no significant difference in cilia density between Groups I and III when compared with the control epithelium. (C) Nonimmunosuppressed orthotopic tracheal allograft at 48 d after transplantation. Nonciliated basal cell epithelium (large arrow), and fibrosis and fibroblasts within the lamina propria (small arrow). (hematoxylin–eosin; original magnification: x25). Note the increased thickness of the lamina propria. (D) Immunosuppressed orthotopic tracheal allografts 48 d after transplantation demonstrating a normal tracheal architecture with a ciliated pseudostratified columnar epithelium (hematoxylin–eosin; original magnification: x25).

View larger version (74K): [in this window] [in a new window]   Figure 2. Graphic demonstration of the lamina propria to cartilage ratio (LCR) on post-transplant day 48.

View larger version (72K): [in this window] [in a new window]   Figure 3. (A) Scanning electron microscopy of the orthotopic isograft 62 d after transplantation, demonstrating a dense population of normal cilia with interspersed mucus cells (magnification: x2,000). (B) Scanning electron microscopy of the nonimmunosuppressed orthotopic allograft 62 d after transplantation, demonstrating a sparse population of blunted cilia with interspersed mucus cells (magnification: x2,000). (C) Scanning electron microscopy of the immunosuppressed orthotopic allograft 62 d after transplantation, demonstrating a dense population of normal cilia with interspersed mucus cells (magnification: x2,000).

View larger version (172K): [in this window] [in a new window]   Figure 4. (A) CD4 lymphocyte subpopulation demonstrating a pronounced peak in Group II (nonimmunosuppressed tracheal allograft). (B) CD8 lymphocyte subpopulation demonstrating a pronounced peak in Group II (nonimmunosuppressed tracheal allograft).

By Day 48, the allograft segment was lined with recipient-derived basal cells at the distal ends of the graft; however, the mid-graft sections demonstrated the persistence of BALB/c epithelium, and an abundant population of fibroblasts and fibrosis developed within the lamina propria (Figure 1C). By Day 62, a sparse population of recipient-derived pseudostratified ciliated columnar epithelium was present in the allograft segment. Scanning electron microscopy of the nonimmunosuppressed allograft demonstrated blunted cilia that were shorter and less densely populated than the epithelium of the control and Groups I and III (Figure 3B). Similarly, transmission electron microscopy demonstrated that the cilia density was significantly less densely populated (5.8 ± 2.3) than the epithelium of the control and Groups I and III (P < 0.05) (Figure 1B).

Group III, Immunosuppressed Orthotopic Allografts
The immunosuppressed mice that received an orthotopic tracheal transplant did not exhibit an audible stridor. Histologic and immunohistochemical assessment at 3, 7, 14, 28, 48, and 62 d after tracheal transplantation demonstrated a more efficient progressive re-epithelialization of the allograft segment than the nonimmunosuppressed group (Group II). Epithelial migration into the allograft segment was consistently demonstrated on Day 3, and by Day 14 the mid-allograft segment was composed of a mixture of both donor- and recipient-derived mucosa (Table 2). The migration of the recipient-derived mucosa can be demonstrated on longitudinal sections of the allograft segment (Figure 5). The immunosuppressed allograft was completely populated by recipient-derived basal cells by Day 28, and by Day 48 the entire allograft had been re-epithelialized with recipient-derived pseudostratified columnar ciliated epithelium (Figure 1D). Scanning electron microscopy performed on the immunosuppressed tracheal allografts on Day 62 demonstrated morphologically normal ciliated epithelium (Figure 3C). Transmission electron microscopy demonstrated that the cilia density (10.1 ± 2.1) was not significantly different from the control epithelium (11.8 ± 2.1) or the isograft (11.13 ± 1.3), but significantly more dense than the nonimmunosuppressed allografts (Figure 1B) (P < 0.05). The LCR remained consistent (0.78 ± 0.26) and did not significantly increase relative to the Group I or the control epithelium (P < 0.05) (Figure 2). Immunohistochemical assessment of the lamina propria confirmed a negligible inflammatory response and demonstrated a lymphocyte subpopulation with mean values that ranged from 0.0/HPF (CD4) and 0.3/HPF (CD8) (Figures 4A and 4B).

View larger version (110K): [in this window] [in a new window]   Figure 5. Longitudinal histologic section of the allograft trachea demonstrating immunohistochemical staining of the recipient-derived basal cells (large arrow), and the nonstaining donor-derived basal cells (small arrow).

View larger version (126K): [in this window] [in a new window]   Figure 6. Histologic section of the immunosuppressed tracheal allograft on Day 28, demonstrating cilia (large arrow) as it progresses laterally and circumferentially toward the nonciliated region (small arrow), (hematoxylin–eosin; original magnification: x25).

View larger version (49K): [in this window] [in a new window]   Figure 7. Three-dimensional computer representation of orthotopic tracheal allograft re-epithelialization with pseudostratified ciliated epithelium. The initial allograft (A) is progressively re-epithelialized from the membranous aspect of the graft (B), and is eventually completely re-epithelialized (C).

View larger version (81K): [in this window] [in a new window]   Figure 8. Composite heterotopic tracheal graft 48 d after transplantation. Sections from the proximal (top left), middle (top middle), and distal (top right) aspect of the allogeneic portion of the composite graft. The allogeneic graft adjacent to the syngeneic graft (sections 0–50) remains patent as a result of syngeneic-derived epithelial migration (top left) Progressive obliteration of the graft occurs in middle (sections 51–100) and distal sections (101–150) (top middle and top right, respectively) (hematoxylin–eosin; original magnification: x10).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Although the mechanisms of tracheal re-epithelialization have been well characterized, a paucity of experimental work exists regarding the behavior of transplanted airway epithelia. A significant body of experimental work using the rodent heterotopic tracheal transplant model has been dedicated to elucidating the mechanisms of tracheal allograft behavior; however, the inability to study the dynamics of airway epithelium in an in vivo orthotopic allograft system has limited experimental progress. The orthotopic tracheal transplant model affords a unique opportunity to study the behavior of airway epithelia in an in vivo allograft system. Using this model, we have elucidated the characteristic pattern and kinetics of tracheal allograft re-epithelialization, a process that may play an essential role in the future of tracheal transplantation.

Irrespective of the state of immunosuppression, we have confirmed earlier work by Ikonen and coworkers (15) demonstrating that orthotopic tracheal allografts undergo re-epithelialization with recipient-derived mucosa; however, for the first time we have demonstrated that immunosuppression positively impacts the kinetics of this process. Re-epithelialization appears to begin at the membranous trachea, which likely reflects the rich blood supply and diffusion of growth factors in this area. Barrow and associates (17) and others (3, 18), have previously suggested that factors such as vascular endothelial growth factor, calcitonin gene–related peptide, and tachykinin substance P are both mitogenic and chemotactic for airway epithelium (3, 18, 19). Persson and colleagues later demonstrated that the re-epithelialization process in autologous epithelia systems is dependent on a variety of microcirculation-derived growth factors which may diffuse from the membranous trachea (20). We have shown that the allograft undergoes a repopulation, first by recipient-derived basal cells, which begins within 72 h after transplantation, then by a progressive differentiation into a pseudostratified ciliated columnar epithelium. During the first 14–21 d, the allograft exists as a chimera composed of both donor- and recipient-derived epithelium; however, over the course of 48 d in the immunosuppressed recipient, the epithelial converts to a recipient-derived phenotype. Although re-epithelialization occurs irrespective of the state of immunosuppression, the process occurs more quickly and more densely in the immunosuppressed recipients. The migration of basal cells into the allograft segment is dependent on the formation of lamellipodia or cell protrusions, which serve to create focal contacts that anchor the basal cell and enable migration (21). Such adhesions can be broken by proteolytic enzymes and inflammatory cell mediators that are abundant during allograft rejection. The administration of anti-inflammatory immunosuppressive agents, such as CsA, likely prevent the secretion of inflammatory factors or modulate the expression of negative mediators, resulting in a more efficient re-epithelialization process as demonstrated by a higher density of morphologically normal ciliated columnar cells in the immunosuppressed recipients.

The heterotopic composite transplant composed of syngeneic and allogeneic trachea, demonstrates the essential role of syngeneic epithelium in preventing a fibroproliferative response. The cellular infiltrate associated with rejection of the heterotopic tracheal allograft is followed by a period of intense fibrosis mediated by macrophages and fibroblasts (10). The syngeneic epithelial cells that lie adjacent to the allogeneic graft likely influence the proliferation of macrophages and fibroblasts. We found in serial sections of the composite heterotopic grafts that the presence of the syngeneic epithelium prevented obliteration of the adjacent allograft; however, the distal allograft, where syngeneic mucosa had not re-epithelialized, became progressively obliterated. This response was not reversed with the addition of CsA 21 d after transplantation, underscoring the essential role of syngeneic epithelium. The protective influence of the syngeneic mucosa on the adjacent allograft may be mediated by such anti-inflammatory cytokines as IL-10. Boehler and coworkers used adenoviral-mediated IL-10 gene transfection to mitigate the obliterative response in heterotopic allografts (22), and subsequently, Dosanjh and colleagues demonstrated that epithelial cell–derived IL-10 can regulate the proliferation of pulmonary fibroblasts in vitro, and suggested that dysregulation of IL-10 may play a role in the fibroproliferative response (23). The specific role of such cytokines remains ill defined.

Prior work demonstrates that the tracheal epithelium is the target of allograft rejection and suggests that a change in the immunologic character of the epithelium may have a significant impact on tracheal allograft rejection (16). In this study, we withdrew immunosuppression after the tracheal allograft had been re-epithelialized with recipient-derived mucosa, and found that the allograft did not manifest histologic evidence of rejection. These findings suggest that the tracheal allograft may be serving as a biologic scaffold, and that the conversion of mucosa from allogeneic to syngeneic mucosa may confer long-lasting allograft tolerance. The presence of ciliated epithelium is essential to a normally functioning tracheal allograft to prevent stasis of secretions and airway obstruction during the interim period of re-epithelialization. In an effort to reduce antigen expression, investigators have attempted such novel approaches as denuding the airway epithelium before transplantation; however, this approach has been unsuccessful, largely because the epithelium protects against a fibroproliferative obliteration of the airway lumen (24).

We have demonstrated that orthotopic tracheal allografts undergo a progressive re-epithelialization with recipient-derived mucosa in a definable pattern, and that this process protects against a fibroproliferative response even after the withdrawal of immunosuppression. These findings may have significant implications regarding the ability to achieve tracheal transplantation without persistent immunosuppression.

	Acknowledgments

 
This study was supported by NIH Training Grant 1K08DC00199-01.

Received in original form October 14, 2002

Received in final form November 27, 2002

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