INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

With improvements in surgical technique, immunosuppression, and postoperative management, early outcome after lung transplantation has improved significantly, such that the major obstacle to long-term survival is chronic graft dysfunction manifest clinically as bronchiolitis obliterans (1). Clinical features include increasing dyspnea and progressive airflow obstruction (2), and pathologic features include inflammation of the small airways followed by airway occlusion with fibrous tissue (3). To date, no effective therapy against this fatal complication has been developed. Alloantigen-dependent (acute rejection) as well as alloantigen-independent factors (ischemia and reperfusion injury, infectious episodes) may contribute to local injury within the airways. This injury triggers an invasion of inflammatory cells and an increase in inflammatory mediators followed by an uncontrolled repair process, with fibroproliferative tissue filling the airway lumen and finally leading to complete fibrous obliteration (4, 5). In other solid organ transplantation (e.g., cardiac) the endothelium seems to be the target of chronic rejection, characterized by vasculopathy with intimal thickening and fibrosis of most graft vessels (6). In contrast, it has been suggested that the injury leading to chronic rejection in the transplanted lung is primarily directed against the bronchiolar epithelium, and we have shown that lymphocytic/mononuclear cell infiltration in the airway might be the precursor of later airway obliteration (5).

Cytokines released by inflammatory cells in an allograft are important mediators in acute allograft rejection (7). Both T-cell products and macrophage products have been associated with acute rejection. T-helper 1 (Th1)-type cytokines (e.g., interleukin [IL]-2, interferon [IFN]-, IL-12) are typically elevated during acute rejection, whereas a Th2-dominant pattern of cytokine expression (IL-4, IL-10) can be found in experimental long-term allograft survival (8). However, little is known about the expression of these cytokines in the setting of bronchiolitis obliterans.

Chemokines, another family of cytokines, activate and attract specific leukocyte subsets and lead to extravasation of these cells at the site of inflammation (9). Chemokines have been divided into different families based on the position of their cysteine residues (10, 11). A subgroup of the  (C-X-C) chemokine family represented by human IL-8 and its functional rodent correlate macrophage inflammatory protein-2 (MIP-2) (12) (containing the sequence glutamic acid-leucin-arginine preceding the C-X-C sequence) primarily attracts neutrophils. In contrast, members of the  (C-C) chemokine family, including monocyte chemoattractant protein-1 (MCP-1) and regulated upon activation, normal T cell expressed and secreted (RANTES), are chemoattractants mainly for monocytes and lymphocytes. RANTES has been found to be upregulated in acute rejection after human kidney transplantation (13), and MCP-1 has been found to be expressed in chronically rejecting murine heart transplants (14).

Since bronchiolitis obliterans is characterized in its early stages by proliferation of itinerant cells (lymphocytes and macrophages), we hypothesized that cytokines and chemokines may play a role in the orchestration of the ongoing recruitment and activation of these cells and act together with growth factors in the fibroproliferative phase.

Thus, the aims of this study were: (1) to characterize the time course of intragraft cytokine gene expression in a heterotopic rat tracheal allograft model of post-transplant bronchiolitis obliterans; and (2) to investigate expression of C-X-C and C-C chemokine genes in the development of post-transplant airway obliteration in this model.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animals

Brown-Norway rats (male, 250 g) were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, IN) and Lewis rats (male, 250 g) from Charles River Canada, Inc. (St. Constant, PQ, Canada). The animals received standard laboratory rat food and water ad libitum. No immunosuppressive drugs were given. All animals received care in compliance with the Principles of Laboratory Animal Care, formulated by the National Society for Medical Research, the Guide for the Care and Use of Laboratory Animals, prepared by the National Academy of Science and published by the National Institutes of Health (NIH Publication No. 85-23, revised 1985), and the Guide to the Care and Use of Experimental Animals, formulated by the Canadian Council on Animal Care. The experimental protocol was approved by the Animal Care Committee of The Toronto Hospital Research Institute.

Heterotopic Tracheal Transplant Model and Experimental Groups

The heterotopic rat tracheal transplant model used has been previously described in detail (5, 15). In brief, Brown-Norway rats served as tracheal donors; tracheas were transplanted subcutaneously into the necks of recipient animals, either isogeneically into Brown-Norway rats or allogeneically into Lewis rats. We have previously described a distinct triphasic time course in the evolution of the allograft lesions (an initial ischemic phase is followed by a lymphocytic infiltrative phase, and finally a phase in which the tracheal lumen is gradually occluded by fibroproliferative tissue) (5). In this model allografts reproducibly develop complete fibrous airway obliteration 3 wk after transplantation, with morphology that is very similar to human bronchiolitis obliterans, whereas isografts remain essentially normal (5). In the present study, tracheal grafts were harvested 3, 7, and 21 d after transplantation as representative time points for the three phases of injury in allografts. For each time point six isografts and six allografts were harvested. Six normal tracheas served as controls. At harvest, each graft was snap frozen in liquid nitrogen and stored at 70° C until RNA extraction.

RNA Extraction

Total RNA was extracted using the guanidium isothiocyanate method as described by Chomczynski and Sacchi (16). Frozen tissue samples were crushed in a liquid nitrogen-cooled mortar. The crushed powder was mixed with 5 ml of 4 M guanidium isothiocyanate buffer (Gibco/ BRL, Gaithersburg, MD) containing 1.5% of freshly added beta-mercaptoethanol, and homogenized with a Polytron tissue homogenizer (Brinkman, Mississauga, ON, Canada) for 1 min at medium speed. The lysate was mixed with 0.1 ml of 2 M sodium acetate, pH 4.2, followed by the addition of 5 ml phenol, pH 4.5, and 1 ml chloroform:isoamyl alcohol (49:1). The aqueous phase was collected after centrifugation at 8,000 rpm for 40 min at 4° C with a high-speed centrifuge (J2-21 centrifuge, JA-17 rotor; Beckman, Fullerton, CA). The supernatant was transferred to a new tube and RNA was precipitated by mixing with equal volume of isopropanol. RNA concentration was measured with a spectrophotometer (Beckman DU 640B).

Reverse Transcriptase-Polymerase Chain Reaction

Total RNA (3 µg) was used for the reverse transcriptase-polymerase chain reaction (RT-PCR), using a Superscript II kit (Gibco/BRL), according to the manufacturer's instructions. The complementary DNA (cDNA) synthesis reaction was performed for all samples simultaneously using the same enzyme batch. The RT reaction product from 0.2 µg of total RNA was used for PCR. PCR primers specific for rat IL-10 (17), and -actin (18) were synthesized by ACGT Corporation (Toronto, ON, Canada). PCR primers for rat IL-2 and IFN- were obtained from Clontech (Palo Alto, CA), and rat MCP-1, MIP-2, and RANTES were purchased from Biosource (Camarillo, CA). The PCR reaction mixture was set up in a total volume of 50 µl, containing 5 µl 10× PCR buffer (200 mM TRIS-Cl, pH 8.4, 500 mM KCl), 1.5 µl 50 mM MgCl₂, 1 µl 10 mM dNTP mix, 0.7 µl of each PCR primer (10 µM), and 0.5 µl Taq polymerase (Gibco/BRL). PCR was performed with a programmable thermal cycler (PTC-100; MJ Research, Watertown, MA). Optimal PCR conditions were determined for each cytokine from pilot experiments. Briefly, the annealing temperature, the number of PCR cycles used, and the predicted molecular size of each cDNA were: -actin (56° C for 1 min, 25 cycles, 539 base pairs [bp]); IL-2 (60° C for 1 min, 38 cycles, 398 bp); IFN- (60° C for 1 min, 38 cycles, 288 bp); IL-10 (56° C for 1 min, 35 cycles, 346 bp); RANTES (60° C for 45 s, 30 cycles, 176 bp); MCP-1 (60° C for 45 s, 25 cycles, 266 bp); and MIP-2 (60° C for 45 s, 30 cycles, 219 bp) (Figure 1).

View larger version (23K): [in this window] [in a new window]   Figure 1.   Representative samples of RT-PCR products for different cytokines and chemokines. Total RNA extracted from rat tracheal tissue was subjected to RT-PCR with primers for -actin, IL-2, IFN-, IL-10, RANTES, MCP-1, and MIP-2. PCR products were resolved on 1.5% agarose gel containing ethidium bromide and were visualized by a gel documentation system. The depicted bands represent typical examples of gene expression in tracheal allografts after transplantation.

For each cytokine examined, all samples were amplified in the same PCR run using the same batch of reaction ingredients (e.g., primer dilution and enzyme) to avoid interassay variations. Ten microliters of the PCR product were loaded on a 1.5% agarose gel with ethidium bromide staining for visualization.

Quantification of RT-PCR Products

The gels were photographed and the optical densities (OD) of each band of the PCR-amplified fragments were measured on a gel documentation system (Gel Doc 1000; Bio-Rad, Mississauga, ON, Canada) with an integrated image analysis software (Molecular Analyst/PC software, Version 1.5; Bio-Rad, Hercules, CA). The system's volume integration feature was used to express total OD in each amplified PCR product band in gel documentation system units. Specific OD of each band was calculated by subtracting the background locally. The relative OD levels of the different mRNA transcripts were then measured in all samples. PCR amplification with the -actin housekeeping gene from the same cDNA preparation was performed to normalize the efficiency of RNA extraction and RT reaction from the same sample. The mean OD value was calculated from duplicate analyses from the same sample. Corrected values were obtained by dividing the measured value for the transcript of interest by the mean -actin value of that specimen.

Statistical Analysis

The data are expressed as mean ± SEM. Differences between groups were analyzed using one-way analysis of variance, followed by Student-Newman-Keuls test for comparisons between two groups. Data analysis was performed with the SigmaStat version 1.0 statistical software (Jandel Scientific, San Rafael, CA).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Distinct histologic features after airway transplantation occurring at the key time points chosen in this study are shown in Figure 2. Previously, we have quantitatively assessed the histologic changes occurring over time in this model and have documented three different phases leading to tracheal fibro-obliteration (5). In the present study, specimens were taken at Day 3 after transplantation, representing the ischemic phase in which both allografts and isografts showed mild loss of surface epithelium, vascular congestion, and edema (Figure 2A). The second time point for harvest was selected at Day 7, to represent the lymphocytic phase in allografts, with a marked mononuclear cell infiltration of the airway wall being most concentrated in the regenerated epithelium (Figure 2B), whereas isografts had recovered to an almost normal-appearing trachea. The third time point examined was 21 d after transplantation. This represented the obliterative phase, where allografts demonstrated circumferential epithelial loss with complete tracheal occlusion by fibroproliferative tissue (Figure 2C). Isografts at Day 21, in contrast, showed a well-preserved tracheal lumen with normal-appearing ciliated epithelium (Figure 2D).

View larger version (129K): [in this window] [in a new window]   Figure 2.   Composite photomicrograph of rat tracheal cross sections at representative time points after tracheal transplantation. (A) Tracheal allograft 3 d after transplantation, showing moderate loss of epithelium, vascular congestion, and edema (hematoxylin and eosin, magnification ×160). (B) Tracheal allograft 7 d after transplantation, showing marked mononuclear cell infiltration of the airway wall being concentrated in the regenerated epithelium (hematoxylin and eosin, magnification ×160). (C ) Tracheal allograft 21 d after transplantation, demonstrating circumferential epithelial loss with complete luminal occlusion by fibroproliferative tissue (elastic trichrome, magnification ×120). (D) Tracheal isograft 21 d after transplantation, showing a well-preserved tracheal lumen with normal-appearing ciliated epithelium (elastic trichrome, magnification ×120). Cart stands for cartilage and eli stands for elastic lamina interna of the mucosa.

RT-PCR Assays

The goal of this study was to examine simultaneous gene expression of several cytokines within the same specimen. Being limited by the yield of total RNA obtained (each tracheal graft provides only a very small amount of total RNA), a semiquantitative PCR technique and not the competitive quantitative PCR technique, which requires significantly more RNA, was applied.

Stable -actin gene expression has been shown in other lung injury models. In this study, the -actin gene was stable and consistently expressed in all the samples (data not shown), and was used for normalization of mRNA levels of cytokine and chemokine gene transcripts among the different study groups.

Intragraft Expression of Cytokine Genes during Post-transplant Airway Obliteration

To investigate the potential role of cytokines during post-transplant airway obliteration, we first examined the expression of cytokine genes during the development of airway obliteration. As representative cytokines of the Th1-type, IL-2 and IFN- were chosen, and IL-10 was examined as an example of a Th2-type cytokine. Results are shown in Figure 3.

View larger version (23K): [in this window] [in a new window]   View larger version (21K): [in this window] [in a new window]   View larger version (32K): [in this window] [in a new window]   Figure 3.   Intragraft cytokine gene expression during the development of post-transplant airway obliteration. Tracheas of Brown-Norway rats were transplanted subcutaneously into the neck of Brown-Norway rats (isografts) or Lewis rats (allografts). The grafts were harvested 3, 7, and 21 d after transplantation (as representative time points for the three phases in the development of airway obliteration: ischemic phase, lymphocytic phase, obliterative phase). Normal tracheas served as control. Total RNA was prepared and was analyzed by RT-PCR. This figure represents a composite picture. On the bottom transcript analysis of the specific cytokine gene is shown. Normalized OD values of the PCR-amplified fragments were obtained by dividing the specific value for the transcript of interest by the mean -actin value of that specimen. The expression of -actin was constant after transplantation and thus served as internal control to assess variations in cDNA or total RNA loading between samples. Normalized cytokine gene transcript levels of each group are presented as bar graphs, and each bar represents the mean ± SEM values of three to four animals. On the top an ethidium bromide-stained 1.5% agarose gel is depicted showing a representative sample of the PCR product from each group. (A) IL-2 gene was significantly upregulated 3, 7, and 21 d after transplantation in allografts compared with isografts and normal tracheas (p < 0.01). (B) IFN- gene was significantly upregulated after transplantation in allografts compared with isografts and normal tracheas (p < 0.05). (C ) IL-10 gene was expressed in normal tracheas; isografts and allografts and transcript levels were not significantly different between groups (p = 0.089). N stands for normal trachea.

At 3, 7, and 21 d after allo-transplantation, IL-2 and (to a lesser degree) IFN- gene transcripts were highly upregulated in the allografts and showed a marked difference in gene expression compared with isografts and normal tracheas, in which these gene transcripts were not at all or only barely detectable. Increasing the number of PCR cycles for these two cytokines up to 38 cycles maintained the obvious and distinct expression pattern of a "black and white picture," reflecting gene expression in allografts versus no gene expression in isografts. Upregulation of Th1-type cytokines in other solid organ allografts (in contrast to isografts) has been shown during acute rejection; however, the striking finding in our study was that the IL-2 and IFN- genes were still expressed late after transplantation, when fibrous airway obliteration was already complete.

In contrast to the Th1-type cytokines, which showed significant differences in gene expression between isogeneic and allogeneic groups, the Th2-type cytokine IL-10 was expressed in native tracheas, in isografts and in allografts at all three time points examined. The levels of the IL-10 gene transcript were similar between groups and did not change significantly over time (p = 0.089).

Intragraft Expression of Chemokine Genes During the Development of Post-transplant Airway Obliteration

Chemokines are involved in many inflammatory diseases; however, their role in post-transplant airway obliteration is unknown. In the second step of our experiments, we investigated the expression of two C-C chemokines (RANTES and MCP-1) known to be involved in lymphocyte and monocyte recruitment, and the expression of one C-X-C chemokine (MIP-2) with neutrophil-attracting properties. Results are shown in Figure 4.

View larger version (30K): [in this window] [in a new window]   View larger version (32K): [in this window] [in a new window]   View larger version (21K): [in this window] [in a new window]   Figure 4.   Intragraft chemokine gene expression during the development of post-transplant airway obliteration. Transcript analysis of the specific chemokine genes are shown. Normalized OD values of the PCR-amplified fragments were obtained by dividing the specific value for the transcript of interest by the mean -actin value of that specimen. Normalized chemokine gene transcript levels of each group are presented as bar graphs, and each bar represents the mean ± SEM values of three to four animals. (A) RANTES gene was expressed in normal tracheas and isografts and significantly upregulated in allografts 21 d after transplantation compared with normal tracheas (p < 0.05). (B) MCP-1 gene was also expressed in normal tracheas and isografts and significantly upregulated in allografts 3, 7, and 21 d after transplantation compared with normal tracheas and isografts (p < 0.05). (C ) MIP-2 gene was not expressed in normal tracheas, but was highly expressed in both isografts and allografts with peak levels early after transplantation. N stands for normal trachea.

Both C-C chemokine gene transcripts (RANTES and MCP-1) were expressed in normal tracheas, and also in isografts and allografts at all time points examined. The transcript levels of RANTES increased over time after transplantation (although not statistically significant) in isografts and allografts and were highest in allografts at 21 d. By contrast, the transcript levels of MCP-1 decreased over time and were highest in allografts 3 d after transplantation. With regard to airway obliteration, both RANTES and MCP-1 were persistently upregulated until and including when luminal airway obliteration was complete.

MIP-2 is an important chemotactic cytokine for polymorphonuclear leukocytes in rodents. In our study, we found that MIP-2 was not constitutively expressed in normal tracheas but was upregulated after transplantation. MIP-2 gene expression in allografts was highest early after transplantation and decreased to barely detectable levels at 21 d after transplantation. This pattern was also seen in the isografts.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Bronchiolitis obliterans is a common disorder, occurring in approximately 50% of recipients surviving more than 3 mo after lung transplantation surgery. It is thought to be a form of chronic allograft rejection and represents the major cause of morbidity and mortality in the long-term follow-up of these patients (19). The etiology and pathogenesis of bronchiolitis obliterans are still incompletely understood (4), in part because clinical studies are difficult to do, because they require repeated human tissue sampling, and lung tissue specimens obtained by transbronchial biopsy are not only tiny, but often do not contain bronchioli, the target of chronic rejection in the lung (20). Therefore, we examined a rodent model of post-transplant bronchiolitis obliterans, which reproduces the bronchiolitis obliterans-like lesions (5, 21, 22). In this model, only the airways are transplanted, and therefore it is very suitable for selectively investigating local cytokine and chemokine expression at the site of post-transplant airway obliteration.

Our data demonstrate a distinct pattern of cytokine and chemokine gene expression in the development of post-transplant airway obliteration. Cytokine genes known to be involved in acute allograft rejection were persistently upregulated, even after fibrous airway obliteration was complete and intragraft C-X-C and C-C chemokine genes were differentially expressed over time. During the development of airway obliteration, a strong Th1-type response in allografts was observed, whereas no significant changes in IL-10 gene expression representing Th2-type cytokines were found.

Selective upregulation of Th1-type cytokines in solid organ allografts (in contrast to isografts) has been shown during acute rejection in clinical (23, 24) as well as experimental organ transplantation (25). In our study, however, strikingly strong expression of IL-2 and (to a lesser degree) IFN- genes were found not only early but also late after transplantation, when fibrous airway obliteration was already complete. Late-peaking IL-2 expression was in contrast to our initial expectation that highest transcript levels of this cytokine would occur at the time of maximal lymphocyte infiltration of the graft. In this model lymphocytic infiltration of the mucosa (most prominent within the epithelium) and the submucosa peaks around Day 7 (5). On Day 21 the allograft has lost its epithelium and "regenerative" intraluminal fibroproliferation with only occasional scattered lymphocytes is seen. The persistence of the Th1 allo-immune response in this late phase of the development of airway obliteration is in accord with the concept that bronchiolitis obliterans could be the result of an ongoing, persistent injury of the foreign tissue, rather than an initial allo-immune injury followed by an immuno-independent repair process. There is one clinical report describing similar findings in a lung transplant patient, who showed a considerable increase in IL-2 and IFN- transcript levels in transbronchial lung biopsy specimens as the patient progressed from normal histologic status to bronchiolitis obliterans (26).

Our observation could have potentially significant clinical consequences. If an early diagnosis of bronchiolitis obliterans could be made, an increase or change in immunosuppressive therapy (including new drugs or anti-cytokine therapy) could potentially be used to prevent the relentless progression of obliteration. However, with regard to clinical transplantation it has to be noted that upregulation of IL-2 is often minimal and not necessarily associated with acute allograft rejection, in contrast to experimental experience (27). This difference is most likely due to the clinical immunosuppressive regimen, which includes cyclosporine, which specifically inhibits IL-2 gene expression. This indicates that allograft rejection can occur in the absence of IL-2 by activation of other pathways. On the other hand, subtherapeutic cyclosporine blood levels can lead to insufficient suppression of IL-2 transcription with detectable levels of IL-2 in these patients, eventually leading to subclinical acute and chronic rejection.

In our study, the IL-10 gene was abundantly expressed in normal tracheas, isografts, and allografts. In other solid organ transplant models, IL-10 gene was not constitutively expressed and was upregulated only in allografts 2 to 7 d after transplantation (25). IL-10 can be produced by a variety of cell types in addition to T cells. Bonfield and colleagues (28) showed constitutive expression of IL-10 in bronchial epithelial cells. In the tracheal transplant modelin contrast to cardiac allografts, for exampleepithelial cells contribute substantially to the overall tissue examined. It is therefore conceivable that one source of IL-10 might have been these epithelial cells.

Whereas cytokines in the transplant setting have been extensively studied over the past few years, chemokines represent a relatively new field of research and their role in (chronic) allograft dysfunction or rejection is unclear. Chemokines induce cell migration and activation by binding to specific cell-surface receptors on target cells. It has been shown that different cells bear different chemokine receptors (29). Selective upregulation of chemokine receptors on T lymphocytes of either the Th1- or the Th2-phenotype, allowing for selective amplification of a Th1-type or Th2-type immune response, might become of crucial interest in the transplant setting. In our study the two C-C chemokine genes examined (RANTES and MCP-1) were both expressed in normal tracheas and upregulated after transplantation, suggesting a potential differential constitutive expression in various solid organs. RANTES mRNA has been shown to be highly expressed during cell-mediated acute rejection in human kidney grafts but was not detected in native human kidney tissue by in situ hybridization (13). On the other hand, RANTES and MCP-1 have been found to be constitutively expressed in human airway epithelial cells (30, 31), making airway epithelial cells a potential source of these chemokines in our model. With regard to airway obliteration, both RANTES and MCP-1 were persistently upregulated, even after luminal airway obliteration was complete.

Rat MIP-2, a member of the C-X-C chemokine family, is one of the most important chemotactic cytokines for polymorphonuclear leukocytes in rodents and is comparable to IL-8 in humans. In our study, we found that MIP-2 was not constitutively expressed in normal tracheas but was upregulated early after transplantation, with a similar pattern in allografts and isografts. In bronchoalveolar lavage fluid from patients with post-transplant bronchiolitis obliterans, elevated IL-8 protein levels have been found (32). However, these elevated levels might simply reflect concomitant local bacterial infectiona frequent complication after lung transplantation, in the presence or absence of bronchiolitis obliteransrather than being a marker of bronchiolitis obliterans itself. In our model, we histologically excluded infection as a confounding factor for upregulation of MIP-2. In contrast to the findings in Th1-type cytokines, upregulation of this C-X-C chemokine after airway transplantation was not restricted to allografts alone, since it also occurred in isografts. Therefore, whereas increased gene expression of IL-2 or IFN- after airway transplantation seems to reflect the allo-immune response, upregulation of chemokine genes might reflect, in part, alloantigen-independent processes present in allografts and isografts.

This in vivo model of post-transplant bronchiolitis obliterans permits the characterization of cytokine and chemokine gene patterns directly in the target organ in which the interaction between local stimuli and infiltrating cells takes place. Our study shows some very interesting patterns of cytokine and chemokine expression. It is important, however, to note the limitations of this study. First, we have observed changes only at the transcriptional level, which may or may not correlate with the protein levels of these cytokines. However, investigations in other solid organ transplant models have demonstrated a good correlation between cytokine transcript measurements by RT-PCR and protein identification by immunostaining (14). Second, we have not delineated the cellular source of the cytokine and chemokine gene transcripts that were found to be upregulated in this study. Cellular localization might be particularly interesting, because in contrast to chronic allograft rejection in other solid organs, it appears that the airway epithelium may be the target in post-transplant bronchiolitis obliterans. Airway epithelium is important in host defense, which might provide a teleologic reason for constitutive expression of several cytokines and chemokines, which are present in other tissues only after stimulation. Therefore, the cascade of cytokines and chemokines activated in chronic airway rejection might indeed be expected to show a distinct and different pattern compared with other organ grafts. Third, the advantage of RT- PCR technique is its sensitivity and its requirement of only small amounts of tissue, allowing the comparison of many mediators in the same specimen. The disadvantage is that quantification of the results remains a concern, particularly if there are only subtle differences between study groups. Ideally, labeling the PCR products with radioisotopes would enhance quantification; however, in this study, due to the dramatic differences in expression, we were readily able to demonstrate these differences in gene expression using a gel documentation system.

This study has provided further clues to the pathogenetic mechanisms involved in bronchiolitis obliterans. Further studies, however, are required to definitively assess the significance of the gene expression of these cytokines and chemokines in post-transplant airway obliteration. Antisense techniques and knockout animals targeting the cytokine and chemokine pathways upregulated in this model may help clarify their roles in the fibro-obliterative process and may ultimately lead to novel interventions in the prevention or treatment of bronchiolitis obliterans.