Growth Factor Upregulation during Obliterative Bronchiolitis in the Mouse Model Robert M. Aris, Sean Walsh, Worakij Chalermskulrat, Vasantha Hathwar, and Isabel P. Neuringer Divisions of Pulmonary and Critical Care Medicine, Department of Medicine and the Cystic Fibrosis Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina

Obliterative bronchiolitis (OB), or chronic allograft rejection, is a major cause of morbidity and mortality after lung transplantation. The goal of these experiments was to determine whether several important growth factors were upregulated during OB in the mouse heterotopic trachea model. Isografts (BALB/c into BALB/c) and allografts (BALB/c into C57BL/6) were implanted in three sets of cyclosporine-treated animals and were harvested from 2 to 10 weeks. Ribonucleic acid was isolated using the cesium chloride-guanidine method and was reverse transcribed and semiquantitated with the polymerase chain reaction using specific primers for platelet-derived growth factor (PDGF)-A and PDGF-B chains, fibroblast growth factor (FGF) isoforms 1 and 2, transforming growth factor-, tumor necrosis factor- (TNF-), edothelin-1, (prepro) epidermal growth factor, insulin-like growth factor-1, and -actin as a control. Transforming growth factor-, TNF-, endothelin-1, and insulin-like growth factor-1 expression were increased 1.5-fold to 5.0-fold (p  0.04 for each) in the allografts compared with the isografts at Weeks 2 through 6. Significantly increased expression of FGF-1, FGF-2, and PDGF-B was noted in the allografts at 4 weeks (p  0.05 for each), which reversed at 6 and 10 weeks. No differences were found with the PDGF-A chain. The isografts expressed more epidermal growth factor than allografts (p  0.001). Treatment with a TNF-–soluble receptor (human TNFR:Fc) significantly reduced epithelial injury (p  0.01) and lumenal obstruction (p  0.037) in this model. We conclude that increased expression of a large number of growth factors occurs during OB in this model. Growth factor blockade (in particular with regard to TNF-) may be useful in ameliorating OB in this model. Keywords: obliterative bronchiolitis; chronic rejection; lung; growth factors; mouse

Although lung transplantation has become a successful clinical therapy for end-stage pulmonary disease as surgical techniques and immunosuppression regimens have improved, long-term survival of lung transplant recipients has been adversely affected by obliterative bronchiolitis (OB) or chronic graft rejection. In fact, OB is largely responsible for the approximately 30% lower 5-year graft survival rates (i.e., 40% versus 56–76%) between lung (or heart–lung) and other solidorgan transplants (1). OB is an inflammatory disorder that leads to airway injury and fibrosis. It affects approximately 50% of lung transplant patients and is the leading cause of late-transplant deaths (2). Therapies for OB are largely ineffective because little is known about the underlying mechanisms of this disease. For these reasons, OB has been considered the perennial “thorn in the side of lung transplantation” (3).

In the past 7 years, a number of animal models of OB have been developed to investigate the pathogenesis of this disorder and have, quite rapidly, expanded the knowledge base on this problem (4–6). Hertz and colleagues first described the histologic changes of OB in a heterotopic mouse model (7), and subsequently demonstrated the efficacy of cyclosporine in slowing the rate of disease progression (8). The present authors and others have characterized the inflammatory cell recruitment during OB in this model (9, 10). Large numbers of CD4 cells, CD8 cells, and macrophages are present during an early phase of “cellular” airway inflammation. Subsequently, T cell numbers decline, and macrophages and myofibroblasts predominate. The elaboration of cytokines from T cells (both Th1 and Th2) and macrophages during OB suggests the pleiotropic nature of the alloimmune response (11). The pathogenesis of the fibropoliferative phase of OB has generated considerable interest as well because antagonism of important fibrotic pathways may prove beneficial in slowing airway scarring and airflow obstruction. Individually, the platelet-derived growth factor (PDGF)-A and PDGF-B chains and the  receptor, fibroblast growth factor (FGF)-2, and transforming growth factor- (TGF-) have all been implicated in the pathogenesis of human and animal model OB (12–15). In the experiments described herein, we simultaneously studied the expression of a large number of profibrotic cytokines, including PDGF, A and B chains, FGF-1 and FGF-2, TGF-1, tumor necrosis factor- (TNF-), endothelin-1, and insulinlike growth factor-1 (IGF-1) to test the hypothesis that these growth factors are upregulated during the fibro-obliterative process that characterizes airway fibrosis in OB. The time course of study was chosen to encompass fully the progression of OB in the mouse model from cellular (acute-type) rejection with epithelial injury and destruction through the fibroproliferative phase marked by mature lumenal scarring. Second, TNFR:Fc, the soluble TNF- receptor, was administered to determine whether it could slow chronic rejection in this model.

METHODS Mice Seventy-two BALB/c (H2-d) and 16 C57BL/6 (H-2b) (Charles River, Raleigh, NC) were obtained from pathogen-free colonies and were housed and used in accordance with the rules of the Institutional Animal Care and Use Committee.

Tracheal Transplantation and Immunosuppression (Received in original form February 26, 2002; accepted in final form April 16, 2002) Funded in part by the National and North Carolina chapters of the American Lung Association, the Cystic Fibrosis Foundation, and the National Heart, Lung, and Blood Institute. Correspondence and requests for reprints should be addressed to Robert Aris, M.D., CB# 7020, 420 Burnett-Womack Building, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7524. E-mail: [email protected] Am J Respir Crit Care Med Vol 166. pp 417–422, 2002 DOI: 10.1164/rccm.2102106 Internet address: www.atsjournals.org

Allografts and isografts were obtained by transplanting BALB/c tracheas into C57BL/6 and BALB/c mice, respectively. Transplantation and immunosuppression (cyclosporine A; Sandoz Pharmaceuticals, East Hanover, NJ; 25 mg/kg intraperitoneally 5 days/week) were performed as previously described (9). Briefly, tracheas were harvested from donor animals, stored in Dulbecco’s modified Eagle’s medium at 4C for 30 minutes, implanted two per recipient into subcutaneous pockets in the dorsum of the neck, and subsequently, harvested at 2, 4, 6, and 10 weeks. One isograft and one allograft from each time point were used for hematoxylin and eosin staining (9).

418

AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE

RNA Isolation from Tracheal Grafts Sixty BALB/c (48 donors and 12 recipients) and 12 C57BL/6 (all recipients) mice were used for the reverse transcription-polymerase chain reaction (RT-PCR). Trachea grafts (two per recipient) from six (three C57BL/6 and three BALB/c) different animals were harvested at each time point and immersed in liquid nitrogen. Total RNA was isolated using the cesium chloride-guanidine method as previously described (11). The purity (260/280 nm absorbance ratio  2) and yield of RNA were determined spectrophotometrically. The integrity of RNA was verified using Nusieve agarose gel electrophoresis. Genomic DNA contamination was removed with RNAse-free DNAase (Promega, Madison, WI) and subsequent ethanol precipitation.

Reverse Transcription and Polymerase Chain Reactions RT-PCR was performed as previously described (11, 16, 17) with minor modifications. PCR was performed using 0.5 M of each target (i.e., PDGF-A and PDGF-B chains, FGF-1 and FGF-2, TNF-, TGF-, IGF-1, endothelin-1, and epidermal growth factor [EGF]) or a control (-actin) 3 and 5 primer pair (see Table 1) and Taq DNA polymerase (Invitrogen Corp., Carlsbad, CA). Water was used for a negative control. The tissues from each (i.e., 2-, 4-, 6-, and 10-week allograft/isograft pair) set of mice were analyzed simultaneously for each growth factor and -actin mRNA using optimal cycle numbers within the linear phase of amplification (IGF-1, TGF-, FGF-1, and endothelin-1: 23– 25 cycles; PDGF-B, TNF-, and EGF-1: 25–27 cycles; PDGF-A and FGF-2: 27–29 cycles; actin: 21–23 cycles). The PCR products were separated by 3% agarose Tris acetate (TAE) gel electrophoresis, stained for 15 minutes in ethidium bromide, digitally photographed under ultraviolet light to quantify the band intensity (ImageQuant software; Molecular Dynamics, Sunnyvale, CA), and normalized to -actin.

VOL 166

2002

and 6 weeks and were examined for the primary endpoint, graft occlusion (using ImageQuant software), and a secondary endpoint, graft epithelialization (morphometric analysis of the percentage of lumenal circumference covered by ciliated epithelium), by two blinded readers.

Statistical Analysis A two-way analysis of variance was used to test the null hypothesis that growth factor transcript levels were not different between allografts and isografts over the 2- to 6-week course of mouse heterotopic trachea OB (18). The 10-week time point was excluded from the analysis of variance because of the marked upregulation of the majority of the growth factors in the isografts. Additionally, isograft/allograft mRNA intensities were compared at each individual time point with unpaired t tests. The TNFR:Fc experiment was analyzed with a repeated-measures analysis of variance (SigmaStat; SPSS Inc., Chicago, IL). A two-sided  of less than 0.05 indicated significance.

RESULTS Histology

Hematoxylin and eosin-stained frozen sections confirmed our previous findings (6) of acute cellular (mononuclear) inflammation in the allografts that peaked at the 2- to 4-week time points and subsequently subsided, followed by progressive lumenal scarring, which began at 4 weeks and culminated at 10 weeks (Figure 1). Allograft epithelial injury in the form of tissue shedding and basement membrane denudation was present at 2 weeks, and epithelial destruction was complete by 4 weeks. Isograft morphology remained normal throughout the study period.

TNFR:Fc Treatment of Mouse Heterotopic Tracheal OB

RNA Isolation and Quantification

Allografts and isografts were generated as previously described. Human TNFR:Fc (a kind gift from Jacques Peschon; Immunex Corp., Seattle, WA), which has a high binding efficiency for mouse TNF-, was administered at a dose of 100 g per mouse subcutaneously every other day from Days 3–21 to isorecipients and allorecipients. Human immunoglobulin G (100 g per mouse subcutaneously every other day, Polygam R; Baxter Healthcare Corp., Glendale, CA) was administered as a negative control. Trachea grafts from 15 different animals (five TNFR:Fc-treated allografts, five TNFR:Fc-treated isografts, and five immunoglobulin G-treated allografts) were harvested at 2, 3, 4,

Mean total RNA yields from two tracheal grafts per sample were 19.0  17.2 g (range 4.1–90.3 g) using the cesium-guanidine method but were much lower, usually immeasurably so, using rapid RNA isolation kits, including the RNeasy Total RNA System (Qiagen Inc., Valencia, CA) and RNAzol B Method (Cinna Scientific Inc., Friendswood, TX). The isolated RNA from each sample lacked digested, low molecular weight RNA bands and displayed the presence of two distinct ribosomal RNA bands after integrity gel electrophoresis (data not shown).

TABLE 1. PCR PRIMER SEQUENCES cDNA of Interest FGF-2 (designed in-house) Genbank #M30644 FGF-1 (designed in-house) Genbank #U67610 PDGF-A Stratagene, La Jolla, CA PDGF-B (designed in-house) from reference 44 TGF- Clonetech, Palo Alto, CA TNF- Stratagene IGF-1 from reference 45 ET-1 (designed in-house) Genbank #U35233 EGF-1 (designed in-house) Genbank #J00380 -actin Clonetech

Mouse Primer Sequences (a) Sense and (b) Antisense (a) 5 AAC TAC AAC TCC AAG CAG AAG AGA GA 3

(b) 5 TTA AGA TCA GCT CTT AGC AGA CAT 3

(a) 5 TGC GGG CGA AGT GTA TAT AAA G 3

(b) 5 GCA GAA ACA AGA TGG CTT TCT G 3

(a) 5 GCC CCT GCC CAT TCG GAG GAA GA 3

(b) 5 GGC CAC CTT GAC GCT GCG GTG G 3

(a) 5 CTG AGC TGG ACT TGA ACA TG 3

(b) 5 TTA AAC TTT CGG TGC TTG CC 3

(a) 5 TGG ACC GCA ACA ACG CCA TCT ATG AGA AAA CC 3

(b) 5 TGG AGC TGA AGC AAT AGT TGG TAT CCA GGG CT 3

(a) 5 ATG AGC ACA GAA AGC ATG ATC 3

(b) 5 TAC AGG CTT GTC ACT CGA ATT 3

(a) 5 CTT CTG AGT CTT GGG CAT GTC AGT 3

(b) 5 TCG TCT TCA CAC CTC TTC TAC CTG 3

(a) 5 TCA GAC ACG AAC ACT CCC TAA G 3

(b) 5 CAC AAC CGA GCA CAT TGA CTA C 3

(a) 5 AAG GAG AAG GGA TTC CTA TCT G 3

(b) 5 TAT TTA GCT GCC TTT CCA GGT C 3

(a) 5 GTG GGC CGC TCT AGG CAC CAA 3

(b) 5 CTC TTT GAT GTC ACG CAC GAT TTC 3

Amplified cDNA Sequence Length 292 bp 250 bp 224 bp 508 bp 525 bp 276 bp 320 bp 392 bp 328 bp 540 bp

Definition of abbreviations: EGF  epidermal growth factor; ET  endothelin; FGF  fibroblast growth factor; IGP  insulin-like growth factor; PCR  polymerase chain reaction; PDGF  platelet-derived growth factor; TGF-  transforming growth factor-; TNF-  tumor necrosis factor.

419

Aris, Walsh, Chalermskulrat, et al.: Growth Factor Expression during Mouse OB

Figure 1. Histologic changes (hematoxylin and eosin) of isografts at (a) 2 weeks and (b) 10 weeks after transplant showing preservation of normal architecture. The 4- and 6-week isograft samples were histologically identical to the 10week sample. Allografts showing (c) epithelial injury, (d) acute inflammation, and (d–f) progressive intralumenal fibrosis (magnification 10).

Increased Expression of Growth Factors in Tracheal Allografts

The mRNA data for TNF-, TGF-, IGF-1, and endothelin-1 during the course of OB are shown in Figures 2 and 3. The largest increase in allograft growth factor mRNA expression was seen for TNF-, 1.5- to 5.2-fold higher than the isografts at 2–6 weeks (p  0.04). This increase was noted to be highest at the 4-week time point and persisted, albeit to a lesser extent, at the 6-week time point. At 10 weeks, allograft expression of TNF- transcripts decreased to slightly less than that of the isografts. TGF- mRNA expression was increased 3-fold over isograft levels at the 4-week time point and then slowly declined over time (p  0.004 by analysis of variance). IGF-1 mRNA expression was also significantly increased, 2.5- to 2.8fold in the allografts in comparison with isografts at the 4- and 6-week time points, respectively (analysis of variance, p  0.02), and expression decreased in the allografts to levels 1.5fold above the isografts at 10 weeks.

Figure 2. PCR products of representative paired samples from isografts and allografts, 2–10 weeks after transplantation. There is significant upregulation of TGF-, TNF-, IGF-1, and endothelin-1 over the time course of chronic rejection, and selected upregulation of FGF-1, FGF-2, and PDGF-B chain at 4 weeks (p  0.04 for each) in the allografts. EGF was significantly downregulated in the allografts (p  0.001).

FGF-1, FGF-2, and PDGF-B mRNA levels were significantly increased in allografts compared with isografts at only the 4-week time point (p  0.04 for each; Figure 2). Lack of Increased Expression of PDGF-A Chain and EGF mRNAs in the Allografts

The expression of PDGF-A chain mRNA was not significantly different between allografts and isografts at any of the time points studied (p 0.2 for all comparisons; Figure 2). EGF mRNA expression was significantly higher in the isografts in comparison with the allografts during the course of OB (p  0.001), a result possibly related to the paucity of source cells (i.e., epithelium) during the evolution of OB in the allografts (Figure 2). Increased Expression of Growth Factors in Isografts at 10 Weeks

With the exception of IGF-1, isograft growth factor mRNA levels were equivalent to or higher that allograft levels at 10

Figure 3. Allograft/isograft intensity ratios (mean  SEM) of the PCR products during the evolution of OB showing significant upregulation of TGF-, TNF-, IGF-1, and endothelin-1 (ET-1). Growth factor data are corrected for actin.

420

AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE

weeks (Figures 2 and 3). Although actin levels showed a similar trend (data not shown), this pattern was more evident with the growth factor mRNA levels. This relative downregulation of growth factor mRNAs in the allografts was associated histologically with the presence of mature intralumenal scar with few cellular constituents. Efficacy of TNFR:Fc in Slowing Mouse Heterotopic Tracheal OB

TNFR:Fc significantly decreased tracheal obliteration (p  0.037) and reduced ciliated epithelial injury (p  0.01) in allografts in comparison with treatment with control (Figure 4). TNFR:Fc-treated isografts had the histologic appearance of untreated isografts.

DISCUSSION The most important finding of this study of mouse trachea allografts was the demonstration of increased mRNA expression during the course of OB of a large number of mesenchymal growth factors (i.e., TNF-, TGF-, endothelin-1, IGF-1, FGF-1, FGF-1, and PDGF-B) that are known to participate in matrix remodeling. These data, temporally coupled with progressive fibrosis in the allografts histologically, strongly suggest a role for not one but a number of potentially profibrotic cytokines in the fibroproliferative phase of OB characteristic of transplanted airway tissue. The loss of the airway epithelium in the allografts at these time points makes it unlikely that the source of such factors is the epithelium (19), although the injured bronchial epithelium can secrete chemoattractants for inflammatory cells. Although unproved by these studies, it is likely that the increased expression of TNF-, TGF-, IGF-1, and endothelin-1 reported herein resulted from macrophage and/or myofibroblast recruitment and activation as these events were temporally associated (9). The decline in allograft mRNA expression at 10 weeks corresponded to the histologic changes of mature scar with minimal cellular infiltrate. Thus, the activation of remodeling pathways is temporally associated with cellular (or acute-type) airway inflammation. The mRNA results are useful in that they reveal a number of pathways that can be targeted for therapeutic intervention to slow fibrosis in OB. Intervention to reduce the activity of TNF- with TNFR:Fc demonstrated the usefulness of antagonizing one of the important growth factors in this fully major histocompatability complex (MHC)-mismatched mouse OB model. In addition, TNFR:Fc reduced epithelial injury in this experiment possibly because TNF- can cause respiratory epithelial cell apoptosis or because of its regulatory activities on other effector cell populations. Recently, similar results have been reported by Smith and coworkers using hamster anti-

VOL 166

2002

mouse TNF- antibodies in a murine heterograft model that develops OB with only a single mismatched human leukocyte antigen A2 molecule (20). Blockade of TNF- has been shown to reduce scarring in animal models of lung fibrosis in vivo (21, 22). Enbrel (Immunex), the Food and Drug Administration– approved drug that is chemically equivalent to TNFR:Fc, may potentially be of benefit to patients with OB, but the concern over lung transplant recipients who are at risk for infection should be taken into consideration. A number of growth factors and cytokines, including those studied here and elsewhere, contribute to matrix remodeling in the lung (23, 24). PDGF, produced by macrophages, smooth muscle cells, fibroblasts, platelets, and endothelial cells, is the most potent mitogen, chemotactic factor, and protein synthesis stimulator for mesenchymal cells yet discovered (25). FGF isoforms 1 (acidic) and 2 (basic) are important factors for angiogenesis, contributing to endothelial cell migration, proliferation, and activation and fibroblast proliferation and collagen synthesis (23). TGF-1, secreted by most mammalian cells, is a potent promoter of extracellular matrix production (26). TGF- is also capable of suppressing the actions of other cytokines and downregulating the inflammatory response. TNF-, secreted predominantly by cells of the macrophage/monocyte lineage, is a pleiotropic cytokine capable of enhancing inflammation as well as causing fibrogenesis (27). IGF-1, a polypeptide secreted by most tissues, regulates fibroblast growth and connective tissue production (28). Finally, EGF, manufactured by epithelial tissues as well as macrophages, can stimulate growth and differentiation of epithelial and mesenchymal cells (29). We chose to survey simultaneously a number of potentially important mesenchymal cell growth factors (PDGF-A and PDGF-B, FGF-1 and FGF-2, TNF-, TGF-, IGF-1, endothelin-1, and EGF) in this experiment in an attempt to define more rigorously the fibroproliferative pathways that are activated during OB. We used the mouse heterograft model of OB because it reliably, albeit rapidly (compared with the events in humans), reproduces the histology of human OB. The mouse tracheal grafts evolve through the processes of airway rejection, namely acute cellular rejection peaking at 2–4 weeks, early fibroproliferation at 4 weeks, and finally, mature airway scarring at 6–10 weeks. Over the 2- to 10-week rejection time course, the production of mesenchymal growth factor transcripts precedes and accompanies the histologic findings of fibrosis. We chose to concentrate on the evolution of OB without being confounding by ischemic changes (present during the first week) by beginning the assessment of growth factor expression at 2 weeks. Using cyclosporine immunosuppression helped recreate the environment that occurs in human OB.

Figure 4. Efficacy of TNFR:Fc treatment (compared with control immunoglobulin G) on the preservation of ciliated epithelium and maintenance of lumenal patency in the fully MHC-mismatched mouse heterograft model of OB. Data represent mean  SEM measurements for five tracheas harvested at each time point. Dashed lines: control allografts. Solid lines with closed circles: TNFR:Fc-treated allografts. Solid lines with closed squares: TNFR:Fc-treated isografts.

Aris, Walsh, Chalermskulrat, et al.: Growth Factor Expression during Mouse OB

The results of our more comprehensive analysis of growth factors during OB are in keeping with animal model data from others supporting a role for one or more of these factors in the fibrosis that accompanies OB. Al-Dossari and colleagues, using the heterotopic mouse model of OB, demonstrated that isografts, which typically engraft without histologic changes, injected with PDGF daily for 30 days developed severe airway scarring (13). Similar changes of OB were found with isografts injected with FGF2. Kallio and colleagues found upregulation of PDGF- and - receptors and increased immunoreactivity of PDGF-A and -B chains in the rat heterotopic model of OB (6). Furthermore, in their experiments, the fibrotic response could be slowed with a specific antagonist of the PDGF receptor. TNF- mRNA increases have been described in ischemic injury to the transplanted lung and in acute lung allograft rejection in rats, the former within the first week of transplantation (30–33), but have not been previously associated with OB. Because TNF- mRNA increases were distant to the phase of graft ischemia, a phenomenon known to upregulate TNF-, they probably played a role in the immunologic and fibrotic processes of OB. The upregulation of a number of growth factors at 10 weeks in the isografts has been previously seen with cytokines in this (11) and other models of chronic graft rejection and is presumed to result from nonalloimmune mechanisms of graft injury or activation (34). Clinical studies demonstrate that fibroblast proliferative activity is increased in bronchoalveolar lavage fluid from patients with OB (35). This activity may be due to a number of possible agonists, as has been suggested in small, selected patient groups. Hertz and colleagues were the first to report that PDGF levels were elevated in the bronchoalveolar lavage of a post-lung transplant patient with OB and in a second patient before the development of OB (12). Magnan and colleagues were the first to report a possible role for TGF- in OB by finding increased bronchoalveolar lavage macrophage production of TGF- isoforms 1 and 2, and that these increases occurred before the onset of airflow obstruction in five patients (15). Subsequently, Hirabayashi and associates found both increased TGF-, and to a lesser extent, PDGF labeling in the lungs explanted from patients undergoing retransplantation for OB (36), and El-Gamel and colleagues found increased immunostaining of TGF- in transbronchial biopsies from patients with OB (37). Elssner and associates found increased levels of TGF- in the epithelial lining fluid of patients with OB (38). Data from Charpin and colleagues indicated a role for IGF-1 in the pathogenesis of OB in three patients who had increased bronchoalveolar lavage cell mRNA levels before the onset of clinical OB (39). Finally, our finding of increased endothelin-1 expression extends the results from Aarnio and associates, who found increased endothelin-1 levels in the bronchoalveolar lavage fluid of lung transplant recipients who were experiencing acute graft rejection (40). The results of our study should be viewed in the light of several potential limitations. First, the demonstration of the increased expression of a number of important growth factors in allografted airway tissue during the evolution of OB does not provide proof that these factors play a role in airway scarring. Antagonism of the TNF- pathway leading to a reduction in OB in this model helps clarify the importance of TNF-. Further proof will have to await antagonism studies of other factors and the impact of that intervention on the course of OB. Additionally, factors other than those studied may be important in fibroproliferation. Second, the molecular events that play a role in the evolution of OB in the heterotopic mouse model may not reflect the biology of human OB. However, the mouse model is well-characterized, histologically re-

421

produces human OB, and affords an opportunity to make considerable progress on the pathogenesis of this condition in a controlled fashion where human tissue is not as readily available. Third, despite the use of semiquantitative methods with correction for a ubiquitous “housekeeping” gene, PCR is not an absolutely quantitative technology. Nonetheless, performing the PCR experiments in triplicate and with good reproducibility lessens the concerns of quantification intrinsic to this technique. Importantly, the role of growth factors that are regulated predominantly by post-transcriptional modification may have been underestimated in these studies. Finally, immunosuppressants themselves may affect growth factor expression (e.g., TGF-) (41) and potentially confound the results of these experiments. In conclusion, our study demonstrated that TGF-, TNF-, IGF-1, endothelin-1, FGF-1, FGF-2, and PDGF-B chain, each capable of stimulating mesenchymal cell growth and/or extracellular matrix production, are significantly upregulated during the course of OB in the mouse model. On the other hand, the expression of EGF was markedly reduced as a result of epithelial damage. The temporal association between the upregulation of the aforementioned growth factors and the histologic changes of fibrosis suggests that these factors are involved in the genesis of airway scarring in OB. In addition, antagonism of the TNF- pathway reduced the severity of OB in this model, confirming the importance of the mRNA results. Blockade of TNF- and TGF- has reduced scarring in other animal models of lung fibrosis as well (42, 43). Therefore, these results provide a number of opportunities to ameliorate OB with therapeutic interventions aimed at antagonizing growth factor production or action. However, because growth factor upregulation occurs before airway scarring, therapeutic efforts to antagonize pathways of fibrogenesis in OB need to be introduced early (quite possibly during “acute” cellular airway rejection in this model) to be effective. Ongoing studies in this arena will help determine the therapeutic success of other antigrowth factor strategies in slowing the fibroproliferative phase of OB in the mouse model. Acknowledgment : The authors thank Susan Hayden for her ongoing support and patience; David Fenstermacher, Ph.D., for providing assistance with RT-PCR; Jacques Peschon, Ph.D., and Kathleen S. Picha, M.S., both at Immunex Corp., for their insights in the planning of the TNFR:Fc experiment; and the investigators and staff in the Cystic Fibrosis Research and Treatment Center core facilities (funded by the Cystic Fibrosis Foundation and the NHLBI), without whose help these experiments would not have been possible.

References 1. United Network for Organ Sharing (UNOS) website, www.unos.org. 2/ 14/01. 2. Hosenpud JD, Bennett LE, Keck BM, Boucek MM, Novick RJ. The registry of the international society for heart and lung transplantation: seventeenth official report-2000. J Heart Lung Transplant 2000;19:909–931. 3. Levine SM, Bryan CL. Bronchiolitis obliterans in lung transplant recipients: the “thorn in the side” of lung transplantation. Chest 1995; 107:967–972. 4. al-Dossari GA, Kshettry VR, Jessurun J, Bolman RM III. Experimental large-animal model of obliterative bronchiolitis after lung transplantation. Ann Thorac Surg 1994;58:34–39. 5. Uyama T, Winter JB, Groen G, Wildevuur CR, Monden Y, Prop J. Late airway changes caused by chronic rejection in rat lung allografts. Transplantation 1992;54:809–812. 6. Kallio EA, Koskinen PK, Aavik E, Buchdunger E, Lemstrom KB. Role of platelet-derived growth factor in obliterative bronchiolitis (chronic rejection) in the rat. Am J Respir Crit Care Med 1999;160:1324–1332. 7. Hertz MI, Jessurun J, King MB, Sazik S, Murray JJ. Reproduction of the obliterative bronchiolitis lesion after heterotopic transplantation of mouse airways. Am J Pathol 1993;142:1945–1951. 8. King MB, Jessurun J, Savik SK, Murray JJ, Hertz MI. Cyclosporine reduces development of obliterative bronchiolitis in a murine heterotopic airway model. Transplantation 1997;63:528–532.

422

AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE

9. Neuringer IP, Mannon R, Coffman T, Parsons M, Burns K, Yankaskas JR, Randell S, Aris RM. Immune cells in the mouse airway model of obliterative bronchiolitis. Am J Respir Cell Mol Biol 1998;19:379–386. 10. Bohler A, Chamberlain D, Kesten S, Slutsky AS, Liu M, Keshavjee S. Lymphocytic airway infiltration as a precursor to fibrous obliteration in a rat model of bronchiolitis obliterans. Transplantation 1997;64:311–317. 11. Neuringer IP, Walsh S, Mannon R, Gabriel S, Aris RM. Enhanced T-cell cytokine gene expression in mouse airway obliterative bronchiolitis. Transplantation 2000;69:399–405. 12. Hertz MI, Henke CA, Nakhleh RE, Harmon KR, Marinelli WA, Fox JMK, Kubo SH, Shumway SJ, Bollman RM, Bitterman PB. Obliterative bronchiolitis after lung transplantation: a fibroproliferative disorder associated with platelet-derived growth factor. Proc Natl Acad Sci USA 1992;89:10385–10389. 13. Al Dossari GA, Jessurun J, Bolman RM, Kshettry VR, King MB, Murray JJ, Hertz MI. Pathogenesis of obliterative bronchiolitis: possible roles of platelet-derived growth factor and basic fibroblast growth factor. Transplantation 1995;59:143–145. 14. King MB, Levrey HJ, Preiner JK, McIvor RS, Hertz MI. PDGF b receptor expression in the heterotopic airway transplant model of obliterative airways disease [abstract]. Am J Respir Crit Care Med 1997;155: A270. 15. Magnan A, Mege JL, Escallier JC, Brisse J, Capo C, Reynaud M, Thomas P, Meric B, Garbe L, Badier M, et al. Balance between alveolar macrophage IL-6 and TGF- in lung-transplant recipients: Marseille and Montreal Lung Transplantation Group. Am J Respir Crit Care Med 1996;153:1431–1436. 16. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. Cold Spring Harbor, NY: Harbor Press; 1989. p. 7–19. 17. Gause WC, Adamovicz J. Use of the PCR to quantitate gene expression. PCR Methods Appl 1994;3:S123–S135. 18. Person R. Using Microsoft Excel 97. Indianapolis, IN: Que Corporation; 1997. 19. Mauck KA, Hosenpud JD. The bronchial epithelium: a potential allogeneic target for chronic rejection after lung transplantation. J Heart Lung Transplant 1996;15:709–714. 20. Smith CR, Jaramillo A, Lu KC, Higuchi T, Kaleem Z, Mohanakumar T. Prevention of obliterative airway disease in HLA-A2-transgenic tracheal allografts by neutralization of tumor necrosis factor. Transplantation 2001;72:1512–1518. 21. Piguet PF, Collart MA, Grau GE, Sappino AP, Vassalli P. Requirement for tumor necrosis factor for the development of silica-induced pulmonary fibrosis. Nature 1990;344:245–247. 22. Piguet P, Collart M, Grau G, Kapanci Y, Vassalli P. Tumor necrosis factor/cachectin plays a key role in bleomycin-induced pneumopathy and fibrosis. J Exp Med 1989;170:655–663. 23. Musaers SE, Bishop JE, McGrouther G, Laurent GJ. Mechanisms of tissue repair: from wound healing to fibrosis. Int J Biochem 1997;29:5–17. 24. Hatamochi A, Mori K, Ueki H. Role of cytokines in connective tissue gene expression. Arch Dermatol Res 1994;287:115–121. 25. Liu JY, Morris GF, Lei WH, Hart C, Lasky J, Brody AR. Rapid activation of PDGF-A and B at sites of lung injury in asbestos-exposed rats. Am J Respir Cell Mol Biol 1997;17:129–140. 26. Border WA, Noble NA. Transforming growth factor  in tissue fibrosis. N Engl J Med 1994;331:1286–1292. 27. Miyazaki Y, Araki K, Vesin C, Garcia I, Kapanci Y, Whitsett JA, Piguet PF, Vassalli P. Expression of a tumor necrosis factor-alpha transgene in murine lung causes lymphocytic and fibrosing alveolitis: a mouse model of progressive pulmonary fibrosis. J Clin Invest 1995;96:250–259. 28. Goldstein RH, Poliks CF, Pilch PF, Smith BD, Fine A. Stimulation of

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

VOL 166

2002

collagen formation by insulin and insulin-like growth factor I in cultures of human fibroblasts. Endocrinology 1989;124:964–970. Lynch SE, Colvin RB, Antoniades HN. Growth factors in wound healing: single and synergistic effects on partial thickness porcine skin wounds. J Clin Invest 1989;84:640–646. Demeester SR, Rolfe MW, Kunkel SL, Swiderski DL, Lincoln PM, Deeb GM, Streiter RM. The bimodal expression of tumor necrosis factor- in association with rat lung implantation and allograft rejection. J Immunol 1993;150:2494–2505. Saito R, Zuo XJ, Marchevesky A, Castracane J, Waters P, Matloff J, Jordan SC. The participation of tumor necrosis factor in the pathogenesis of lung allograft rejection in the rat. Transplantation 1993;55:967–972. Zuo XJ, Matsumura Y, Prehn J, Saito R, Marchevesky A, Matliff J, Jordan SC. Cytokine gene expression in rejecting and tolerant rat lung allografts: analysis by RT-PCR. Transpl Immunol 1995;3:151–161. Sumitomo M, Sakiyama S, Tanida N, Fukumoto Y, Monden Y, Uyama T. Difference in cytokine production in acute and chronic rejection of rat lung allografts. Transpl Int 1996;9(Suppl.):S223–S225. Nadeau K, Azuma H, Tilney N. Sequential cytokine dynamics in chronic rejection of rat renal allografts: role of cytokines RANTES and MCP-1. Proc Natl Acad Sci USA 1995;92:8729–8734. Jonosono M, Fang KC, Keith FM, Turck CW, Blanc PD, Hall TS, Fukano AK, Rifkin CJ, Gold WM, Webb WR, et al. Measurement of fibroblast proliferative activity in bronchoalveolar lavage fluid in the analysis of obliterative bronchiolitis among lung transplant recipients. J Heart Lung Transplant 1999;18:972–985. Hirabayashi T, Demertzis S, Schafers J, Hoshino K, Nashan B. Chronic rejection in lung allografts: immunohistological analysis of fibroagenesis. Transpl Int 1996;9(Suppl 1):S293– S295. El-Gamel A, Sim E, Hasleton P, Hutchinson J, Yonan N, Egan J, Campbell C, Rahman A, Sheldon S, Deiraniya A, et al. TGF and obliterative bronchiolitis following pulmonary transplantation. J Heart Lung Transplant 1999;18:828–837. Elssner A, Jaumann F, Dobmann S, Behr J, Schwaiblmair M, Reichenspurner H, Furst H, Briegel J, Vogelmeier C. Elevated levels of interleukin-8 and transforming growth factor-beta in bronchoalveolar lavage fluid from patients with bronchiolitis obliterans syndrome: proinflammatory role of bronchial epithelial cells: Munich Lung Transplant Group. Transplantation 2000;70:362–367. Charpin J, Stern M, Grenet D, Israel-Biet D. Insulinlike growth factor-1 in lung transplants with obliterative bronchiolitis. Am J Respir Crit Care Med 2000;161:1991–1998. Aarnio P, Tukiainen P, Taskinen E, Harjula A, Fyhrquist F. Endothelin in bronchoalveolar lavage fluid is increased in lung-transplanted patients. Scand J Thorac Cardiovasc Surg 1996;30:113–116. Wolf G, Zahner G, Ziyadeh FN, Stahl RA. Cyclosporin A induces transcription of transforming growth factor beta in a cultured murine proximal tubular cell line. Exp Nephrol 1996;4:304–308. Giri SN, Hyde DM, Hollinger MA. Effect of antibody to transforming factor beta on bleomycin induced accumulation of lung collagen in mice. Thorax 1993;48:959–966. Denis M. Neutralization of transforming growth factor-beta (1) in a mouse model of immune-induced lung fibrosis. Immunology 1994;82: 584–590. Bonthron DT, Sultan P, Collins T. Structure of the murine c-sis protooncogene (Sis, PDGFB) encoding the B chain of platelet-derived growth factor. Genomics 1991;10:287–292. Bell GI, Stempien MM, Fong NM, Rall LB. Sequences of liver cDNAs encoding two different mouse insulin-like growth factor 1 precursors. Nucleic Acids Res 1986;14:7873–7882.