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American Journal of Respiratory and Critical Care Medicine Vol 166. pp. 417-422, (2002)
© 2002 American Thoracic Society

Original Article

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

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: aris{at}med.unc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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-ß, tumor necrosis factor-{alpha} (TNF-{alpha}), edothelin-1, (prepro) epidermal growth factor, insulin-like growth factor-1, and ß-actin as a control. Transforming growth factor-ß, TNF-{alpha}, 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-{alpha}–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-{alpha}) may be useful in ameliorating OB in this model.

Key Words: obliterative bronchiolitis • chronic rejection • lung • growth factors • mouse


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 solid-organ 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 ß receptor, fibroblast growth factor (FGF)-2, and transforming growth factor-ß (TGF-ß) 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-ß1, tumor necrosis factor-{alpha} (TNF-{alpha}), endothelin-1, and insulin-like 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-{alpha} receptor, was administered to determine whether it could slow chronic rejection in this model.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
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
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 4°C 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).

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 µM of each target (i.e., PDGF-A and PDGF-B chains, FGF-1 and FGF-2, TNF-{alpha}, TGF-ß, IGF-1, endothelin-1, and epidermal growth factor [EGF]) or a control (ß-actin) 3' and 5' primer pair (see Table 1) and Taq DNA polymerase (Invitrogen Corp., Carlsbad, CA). Water was used for a negative control.


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  		TABLE 1. Pcr primer sequences

 
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 ß-actin mRNA using optimal cycle numbers within the linear phase of amplification (IGF-1, TGF-ß, FGF-1, and endothelin-1: 23–25 cycles; PDGF-B, TNF-{alpha}, 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 ß-actin.

TNFR:Fc Treatment of Mouse Heterotopic Tracheal OB
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-{alpha}, was administered at a dose of 100 µg per mouse subcutaneously every other day from Days 3–21 to isorecipients and allorecipients. Human immunoglobulin G (100 µg 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, 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 {alpha} of less than 0.05 indicated significance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
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.



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  		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 10-week sample. Allografts showing (c) epithelial injury, (d) acute inflammation, and (d–f) progressive intralumenal fibrosis (magnification x10).

 
RNA Isolation and Quantification
Mean total RNA yields from two tracheal grafts per sample were 19.0 ± 17.2 µg (range 4.1–90.3 µg) 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).

Increased Expression of Growth Factors in Tracheal Allografts
The mRNA data for TNF-{alpha}, TGF-ß, 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-{alpha}, 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-{alpha} transcripts decreased to slightly less than that of the isografts. TGF-ß 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 2.8-fold 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 1.5-fold above the isografts at 10 weeks.



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  		Figure 2. PCR products of representative paired samples from isografts and allografts, 2–10 weeks after transplantation. There is significant upregulation of TGF-ß, TNF-{alpha}, 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).

 


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

 
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 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.



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  		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.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
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-{alpha}, TGF-ß, 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-{alpha}, TGF-ß, 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-{alpha} 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-{alpha} 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-mouse TNF-{alpha} antibodies in a murine heterograft model that develops OB with only a single mismatched human leukocyte antigen A2 molecule (20). Blockade of TNF-{alpha} 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-ß1, secreted by most mammalian cells, is a potent promoter of extracellular matrix production (26). TGF-ß is also capable of suppressing the actions of other cytokines and downregulating the inflammatory response. TNF-{alpha}, 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-{alpha}, TGF-ß, 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.

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-{alpha} and -ß 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-{alpha} 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-{alpha} mRNA increases were distant to the phase of graft ischemia, a phenomenon known to upregulate TNF-{alpha}, 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-ß in OB by finding increased bronchoalveolar lavage macrophage production of TGF-ß 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-ß, 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-ß in transbronchial biopsies from patients with OB (37). Elssner and associates found increased levels of TGF-ß 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-{alpha} pathway leading to a reduction in OB in this model helps clarify the importance of TNF-{alpha}. 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 reproduces 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-ß) (41) and potentially confound the results of these experiments.

In conclusion, our study demonstrated that TGF-ß, TNF-{alpha}, 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-{alpha} pathway reduced the severity of OB in this model, confirming the importance of the mRNA results. Blockade of TNF-{alpha} and TGF-ß 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.


   Acknowledgments
 
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.

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.

Received in original form February 26, 2002; accepted in final form April 16, 2002


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 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
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