Insulinlike Growth Factor-1 in Lung Transplants with ... - ATS Journals

Insulinlike Growth Factor-1 in Lung Transplants with Obliterative Bronchiolitis JEAN-MARIE CHARPIN, MARC STERN, DOMINIQUE GRENET, and DOMINIQUE ISRAËL-BIET Laboratoire d’Immunologie Pulmonaire and Service de Pneumologie, Hôpital Laënnec, Paris; Service de Pneumologie, Hôpital Foch, Suresnes, France

Bronchiolitis obliterans syndrome (BOS) is the major complication limiting survival of lung transplant recipients (Tx patients). The mechanisms underlying this fibrotic process are not known. We assessed IGF-1 and IGFBP-3 expression, critical mediators in different models of pulmonary fibrosis, in nine Tx patients. Three of them developed a BOS at 8, 14, and 17 mo postgraft, respectively. Two of the remaining six displayed a recurrent cytomegalovirus (CMV) infection, and four are in stable condition. IGF-1 mRNA expression was quantitated by RT-PCR in cells from four to six BAL per patient performed during the first 6 mo postgraft. Contrasting with a constantly low expression of IGF-1 mRNA in BAL cells from the six patients without BOS, the three patients with BOS presented marked peaks of IGF-1 on two to five occasions during the study period. These peaks, 3- to 13-fold increased compared with values from the former patients, preceded the diagnosis of BOS by 7, 13, and 17 mo, respectively. On the other hand, IGFBP-3 was highly and exclusively expressed in the three patients with BOS, the mRNA as well as the gene product as demonstrated by Western blotting. Our data strongly argue for a role of IGF-1 and IGFBP-3 in the fibrotic process underlying BOS, and for their possible value as an early marker of this complication.

Lung transplantation is an established modality for the treatment of selected patients with end-stage pulmonary disease. Despite improvement in surgical and medical management, chronic rejection has emerged as a major factor limiting the long-term survival of lung allograft recipients (1). Chronic lung rejection (CLR) is a clinicopathologic syndrome of graft dysfunction that is characterized histologically by obliterative bronchiolitis (OB) and physiologically by airflow limitation. Pathologic examination of transplanted lung tissue definitely establishes the diagnosis of OB (2). However, the very poor sensitivity of transbronchial biopsies has led to the diagnosis of CLR on spirometric parameters instead of histologic ones. Airflow limitation, defining the bronchiolitis obliterans syndrome (BOS), is graded according to a standardized working formulation (3). Chronic rejection occurs in 25 to 50% of longterm survivors of lung transplantation, and its prognosis is still very dark (4). Early predictive markers of this condition are lacking and the diagnosis is generally ascertained when the fibrosis process is irreversible and the respiratory failure inescapable. The establishment of such markers would be critical to the management of lung transplant recipients, allowing for earlier therapeutic interventions and hence for a potentially better prognosis.

(Received in original form May 14, 1999 and in revised form November 11, 1999) Supported in part by the Délégation à la Recherche Clinique de l’Assistance Publique-Hôpitaux de Paris (Contrat de recherche Clinique), and by l’Association Française de Lutte contre la Mucoviscidose. J.-M. Charpin is the recipient of a scholarship from the Société de Pneumologie de Langue Française. Correspondence and requests for reprints should be addressed to D. Israël-Biet, M.D., Service de Pneumologie, Hôpital Laënnec, 42 rue de Sèvres, 75007 - Paris, France. E-mail: [email protected] Am J Respir Crit Care Med Vol 161. pp 1991–1998, 2000 Internet address: www.atsjournals.org

Histologically, the BOS is characterized by an OB, a fibroproliferative process involving the accumulation of mesenchymal cells and their connective tissue products in the bronchiolar lumina, resulting in progressive airflow obstruction and graft failure (5). The pathogenesis of this process has not been elucidated. It may involve both alloantigen-dependent and -independent mechanisms. There is some evidence that BOS occurs partly as the result of injuries immunological in nature and is a response to CLR. Indeed, frequent and severe episodes of acute lung rejection (ALR) have been clearly associated with a subsequent development of BOS (6–9). Nevertheless, some other factors such as cytomegalovirus infection have been involved through the cytokine release they trigger (10). Finally, early inflammatory events, related for instance to ischemia, can also lead to BOS (11). In any case, OB is the result of tissue repair and remodeling in response to graft injury, whatever the offending agent. Fibroblasts (FB) are the main cells involved in these structural changes. Their proliferation and their secretion of collagen and extracellular matrix components are regulated by many cytokines (CK) and growth factors (GF) (12). These mediators capable of modulating FB functions are released mainly by alveolar macrophages (AM) in the lung and may be involved in the early stage of OB (13). Insulin-like growth factor-1 (IGF-1) is a profibrogenic mediator acting as a potent mitogen (14) and stimulator of collagen synthesis by FB (15). It is secreted mainly by alveolar macrophages in the lung (16), but it can be expressed by other cell types such as FB and epithelial or endothelial cells (14, 17). Increased IGF-1 expression and release has been demonstrated in patients with idiopathic pulmonary fibrosis (18), systemic sclerosis (19), coal-worker pneumoconiosis (20), and pulmonary sarcoidosis (21) in adults, as well as in interstitial lung disease in children (22). Although the mechanisms initiating pulmonary fibrosis on the one hand and OB in the context of CLR on the other hand are certainly different in nature, we hypothesized that IGF-1 might be involved in the fibroproliferative process that characterizes OB. The local bioavailability of IGF-1 in the lung is regulated by a system of at least six specific high-affinity IGF-binding proteins (IGFBP) (23). The functional roles of IGFBP are relatively poorly understood. Their actions are generally inhibitory through an interference with IGF-1 binding to its receptor, but they can also potentiate or enhance IGF-stimulated cell growth. To determine whether IGF-1 might be involved in the development of OB, we serially evaluated its expression in the alveolar cells of lung transplant recipients during the first 6 mo postgraft. We also assessed the expression of IGFBP-2 and -3, described as the major IGFBP expressed in the lung (23). Our data suggest a strong relationship between an early increased expression of IGF-1 and IGFBP-3 and a subsequent development of BOS.

METHODS Study Population Nine lung transplant recipients grafted between February 1996 and April 1998 were prospectively included in this study. Their initial di-

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agnosis was mucoviscidosis (n ⫽ 5), primary pulmonary hypertension (n ⫽ 1), lymphangiomyomatosis (n ⫽ 2) and emphysema (n ⫽ 1). All patients were initially maintained on an immunosuppressive regimen of cyclosporin, azathioprine, and prednisone. FK506 was substituted for cyclosporin after three consecutive ALRs in seven patients. The postgraft follow-up (15 to 30 mo) and the acute complications of transplantation such as ALR and CMV infections are reported in Table 1. Three of the patients (patients 7 to 9) developed a BOS at 8, 14, and 17 mo postgraft, respectively. Patient 8 died of this complication 7 mo after the diagnosis of BOS.

RNA Extraction and Reverse Transcription Total RNA was isolated and purified using the Qiagen RNeasy Mini Kit (Qiagen, Germany). RNA was recovered in 50 ␮l of RNase-free water, and submitted to DNase digestion in a 100-␮l final volume with 2 U of RQ1 RNase-free DNase (Promega, Madison, WI) at 37⬚ C for 15 min to discard eventual residual genomic DNA. DNase was then removed according to the Quiagen Clean’up protocol of the Quiagen RNeasy Mini Kit. Reverse transcription of the purified RNA was performed by adding 5 ␮l of 5X hexanucleotides (Boehringer Mannheim, Mannheim, Germany) and incubation at 65⬚ C for 5 min for random priming and then incubation at 37⬚ C for 1 h with 1,000 U of M-MLV reverse transcriptase RNase H minus (Promega), 50 mM TRIS-HCl at pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 1 mM dNTP, and 100 U of rRNasin ribonuclease inhibitor (Promega). The reaction was stopped by incubation at 95⬚ C for 5 min. The cDNA was stored at ⫺20⬚ C. For each sample, a negative control (without reverse transcriptase) was included (RT⫺).

PCR Primers and Competitors PCR primers were deduced from published sequences using the OLIGO system software and were synthesized by Genset (Paris, France). They were GGT GAA GGT CGG AGT CAA CGG (sense) and GAG GGA TCT CGC TCC TGG AAG A (antisense) for GAPDH to yield an expected PCR product of 240 pb for cDNA, TGT CCT CCT CGC ATC TCT TC (sense) and ACT TGG CAG GCT TGA GGG GT (antisense) for IGF-1 (25) (PCR product: 134 pb), GCT AGT GAG TCG GAG GAA GA (sense) and TTC TGG GGT ATC TGT GCT CTG (antisense) for IGFBP-3 (26) (PCR product: 188 pb) and ATG AAG GAG CTG GCC GTG TT (sense) and AAG AGA TGA CAC TCG GGG TC (antisense) for IGFBP-2 (PCR product: 374 pb) (Genebank, M35410). GAPDH competitor was synthesized by deletion of 12 pb from the 240 pb PCR product using Nco1 restriction enzyme (27). IGF-1 competitor was synthesized by PCR using a composite primer (TGT CCT CCT CGC ATC TCT TCT CAC CTT CAC CAG CTC TGC C) and the IGF-1 antisense primer (see above), which lead to a deletion of 18 pb from the 134 pb IGF-1 PCR product

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TABLE 1 PATIENTS WITH AND WITHOUT BOS: CLINICAL DATA Months Postgraft

CMV (Y/⫺)

ALR

Macrophages in BAL (%)

2 3 4 5 6

⫺ ⫺ ⫺ ⫺ ⫺

⫺ A2 ⫺ A2 ⫺

84 91 95 95 82

2

2 3 4 6

⫺ ⫺ ⫺ ⫺

⫺ A1 ⫺ ⫺

98 99 97 98

3

1 2 3 4 6

⫺ ⫺ ⫺ ⫺ ⫺

⫺ ⫺ A1B1 ⫺ ⫺

77 85 86 81 75

4

1 2 3 4 6

Y ⫺ Y ⫺ Y

⫺ ⫺ ⫺ A1 ⫺

76 89 75 85 93

5

2 3 4 5 6

Y Y Y Y Y

⫺ ⫺ A1 A2B2 A1

89 95 93 75 79

6

1 2 3 4 6

⫺ ⫺ ⫺ ⫺ –

A2B1 A1 ⫺ ⫺ –

75 91 99 90 88

1 3 4 5 6

⫺ ⫺ ⫺ ⫺ ⫺

A2 A1 B1 A2 A2

80 75 87 89 80

1/8 mo 2/12 mo

1 2 3 4 5 6

Y ⫺ ⫺ ⫺ ⫺ ⫺

A2 A2 ⫺ ⫺ ⫺ B1

92 88 96 97 83 90

1/17 mo 2/18 mo

1 2 3 4 5 6

Y ⫺ Y Y ⫺ ⫺

A2 ⫺ ⫺ ⫺ ⫺ A1

100 100 65 88 88 96

1/14 mo 2/18 mo

Patient No. Patients without BOS 1

Transbronchial Biopsies and Bronchoalveolar Lavage In addition to clinical and functional evaluation, BAL and transbronchial biopsy (TBB) were routinely performed in each patient either to detect asymptomatic complications at Day 30 after surgery (then every month during the first 6 mo and every 6 mo thereafter) or as part of the routine procedure for the diagnosis of complications suspected on clinical and/or functional grounds. Chronic rejection, reflected by the airflow limitation that defines the BOS, was graded according to a standardized working formulation (3). TBB specimens were routinely processed to search for (1) opportunistic agents by appropriate stainings and (2) lung rejection, classified in Grades 1–4 of increasing severity (24). BAL cells were separated into four aliquots. The first one was routinely processed for virologic and bacteriologic evaluation. The second one served to prepare cytospins which, after staining with May Grunwald Giemsa, allowed the establishment of cell differentials. In addition, 1 ⫻ 106 cells were pelleted for RNA extraction (see below). Finally, another 1 ⫻ 106 cells were cultured in RPMI 1640 (ATGC, Noisy-le-Grand, France) supplemented with 1% FCS for 20 h in an incubator (5% CO2, 37⬚ C). Cell free supernatants were kept frozen at ⫺80⬚ C until use for Western blotting studies (see below). Only the BAL performed during the first 6 mo postgraft were included in this study. Altogether, a total of 45 BAL (four to six per patient) were prospectively studied for IGF-1 and IGFBP expression.

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Patients with BOS 7

8

9

BOS (grade/months postgraft)

Definition of abbreviations: ALR ⫽ acute lung rejection graded according to the International Working Formulation (Reference 24); BOS ⫽ bronchiolitis obliterans graded according to the International Working Formulation (Reference 3); CMV ⫽ cytomegalovirus.

(28). Competitor were then amplified by PCR, electrophoresed, purified, and quantified by spectrometry.

Quantitation of IGF-1 Expression by Competitive Polymerase Chain Reaction For each BAL or PBMC analyzed, 2 ␮l of cDNA were amplified in presence of five 2-fold dilutions of GAPDH or IGF-1 competitor using a range of competitor concentrations close to the expected target cDNA concentration (29). PCR conditions were 10 mM TRIS-HCl at pH 9.0, 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 0.01% (wt/ vol) gelatin (10⫻ buffer for Taq DNA polymerase) (ATGC), 0.4 mM dNTP (Promega), 0.2 ␮M of each primer, 1 U of Taq DNA poly-

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Charpin, Stern, Grenet, et al.: IGF-1 and Chronic Lung Rejection merase (ATGC), in a final volume of 50 ␮l. For IGF-1 PCR amplification, MgCl2 concentration was increased to 2 mM. PCR cycles were: preincubation for 3 min at 95⬚ C, then 35 cycles (94⬚ C 30 s, 59⬚ C 40 s, 72⬚ C 40 s) for GAPDH, 45 cycles (94⬚ C 30 s, 61⬚ C 50 s, 72⬚ C 40 s) for IGF-1, and a final incubation at 72⬚ C for 10 min. A RT control and a PCR control (without cDNA) were included in each experiment. PCR products were electrophoresed on a 7% polyacrylamide gel for GAPDH and on an 8% one for IGF-1, in denaturing conditions to avoid heteroduplex formation (40% [wt/vol] urea, heating at 65⬚ C and high voltage migration). Gels were stained with SYBR Green II RNA (FMC BioProducts, Rockland, ME), and photographed in UV light with Polaroid type 665 positive/negative films. The density ratios of target cDNA PCR product (tcDNA) to competitor PCR product was determined by laser densitometric analysis for each of the five 2-fold competitor dilutions. The molar ratio was obtained by multiplying the density ratio by 228/240 (competitor/tcDNA size) for GAPDH and 116/135 for IGF-1. Data were plotted as log molar ratio (tcDNA/competitor) versus log input competitor. The equivalence point of unknown (tcDNA) and known (competitor) template was determined by linear regression to deduce the initial amount of target cDNA in the sample. Both GAPDH and IGF-1 quantitative competitive PCR were performed for each BAL and PBMC to normalize IGF-1 expression for RNA recovery and RT efficiency by comparison with the GAPDH domestic gene expression.

Semiquantitation of IGFBP Expression cDNA of each sample was diluted to adjust for equal amount of GAPDH evaluated as described above. PCR conditions for IGFBP-2 and IGFBP-3 amplification were similar to those used for GAPDH. PCR cycles were: preincubation for 3 min at 95⬚ C, 38 cycles (94⬚ C 40 s, 59⬚ C 60 s, 72⬚ C 40 s) for IGFBP-2 and 40 cycles (94⬚ C 40 s, 58⬚ C 60 s, 72⬚ C 40 s) for IGFBP-3 and a final incubation at 72⬚ C for 7 min. The number of cycles sufficient to reach the PCR exponential phase was determined for each IGFBP amplification in preliminary experiments. PCR products were electrophoresed on a 3% NuSieve/agarose 3/1 gel (FMC Bioproducts), stained with ethidium bromide (Sigma Chemical, St. Louis, MO), and photographed in UV light. A densitometric analysis was then performed; density values ⬍ 150 arbitrary units were considered as a low level gene expression and noted “⫹”. Values ⬎ 300 arbitrary units were considered as a high level gene expression and noted “⫹⫹⫹”. Intermediate values were noted “⫹⫹”.

IGFBP-3 Western Blot BAL cells culture supernatants were concentrated 10⫻ by a centrifugation filtration using a membrane with a 3 kD cutoff (Amicon, Danvers, MA). Proteins were quantified using the BioRad Protein Assay (BioRad Laboratories, Richmond, CA) according to the manufac-

turer’s instructions. An aliquot of 100 ␮g proteins was incubated with 1/6 volume of 6⫻ Laemmli buffer for 5 min at 100⬚ C and then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Normal human serum served as control. Sera obtained from the patients concurrently with their BAL cells were evaluated in parallel. PAGE was then blotted on 0.45 ␮m nitrocellulose membrane (Schleicher and Schrell, Munich, Germany) overnight at 14 V. The nitrocellulose sheet was then saturated for 4 h at room temperature in phosphate-buffered saline (PBS) containing 10% powdered milk and 0.2% Tween 20 (Sigma). The saturated membrane was then incubated for 20 h at 4⬚ C with rabbit antihuman IGFBP-3 antiserum (UBI, Lake Placid, NY) diluted at 1/1,000 in 5% milk-PBS. It was then washed three times in 0.2% Tween-PBS and incubated for 1 h at 37⬚ C with antirabbit peroxidase-linked species-specific whole antibody (from donkey) (UBI) diluted at 1/6,000 in 5% milk-PBS. After three washes in 0.2% Tween-PBS, the membrane was incubated for 2 min at room temperature with chemiluminescence reaction detection reagents and exposed to autoradiography film (ECL Western Blotting and Hyperfilm-ECL; Amersham, Buckinghamshire, UK). A densitometric analysis based on the specific IGFBP-3 doublet obtained from 100 ␮g of total protein was then performed. Density values ⬍ 200 arbitrary units were considered as low level protein expression and noted “⫹” in Table 2; values ⬎ 400 arbitrary units were considered as high level protein expression and noted “⫹⫹⫹” and intermediate values were noted “⫹⫹”.

Statistical Analysis We compared the IGF-1/GAPDH ratios observed in patients with BOS and in those without BOS at a similar time point, i.e., 3 mo, using a Mann-Whitney U test. We also compared the repartition of individual ratios in the two groups using a ␹2 test; p values ⬍ 0.05 were considered significant.

RESULTS IGF-1 mRNA Expression

For each experiment, the RT- and PCR- controls remained negative. In the study population as a whole, IGF-1 mRNA was detected in every BAL sample examined. However, IGF-1/ GAPDH ratios showed marked variations between patients (Figure 1), whereas they appeared to be relatively stable in each individual subject (Figure 2 and Table 2). There were two distinct profiles of IGF-1 expression. Six patients displayed constantly low IGF-1/GAPDH ratios (⬍ 0.2) over all of the study period (Months 1 to 6 postgraft), whereas the three remaining patients displayed elevated ratios either at each time point stud-

TABLE 2 IGF-1/GAPDH RATIOS IN BAL CELLS Months Postgraft Patient No.

1 mo

2 mo

3 mo

4 mo

5 mo

6 mo

Patients without BOS 1 2 3 4 5 6

ND ND 0.05 0.14 ND 0.18

0.07 0.13 0.04 0.06 0.01 0.03

0.01 0.03 0.11 0.08 0.01 0.10

0.03 0.09 0.09 0.13 0.01 0.17

0.02 ND ND ND 0.03 ND

0.03 0.15 0.15 0.06 0.01 0.18

0.12 ⫾ 0.07

0.06 ⫾ 0.04

0.06 ⫾ 0.04

0.09 ⫾ 0.06

0.02 ⫾ 0.01

0.10 ⫾ 0.07

0.75 0.52 0.55

ND 0.18 0.31

1.12 0.25 0.29

1.81 0.49 ND

1.38 0.06 0.50

2.25 0.07 0.23

0.61 ⫾ 0.12

0.25 ⫾ 0.09

0.55 ⫾ 0.49*

1.15 ⫾ 0.93

0.65 ⫾ 0.67

0.85 ⫾ 1.21

Mean ⫾ SD Patients with BOS 7 8 9 Mean ⫾ SD

* Patients with BOS versus those without BOS; p ⬍ 0.02, Mann-Whitney test.

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Figure 1. GAPDH and IGF-1 mRNA quantitation by competitive RT-PCR in BAL cells recovered at 3 mo postgraft from two patients without BOS (Patient 1: lanes 1–5, Patient 2: lanes 6–10) and two patients with BOS (Patient 8: lanes 11– 15, Patient 7: lanes 16–20). Panel A represents gel electrophoresis of the GAPDH PCR products. For each sample, a constant amount of cDNA was amplified by PCR in presence of five 2-fold dilutions of GAPDH competitor (35.10⫺12 to 2.10⫺12 M). Panel B represents gel electrophoresis of the IGF-1 PCR products. For each sample, a constant amount of cDNA was amplified by PCR in presence of five 2-fold dilutions of IGF-1 competitor (5.10⫺12 to 0.3.10⫺12 M). Arrows indicate visual estimate of each equivalence point.

ied (Patient 7) (range: 0.75 to 2.25) or at least during the first four or five months (Patients 8 and 9) (range: 0.18 to 0.55). It should be emphasized that these last three patients were those who subsequently developed a BOS, whereas the six former patients did not. The comparison of equivalent individual samples at a similar time point (3 mo postgraft) showed a significant dif-

ference between patients with BOS and those without (IGF-1/ GAPDH ⫽ 0.55 ⫾ 0.5 versus 0.06 ⫾ 0.04, respectively, p ⫽ 0.02 Mann-Whitney U test) (Table 2). This difference was present at each time point studied between Months 1 and 4, with values in patients with BOS being, respectively, 5-, 4-, 9- and 13-fold increased compared with those in patients without BOS at the

Figure 2. IGF-1 mRNA expression in BAL cells from nine lung transplant recipients during the first 6 mo postgraft. Each bar represents the IGF-1/ GAPDH ratio obtained by competitive RT-PCR.

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considered intervals (Table 2). Among the three patients with BOS, it is noteworthy that the highest IGF-1 values were observed in Patient 7 who developed BOS within the shortest period (8 mo postgraft). With respect to the repartition of high IGF-1 values among the patients, ratios ⬎ 0.2 were observed only in 13 of 45 samples. Those samples were recovered exclusively in patients with BOS (p ⬍ 0.001, ␹2 test). Of note is the fact that when present this increased expression of IGF-1 was observed from the very beginning of the evolution postgraft, as early as at 1 mo, largely preceding the diagnosis of BOS, ascertained at 8, 14, and 17 mos postgraft, respectively, in the three affected patients. Also of importance, the increased expression of IGF-1 did not appear to be related to episodes of acute lung rejection or of CMV infection, as showed in Table 1. Indeed, despite their significant incidence of acute complications of the transplantation process, Patients 1 to 6 (with no BOS) had a remarkably low and stable expression of IGF-1. Altogether, an early, marked, and prolonged overexpression of IGF-1 transcripts was present in the lung of patients with a subsequent BOS, and only in these patients. Pulmonary Compartmentalization of IGF-1 Expression

To ensure the tissue-specific nature of IGF-1 mRNA expression in lung transplant recipients, we quantitated these transcripts in the cellular extracts from BAL cells and peripheral blood mononuclear cells obtained in parallel in each patient. Unrespective of the data obtained from BAL cells, in which IGF-1/GAPDH ratios ranged between 0.01 and 1.81 in the three representative experiments shown in Figure 3, PBMC displayed a very low IGF-1 expression in all instances (range: 0.01 to 0.02). These data clearly argue for a marked compartmentalization of IGF-1 expression in lung cells, with a very poor expression in mononuclear peripheral blood cells.

Figure 3. IGF-1 mRNA levels assessed by competitive RT-PCR in BAL cells and corresponding PBMC. Data shown here were obtained from two patients without BOS and one patient with BOS.

The next step was to seek for the expression of two major IGF-1 regulatory gene transcripts, IGFBP-2 and -3. For this purpose, we used a semiquantitative PCR according to our preliminary results, which showed clear-cut results. Regarding IGFBP-2, it could be detected in every BAL sample studied. Furthermore, no clear quantitative difference between the patients was observed, as reported in Table 3 and Figure 4. In marked contrast, IGFBP-3 was highly variable among the patients. It was either undetected or at a very low level in six patients, whereas it was strongly expressed in three (Table 3 and Figure 4). Again, it should be underlined that the latter are those who subsequently developed a BOS (Patients 7 to 9). All the negative results were obtained in patients with no BOS (p ⬍ 0.001, ␹2 test). Altogether, there seemed to be a strong relationship between the high expression of IGF-1 and that of IGFBP-3. This was not observed for IGFBP-2, present in all patients irrespective of their clinical status.

all of the samples with highly expressed transcripts were positive for the protein. Finally, among the 14 samples with intermediate results in RT-PCR, eight of 14 (57%) exhibited the IGFBP-3 protein in BAL cells culture supernatants. We performed a densitometric analysis of the bands observed, and reported the results of this semiquantitative analysis in Table 3. BAL cells of Patients 7 and 8, who subsequently developed a BOS, expressed the highest levels of IGFBP-3 protein. As a whole, these data seemed to argue for a poor post-transcriptional regulation of IGFBP-3 in the lung of our patients. However, regarding the size of the protein, there was a clear difference between that observed in control sera and that produced by BAL cells. Indeed, the protein detected in control sera as well as in sera from our patients (Figure 5) appeared as a 39–43 kD doublet, corresponding to the two glycosylation forms of the circulating IGFBP-3 and a 29-kD protein corresponding to a degraded fragment of IGFBP-3 (30). In contrast, IGFBP-3 present in BAL cells culture supernatants of our patients migrated as a doublet with a size between 32.5 and 41.8 kD, arguing for a tissue-specific regulation of IGFBP-3 in the lung of our patients.

IGFBP-3 Protein Expression

DISCUSSION

Given the fact that the regulatory effects on IGF-1 in the lung, if any, are due to its binding proteins gene products, and having demonstrated the preponderance of IGFBP-3 expression in the lung of our patients, we sought for the corresponding protein in their BAL cells culture supernatants, using the Western blotting technique. Normal human serum served as a positive control for the detection of the IGFBP-3 protein. As a whole, there was a nice relationship between the high expression of IGFBP-3 transcripts and the detection of the corresponding protein. Indeed, BAL cells in which no IGFBP-3 mRNA could be demonstrated did not produce any detectable IGFBP-3 in culture. Conversely,

In this study, we have prospectively quantified the IGF-1 mRNA expression in sequential BAL obtained from nine lung transplanted patients during their first 6 mo postgraft. This type of sample appeared as relevant to assess the pulmonary IGF-1 production since the main cellular source of IGF-1 in the lung is the alveolar macrophages (16), which accounted for 75 to 99% of cells recovered by BAL (Table 1). IGF-1 transcripts were detected in all samples, suggesting a constitutive expression in the lung of these subjects. However, the quantitative assessment of this expression revealed important features. First, the three patients with BOS displayed IGF-1 mRNA

IGFBP-2 and IGFBP-3 mRNA Expression

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AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE TABLE 3 IGFBP-2 AND IGFBP-3 IN BAL CELLS

Patient No.

Months Postgraft

IGFBP-2 mRNA*

IGFBP-3 mRNA*

IGFBP-3 Protein†

1

2 3 4 5 6

⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹

⫹ ⫺ ⫹⫹ ⫹ ⫺

⫹ ⫺ ⫹⫹

2

2 3 4 6

⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹

⫺ ⫺ ⫺ ⫺

⫺ ⫺

3

1 2 3 4 6

⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹

⫺ ⫹ ⫹ ⫺ ⫹

⫺ ⫺ ⫹

4

1 2 3 4 6

⫹⫹ ⫹ ⫹⫹ ⫹⫹ ⫹⫹

⫺ ⫹ ⫺ ⫹ ⫹⫹

⫺ ⫺ ⫺ ⫹ ⫹

5

2 3 4 5 6

⫹⫹ ⫹ ⫹ ⫹⫹ ⫹⫹

⫺ ⫹ ⫺ ⫺ ⫺

⫺ ⫹ ⫺

6

1 2 3 4 6

7

1 3 4 5 6

⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹

⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹

⫹ ⫹⫹ ⫹⫹

8

1 2 3 4 5 6

⫹⫹ ⫹ ⫹⫹ ⫹ ⫹⫹ ⫹⫹

⫹⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹

⫹⫹⫹ ⫹ ⫹⫹ ⫹⫹⫹ ⫺ ⫹

9

1 2 3 4 5 6

⫺

⫺

⫺

⫹ ⫺ ⫺ ⫹

⫺

⫹⫹ ⫹⫹⫹ ⫹⫹

* mRNA were quantified by semiquantitative PCR. Density values ⬍ 150 arbitrary units (a.u.) are noted ⫹, ⬎ 300 a.u. are noted ⫹⫹⫹, and intermediate values are noted ⫹⫹. Undetectable values are noted ⫺. † IGFBP-3 protein semiquantitation was performed by Western blot on 100-␮g total protein obtained from BAL cell culture supernatants. Density values ⬍ 200 a.u. are noted ⫹, ⬎ 400 a.u. are noted ⫹⫹⫹, and intermediate values are noted ⫹⫹.

levels 4- to 13-fold higher than those of patients without BOS at each time point studied between Months 1 to 4. Moreover, the patient who developed a BOS within the shortest delay presented the highest IGF-1 values as early as at 1 mo postgraft, and over all of the study period (Patient 7). Lastly, this overexpression of IGF-1 was observed exclusively in those patients with BOS. Altogether these data might suggest a role for IGF-1 in the fibrotic process leading to the obliterative bronchiolitis that characterizes chronic lung rejection. IGF-1 has been shown to be involved in pulmonary fibrotic conditions such as coal worker’s pneumoconiosis (20), systemic sclerosis (19), idiopathic pulmonary fibrosis (18), and sarcoi-

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dosis (21). These studies focused on the IGF-1 protein content of BAL fluid. Our approach using RT-PCR, in addition to being quantitative, is also far more sensitive, and could serve to detect a small increase in IGF-1 earlier than the protein measurements in BAL. This could prove to be of importance in the follow-up of these patients. Indeed, we have also demonstrated an important characteristic of IGF-1 expression in the context of lung transplantation, i.e., its early and sustained expression over the first 4 to 6 mo postgraft in patients who subsequently develop a BOS. This thereby defined a distinct profile of expression that might be very indicative of this severe complication. Such a profile has never been, to our knowledge, described in the context of human pulmonary fibrosis. In contrast, the role of such an early and sustained expression of this profibrotic agent has been emphasized in an animal model of bleomycin induced pulmonary fibrosis where it could be serially assessed (31). The investigators had shown that the IGF1 transcripts were increased 3- to 8-fold in the bleomycin-treated animals compared with those in a control group, and that they could be detected for as long as 4 wk after exposure to the drug, whereas fibrosis was demonstrated thereafter at 4 wk postexposure (31). On the other hand, we were able to detect an IGF-1 overexpression in three of our patients several months before the diagnosis of BOS was ascertained. We had shown in a previous study that peaks of TGF-␤ mRNA in alveolar cells could serve as an early marker of BOS (32). However, the peaks were transient in some patients, and appeared occasionally very late in the follow-up. In the context of the requirement of early therapeutic modifications before the fibrotic changes have become irreversible, IGF-1 monitoring in lung transplant patients might prove to be more reliable than TGF-␤. This is currently investigated in our institution through a prospective study evaluating the role of several cytokines in the development of BOS. Finally, a striking feature of IGF-1 expression in our patients was its preponderance in the lung. Indeed, in contrast to its significant variations in BAL cells according to the patients’ clinical status (BOS or no BOS), it appeared to be remarkably low in PBMC, irrespective of the patients’ status. Clearly, this phenomenon is locally regulated, and the production of IGF-1 is tissue-specific. The physiopathologic significance of this phenomenon is still speculative. We have shown that it did not appear to be related to identified acute complications of transplantation such as episodes of ALR or CMV infection. Several mechanisms could underlie IGF-1 increased expression in the lung of transplant recipients. The triggering event might be allogeneic in nature and undetected by conventional clinical and pathologic screening methods. It might also be due to an unidentified pathogen. A so far unidentified inflammatory event occurring in the very early post-operative period should be also be considered in the context of the early and sustained increase of IGF-1 expression in affected patients. Ischemia for instance has been underlined as a possible mechanism leading to BOS (11). In any case, whatever the origin of IGF-1 expression, some arguments suggest that it might actually be involved in the fibrotic process leading to BOS. Recently, it has been shown in a model of aortic transplantation in the rat, that the ex vivo treatment of donor graft with IGF-1 before transplantation increased the local vascular expression of IGF-1 during several weeks and accelerated transplant arteriosclerosis (33). Accordingly, we could hypothesize that the high intrapulmonary expression of IGF-1, whatever its origin, may indeed be involved in the tissue-repair process that follows lung injury in these subjects. The bioavailability of IGF-1 in the lung is regulated by at least six highly specific binding proteins. The present study fo-

Charpin, Stern, Grenet, et al.: IGF-1 and Chronic Lung Rejection

1997

Figure 4. IGFBP-2 and IGFBP3 mRNA semiquantitation in BAL cells from two patients without BOS (Patient 1: lanes 1–5, Patient 2: lanes 6–9) and two patients with BOS (Patient 7: lanes 10–14, and Patient 8: lanes 15–18). Panel A represents gel electropheresis of the GAPDH PCR products. Panel B represents gel electropheresis of the IGFBP-2 PCR products. Panel C represents gel electropheresis of the IGFBP-3 PCR products.

cuses on two of them, IGFBP-2 and IGFBP-3, because of their reported preponderance in the human lung. Indeed, IGFBP-2 is considered as the major IGFBP expressed in the lung and has been involved in the fibrosis process that characterizes lung interstitial fibrosis in children (22). IGFBP-3 is the major circulating IGFBP and has been involved in idiopathic pulmonary fibrosis (18) and in sarcoidosis (21). In the present study, we have shown that IGFBP-2 was highly expressed in all the BAL studied. In contrast to the increased IGFBP-2 expression observed in the context of interstitial lung disease in children (22), we did not find any overexpression related to the BOS in lung transplant recipients. In contrast, IGFBP-3 expression did not appear as constitutive in these patients. It remained undetected by our sensitive RT-PCR method in 16 of 42 BAL samples, whereas it was highly expressed in the 13 of 14 samples recovered in the three patients with a subsequent BOS. Furthermore, the corresponding gene product was present at high levels in the lung of the same patients. A similar increased expression of IGFBP-3 in the fibrotic lung has been previously described in idiopathic pulmonary fibrosis and in sarcoidosis (18, 21). However, the role of IGFBP-3 in the lung remains uncertain. Some in vitro studies have shown that IGFBP-3 can either potentiate or inhibit IGF-1 profibrotic actions depending on the experimental conditions (30). In an impaired wound healing animal model, the corticosteroid-treated rat, an application of equimolar concentrations of IGFBP-3 and IGF-1 on the wound resulted in a wound-healing acceleration, whereas

IGF-1 alone had no such effects (34). In our patients, IGF-1 and IGFBP-3 were concurrently and highly expressed markedly before the diagnosis of BOS. This might indicate that IGFBP-3 is positively regulated in these patients to potentiate the effects of IGF-1. The IGFBP-3 staining protein observed on immunoblotting of BAL cells culture supernatants migrated as a doublet with a size between 32.5 and 41.8 kD. This size was lower than the doublet observed on immunoblots from normal human serum, as well as from patients’ sera, which was 39 to 43 kD. The two bands of the IGFBP-3 doublet observed in the sera have been previously described and represent different glycosylation forms of IGFBP-3 (30). The doublet observed in the culture supernatants might represent different glycosylation forms of a tissue-specific IGFBP-3, arguing for different mechanisms of protein maturation in the lung and in the peripheral blood. A similar phenomenon has already been reported with no elucidation of the biochemical structure of this tissue specific IGFBP-3 (18, 21). To conclude, we have shown in the present study that the development of BOS in lung transplant recipients might be associated with a very early, marked, and sustained expression of IGF-1 and IGFBP-3 in the lung. These data led us to consider the relevance of the increase of these two mediators as an early marker of BOS. On the other hand, our data might appear as an original insight into the pathogenesis of BOS, largely unknown so far. It might be the common expression of allogeneic events occur-

Figure 5. Western immunoblot analysis of IGFBP-3. Lanes 1 and 2: normal human serum used as control; lanes 3 and 4: BAL cells culture supernatants at 4 mo postgraft from Patients 1 and 2, respectively; lanes 5 and 6: BAL cells culture supernatants at 4 mo postgraft from Patients 8 and 7, respectively.

1998


ring during ALR episodes, and of inflammatory ones occurring either very early in the process or later in the course of the postgraft evolution. Among early events, the role of ischemia has been pointed to, but other critical factors probably remain unidentified so far. In any case, the IGF-1/IGFBP-3 pair could have a crucial role in the pathogenesis of the fibroproliferative disorder that underlies the BOS through the potentiation of IGF-1 profibrotic activity by its major regulatory protein, IGFBP-3. It is clear that these data need to be confirmed by a larger prospective study. The value of sequential determinations of IGF-1/IGFBP-3 expression in BAL cells from lung transplant recipients is currently being investigated in our institution. If our data are confirmed, this could lead to new recommendations for the management of these patients and to the delineation of more aggressive and more specific therapeutic strategies in the context of chronic lung rejection. Acknowledgment : The writers gratefully acknowledge Pr. Annick Clement and Dr. Katarina Chadelat (INSERM U515, Hôpital Trousseau, Paris). They also wish to thank Dany Kucharczkyk, Anne Dumoncel, and Anne-Marie Laval for excellent technical assistance.

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