INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Insulin-like growth factor-1 (IGF-1) is a highly mitogenic polypeptide produced by most tissues, but predominantly by the liver. It may act as an autocrine or paracrine growth factor (1) and mediates most of the peripheral actions of growth hormone (GH). There is evidence that increased numbers of alveolar macrophages (AM) and interstitial cell types (2, 3) demonstrate expression of IGF-1 in patients with idiopathic pulmonary fibrosis (IPF) and pulmonary sarcoidosis; increased messenger RNA (mRNA) expression has been reported in murine lung tissues after induction of bleomycin-induced fibrosis (4). Because IGF-1 induces fibroblast proliferation and increased fibroblast transcription of collagen and laminin genes, this increased IGF-1 expression may be involved in the pathogenesis of these diseases. Indeed, bronchoalveolar lavage fluid (BALF) from patients with fibrotic lung disease, such as pulmonary sarcoidosis (5), pneumoconiosis (6), and systemic sclerosis (7) exhibited IGF-1-mediated increased fibrogenic activity.

The human IGF-1 gene comprises six exons spanning a region of over 90 kilobases of genomic DNA located on the long arm of chromosome 12. Exons 1 and 2 are alternatively spliced leader exons (1) coding for the 5' untranslated region and contain distinct transcription start sites. These are differentially spliced to the common exon 3 to produce Class 1 (exons 1 to 3) or Class 2 (exons 2 to 3) IGF-1 mRNA transcripts. The mature IGF-1 peptide (B, C, A, and D domains) and the first 16 amino acids of the E domain are coded by exons 3 and 4. The 5' end of exon 3 also encodes the signal peptide on the prohormone. Exons 5 and 6 are alternatively spliced and encode for part of a distinct extension peptide, called the E domain. Use of exon 5 in either Class 1 or Class 2 transcripts produces an IGF-1Eb transcript, whereas splicing of exon 5 (exon 4-6 transcript) gives Class 1 or 2 IGF-1Ea transcripts. The IGF-1Ea and IGF-1Eb type complementary deoxyribonucleic acids (cDNAs) code for distinct 3' untranslated regions and different E domain coding sequences (8).

The biologic significance of these splice variants is unknown. It has been proposed that preferential use of exon 1- derived leader transcripts could be linked to synthesis of paracrine IGF-1, and may influence interaction with insulin-like growth factor binding protein (IGFBP), or promote the formation of the truncated IGF-1 peptide. Furthermore, use of exon 5 (IGF-1Eb) may favor an endocrine IGF-1 fate, rather than exon 6 (IGF-1Ea) which may be associated with local IGF-1 action (9, 10). There is some evidence that local concentrations of both GH and IGF-1 itself may regulate the proportions of the different transcripts.

This study investigates whether the differences in IGF-1 expression reported in lungs of patients with fibrotic pulmonary disease are associated with changes in the relative expression of particular IGF-1 splice variants. A comparison has been performed between the expression of Class 1 or 2 and IGF-1Ea or IGF-1Eb transcripts in fibroblast strains from normal and fibrotic lung, and bronchoalveolar lavage cells (BALC) obtained from pulmonary sarcoidosis patients, including those with confirmed fibrosis, IPF, and healthy control subjects using competitive reverse transcriptase/polymerase chain reaction (RT-PCR). The data show that BALC from patient groups exhibit a paradoxical reduction in total levels of IGF-1 mRNAs. However, this is more than compensated for by the increased IGF-1 expression found in fibroblast strains. Fibrotic lung fibroblasts constitutively express more IGF-1 than normal cells and show enhanced IGF-1 responsiveness to transforming growth factor- (TGF-) treatment. When the relative expression of IGF-1 transcripts was compared by the ratios of Class 1 to Class 2, and of IGF-1Ea to IGF-1Eb, it was found that IPF BALC expressed significantly more Class 1 over Class 2 and significantly less IGF-1Ea over IGF-1Eb compared with fibrosis-free subjects. Furthermore, the sarcoidosis patients with fibrosis did not express IGF-1Eb in either the Class 1 or Class 2 form. Class 1 IGF-1Ea was the dominant form to be expressed by all fibroblasts. Uniquely, Class 2 IGF-1Ea, Class 1 IGF-1Eb, and Class 2 IGF-1Eb transcripts were only expressed by fibroblasts after TGF- stimulation. Thus, these findings suggest that IGF-1 expression in the fibrotic lung is altered in response to changes in local cell phenotypes and mediators, and this is exerted through the pattern of alternative mRNA splicing. We speculate that variants could have different fibrogenic properties to account for this.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Subject Group

Seven patients (2 male, 5 female; mean age 43.9 yr, range 29 to 60 yr) with biopsy-proven sarcoidosis presented with acute symptoms of cough, dyspnea, or chest pain. Radiographic studies (conventional chest radiograph and high-resolution computed tomography [HRCT] scan) confirmed that all seven patients had radiologic stages I/II with no evidence of established parenchymal fibrosis. Four of these patients had normal lung function, whereas three patients had reduction in lung volume and carbon monoxide transfer factor. All seven patients with sarcoidosis had high serum angiotensin-converting enzyme levels, mean 104 ± 19 IU/L (normal range 18 to 55). Six patients (5 male, 1 female; mean age 50.8 yr, range 34 to 63 yr) with biopsy-proven sarcoidosis had evidence of radiographic stage III/IV sarcoidosis with bilateral nodular parenchymal infiltration, including areas of fibrosis and honeycombing; all exhibited decreases in lung volume carbon monoxide transfer factor.

Seven patients (4 male, 3 female, mean age 67.4 yr, range 47 to 77 yr) with IPF were investigated. They presented with cough or dyspnea (duration 9 ± 2 mo), bilateral basal to midzone inspiratory crackles, and radiographic bilateral interstitial fibrosis, supported by pathognomonic HRCT scan patterns of varying degrees of reticular infiltrates, diffuse honeycombing, and sparse ground-glass opacification in a predominately subpleural and basal distribution. Of the seven patients, five had lung biopsies with histologic confirmation of usual interstitial pneumonia. All seven patients showed significant reduction in total lung capacity and transfer factor. None of the sarcoidosis and IPF patients had received any prior steroid or immunosuppressive therapy. None of these patients were current smokers, or had any documented evidence of previous respiratory disease.

The control group comprised eight healthy nonsmoking volunteers (4 male, 4 female; mean age 21.6 yr, range 19 to 25 yr) with normal pulmonary function. None of the recruited subjects had suffered any respiratory symptoms in the 6 wk preceding the study. Prior study approval was obtained from the Research Ethics Committee of the North Staffordshire Hospital Trust, Stoke-on-Trent, UK. All subjects gave informed written consent.

Several investigators have demonstrated a decrease in plasma/serum concentrations and mRNA expression of IGF-1 with increasing age (11). Therefore, in an effort to minimize bias arising from control/patient age differences, the age range of the stage III/IV sarcoidosis patient group overlaps both the stage I/II sarcoidosis group and the patients with IPF. The ages of each group ranged between, 19 and 25 yr for normal subjects, 29 to 60 yr for sarcoidosis stage I/II patients, 34 to 63 yr for sarcoidosis stage III/IV patients, and 47 to 77 yr for patients with IPF.

Bronchoalveolar Lavage (BAL)

BAL was performed on all subjects as described previously (5) using flexible fiberoptic bronchoscopy under local anaesthesia. Briefly, the right middle lobe was instilled with successive aliquots of sterile 0.9% isotonic saline, to a volume of 180 ml. The lavage fluid aliquots were immediately gently aspirated by suction, and collected into sterile siliconized glass bottles maintained at 4° C for processing.

Sample Processing

The BALF was filtered through sterile surgical gauze, and centrifuged at 400 × g/4° C/10 min; the retrieved cell pellet was washed with RPMI 1640 culture medium (Life Technologies, Paisley, UK). Cell viability was assessed by cell exclusion of trypan blue. The cell concentration in RPMI 1640 was adjusted to 1 × 10⁶ cells/ml. 3 × 10⁵ cells were removed for cytospin preparations (Cytospin 2; Shandon, Basingstoke, UK). Cytospins were stained for morphology (Giemsa stain plus May-Grunwald stain; both Merck Ltd., Lutterworth, UK) and a differential cell count was performed. The remaining cell suspension was recentrifuged 550 × g/10° C/10 min and the cell pellet was homogenized using 1 ml of Trizol per 10⁶ cells (Life Technologies) and stored at 80° C.

Fibroblast Cell Culture

Primary fibrotic lung fibroblasts isolated from a patient with IPF were kindly provided by Dr. R. McAnulty (Centre for Respiratory Research, Royal Free and University College Medical School, London). The strain was designated HIPF-1. These cells are characterized by a more myofibroblastlike phenotype than normal cells. In culture they have a more flattened, dendritic morphology and show increased -smooth muscle actin expression. HIPF-1 and normal human lung fibroblasts (strain IMR90; American Type Culture Collection, Rockville, MD) were routinely cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% (vol/vol) fetal calf serum (FCS) and 1× antibiotic/ antimycotic (Sigma, Poole, UK). Cells were seeded at a density of 10⁵ per 25-cm² flask and cultured for 3 d to near-confluence. Medium was replaced with fresh DMEM containing 0.2% (vol/vol) FCS (low-serum media), for 24 h. Quiescent cells were then stimulated with low-serum media containing 10 ng/ml of TGF- (R&D Systems, Abingdon, UK) for a further 24 h, whereas controls remained in the low-serum media alone. Fibroblasts were then homogenized in Trizol for RNA extraction.

RT-PCR

Total RNA was purified from the Trizol homogenates according to the manufacturer's protocol previously described by Allen and associates (5). For cDNA synthesis, 2 µg total RNA was used in a 20-µl reaction, incubated for 1 h at 42° C with the following: 20 pmol oligo dT primer; 75 mM KCl, 3 mM MgCl₂, 50 mM Tris-HCl, pH 8.3, 0.5 mM each deoxynucleoside triphosphate (dNTP), 0.5 U ribonuclease (RNase) inhibitor, and 200 U murine Moloney leukemia virus (MMLV) reverse transcriptase (Clontech, Cambridge Bioscience, Cambridge, UK). The reaction was terminated by heating at 95° C for 5 min, then chilled on ice. cDNA was diluted in 100 µl deionized water (dH₂O) and stored at 80° C. For the BALC samples, 15 µl of a further 1:100 dilution was used in each IGF-1 transcript RT-PCR reaction, whereas 2 µl of undiluted cDNA was used for the fibroblast cell lines, and 5 µl of 1:100 diluted cDNA was used for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

RT-PCR Primers

Specific RT-PCR primers were designed with computer-aided assistance (Primer, version 0.5; Whitehead Institute for Biomedical Research, Cambridge MA). Native product oligonucleotides and competitive template (CT) primers (Table 1) were synthesized commercially (Cruachem Ltd., Glasgow, UK). Primer pairs were selected from different exons to identify the different IGF-1 transcripts. To investigate the expression of Class 1 transcripts the primer for exon 1 was used, whereas for Class 2 the exon 2 primer was used, in combination with either the exon 5 primer for IGF-1Eb transcripts or the exon 6 primer for IGF-1Ea splice variants (Figure 1). For the housekeeping gene GAPDH primers were designed that spanned introns in the genomic template, so any genomic contamination could be identified by increased product size. Primers for both the native and CT amplifications were optimized for use under the same RT-PCR conditions, and were designed to give shorter CT RT-PCR products when the native sense and CT primers were used. The product sizes were sufficiently different to measure the density of product bands on 2% agarose gel. The CT and native sense primer were the same whereas the CT antisense primer was specifically synthesized to produce CT products.

View larger version (12K): [in this window] [in a new window]   Figure 1.   Schematic representation of genomic DNA gene structure, for human IGF-1. The exons are depicted as numbered boxes. The black boxes illustrate the mature coding region. The white boxes represent the untranslated 5' and 3' regions and the gray areas are the precursor-unprocessed peptide. The thick black lines indicate the positions where alternative splicing occurs. Finally, the arrows and labels a-d on the diagram correspond with our RT-PCR primer locations used to investigate the IGF-1 transcripts.

RT-PCR Reaction Conditions

Each RT-PCR reaction had a total volume of 50 µl and consisted of 25 pmol primers, an aliquot of master mix containing 100 µM dNTPs, 1.5 mM MgCl₂, 5 µl 10 × PCR buffer, and 2.5 U Amplitaq Gold DNA polymerase (Perkin-Elmer, Warrington, UK) and an aliquot of cDNA as previously described. A "hot start" RT-PCR was employed and cycling conditions were as follows: 95° C enzyme activation for 12 min, then 1 min 15 s at 94° C denaturation, 1 min at 55° C (IGF-1 transcripts) or 1 min at 60° C (GAPDH) annealing and 1 min at 72° C primer extension, for 41 cycles (IGF-1 transcripts) or 31 cycles (GAPDH), then a final extension of 10 min at 72° C. The cycle numbers and conditions were established from preliminary experiments so that amplifications remained in the exponential phase (results not shown).

CT Preparation

The CT RT-PCR reactions consisted of 25 pmol of the sense and CT primer under the conditions previously described. A 15-µl aliquot of the RT-PCR product was analyzed by electrophoresis through a 2% Seakem agarose gel (Flowgen Instruments, Lichfield, UK) and stained with 2 µg/ml ethidium bromide, then identified by predicted product size. The CT RT-PCR products were extracted and purified using Microspin S-300HR columns (Pharmacia Biotech, St. Albans, UK). The concentration of the pure CT was quantified by electrophoresis as previously described using 4 or 8 µl of CT, adjacent to a 7.5 to 30 ng range of a prepared size standard and analyzed densitometrically (Bio-Rad GS-670 densitometer and Molecular Analyst software, Hemel Hempstead, UK). Size standards were prepared from pBR322 (Life Technologies, Paisley, UK) cut with restriction enzymes (Roche Diagnostics, Lewes, UK): Ssp I with Pvu I to give 434 bp (for 440 bp exon 1/6 CT), or Sph I with Nhe I to give 345 bp (for 349 bp exon 2/6 CT), or Pvu I with Eco RV to give 814 bp (for 805 bp exon 1/5 CT), or Bsm I with Sal I to give 703 bp (for 714 bp exon 2/5 CT), or Hind III to give 909 bp (for 903 bp GAPDH CT). Serial dilutions of each CT were prepared from the same original stock solution. Either 1 or 5 µl of the required CT dilution was included to each 50 µl RT-PCR, with the addition of 4 µl dH₂O to the reactions with only 1 µl of CT.

Competitive RT-PCR

All RT-PCR reactions were performed in triplicate under the conditions previously described and visualized using 2% agarose gels and ethidium bromide staining as before. Both the CT and native RT-PCR products were quantified for each reaction using digital image analysis. The product bands were not saturated, therefore within the dynamic linear range of the system. They were quantified densitometrically on the basis of total ethidium bromide staining, where the intensity was dependent on both the number and size of molecules present in each band. Thus, it was necessary to employ a correction factor to account for the differences in size between native and CT products. Therefore, the staining intensity of the native band was corrected to the size of the CT product band. Gene expression was calculated as we have previously described (14) using Equation (1), where iN is the initial number of native molecules, ND is the native band density, CTD is the CT band density, CTs is the CT size in base pairs, Ns is the native size in base pairs, and iCT is the initial number of CT molecules.

(1)

Results were then expressed as the mean number of molecules for each IGF-1 splice variant mRNA per 10⁶ molecules of GAPDH mRNA for BALC or fibroblasts. Expression of the housekeeping gene (GAPDH) was used to provide an index for equality of RNA loading into the RT reaction and to verify the success of the cDNA synthesis, thus reducing errors incurred for sample variation in data analysis.

RT-PCR Product Analysis

The native IGF-1 transcripts and GAPDH RT-PCR products were verified by their predicted product sizes of 502 bp for exon 1/6, 411 bp for exon 2/6, 863 bp for exon 1/5, 772 bp for exon 2/5, and for GAPDH 983 bp. For further confirmation the RT-PCR products underwent restriction digests at unique cutting sites. Exon 1/6 products were digested with RsaI, exon 2/6 with BanI, and exon 1/5 and 2/5 RT-PCR products were restricted at a PstI site.

Statistical Analysis

Data were analyzed using Stata software (Intercooled Stata, version 6; Timberlake Consultants, West Wickham, UK). Results from competitive RT-PCR were expressed as the mean of three independent experiments. A Kruskal-Wallis test was performed to test for differences in variant expression between and within subject groups. Where a significant difference was indicated, each pairing was examined using Student's t test. The effects on IGF-1 variant expression of cell type (AM, neutrophils, lymphocytes) and cell number (cells per milliliter of BALF) were evaluated by calculating the Pearson product-moment correlation coefficient. In all cases the level of significance was p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Characterization of BALC

Distribution of BALC cell types for each subject group is presented in Figure 2. The most important features observed were that patients with stage I/II sarcoidosis showed significant (p < 0.02) alveolar lymphocytosis (30% of total cells), patients with stage III/IV sarcoidosis demonstrated significantly increased numbers of neutrophils (p < 0.01), and patients with IPF had significantly (p < 0.05) increased numbers of neutrophils (19% of total cells) and eosinophils (p < 0.003) (4% of total cells) in comparison to normal volunteers. IGF-1 transcript expression failed to correlate with individual cell types, although it is most likely that the majority is AM-derived.

View larger version (41K): [in this window] [in a new window]   Figure 2.   BALC differential cell counts for all subjects, expressed as total number cells/106 cells ± SEM, including alveolar macrophages (AM), neutrophils (N), lymphocytes (L), and eosinophils (E). *Patient cell distribution was significantly different from normal subjects (p < 0.05).

Expression of IGF-1 mRNA Splice Variants

A competitive RT-PCR method was implemented to amplify the four major classes of IGF-1 splice variants and the housekeeping gene GAPDH. The investigation was performed on BALC isolated from pulmonary sarcoidosis patients, IPF patients, and normal volunteers. IGF-1 transcript expression was also determined in lung fibroblast strains, from normal (IMR90) and fibrotic lung (HIPF-1). Expression was examined in the presence or absence of TGF-, an important fibrogenic mediator known to be increased in fibrosis. To investigate the expression of IGF-1 alternatively spliced transcripts, specific sense primers for exon 1 or 2 and antisense primers for exon 5 or 6 (Table 1) were used in the combinations outlined in METHODS. Figure 3 is a representative agarose gel demonstrating RT-PCR products of IGF-1 transcript expression from a selection of normal and patient BALC samples, and fibroblast strains. Under the conditions employed, the RT-PCR products amplified from GAPDH and all the four IGF-1 mRNA transcripts could be detected by ethidium bromide staining in BALC from normal and stage I/II sarcoidosis patients and in both fibroblast cell lines only after TGF- stimulation. However, Class 2 IGF-1Eb transcripts were not detected in BALC from IPF and sarcoidosis stage III/IV patients. The latter also failed to express Class 1 IGF-1Eb. The amplification of both the native and synthesized products corresponded with the predicted sizes calculated from published sequence information (Table 1). For further verification restriction analysis was performed. IGF-1 products were restricted at unique cutting sites to give the following products. Class 1 IGF-1Ea cut with RsaI gave 81 bp and 421 bp products, Class 2 IGF-1Ea cut with BanI gave products of 135 bp and 276 bp fragments, whereas Class 1 IGF-1Eb and Class 2 IGF-1Eb cut with PstI produced 262 bp/600 bp and 262 bp/510 bp respectively. The products complied with the expected cutting pattern predicted from sequence data, therefore confirming the RT-PCR product identities.

View larger version (46K): [in this window] [in a new window]   Figure 3.   Representative agarose gel showing RT-PCR products from the four IGF-1 transcripts. Lanes A to D contain Class 1 IGF-1Ea transcripts (502 bp), lanes E to H contain Class 2 IGF-1Ea transcripts (411 bp), lanes I to L contain Class 1 IGF-1Eb transcripts (863 bp), and lanes M to P contain Class 2 IGF-1Eb transcripts (772 bp). Lanes C, E, J, and P are BALC from normal subjects; lanes F, K, and N are BALC from patients with stage I/II sarcoidosis; lanes B, H, and I are BALC from patients with stage III/IV sarcoidosis; lanes A and O are BALC from patients with IPF. Lane D contains IMR90 control, lane G contains HIPF-1 control, lane M contains IMR90 with TGF-, and lane L contains HIPF-1 with TGF-. The markers at either side of the gel are a 100 bp ladder.

Quantification of IGF-1 mRNA Splice Variants

Differential expression of the variant forms was observed within and between each of the BALC subject groups (Figure 4A). First, we summarize comparisons within BALC groups; in normal subjects, compared with the most abundant transcript, Class 1 IGF-1Ea, significant reductions in expression of the other variant transcripts were observed. In descending order these were Class 1 IGF-1Eb (p < 0.04) > Class 2 IGF-1Ea (p < 0.03) > Class 2 IGF-1Eb (p < 0.004). In patients with stage I/II sarcoidosis only Class 2 IGF-1Eb expression was significantly lower than the Class 1 IGF-1Ea (p < 0.02). In patients with stage III/IV sarcoidosis Class 1 IGF-1Ea and Class 2 IGF-1Ea were the only transcripts to be expressed. In patients with IPF expression of Class 1 IGF-1Ea and IGF-1Eb transcripts were not significantly different from each other but were significantly higher than Class 2 IGF-1Ea (p < 0.009). Class 2 IGF-1Eb transcripts were not detected.

View larger version (52K): [in this window] [in a new window]   Figure 4.   (A) Relative mRNA expression of IGF-1 splice variants in unfractionated BALC from normal subjects and patients with ILD. Results from all samples are presented as the log of the mean number of IGF-1 molecules/106 GAPDH ± SEM. Asterisk indicates that normal subjects had significantly higher IGF-1 transcript expression than the patients (p < 0.03). (B) Relative mRNA expression of IGF-1 splice variants in fibroblast cell lines isolated from normal lung (IMR90) and from IPF lung (HIPF-1) with TGF- (T) stimulation and without (C). The stimulated fibroblasts expressed significantly higher amounts of each transcript than the controls (p < 0.05).

Second, we consider comparisons between groups; expression of all the variants was higher in normal subjects compared with the patient groups. Expression of Class 1 IGF-1Ea was significantly higher in normal subjects than each patient group (p < 0.03) but there were no significant differences in expression of Class 1 IGF-1Ea between patient groups. Class 2 IGF-1Ea expression was also significantly higher in normal subjects compared with the patient groups (p < 0.01). However, expression of Class 2 IGF-1Ea was significantly higher in stage I/II sarcoidosis patients compared with stage III/IV sarcoidosis patients and patients with IPF (p < 0.01). Class 1 IGF-1Eb expression was not significantly different between any of the subject groups except patients with stage III/IV sarcoidosis where there was no expression. Finally, Class 2 IGF-1Eb was not expressed by either the patients with stage III/IV sarcoidosis or the patients with IPF, and expression was not significantly different from zero in the stage I/II sarcoidosis patients, but was significantly higher in normal subjects compared with each patient group (p < 0.02). Fibroblast strains constitutively expressed only Class 1 IGF-1Ea, and that expression was significantly enhanced (p < 0.05) with TGF- stimulation (Figure 4B), which also led to expression of Class 2 IGF-1Ea, Class1 IGF-1Eb, and Class 2 IGF-1Eb.

Expression of Leader Exons and E-domain Sequences

Use of the alternative exons 1 or 2, coding for Class 1 or Class 2 pro-IGF-1 respectively, is shown in Figure 5A. Expression of Class 1 transcripts predominated in BALC of normal subjects and patients with interstitial lung disease (ILD). Overall, abundance of exon 1 and exon 2 transcripts was significantly reduced in patient groups (p < 0.01) compared with normal subjects. The ratio of exon 1:exon 2 transcripts, which shows their relative abundance, remains similar at 2:1 in the normal subjects and 2:1 in patients with stage I/II sarcoidosis. However, this ratio rises steadily in sarcoidosis stage III/IV BALC to 5:1 and increases dramatically to 23:1 in patients with IPF. Fibroblasts express high constitutive levels of Class 1 transcripts (Figure 5B), increased further by TGF- treatment. Interestingly, there is significant (p < 0.004) dramatic induction of Class 2 expression only in response to TGF- stimulation. There were no significant differences between TGF--stimulated IMR90 and HIPF-1 Class 2 expression.

View larger version (40K): [in this window] [in a new window]   Figure 5.   (A) Relative mRNA expression of Class 1 and Class 2 IGF-1 splice variants in unfractionated BALC. The data are expressed as the log of the mean number of IGF-1 molecules/106 GAPDH ± SEM. Asterisk indicates that the expression of each transcript was significantly reduced in the patient groups compared with normal subjects (p < 0.01). (B) Relative mRNA expression of Class 1 and Class 2 IGF-1 splice variants in fibroblast cell lines isolated from normal lung (IMR90) and IPF lung (IPF-1) with TGF- stimulation (T) or without (C). Class 1 expression was significantly higher than Class 2 for all the fibroblasts (p < 0.03), and Class 2 expression was significantly higher in both cell lines after TGF- stimulation compared with the controls (p < 0.004).

Figure 6A shows the expression of transcripts encoding either the IGF-1Ea or IGF-1Eb forms of pro-IGF-1 in BALC. IGF-1Ea was abundantly expressed by normal subjects and all patients with sarcoidosis, but with notable differences. In comparison to normal subjects, IGF-1Ea transcript numbers were significantly lower in BALC from patients with IPF (p < 0.005), stage I/II sarcoidosis (p < 0.009), and stage III/IV sarcoidosis (p < 0.004). Expression of IGF-1Ea was significantly further reduced in IPF patients (p < 0.03) and sarcoidosis stage III/IV (p < 0.02) compared with the stage I/II sarcoidosis group. The ratio of IGF-1Ea:IGF-1Eb transcripts again remains similar for normal subjects and patients with stage I/II sarcoidosis, both at 2:1. However, in patients with IPF this ratio falls to 0.4:1, indicating the predominance of IGF-1Eb transcripts in these patients. Patients with stage III/IV sarcoidosis failed to express IGF-1Eb. Figure 6B shows the expression of E-domain transcripts in the fibroblast cell lines. IGF-1Ea was the predominant transcript expressed constitutively and was increased further after TGF- treatment. Under basal conditions, fibroblasts failed to express IGF-1Eb, but interestingly expression was induced after TGF- stimulation. Expression of IGF-1Ea was significantly higher (p < 0.04) in HIPF-1 cells compared with IMR90 under basal and TGF--stimulated conditions. However, there was no significant difference in TGF--stimulated IGF-1Eb expression between IMR90 and HIPF-1, but both were significantly higher than their respective controls (p < 0.02).

View larger version (38K): [in this window] [in a new window]   Figure 6.   (A) Relative mRNA expression of IGF-1Ea and IGF-1Eb splice variants in unfractionated BALC. The data are expressed as the log of the mean number of IGF-1 molecules/106 GAPDH ± SEM. Asterisk indicates that expression of each transcript was significantly reduced in the patient groups compared with normal subjects (p < 0.01). (B) Relative mRNA expression of IGF-1Ea and IGF-1Eb transcripts in fibroblast cell lines isolated from normal lung (IMR90) and IPF lung (HIPF-1) stimulated with TGF- (T) or without (C). There was a significant increase in IGF-1Ea expression between the IMR90 C and HIPF-1 C and between IMR90 T and HIPF-1 T (p < 0.04). There was also a significant increase in IGF-1Eb expression within each cell line between control and stimulated conditions (p < 0.02).

Total IGF-1 mRNA Expression

Figure 7A shows the total IGF-1 expression in BALC for each subject group. All the patients showed significantly reduced IGF-1 expression compared with normal subjects. There was a 20-fold decrease in stage III/IV sarcoidosis BALC (p < 0.004), a 15-fold decrease in patients with IPF (p < 0.01), and 3-fold decrease in sarcoidosis stage I/II patients (p < 0.04). There was also a significant difference between the total IGF-1 expression of stage I/II and stage III/IV sarcoidosis BALC (p < 0.01). There were no significant differences between sarcoidosis and IPF patients. Figure 7B shows that normal fibroblasts constitutively express significantly less (p < 0.03) total IGF-1 compared with fibrotic lung fibroblasts. TGF- treatment only increased expression significantly in HIPF-1 (p < 0.03) and not IMR90. Furthermore, this increase was significantly higher (p < 0.02) compared with TGF--stimulated IMR90.

View larger version (20K): [in this window] [in a new window]   Figure 7.   (A) Total IGF-1 mRNA expression in BALC from normal subjects, patients with stage I/II and stage III/IV sarcoidosis and patients with IPF. Asterisk indicates that when compared with normal IGF-1 expression there was a significant reduction observed in patients (p < 0.04). (B) Total IGF-1 mRNA expression in fibroblast cell lines isolated from normal lung (IMR90) and IPF lung (HIPF-1) stimulated with TGF- (T) or without (C). Constitutive total IGF-1 expression was significantly increased in HIPF-1 C (p < 0.003) compared with IMR90 C. After TGF- treatment, IGF-1 expression was significantly increased in HIPF-1 T compared with HIPF-1 C (§p < 0.03) and IMR90 T (*p < 0.02) respectively, but not in IMR90 T compared with IMR90 C.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

IGF-1 is a profibrogenic growth factor, promoting fibroblast proliferation and extracellular matrix deposition, whose increased expression may be critical to the pathogenesis of pulmonary fibrosis. Paradoxically this study shows that mRNA expression of total IGF-1 decreases dramatically, compared with normal subjects, in BALC from patients with IPF and sarcoidosis having established fibrosis or a nonfibrotic phenotype. However, in contrast to BALC, fibrotic lung fibroblasts demonstrate increased constitutive and further, dramatically increased TGF--stimulated IGF-1 expression, compared with normal lung fibroblasts.

Simultaneous changes take place in the pattern of splice variant expression. Healthy subjects and sarcoidosis patients without fibrosis showed similar levels of expression of Class 1/Class 2 and IGF-1Ea/IGF-1Eb transcripts. However, sarcoidosis patients with fibrosis and patients with IPF demonstrated respective 5-fold and 10-fold increases in the abundance of Class 1 forms, and patients with IPF a 6-fold decrease in IGF-1Ea forms. These switches in IGF-1 transcript expression appear to be associated with the presence of fibrosis, as they were not observed in either the normal volunteers or the fibrosis-free sarcoidosis patients. In parallel, all fibrosis patients failed to express Class 2, IGF-1Eb forms and sarcoidosis patients with fibrosis did not express the Class 1, IGF-1Eb variant either. Fibrotic fibroblasts expressed higher constitutive levels of Class 1, IGF-1Ea transcripts compared with normal fibroblasts. Class 2, IGF-1Eb forms were highly expressed by fibroblasts only after stimulation with TGF-, which also further increased levels of Class 1, IGF-1Ea transcripts.

Previous studies have demonstrated that IGF-1 in the lungs of normal subjects appears to be produced solely in AM (15). Conflicting reports suggest spontaneous enhanced release of IGF-1 in IPF AM compared with normal AM (15), or that IPF and normal AM release similar amounts (16). Although correlation of expression with BALC cell type was attempted in this study, we were unable to link the expression to a single cell type. Homma and coworkers (3) have detected IGF-1 and IGF-1 receptor protein in AM from patients with IPF, sarcoidosis, and normal subjects. However, they have also shown in lung tissue specimens that alveolar type II epithelial cells, vascular endothelial cells, vascular smooth-muscle cells, and fibroblasts all express IGF-1 and IGF-1 receptor in patients with early-stage IPF but not in normal subjects (3). There is no published evidence to support IGF-1 expression in neutrophils; however, there is evidence that lymphocytes can express IGF-1 (17). Thus, in patients with sarcoidosis (where the underlying disease is driven predominately by lymphocytic alveolitis) some contribution to IGF-1 expression from lymphocytes is possible. We can however assume that the majority of IGF-1 mRNA expression detected in BALC from normal lung is derived from AM. However, within the BALC of patients with IPF and sarcoidosis, the dramatic reduction in putatively AM-derived IGF-1 expression cannot be accounted for by the relatively small change in AM total number. Rather, it is suggestive of a change within the local AM population, with a proportion of these AM downregulating or failing to express IGF-1. Indeed the AM population represents a dynamic system of phenotypically and functionally distinct subpopulations of immune inducers and suppressor macrophages (18).

Our group has previously investigated distinct subpopulations in BAL from patients with active sarcoidosis and normal subjects. We demonstrated that with the onset of sarcoidosis inflammation, the numbers of a specific AM subset increased in direct proportion to the lavage lymphocytosis and was capable of suppressing T-cell responses (18). Accordingly, with the onset of fibrosis it has been shown that in patients with IPF "small," but not the "large" AM typical of fibrosis (19), on BAL cytospins express Class 1, IGF-1Eb mRNA (16). Therefore, phenotype and functional shifts within AM subpopulations induced with the onset of local inflammation and subsequent progression to fibrosis could have a direct influence on IGF-1 transcript expression on retrieved BALC. This may also help to explain the observed switch toward expression of Class 1 and IGF-1Eb transcripts in IPF.

Our findings of increased IGF-1 expression from fibrotic fibroblasts could account for much of the reported increases in IGF-1 found in fibrosis. These cells appear to have increased responsiveness to the profibrogenic mediator TGF- that results in enhanced expression of IGF-1. The mechanism responsible for this remains unknown and only a very limited literature exists in relation to this. In human osteoblasts TGF- has been shown to induce all four classes of IGF-1 transcript in a time- and dose-dependent manner (20), whereas here we found specific induction of Class 2 and IGF-1Eb transcripts. The precise relationship between IGF-1 mRNA expression and IGF-1 peptide remains poorly understood. Although there is a pressing need to demonstrate the functional roles of IGF-1 transcripts, there is evidence to suggest that Class 1-derived transcripts have autocrine/paracrine properties, whereas Class 2 transcripts have endocrine behavior (21). These transcripts can be translated (22); the alternative E peptides generated from the pre-pro-IGF-1s, IGF-1Ea or IGF-1Eb encode the same mature peptide and 16 amino acids of E domain with different C-terminals.

It has been suggested that these C-terminal peptides may influence processing or secretion of the mature IGF-1 peptide from cells (23). An additional variant from liver, containing an exon 4-5-(52 bp only)-6 splice has also been described and termed IGF-1Ec (24). It has putative autocrine/paracrine functions and is induced in muscle in response to hypertrophy (25). Our primers would detect this transcript but we failed to find its expression in any of our samples. The transcripts potentially affect the interaction of IGF-1 with IGF-1 receptor or IGFBP because it has been shown by functional epitope mapping that the carboxy terminus (3' end) of IGF-1 is an important determinant of the affinity of the peptide for a particular receptor or binding protein (26). However, the E peptide is translated, and cleaved during processing of IGF-1 prohormone; therefore, E peptides themselves may have distinct biologic roles after being cleaved from the prohormone (27). It is unclear if the amounts and types of IGF-1 may be influenced by local expression of other peptides. For example, the translation efficiency of IGF-1 is dependent on the length of the 5' untranslated region (27) and factors influencing choice of leader exon and transcriptional start site may affect local concentrations of IGF-1 protein (1, 23, 28).

Biologic activity of IGF-1 may be modulated through association with IGFBP. We have previously demonstrated expression of IGFBP-3 in BALC and increased IGFBP-3 in BALF from sarcoidosis patients with established fibrosis (5), and IGFBP-3 release is increased from cultured IPF BALC (16). At the cellular level, proteolysis of IGFBPs is believed to play an important role in controlling the bioavailability of IGF-1 to receptors. Thrombin is a serine protease, shown to be located in the extracellular matrix (ECM) of human tissues and macrophages (31). Furthermore, it has been demonstrated that thrombin can cleave IGFBP-5 and to a lesser extent IGFBP-3 in smooth-muscle cells (32). Thrombin has been found to cleave ECM proteins, for example plasminogen activator inhibitor-1 (PAI-1), which is thought to be one of the major binding sites for IGFBP-5 within the ECM (33). Therefore, it is possible that thrombin could also release IGF-1 from IGF-1/IGFBP-5 complexes associated with the ECM (33).

It has been previously demonstrated that 24 h after exposure to thrombin, porcine smooth muscle cells showed significantly reduced IGF-1 mRNA, but IGF-1 peptide levels in conditioned media remained the same, suggesting that IGF-1 might be released from IGFBPs (34). This phenomenon may provide a mechanism to reconcile the paradoxical reduction in total mRNA expression in the patient BALC observed in this study. Other IGFBP proteases may also fulfil this role with the reported increases in IGF-1-mediated fibroblast mitogenicity of patient BALF (6, 7). It is possible that IGFBPs could therefore alter the mitogenic effects of IGF-1: indeed the IGF-1/ IGFBP-3 complex may be more fibrogenic than IGF-1 alone, and may account for the increased IGF-1-mediated fibrogenic activity observed in BALF from a number of studies. For example, in patients with systemic sclerosis, the major peak of IGF-1-mediated fibroblast growth promoting activity in BALF corresponds to a molecular size of 67 kD (7).

In conclusion, we have shown that IGF-1 mRNA transcripts are differentially expressed in BALC from healthy subjects, patients with pulmonary sarcoidosis and IPF, and in fibroblasts from normal and fibrotic lung. Total IGF-1 mRNA expression is downregulated in BALC, associated with disease onset. In contrast, IGF-1 is overexpressed by fibrotic fibroblasts and by fibroblasts exposed to a profibrogenic mediator. We hypothesize that differences within AM subpopulations of the host lung could account for switches in BALC IGF-1 transcript expression. The data suggest that the type of transcript expressed may have different functional implications. Treatment of fibroblasts with profibrogenic TGF- induces high expression of Class 2 and IGF-1Eb transcripts which are not expressed by normal fibroblasts and may have a unique role in the fibrogenic process. Studies are now urgently required to focus on the precise role of specific IGF-1 splice variants and modulating factors such as IGFBP, and their interactions with the changing cell profile during the process of tissue remodeling after lung injury that may result in fibrosis.