Insulin-like growth factor binding proteins in air- and 85% oxygen-exposed adult rat lung

Robin N. N. Han1, Victor K. M. Han2, Shilpa Buch1, Bruce A. Freeman3, Martin Post1, and A. Keith Tanswell1

1 Medical Research Council Group in Lung Development, Hospital for Sick Children Research Institute and Department of Paediatrics, University of Toronto, Toronto, Ontario M5S 1A8; 2 Medical Research Council Group in Fetal and Neonatal Health and Development, Lawson Research Institute, St. Joseph's Health Centre and Departments of Paediatrics, Anatomy, and Biochemistry, University of Western Ontario, London, Ontario, Canada N6A 4V2; and 3 Departments of Anesthesiology, Biochemistry, and Pediatrics, University of Alabama at Birmingham, Birmingham, Alabama 35294

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Expression of insulin-like growth factor (IGF) I and its type I receptor is increased in the adult rat lung exposed to 85% O2. We hypothesized that there would be a parallel up- and downregulation of growth-stimulating and growth-inhibiting IGF binding proteins (IGFBPs), respectively. The normal adult rat lung expresses mRNAs for IGFBP-2, -3, -4, -5, and -6 but not for IGFBP-1. O2 exposure for 6 or 14 days reduced IGFBP-3 and -6 and increased IGFBP-4 mRNA abundance. IGFBP-5 mRNA was reduced at 6 days but increased at 14 days. IGFBP-4 mRNA was localized to perivascular and peribronchial interstitial cells and IGFBP-5 mRNA to airway and alveolar epithelial cells. IGFBP-2, -4, and -5 immunolocalized to airway epithelial cells in normal lung and to perivascular exudates after 6 days in 85% O2. IGFBP-2 was diffusely increased throughout the lung tissue only after a 6-day exposure. IGFBP-5 was reduced after a 6-day exposure but was increased and widely distributed after 14 days. IGFBP-4 increased over airway epithelium and subepithelial cells after 6 days and over perivascular interstitial cells after 14 days of 85% O2. These data are consistent with the predicted changes for IGFBPs on O2 exposure except that the generally growth-inhibitory IGFBP-4 was increased at sites of active cell proliferation.

pulmonary oxygen toxicity; cell interactions; lung injury

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

SUSTAINED INHALATION of elevated O2 concentrations results in a pattern of diffuse lung injury that includes both type II pneumocyte and pulmonary fibroblast hyperplasias (2). In addition, there is pulmonary hypertension with increased muscularization of both preacinar and intra-acinar arteries (27, 28) and increased airway reactivity with peribronchial smooth muscle hyperplasia (39). These changes are observed not only in animal models but also in adult and neonatal humans exposed to prolonged elevated O2 concentrations (2).

Adult rats exposed to 85% O2 for 14 days have a proliferative lung injury that has been well characterized by morphometric analysis (12). Our laboratory has found the lung injury in this animal model to be highly reproducible and has used it to characterize O2-mediated changes in the expression of several growth factors including platelet-derived growth factor (PDGF) (20), basic fibroblast growth factor (4), and transforming growth factor-beta 1 (32) during the proliferative response to lung injury. Most recently, we (21) examined the O2-mediated changes in the expression of insulin-like growth factor (IGF) I and the IGF-I receptor (IGF-IR). A summary of the relationship between changes in cell number and growth factor expression is shown in Fig. 1. Based on studies of IGF-related gene expression during normal lung growth (31, 41), we had anticipated that we would observe increased IGF-I and IGF-IR expression in the lung parenchyma as part of an autocrine and paracrine growth factor response to 85% O2-mediated lung injury. Although a widespread increase in IGF-I expression was observed (21), this was not the case for the IGF-IR. Contrary to our expectations, there was no evident localization of high-abundance IGF-IR to interstitial lung fibroblasts, whereas localization of high-abundance IGF-IR to alveolar epithelium was evident after a 14-day exposure to 85% O2 but not after a 7-day exposure (21). IGF-IR immunoreactivity was primarily localized to perivascular interstitial cells in air-exposed animals. This perivascular IGF-IR immunoreactivity increased with 85% O2 exposure and also became evident in peribronchial interstitial cells and large- and small-vessel endothelial cells (21), all of which were sites of active DNA synthesis.


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Fig. 1.   A: changes in total lung fibroblast (bullet ), type II pneumocyte (square ), endothelial cell (open circle ), and alveolar macrophage (black-square) numbers. [Modified from Crapo et al. (12).] B: relative immunoreactivity of basic fibroblast growth factor (triangle ), insulin-like growth factor (IGF) I (bullet ), platelet-derived growth factor BB homodimer (open circle ), IGF-I receptor (black-square), and transforming growth factor-beta (square ) with time after exposure to 85% O2. [Modified from previously reported data (4, 20, 21, 32).] -/-, No immunoreactivity; -/+, minimal immunoreactivity; +, limited immunoreactivity; ++, highly immunoreactive (localized); +++, highly immunoreactive (diffuse).

These data are consistent with IGF-I and IGF-IR playing a role in the smooth muscle hyperplasias of pulmonary O2 toxicity. A critical role for the IGF-IR has been suggested in other experimental smooth muscle cell hyperplasias in vivo and in vitro (6, 15, 33). As recently reviewed (25), the mitogenic effects of IGF-I may be modulated by the presence of IGF binding proteins (IGFBPs). We therefore elected to continue our investigation of the role of IGF-related gene expression in the 85% O2-exposed rat lung by examining changes in the expression of IGFBP mRNAs and peptide immunoreactivity. Of the six known IGFBPs (IGFBP-2, -3, -4, -5, and -6), mRNAs are present in normal lung tissue (31, 42). Whether their expression is altered during the proliferative stages of lung injury is unknown, although experimental smooth muscle hyperplasia of the urinary tract is associated with altered IGFBP expression (6). Increased IGFBP-2 secretion by an immortalized type II pneumocyte cell line, growth arrested by exposure to hyperoxia, has been reported (5), but these data cannot be extrapolated to an in vivo situation in which there is type II pneumocyte hyperplasia. Our hypothesis was that those IGFBPs that enhance the mitogenic effect of IGF-I would be upregulated and inhibitory IGFBPs downregulated in parallel with upregulation of IGF-I and IGF-IR to facilitate smooth muscle hyperplasia.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. Radioisotopes and nylon membrane were purchased from Amersham Canada (Oakville, Ontario), and restriction enzymes and dextran sulfate were from Pharmacia Canada (Baie D'Urfé, Québec). BSA type V, Ficoll 400, polyvinylpyrrolidone, guanidinium thiocyanate, cesium chloride, poly-L-lysine, and salmon sperm DNA were from Sigma (St. Louis, MO). Organic solvents were of HPLC grade. Porcine trypsin (1:250) and RNase A were from GIBCO BRL (Life Technologies, Burlington, Ontario). The cDNA for 18S rRNA was a gift from Dr. D. Denhardt (Rutgers University, Piscataway, NJ). Rat cDNAs encoding IGFBP-I (insert size 1.5 kb) and IGFBP-II (insert size 0.6 kb) were gifts from Drs. G. T. Ooi and M. M. Rechler (National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD). A rat IGFBP-3 cDNA (insert size 2.0 kb) was a gift from Dr. A. C. Herrington (Prince Henry's Institute of Medical Research, Melbourne, Australia). Rat IGFBP-4 (insert size 0.4 kb), IGFBP-5 (insert size 0.3 kb), and IGFBP-6 (insert size 0.2 kb) cDNAs were gifts from Dr. S. Shimasaki (University of California, San Diego). Bovine IGFBP-2, human IGFBP-5, and rabbit antisera to bovine IGFBP-2, recombinant human IGFBP-4, and human IGFBP-5 were from Upstate Biotechnology (Lake Placid, NY). Antiserum to human IGFBP-3, from this supplier, is listed as not having cross-reactivity to rat IGFBP-3. A kit for avidin-biotin-peroxidase complex immunocytochemical staining was purchased from Vector Laboratories (Burlingame, CA), and alpha -aminopropyltriethoxysilane was from Pierce Chemical (Rockford, IL). Superfrost Plus slides for in situ hybridization studies were from Fisher Scientific (Unionville, Ontario).

Exposure system. Pathogen-free virgin female Sprague-Dawley rats of 200-250 g were obtained from Charles River Laboratories (Raleigh, NC). The animals were maintained in 60 × 48 × 25-cm plastic chambers with a 12:12-h light-dark cycle. Food and water were available ad libitum.

Rats (n = 4) were exposed to air or 85% O2 for 0, 6, or 14 days for collection of tissue for total RNA extraction. Other groups of four animals at each time point were used for immunohistochemistry and in situ hybridization studies. O2 concentrations in the exposure chambers were calibrated daily with a Beckman Instruments (Schiller Park, IL) O2 analyzer. Gas flow was set to maintain minimal chamber humidity, with a CO2 concentration < 0.5%.

The animals were anesthetized with intraperitoneal ketamine (80 mg/kg) and xylazine (20 mg/kg). A catheter was inserted into the trachea and sutured in place to facilitate lung inflation. The anterior part of the chest wall was reflected upward. While the heart was beating, a catheter was inserted through the right ventricle into the main pulmonary artery, and an incision was made in the left atrial appendage to allow drainage. The pulmonary circulation was then flushed with phosphate-buffered saline containing 1 unit/ml of heparin during intermittent lung inflations with air until the lungs became white. For these inflations, the lungs were expanded to the point at which they touched the inner walls of the rib cage.

Isolation of lung RNA. Total lung RNA was isolated as previously described (4, 20, 21, 32). Briefly, the thoracic contents were removed en bloc, and the lungs were dissected away from vessels and large airways to be flash-frozen in liquid nitrogen after weighing. Total (nuclear and cytoplasmic) RNA was isolated by lysing the tissue in 4 M guanidinium thiocyanate, followed by centrifugation on a 5.7 M cesium chloride cushion to pellet RNA (8). After extraction with phenol-chloroform (1:1 vol/vol), the RNA was ethanol precipitated and collected by centrifugation. This RNA was lyophilized and dissolved in water. RNA integrity was confirmed after fractionation on 1.2% (wt/vol) agarose-formaldehyde gels by staining the rRNA bands with ethidium bromide.

Radiolabeling of cDNAs. cDNAs were labeled with [32P]dCTP by random priming with an oligo-labeling kit (Pharmacia Canada) to specific activities of 1 × 109 counts · min-1 · µg-1. The same cDNAs were subcloned into a pBluescript-SK vector (Stratagene, La Jolla, CA) and linearized with appropriate restriction enzymes. 35S-labeled antisense and sense RNA probes were transcribed with either T3 or T7 promoters (RNA transcription kit, Promega, Madison, WI) according to the manufacturer's instructions.

Northern blot analyses. Total RNAs from air- and O2-exposed lungs (20 µg/lane) were denatured in formaldehyde for 30 min at 65°C, then electrophoresed in 1% (wt/vol) agarose gels containing 2.2 M formaldehyde. The RNAs were then transferred to a Zeta-Probe membrane (Bio-Rad Laboratories, Richmond, CA) with the capillary-transfer technique. After transfer, the blots were baked at 80°C for 1 h. Blots were prehybridized in a Zeta-Probe hybridization buffer [50% (vol/vol) formamide, 5× saline-sodium phosphate-EDTA (0.75 mM NaCl, 44 mM Na2HPO4 · 2H2O, and 5 mM EDTA), 7% (wt/vol) SDS, and 0.1 mg/ml of denatured salmon sperm DNA] at 42°C for 2 h, followed by overnight hybridization with a gel-eluted riboprobe (1-2 × 106 counts · min-1 · ml-1) in the same buffer at 42°C. The blots were washed two times (30 min each) in 1× saline-sodium citrate (SSC; 1.5 M NaCl and 0.15 M sodium citrate)-0.1% SDS at 42°C, followed by two washes (30 min each) in 0.1× SSC-0.1% SDS at 42°C. After the washes, the blots were exposed for 14 h to 10 days at -70°C to Kodak XAR-5 film using Dupont Cronex intensifying screens. Between hybridizations, the blots were stripped by washing two times for 30 min at 80°C in 0.01× SSC-0.5% SDS. The blots were then hybridized with a radiolabeled 18S ribosomal cDNA probe to assess minor inconsistencies in RNA loading and/or transfer.

Immunohistochemistry. Lungs from rats that had been exposed to air or 85% O2 for 6 and 14 days were perfusion fixed with a freshly prepared fixative [4% (wt/vol) paraformaldehyde-0.2% (vol/vol) glutaraldehyde] after the blood had been flushed from the pulmonary circulation. During perfusion, the lungs were held inflated at a pressure of 12 cmH2O. The tissues were processed and embedded in paraffin, 5-µm sections were cut, and the tissue sections were mounted on alpha -aminopropyltriethoxysilane-coated slides. Immunohistochemical studies were conducted with an avidin-biotin-peroxidase complex method as previously described (41). Briefly, tissue sections were deparaffinized in xylene, rehydrated, and washed in PBS. Endogenous peroxidase activity was quenched by incubating tissue sections in 3% (vol/vol) hydrogen peroxide in PBS for 10 min. After a 5-min exposure to 0.0625% (wt/vol) trypsin in PBS, sections were incubated with specific primary antisera for 20-24 h at 4°C. Antiserum dilutions were 1:500 for all IGFBPs. Antibody specificity was verified by demonstrating an absence of immunostaining when either nonimmune serum or a blocking solution [5% (vol/vol) normal goat serum-1% (wt/vol) BSA] without antibody was substituted for the primary antibody. The specificity of immunostaining for IGFBP-2 and IGFBP-5 was demonstrated by the loss of immunoreactivity with primary antisera that had been immunoabsorbed with 1 µM bovine IGFBP-2 or 100 ng/ml of human IGFBP-5. No peptide was available to confirm the specificity of immunostaining for IGFBP-4. After completion of the immunohistochemical procedures, the slides were lightly counterstained with Carazzi hematoxylin.

In situ hybridization histochemistry. This was performed as previously described (41). All slides for each probe were processed together. Briefly, tissue sections mounted on poly-L-lysine-coated slides were deparaffinized in xylene, rehydrated in a descending ethanol series, and incubated sequentially in 0.2% (vol/vol) Triton X-100 in PBS with 10 µg/ml of proteinase K, followed by 0.25% (wt/vol) acetic anhydride in triethanolamine buffer. Sections were then dehydrated in an ascending ethanol series and prehybridized with an in situ hybridization buffer [0.3 M NaCl, 20 mM Tris · HCl, pH 8.0, 1 mM EDTA, 1× Denhardt's solution, 500 µg/ml of yeast tRNA, 100 µg/ml of denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate, 0.1% (wt/vol) SDS, 100 mM dithiothreitol, and 50% (vol/vol) formamide] at 42°C for 2 h. Tissue sections were then hybridized overnight at 42°C with 35S-labeled antisense cRNA probes for IGFBP-4 or IGFBP-5 at concentrations of 1 × 106 disintegrations · min-1 · ml-1 in the in situ hybridization buffer. Sections were then sequentially treated with the in situ hybridization buffer at 55°C for 10 min, RNase A for 30 min at 37°C, two times with 2× SSC at 20°C for 30 min, four times with 2× SSC at 42°C for 30 min, and finally two times with 0.1× SSC at 20°C for 15 min. For the purposes of illustration, the control sections with radiolabeled sense RNA probes that are shown were all from animals exposed to 85% O2 for 6 days.

Tissue sections were then dehydrated in an ascending ethanol series, coated with NTB-2 nuclear track photoemulsion (Kodak Laboratories, Rochester, NY), and exposed at 4°C for 3-21 days. The photoemulsion was developed with a D-19 developer (Kodak Laboratories), fixed, stained with Carazzi hematoxylin and eosin, and mounted with Permount. The slides were viewed with both bright- and dark-field photomicroscopy. The specificity of in situ hybridization was demonstrated by the absence of hybridization signals with radiolabeled sense RNA probes.

Autoradiography. This was performed as previously described (4, 20, 21). Briefly, the animals received 1 µCi/g of intraperitoneal [3H]thymidine 2 h before lung fixation. The lungs were cleared of blood as described in Exposure system. The lungs were fixed with a paraformaldehyde-based fixative, and 5-µm sections were prepared. Sections were mounted on alpha -aminopropyltriethoxysilane-coated slides and lightly stained with Carazzi hematoxylin. The slides were coated with Kodak NBT-3 emulsion for autoradiography and developed after 2 wk for examination by light microscopy to identify dense areas of silver granule concentration over cell nuclei undergoing active DNA synthesis.

Data presentation. The abundance of the various IGFBP mRNAs was quantified by subjecting Northern blots to phosphorimage analysis (Phosphorimager SI, Molecular Dynamics, Sunnyvale, CA). The amount of 18S rRNA in the total RNA samples in the same blots was also quantified with the same method and was used to correct for minor inconsistencies in loading and transfer. The relative abundance of IGFBP mRNAs in the air-exposed and 85% O2-exposed (6- and 14-day) animals (n = 4/group) was then calculated, with mRNA abundance in air-exposed animals as 100%, and is expressed as the mean ± SE of each group. No differences were observed between preexposure animals or animals exposed to air for various periods as control animals. For the purposes of presentation, only preexposure values are shown. Statistical significance (P < 0.05) was determined by an analysis of variance followed by assessment of differences with Dunnett's two-sided test (18).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

To determine which of the currently recognized IGFBPs, if any, were affected by exposure of the lung to 85% O2, we assessed whole lung total RNAs for IGFBP abundance by Northern blot hybridization. The blots are shown in Fig. 2, and the results of quantitation are shown in Fig. 3. Consistent with previous reports (31, 42), we were unable to detect IGFBP-1 mRNA in any of our lung samples. IGFBP-2 mRNA did not change, whereas both IGFBP-3 and IGFBP-6 mRNAs declined significantly by day 6 of exposure to 85% O2 (P < 0.05). The decline in IGFBP-3 mRNA was still significant on day 14 of exposure to 85% O2 (P < 0.05). The only IGFBPs to show increased mRNA abundance in response to 85% O2 were IGFBP-4 and IGFBP-5. IGFBP-4 mRNA abundance was significantly increased at both 6 and 14 days (P < 0.05), whereas IGFBP-5 was significantly reduced on day 6 (P < 0.05) and then significantly increased on day 14 (P < 0.05). The latter two IGFBPs were specifically selected for further investigation by in situ hybridization because the normal distribution of IGFBP mRNAs in the adult rat lung has been described elsewhere (42).


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Fig. 2.   Northern blots of total RNA from lungs of 4 animals/group exposed to air or 85% O2 for 6 or 14 days and probed sequentially with radiolabeled IGF binding protein (IGFBP)-2 to IGFBP-6 cDNAs. Blots were exposed for 5 days (IGFBP-4) to 2 wk (IGFBP-2) to XAR film.


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Fig. 3.   Composite Northern analyses for IGFBP RNAs from lungs of 4 animals/group exposed to air or 85% O2 for 6 (D6) or 14 (D14) days. Values are means ± SE. * P < 0.05 by ANOVA compared with values in air-exposed animals.

The intensity of the in situ hybridization signals for IGFBP-4 and IGFBP-5 was consistent with the Northern analyses. An increase in IGFBP-4 mRNA was evident at both 6 and 14 days of exposure to 85% O2 and was localized to the interstitium, particularly the perivascular and peribronchial regions (Fig. 4). The signal for IGFBP-5 was reduced on day 6 and increased on day 14 of exposure to 85% O2. IGFBP-5 mRNA was localized primarily to bronchial and alveolar epithelial cells (Fig. 5). The localization of IGFBP-4 mRNA to subepithelial airway cells and of IGFBP-5 mRNA to airway epithelial cells was most evident when viewed at high power (Fig. 6).


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Fig. 4.   Bright-field (A, C, E, and G) and dark-field (B, D, F, and H) photomicrographs of in situ hybridization for IGFBP-4 mRNA. A and B: control lung tissue studied with a sense probe. Original magnification, ×800. C and D: lungs from animals exposed to air. Original magnification, ×800. E and F: lungs from animals exposed to 85% O2 for 6 days showing increased hybridization, particularly around airways (a) and vessels (v). Original magnification, ×200. G and H: lungs from animals exposed to 85% O2 for 14 days had even greater hybridization around airways and vessels as well as widespread hybridization in parenchymal tissue. Original magnification, ×100.


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Fig. 5.   Bright-field (A, C, E, and G) and dark-field (B, D, F, and H) photomicrographs of in situ hybridization for IGFBP-5 mRNA. A and B: control lung tissue studied with a sense probe. Original magnification, ×800. C and D: lungs from animals exposed to air showing hybridization to airway epithelium. Original magnification, ×800. E and F: a similar appearance was observed in lungs from animals exposed to 85% O2 for 6 days. Original magnification, ×800. G and H: lungs from animals exposed to 85% O2 for 14 days had increased hybridization over bronchial epithelium (a) but not over vascular (v) endothelium. Original magnification, ×200.


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Fig. 6.   Bright-field photomicrographs of in situ hybridization of airway epithelium for IGFBP-4 (A) and IGFBP-5 (B) viewed under high magnification (×1,000). IGFBP-4 mRNA is localized in subepithelial cells, whereas IGFBP-5 is localized to epithelial cell layer (arrows).

Because of antibody availability, lung tissue could only be examined for IGFBP-2, -4, and -5 immunoreactivity. Weak immunoreactivity for IGFBP-2 was evident over airway epithelial cells and some interstitial cells, particularly those surrounding vessels and airways, of animals breathing air (Fig. 7B). After exposure to 85% O2 for 6 days, staining for IGFBP-2 increased in intensity throughout the lung, with the exception of nonimmunoreactive endothelial cells, and was also evident in perivascular exudates (Fig. 7C). After exposure to 85% O2 for 14 days, the intensity of IGFBP-2 immunoreactivity was similar to that seen in animals breathing air except for the presence of a small number of intensely immunoreactive cells adjacent to the blood vessels (Fig. 7D). Weak immunoreactivity for IGFBP-4 was evident on airway epithelial cells of animals breathing air (Fig. 8B). After a 6-day exposure to 85% O2, staining for IGFBP-4 was increased over the airway epithelium and was also seen both over subepithelial cells (Fig. 8C) and in isolated cells found in perivascular exudates (Fig. 8D). After a 14-day exposure to 85% O2, intense IGFBP-4 immunoreactivity was evident over airway and alveolar epithelial cells (Fig. 8E) and in cells in close proximity to the vascular endothelium (Fig. 8F). IGFBP-5, like IGFBP-4, was observed on the airway epithelium in the lungs of animals breathing air (Fig. 9B). In contrast to IGFBP-4, a 6-day exposure to 85% O2 was associated with a loss of IGFBP-5 immunoreactivity over the airway epithelium and the appearance of diffuse staining in perivascular exudates (Fig. 9C). By the end of the 14-day exposure to 85% O2, airway epithelial immunoreactivity for IGFBP-5 was restored along with diffuse staining of the parenchyma, particularly in the perivascular and peribronchial areas (Fig. 9D). The major sites of IGFBP immunolocalization were airway epithelium and both perivascular and peribronchial cells. That these are sites of cell proliferation after exposure to 85% O2 is illustrated in Fig. 10. As previously reported, air-exposed lungs have minimal cell division, whereas there are quantitative differences, but no differences in distribution, in the dividing cells between lungs exposed to 85% O2 for 6 days and those exposed for 14 days (21).


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Fig. 7.   Immunohistochemistry for IGFBP-2. A: negative control. B: air-exposed animals showing IGFBP-2 localized to airway epithelium as well as to occasional parenchymal cells (arrows). C: after a 6-day exposure to 85% O2, immunoreactive IGFBP-2 was widely distributed throughout lung and in perivascular exudates (star ), with exception of endothelial cells (arrow). D: after a 14-day exposure, parenchymal IGFBP-2 immunoreactivity was similar in distribution to air-exposed animal, with exception of intense immunoreactivity on occasional perivascular cells (arrows). Original magnifications: A, B, and D, ×250; C, ×400.


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Fig. 8.   Immunohistochemistry for IGFBP-4. A: negative control. B: air-exposed animals have IGFBP-4 localized to airway epithelium as well as to occasional parenchymal cells. C and D: after a 6-day exposure to 85% O2, an increased number of airway epithelial cells are immunoreactive, and immunoreactive cells are also present in cells surrounding airway (C, arrows) and on large single cells in perivascular exudates (D, arrows). E and F: after a 14-day exposure, IGFBP-4 immunoreactivity was increased on airway and alveolar (E, arrow) epithelium as well as in perivascular cells (F, arrows). Original magnifications: A, B, E, and F, ×250; C and D, ×400.


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Fig. 9.   Immunohistochemistry for IGFBP-5. A: negative control. B: air-exposed animals have IGFBP-5 localized to airway epithelium. C: after a 6-day exposure to 85% O2, IGFBP-5 is no longer evident on airway epithelial cells but is found in exudates around vessels. D: after a 14-day exposure, IGFBP-5 immunoreactivity was widely distributed throughout lung but was particularly intense on airway epithelium and on parenchymal cells surrounding airways and vessels. Original magnification, ×250.


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Fig. 10.   [3H]thymidine autoradiography of a tissue section containing an airway and vessels from lung of a rat exposed to 85% O2 for 14 days. Cells undergoing DNA synthesis can be identified by collections of silver granules over their nuclei. As previously reported (21), air-exposed animals have few dividing cells, and animals exposed to 85% O2 for 6 days have a similar distribution of dividing cells. Straight arrows, dividing perivascular and peribronchial cells; curved arrows, dividing airway cells. Original magnification, ×250.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In our previous study (21) of IGF-I and IGF-IR expression in an 85% O2-exposed lung, we observed an increase in lung IGF-I expression, whereas IGF-IR was immunolocalized to the peribronchial and perivascular smooth muscle. These findings are consistent with a role for IGF-I in the airway and vessel smooth muscle hyperplasias seen in this lung injury. Injury-induced smooth muscle hyperplasia has been shown to be associated with increased IGF-I expression in other organs (7). Because the interaction of IGF-I with its receptor is modulated by the amount and the type of IGFBP that is present in the intercellular milieu of tissues, the final biological effect of this increase in IGF-I may be modulated by the IGFBPs present.

The observed pattern of expression of IGFBP mRNAs in the air-exposed adult rat lung is consistent with previous reports (1, 13, 17, 31, 37, 42) in that IGFBP-1 mRNA was absent, IGFBP-2, -3, and -4 mRNAs were expressed in similar abundance, and IGFBP-6 had the greatest abundance. The expression of all IGFBP mRNAs, with the exception of IGFBP-2 mRNA, was modified by 85% O2 exposure. Although there was no change in IGFBP-2 mRNA abundance, immunohistochemistry revealed a marked increase in IGFBP-2 immunoreactivity throughout the lung, with the exception of endothelial cells, after a 6-day exposure to 85% O2, the exposure time at which maximum proliferation is seen in this model (12, 20). The discrepancy between IGFBP-2 mRNA abundance and the increase in IGFBP-2 observed with immunohistochemistry suggests that either an increase in mRNA occurred before the 6-day time point examined, that IGFBP-2 was derived from nonpulmonary sources, or that there had been a reduction in the activity of protease(s) against IGFBP-2 (19). The stimulus for increased IGFBP-2 deposition is unknown, but PDGF has been reported to increase IGFBP-2 secretion in vitro, and under some culture conditions, an increase in IGFBP-2 was seen in the absence of increased IGFBP-2 mRNA (11). After a 6-day exposure to 85% O2, adult rat lungs have increased parenchymal PDGF deposition (20). By the end of the 14-day exposure to 85% O2, at which time lung cell proliferation had declined significantly (12, 20), there was a general reduction in immunoreactive IGFBP-2. An increase in IGFBP-2 expression in association with increased cell proliferation would not have been predicted from in vitro studies with isolated type II pneumocytes (5) but is consistent with findings in regenerating kidney in vivo (22) and proliferating colon epithelial cells in vitro (23).

IGFBP-3 mRNA decreased significantly after both a 6-day and a 14-day exposure to 85% O2. In the absence of an appropriate IGFBP-3 antiserum, we cannot automatically assume that a decrease in IGFBP-3 mRNA is accompanied by a parallel reduction in tissue IGFBP-3. IGFBP-3 can inhibit the actions of IGFs in vitro, suggesting that it may modulate the actions of IGFs in vivo (14, 43). This may be a direct or indirect effect because IGFBP-3 can downregulate the secretion of other IGFBPs (29) or directly inhibit cell growth independent of IGFs (34) and can also inhibit the increase in DNA synthesis elicited by growth factors other than IGF-I (40). If IGFBP-3 acts in an inhibitory fashion in the airways of the nonproliferating adult lung, any reduction after exposure to 85% O2 would facilitate the active cell division that is seen in adult rat lungs after exposure to 85% O2. IGFBP-6 mRNA was, like IGFBP-3 mRNA, reduced in 85% O2-exposed lungs. We did not have an antibody suitable for IGFBP-6 immunohistochemistry available to us. Although IGFBP-6 inhibits both IGF-I and IGF-II bioactivity, it binds IGF-I 50- to 70-fold less strongly than IGF-II (38). From our previous findings (21), IGF-II does not appear to play any significant role in 85% O2-mediated lung injury in the adult rat. This suggests that any effect of a reduction in IGFBP-6 expression on IGF-I bioactivity would be minimal relative to the effect of reduced IGFBP-3 expression.

IGFBP-4 is generally considered to have a consistently inhibitory effect on IGF-mediated cell proliferation (9). Perivascular smooth muscle cells are known to contain IGFBP-4 mRNA (11) and to have IGF receptors (10, 16). Our previous observation (21) that exposure to 85% O2 increased immunoreactive perivascular and peribronchial IGF-IRs suggested a role for IGFs in the perivascular and peribronchial smooth muscle hyperplasias seen with pulmonary O2 toxicity. It therefore seems counterintuitive that increased inhibitory IGFBP-4 mRNA and protein should be localized to sites of smooth muscle hyperplasia after 85% O2 exposure. Similarly counterintuitive are reports (3, 13) correlating IGFBP-4 mRNA expression with sites and stages of active proliferation in early and late fetal lung development and a recent report (6) that increased IGFBP-4 gene expression is closely associated with smooth muscle hypertrophy. The biological relevance of the increased expression of the IGFBP-4 gene in O2-induced lung cell hyperplasia is therefore unknown. Possible explanations include overexpression of the IGFBP-4 gene to counterbalance an unmitigated action of IGF-I or a potentiating action of other IGFBPs (30).

IGFBP-5 mRNA was reduced after a 6-day exposure to 85% O2. Because IGFBP-5 is inhibitory for IGF-I and IGF-II bioactivity (38), the observed reduction in mRNA and protein would favor IGF-mediated DNA synthesis. After a 14-day exposure to 85% O2, the observed increase in IGFBP-5 mRNA and protein would tend to inhibit IGF-I-mediated cell proliferation. Morphometric analysis has shown that although there is still an increased total lung cell number relative to air-exposed control lungs, the total cell number after a 14-day exposure to 85% O2 is less than that seen after a 6-day exposure (12). This suggests that even though there is still active DNA synthesis occurring (20), the proliferative response to injury is lessened late in the exposure period. The primary target for such an inhibitory effect of IGFBP-5 is likely to be the epithelium because secreted IGFBP-5 appears to be rapidly incorporated intact into the extracellular matrix (26). An increased expression of specific binding proteins may be one mechanism by which aberrant cell proliferation during lung injury is controlled. It remains to be determined whether 85% O2-mediated changes in IGF-I and IGFBP gene expression are a direct response to oxidant stress or are secondary to increased intrapulmonary concentrations of PDGF and basic fibroblast growth factor (4, 20). Both growth factors have been reported to modulate both IGF-I and IGFBP gene expression in vitro (24, 36). Increased matrix collagen, as seen in this model after a 14-day exposure to 85% O2 (32), has also been reported to increase cellular release of IGFBPs (35).

Peribronchial and perivascular parenchymal cells were the sites of high-abundance IGF-IR and sites of active DNA synthesis in the lungs of animals exposed to 85% O2 (21). Our original hypothesis was that IGFBPs that inhibit the mitogenic effects of IGFs would be downregulated and that stimulatory IGFBPs would be upregulated in parallel with an upregulation of IGF-IR and active DNA synthesis. IGFBP-2, -4, and -5 were immunolocalized to airway epithelium, IGFBP-4 and -5 to perivascular parenchymal cells, and IGFBP-5 to peribronchial parenchymal cells, all sites of active DNA synthesis. Taken as a whole, these data are consistent with a role for lung-derived IGFBPs in the modulation of IGF-I-mediated lung cell proliferation during pulmonary O2 toxicity. As previously reported (21), the distribution of macrophages in the lungs of 85% O2-exposed adult rats is inconsistent with their being the primary source of IGFBP-4 or IGFBP-5 as determined by in situ hybridization. However, extrapulmonary IGFBPs may play a contributory role because they, as well as IGF-I and IGF-II (21), are found in the perivascular exudates seen after a 6-day exposure to 85% O2.

    ACKNOWLEDGEMENTS

We thank K. Nygard and A. Rivera for expert technical assistance.

    FOOTNOTES

This work was supported by Group Grants from the Medical Research Council of Canada; National Heart, Lung, and Blood Institute Grant RO1-HL-51245; and by an equipment grant from the Ontario Thoracic Society.

A. K. Tanswell holds the Hospital for Sick Children Women's Auxiliary and University of Toronto Chair in Neonatal Medicine.

Address for reprint requests: A. K. Tanswell, Division of Neonatology, Hospital for Sick Children, 555 Univ. Ave., Toronto, Ontario, Canada M5G 1X8.

Received 27 June 1997; accepted in final form 7 January 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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