1Second Department of Internal Medicine, Nagasaki University School of Medicine, 2Department of Pathology, Nagasaki University Hospital, Nagasaki 852-850; and 3Second Department of Internal Medicine, Oita Medical University, Oita 879-5593, Japan
Submitted 11 September 2002 ; accepted in final form 2 July 2003
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ABSTRACT |
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pulmonary fibrosis; bleomycin
The collagen-specific stress protein, 47-kDa heat shock protein (HSP47), localized in the endoplasmic reticulum, is involved in synthesis/assembly of various collagens as a collagen-specific molecular chaperone (5, 13, 25). HSP47 has been reported to play a key role in increased deposition of collagens in allograft renal tissues (1) and the peritoneum of patients on continuous ambulatory peritoneal dialysis (20). With the use of Northern blotting analysis, Masuda and colleagues (12) also showed that the level of HSP47 mRNA correlated with that of collagen type I and type III mRNAs during the progression of rat experimental liver fibrosis. Thus HSP47 has been demonstrated to be involved in fibrotic diseases. In pulmonary fibrosis, it also has been reported that HSP47 is highly expressed on myofibroblasts or fibroblasts in autopsied human pulmonary fibrosis (16) and bleomycin (BLM)-treated rats (15). Also, we previously reported that HSP47 was expressed abundantly on type II pneumocytes in addition to myofibroblasts in active fibrotic areas of usual interstitial pneumonia, which is the most common pathological form of IPF (8). Thus HSP47 seems to be important in pulmonary fibrosis, similar to its role in other fibrotic diseases, but the association between HSP47 and pulmonary fibrosis remains obscure. In the present study, we used a murine BLM-induced fibrosis model to assess the temporal changes in localization of HSP47 proteins and in expression of HSP47 mRNA during development of inflammatory and fibrotic lesions.
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MATERIALS AND METHODS |
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Bronchoalveolar lavage. Bronchoalveolar lavage (BAL) was performed (n = 5 in both groups at each time point) at 2, 4, 5, 6, 7, and 8 wk after the BLM or saline administration. The trachea was cannulated with a 25-gauge Teflon needle (Terumo, Tokyo, Japan) under deep anesthesia, an aliquot of saline (0.9% NaCl at room temperature) was injected into the lung, and 5.0 ml of the total volume was recovered. The recovered fluid was centrifuged at 700 g for 10 min to sediment the cells. After being washed twice with PBS solution, cells were suspended with PBS containing 10% heat-inactivated fetal calf serum and counted with a hemocytometer. Differential cell counts were determined from cell suspensions displayed on slides using a cytocentrifuge (Cytospin 2; Shandon, Sewickley, PA). Cells on a slide were dried, fixed, and then stained by the May-Giemsa method. Two hundred cells were identified under a photomicroscope.
Antibodies. Primary antibodies used for the immunohistochemical studies included anti--smooth muscle actin (
-SMA; Neomarkers, Fremont, CA), anti-surfactant protein A (SP-A; Santa Cruz Biotechnology, Santa Cruz, CA), anti-F4/80 (Serotec) pretreated with 1% trypsin for 20 min at room temperature before use, and anti-HSP47 (Biotechnologies, Victoria, British Columbia, Canada).
-SMA, SP-A, and F4/80 were used as markers for myofibroblasts, type II pneumocytes, and macrophages, respectively.
Histology and immunohistochemistry. At 2, 4, 5, 6, 7, and 8 wk after BLM or saline administration (at least 3 mice in both groups at each time point), mice were killed by exsanguination under deep anesthesia. Lungs were then removed from each mouse via a midline incision, inflated, fixed in 10% formaldehyde/neutral buffer solution, embedded in paraffin, and processed to obtain 4-µm sections for staining with standard hematoxylin-eosin. Sequential or pairs of mirror image lung sections were placed on glass slides for immunohistochemical analysis.
The severity of fibrosis was evaluated semiquantitatively using a predetermined scale of severity (Ashcroft score) (2). The entire lung section was examined at a magnification of x100. For each of >25 microscopic fields required to review the section, a numerical fibrosis score ranging from 0 (normal) to 6 (most severe) was assigned. The degree of cell infiltration and pneumonitis/fibrosis in the microscopic lung sections was assessed as the mean score for the observed fields in each sample.
To confirm the localization of HSP47, immunohistochemistry was performed with the conventional avidin-biotinperoxidase histochemical technique using Histomouse-Plus kits (Zymed Laboratories, South San Francisco, CA) for HSP47 and -SMA and Vecstain Elite ABC kit (Vector Laboratories, Burlingame, CA) for SP-A and F4/80. Briefly, paraffin sections were deparaffinized with toluene and rinsed thoroughly with ethanol. Sections were then soaked in 0.3% H2O2 in methanol for 20 min at room temperature to inactivate endogenous peroxidases. They were incubated with blocking serum for 30 min and then covered with primary antibodies and incubated for 1 h. After being washed in 0.075% Brij (Sigma Chemical, St. Louis, MO) in PBS, sections were processed further using kits according to the protocols provided by the manufacturers and then developed with 3,3'-diaminobenzidine and H2O2. The standard Mayer's hematoxylin staining method was used to localize HSP47 and other proteins in lung sections. For immunoperoxidase labeling, we controlled for the development of the peroxidase-based color change, and all sections were developed using the same amount of time.
Measurement of lung hydroxyproline content. To estimate the total amount of collagen deposited in the lung as an indicator of pulmonary fibrosis, we measured the hydroxyproline content of the right lung of mice (n = 5 in both groups at each time point) at 1, 2, 4, 5, 6, 7, and 8 wk after the BLM or saline administration. After measurement of wet weight, a 10-mg aliquot of dried lung homogenate was hydrolyzed in 6 N HCl at 110°C for 24 h. The resulting hydrolysate was fixed by nitrogen at 60°C for 20 min after being filtered through a 0.45-µm nylon membrane, dissolved in 12.5 mM sodium borate, and prepared for derivatization using fluorescamine solution. After the resulting solution was centrifuged at 600 g for 10 min, the amount of hydroxyproline in the supernatant was determined with a capillary electrophoresis system (P/ACE MDQ; Beckman Coulter).
Semiquantification of HSP47 mRNA by RT-PCR. RT-PCR was performed using homogenized lung tissue. For RNA isolation, the whole left lungs, which had been frozen at -80°C immediately after removal from animals in both groups, were homogenized in Isogen (Nippon Gene, Tokyo, Japan). Total RNA was extracted from the lungs (at least 3 mice in both groups at each time point) at 1, 2, 4, 6, 7, and 8 wk after the BLM or saline administration using the acid guanidium-thiocyanate-phenol-chloroform method. Approximately 2 µg of total RNA was reverse-transcribed with random hexamer according to the instructions provided by the manufacturer (Thermoscript RT-PCR System; GIBCO BRL, Gaithersburg, MD). The primers for HSP47 were forward 5'-ACCACAGGATGGTGGACAACCGT-3' and reverse 5'-ATCTCGCATCTTGTCTCCCTTGGG-3'. The constitutive control was glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The optimal number of PCR cycles for each primer set was determined in preliminary experiments so that the amplification process was conducted during the exponential phase of amplification. The target sequences of HSP47 and GAPDH genes were separately amplified for 30 cycles on a thermal cycler (GeneAmp PCR System 9600; Perkin Elmer, Norwalk, CT). Samples of each reaction product were separated by agarose gel electrophoresis, visualized by ethidium bromide staining, and photographed with 290-nm ultraviolet illumination. The density of each band was measured by densitometry. Relative expression of HSP47 mRNA at each time point was normalized to the expression of the internal control, GAPDH.
Statistical analysis. All values were expressed as means ± SD. The Mann-Whitney U-test was used to examine differences between unpaired samples. The correlation coefficient was tested for statistical significance with the Spearman's rank test. Statistical analysis was performed using Stat-View-J 4.5 software (Abacus Concepts, Berkeley, CA). Significance was defined by a P value of <0.05.
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RESULTS |
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Histological examination revealed no significant changes in control lungs (Fig. 1A), whereas marked interstitial and intra-alveolar pneumonia and/or fibrosis were noted in the lungs between 2 and 8 wks after the BLM administration (Fig. 1B). The extent of pneumonitis/fibrosis, localized mainly in the subpleural regions, increased gradually with time from 2 to 6 wk after BLM administration. The mean fibrosis score in BLM-treated mice (2.28 ± 0.96) was significantly higher than in control mice (0.94 ± 0.73, P = 0.0002; Fig. 2). Collagen content was also assessed by measuring hydroxyproline in the right lung in both groups at 1, 2, 4, 5, 6, 7, and 8 wk after BLM or saline administration. The mean hydroxyproline contents for the BLM-treated mice and control mice were 66.0 ± 10.7 µmol/g and 58.2 ± 4.8 µmol/g, respectively (P = 0.0003). There were significant differences between these two groups at 5, 6, and 7 wk after BLM or saline administration (Fig. 3).
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Immunohistochemistry. We next examined the localization of HSP47 in BLM-treated lungs. Weak immunoreactive HSP47 expression was detected in airway epithelial cells and interstitial cells in control lungs (Fig. 4A). In contrast, markedly increased HSP47 immunostaining was noted in BLM-treated lungs (Fig. 4B), and the level of HSP47 expression increased microscopically with time from 2 until 6 wk after BLM administration. This increment of HSP47 paralleled the changes in fibrosis score and hydroxyproline content (Figs. 2 and 3). To determine cells expressing HSP47 in BLM-treated lungs, immunostaining for HSP47 and SP-A, F4/80, or -SMA was performed in sequential or pairs of mirror image lung sections. Around 4 wk after BLM administration, SP-A-positive cuboidal cells located in alveolar walls began to increase in fibrotic areas, and some of them were HSP47 positive. At 5 wk after treatment, the alveolar walls were frequently lined by abundant SP-A-positive type II pneumocytes (Fig. 5A), and some of them were also HSP47 positive (Fig. 5B). At the same time interval, we also noted increases of F4/80-(Fig. 5C) and HSP47-(Fig. 5D) positive macrophages in fibrotic areas. Interstitial cells expressing
-SMA (Fig. 5E) and HSP47 (Fig. 5F) further increased at 6 wk after the treatment. After that period, these changes of pneumonitis/fibrosis appeared to diminish (data not shown).
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Semiquantification of HSP47 mRNA. HSP47 mRNA levels in the lung tissue samples harvested at 1, 2, 4, 5, 6, 7, and 8 wk after treatment were analyzed using RT-PCR and compared between the two experimental groups. At 6 and 7 wk after treatment, the relative amounts of HSP47 mRNA were significantly higher in the BLM-treated lungs than in control lungs (Fig. 6). Moreover, the relative expression of HSP47 mRNA correlated significantly with the hydroxyproline content in BLM-treated lungs (r = 0.406, P < 0.05; Fig. 7).
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DISCUSSION |
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In the present study, we have shown that both HSP47 immunoreactivity and mRNA were markedly induced during lung fibrosis in mice treated with BLM. The relative expression of HSP47 mRNA also correlated significantly with the hydroxyproline content, which reflects total collagen content. HSP47, which has a molecular chaperone-like function under stress conditions, is involved in the processing and transporting of procollagen in the endoplasmic reticulum (5, 13, 25). Recent reports demonstrating that expression of HSP47 correlates well with collagen expression (1, 8, 12, 15-17, 20, 23) indicate that HSP47 possibly plays a key role in increased deposition of collagens during the progression of human fibrotic diseases, including pulmonary fibrosis. Previous studies have shown overexpression of HSP47 in myofibroblasts and fibroblasts in BLM-treated rats and autopsy examination of cases with various pulmonary fibrosis, along with increased deposition of types I and III collagen in fibrotic areas (15, 16). The present findings confirmed and extended these early findings by demonstrating overexpression of HSP47 at both the protein and mRNA levels. In addition, the relative expression of HSP47 mRNA correlated significantly with hydroxyproline content. These findings suggest that HSP47 may play an important role in the pathogenesis of pulmonary fibrosis, similar to other fibrotic diseases (1, 12, 20).
It is of interest to know which cells contribute to fibrogenesis in the lung. Razzaque and colleagues (15, 16) noted colocalization of collagens and HSP47 in regions of pulmonary fibrosis and that HSP47-expressing cells were found to be mainly -SMA-positive interstitial cells (myofibroblasts) and vimentin-positive cells (fibroblasts) by double immunostaining of lung sections from autopsies of patients with various pulmonary fibrotic diseases and BLM-induced pulmonary fibrosis in rats (15, 16). In addition, we recently demonstrated that HSP47 was coexpressed with type I procollagen on both myofibroblasts and the abundant type II pneumocytes in active fibrotic areas of lung biopsy specimens from patients with IPF. We speculated that these cells play an important role in pulmonary fibrosis through HSP47-associated regulation of type I procollagen (8). In the present study using an animal model, HSP47 was localized predominantly in
-SMA-positive myofibroblasts, intra-alveolar F4/80-positive macrophages (Fig. 5), and F4/80-negative, SPA-positive type II pneumocytes, similar to the pattern observed in human pulmonary fibrosis (8). Therefore, alveolar macrophages and type II pneumocytes, in addition to fibroblasts and myofibroblasts, may play an important role in the fibrotic process in BLM-treated lungs through the induction of HSP47. The exact mechanism of intracellular processing of collagen in those cells remains unclear. Furthermore, there is no evidence that type II pneumocytes and alveolar macrophages synthesize type I procollagen in BLM-treated lungs. Further studies are necessary to elucidate whether these cells can synthesize HSP47 and procollagen.
Pulmonary fibrosis remains a devastating clinical disorder for which there are limited therapeutic options. A number of experimental approaches have been investigated in animal models, including the inhibition of key cytokines and growth factors (3, 4, 6, 24, 26, 28), but to date none of these approaches has come to fruition in the clinic. Therapeutic intervention directed against HSP47 might alter the fibrotic process, which might be of clinical value. Experimental studies have shown that an antisense oligodeoxynucleotide against HSP47 inhibited both HSP47 production and consequently diminished the levels of 1(I) procollagen chains in vitro (18). Furthermore, in a recent in vivo study, Sunamoto et al. (23) reported that inhibition of HSP47 by antisense oligodeoxynucleotides markedly suppressed collagen accumulation and subsequently attenuated the histological manifestations in experimental glomerulonephritis. Further studies using an antisense oligonucleotide against HSP47 are under way in our laboratories to examine whether this antisense suppresses collagen accumulation in an experimental pulmonary fibrosis/pneumonitis induced by BLM-sulfate. However, it is possible that the protective effect of antisense-HSP47 is specific to BLM-induced pulmonary fibrosis, since BLM-induced pulmonary fibrosis and IPF are not the same disease.
In summary, we have demonstrated, in the present study, the role of increased expression of HSP47 mRNA and protein levels in the progression of pneumonitis/fibrosis in BLM-treated lungs. Our results suggest that alveolar macrophages and type II pneumocytes, in addition to myofibroblasts, may play an important role in the fibrotic process of BLM-treated lungs through upregulation of HSP47. Further studies are required to investigate the therapeutic effects of antisense against HSP47 in BLM-treated lungs.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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