Connective tissue growth factor (CTGF) is a growth and chemotactic factor for fibroblasts encoded by an immediate early gene that is transcriptionally activated by transforming growth factor-β. Previous studies have shown that both CTGF messenger ribonuclear acid (mRNA) and protein are expressed in renal fibrosis and bleomycin-induced pulmonary fibrosis in mice. The aim of the present study was to investigate the localization of CTGF protein and its mRNA expression in the fibrotic lung tissue of patients with idiopathic pulmonary fibrosis (IPF).
Using human fibrotic lung tissue obtained from eight autopsy cases and four biopsy cases with IPF, immunohistochemical staining, in situ hybridization, and reverse transcription-polymerase chain reaction (RT-PCR) were performed.
The cellular immunoreactivity for CTGF was markedly increased in the lung tissue of patients with IPF, compared to normal lungs. The immunolocalization of CTGF was confined predominantly to proliferating type II alveolar epithelial cells and activated fibroblasts. In the normal lung, type II alveolar epithelial cells stained for CTGF were sparsely distributed. CTGF mRNA was localized in proliferating type II alveolar epithelial cells and activated fibroblasts in the interstitium of fibrotic lung tissues. RT-PCR analysis showed that CTGF mRNA was expressed at a higher level in fibrotic lungs than in normal lungs.
In both an autocrine and a paracrine manner, type II alveolar epithelial cells and activated fibroblasts may play a critical role in pulmonary fibrosis by producing connective tissue growth factor which modulates fibroblast proliferation and extracellular matrix production.
- connective tissue growth factor
- idiopathic pulmonary fibrosis
- in situ hybridization
- transforming growth factor-β
Supported by the Ministry of Education, Science and Culture, Japan.
Pulmonary fibrosis proceeds in a cascade fashion beginning with an inflammatory phase, followed by the proliferation of type II alveolar epithelial cells and fibroblasts, and deposition of extracellular matrix (ECM) protein, such as collagen. Previous studies have demonstrated that alveolar macrophages and type II alveolar epithelial cells synthesize various growth factors and cytokines, such as tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β), platelet derived growth factor (PDGF), and transforming growth factor-β (TGF-β), all of which may play important roles in the process of pulmonary fibrosis 1–8. Among these growth factors, TGF-β has been thought to be a critical molecule in pulmonary fibrosis 9–14. Connective tissue growth factor (CTGF), a newly described growth factor which is a cysteine-rich 38-kDa mitogenic peptide, functions as a downstream mediator of the TGF-β action in connective tissue cells 15–17. Previous studies demonstrated that there were CTGF-dependent and CTGF-independent signalling pathways activated by TGF-β in fibroblasts 18–20. It is known that CTGF expression is upregulated in bleomycin-induced lung fibrosis, skin fibrosis, renal fibrosis, and other disorders 19, 21–25. Recently, Allen et al. 22 reported that the messenger ribonucleic acid (mRNA) level of CTGF in bronchoalveolar lavage cells of patients with idiopathic pulmonary fibrosis (IPF) and sarcoidosis was significantly higher than that in healthy control subjects 22. However, to date, there is little information on the role of CTGF in interstitial lung diseases, especially in the epithelium and fibroblasts. To extend the findings and investigate whether CTGF is involved in IPF, the immunohistochemical localization of CTGF protein, and CTGF mRNA expression by in situ hybridization in autopsy and biopsy specimens obtained from patients with IPF were studied.
Materials and methods
Autopsy and biopsy specimens
Fibrotic lung tissue specimens were obtained from a total of 12 patients (six males, six females, mean age 65.2 yrs, range 36–80 yrs), including eight cases in which autopsy was performed within three post mortem hours at Tohoku University Hospital, and four cases of biopsy by video-assisted thoracic surgery performed at Iwate Medical University Hospital. According to their pathological and clinical features, 12 cases of IPF are compared in table 1⇓. The pathological classification of IPF cases into acute interstitial pneumonia (AIP) (n=2), and usual interstitial pneumonia (UIP) (n=10) was according to Katzenstein and Myers 26. Four normal lung specimens obtained from autopsy and two normal lung specimens from resection of cancer were used as controls. The specimens were fixed in periodate-lysine-paraformaldehyde (PLP) for 4–8 h at 4 °C and routinely embedded in paraffin wax. Sections, 3 µm in thickness, were prepared for immunohistochemical and in situ hybridization studies. For immunohistochemical study, consecutive serial sections were used for all cases. A part of the specimens was snap frozen in liquid nitrogen and stored at −80 °C.
Human foetal lung cell line (HFL-1) and human alveolar epithelial cell carcinoma cell line (A549) were purchased from American Tissue Culture Collection (Rockville, MD, USA) and cultured in Roswell Park Memorial Institute (RPMI) 1640 containing 100 U·mL−1 of penicillin, 100 µg·mL−1 streptomycin, 2 mM l-glutamine and 10% foetal calf serum. Cells were used after the second to third passage. Human bronchial epithelial cells were obtained from healthy volunteers by bronchial brushing under bronchoscopy. This was approved by the Ethics Committee of the Iwate Medical University School of Medicine. The cells were cultured in four wells of rat collagen I-coated 12 well tissue culture plates (Iwaki, Funabashi, Japan) in sodium chloride, adenine, glucose, mannitol (SAGM) medium (Clonetics, San Diego, CA, USA) containing 0.5 mg·mL−1 epidermal growth factor (EGF), 0.5 mg·mL−1 insulin, 0.1 mg·mL−1 retinoic acid, 0.5 mg·mL−1 epinephrine, 0.5 mg·mL−1 hydrocortisone, 10 mg·mL−1 transfferin, 6.5 mg·mL−1 triiodide thyronine, 50 mg·mL−1 bovine serum albumin-fatty acid free (BSA-FAF), and 7.5 mg·mL−1 bovine pituitary extract (BPE). These cells were used for ribonucleic acid (RNA) extraction.
Ribonucleic acid extraction
RNA was extracted from the frozen lung tissues and cultured cells previously described. Healthy lung tissues were obtained from the healthy parts of excised lung tissues of the patients with lung cancer who underwent surgical operation. The healthy lung specimens were evaluated microscopically to be free of cancer cells. Fibrotic lung tissues were obtained from patients with IPF by video-assisted thoracic surgery. Total RNA was isolated by the single-step acid guanidium thiocyanate-phenol-chloroform extraction method as described elsewhere 27.
Antibodies and immunohistochemical staining
The biotin-streptavidin system was adopted using a Histofine Kit (Nichirei, Tokyo, Japan) for immunohistochemical staining. The sections were deparaffinized and treated with 0.3% hydrogen peroxide in methanol for 15 min to block endogenous peroxidase activity. The sections were incubated with 10% normal goat serum for 30 min at room temperature to block the nonspecific antibody reaction. A specific CTGF polyclonal antibody was used as the primary antibody in the immunohistochemical staining (Okayama University, Japan) 28. Anti-cytokeratin, CD68, and α-smooth muscle actin (α-SMA) (Nichirei, Tokyo, Japan) were also used for distinguishing epithelia, macrophages, and activated fibroblasts, respectively. The antibodies used, working dilution, and sources are listed in table 2⇓. The sections were incubated overnight at 4 °C with the primary antibodies. 3′3-diaminobenzidine (DAB) was used as the chromogenic substrate.
In situ hybridization
Paraffin sections of the samples fixed in PLP were investigated as described previously, by using a slightly modified nonradioactive in situ hybridization technique with digoxigenin-labelled RNA probes 29, 30. Briefly, paraffin-embedded tissues were cut into 3-µm thin sections, mounted onto silane-coated slides, deparaffinized, and treated with pronase (0.25 mg·mL−1 in 50 mM Tris-hydrochloric acid (HCl), pH 7.6, containing 5 mM ethylendiaminetetraacetic acid (EDTA)) digestion for 10 min at room temperature followed by 0.05 N HCl for 10 min. The sections were then post-fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) for 15 min and treated with glycine (2 mg·mL−1 in PBS) twice for 3 min each. After washing with PBS, the samples were acetylated with a freshly prepared mixture of 0.25% acetic anhydride in triethanolamine buffer for 10 min.
A 2100-base pair fragment of CTGF complementary deoxyribonucleic acid (cDNA) was subcloned into the EcoRI site of Bluescript phagemid and used to prepare probes. The DNA was linearized by using XbaI to prepare the antisense strand and XhoI for the sense strand. The probes were labelled with digoxigenin-11-UTP by using a digoxigenin (DIG) RNA-labelling kit (Boehringer Mannheim Biochemical, Germany) 16, 28.
The labelled RNA probes (1 µg·mL−1 ), in a mixture containing 50% formamide, 10% dextran sulphate, 1×Denhardt's solution, 500 µg·mL−1 transfer ribonucleic acid (tRNA), 80 µg·mL−1 sermon sperm DNA, 0.3 M sodium chloride (NaCl), 1 mM EDTA, and 10 mM Tris-HCl, pH 7.0 were placed on the slides and cover-slipped.
Hybridization was performed in a humidified chamber for 18 h at 45 °C. For the negative control, sense probes were used. They were then rinsed for 10 min in 2×sodium citrate sodium chloride (SSC) twice and in 0.5×SSC twice at 45 °C. The digoxigenin-labelled probes were visualized as per the protocol described in the DIG nucleic acid detection kit (Boehringer Mannheim Biochemical, Germany). Methyl green was used as the counter-stain.
Reverse transcription-polymerase chain reaction for connective tissue growth factor messenger ribonucleic acid
The primers were designed from the published sequences from human CTGF cDNA 31, as follows: sense, 5′-TTCCAGAGCAG-CTGCAAGTACCA-3′; antisense, 5′-TTGTCATTGGTAACCCGGGTGGA-3′. As a control, the primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA were also designed as follows: sense, 5′-TAAAGGTGGAGTCAAACGGATTTGGT-3′; antisense, 5′-CATGTGGGCCA-TGAGGTCCACCAC-3′ 32. cDNA was generated by reverse transcription (RT) of 0.5 µg of aliquots of total RNA in a total volume of 20 µL containing 1×polymerase chain reaction (PCR) buffer (10 mM Tris-HCl, pH 8.3; 50 mM potassium chloride (KCl); 5 mM magnesium chloride (MgCl2); 1 mM of each deoxynucleotide triphosphate (dNTP), 1 U ribonuclease (RNase) inhibitor, and 2.5 U murine leukaemia virus (MuLV) reverse transcriptase) at 42 °C for 15 min (PCR reagents were purchased from Perkin Elmer, Applied Biosystems Division, Foster City, CA, USA). Twenty µL of the RT reaction were used for PCR amplification in a total volume of 100 µL using 0.5 U Taq-polymerase and primers (20 pmol each).
Amplification was done with 35 cycles, with denaturation at 95 °C for 15 s, primer annealing and extension at 60 °C for 30 s, followed by a final extension at 72 °C for 7 min using the GeneAmp PCR System 2400 (Perkin Elmer). The cycle number of PCR was selected where the reaction was exponential on the basis of preliminary experiments. The expected fragment from this process should exhibit 329 base pairs. Aliquots of 10 µL of PCR reaction mixture were size-fractionated by electrophoresis on 1.5% agarose gels (NuSieve GTG; FMC BioProducts, Rockland, ME, USA) in TBE-buffer (i.e. 89 mM Tris base, 89 mM boric acid, 2 mM EDTA). PCR products were visualized by staining gels with ethydium bromide.
Immunostaining for connective tissue growth factor in normal lung tissue
Normal control tissues were characterized by a thin alveolar wall with few intra-alveolar macrophages. Type II alveolar epithelial cells were observed sparsely. Staining for type IV collagen revealed a uniform, smooth linear pattern along the epithelial and capillary basement membranes (data not shown). Immunoreactivity for CTGF was observed sparsely in a few type II alveolar epithelial cells (fig. 1a⇓). In these cells, type II alveolar epithelial cells were identified by cytokeratin immunoreactivity (fig. 1b⇓), whereas macrophages stained positive for CD68 (fig. 1c⇓).
Immunostaining for connective tissue growth factor in the lung tissue of patients with idiopathic pulmonary fibrosis
Morphologically, the changes observed in specimens of fibrotic lung disease (AIP and UIP with acute exacerbation) were focal hyaline membrane formation apposed to the alveolar walls, intra-alveolar accumulation of macrophages and activated fibroblasts (myofibroblasts) infiltrated in the eosinophilic oedematous matrix (fig. 2a⇓). Variable thickening of the alveolar septa with focal discontinuities in the alveolar basal lamina were visualized by immunostaining for type IV collagen (data not shown). Type II alveolar epithelial cells (hypertrophy pneumocytes) were positive for CTGF (fig. 2b, 3a⇓⇓), identified by immunostaining for cytokeratin (fig. 2c, 3b⇓⇓). Most alveolar macrophages were negative for CTGF, identified by immunostaining for CD68 (fig. 2d⇓). Activated fibroblasts containing α-SMA in the interstitium were positive for CTGF (fig. 3c⇓). At the foci of old fibrosis in UIP, the normal alveolar architecture was lost and replaced by areas with deposition of collagen fibre, where activated fibroblasts were sparse. Alveolar macrophages and proliferated type II alveolar epithelial cells were sparsely distributed, and they were weakly positive or negative for CTGF (data not shown).
Localization of connective tissue growth factor detected by in situ hybridization
In the present study, four cases were examined by in situ hybridization and a positive purple staining was observed in proliferating type II alveolar epithelia lining the alveolar walls and activated fibroblasts of the interstitium (fig. 4a, 4b⇓). Control sections hybridized with the CTGF sense probe showed no signals (fig. 4c⇓).
Connective tissue growth factor expression analysed by reverse-transcription polymerase chain reaction
CTGF mRNA expression was evaluated by RT-PCR in normal lung tissues and in the lung tissues of IPF. In addition, CTGF mRNA expression was also evaluated in HFL-1, A549 and human cultured bronchial epithelial cells (lane 7–9). Weak bands corresponding to CTGF mRNA were observed in normal lung tissue compared to those of IPF (fig. 5⇓, panel A lane 1–6). Conversely, the amount of RT-PCR products from GAPDH mRNA in normal lung tissues appeared to be at the same level as in IPF (fig. 5⇓, panel B), suggesting that the CTGF mRNA level in the lung tissue of IPF might be higher than that in the normal lung tissue.
In the present study using fibrotic lung tissues of IPF demonstrated the expression and localization of CTGF mRNA and protein by in situ hybridization and immunohistochemical staining, respectively, in both a large number of proliferating type II alveolar epithelial cells and activated fibroblasts of the interstitium. In addition, CTGF mRNA was detected by RT-PCR in the lung tissue of IPF, HFL-1 cells, A549 and cultured human bronchial epithelial cells.
Lasky et al. 19 reported that there was an increase in CTGF mRNA expression in both human and murine lung fibroblasts stimulated with TGF-β in vitro. In addition, they demonstrated that CTGF mRNA expression was upregulated in bleomycin-induced lung fibrosis in mice in vivo. Allen et al. 22 used a multiplex competitive RT-PCR to quantify insulin-like growth factor binding protein-related protein 2 (IGFBP-rP2) (CTGF) transcripts in bronchoalveolar lavage cells from healthy subjects and patients with IPF. IGFBP-rP2 messenger RNA expression was enhanced >10-fold in patients with IPF. They also suggested that the cellular sources of the elevated level of CTGF mRNA were neutrophils and lymphocytes. The present study could not evaluate this possibility because few such cells were seen. It has been demonstrated that CTGF is secreted by fibroblasts in human organs other than the lung and have a role in stimulating fibroblast cell growth, matrix production, and granulation tissue formation 21, 23–25, 33. In addition, CTGF mRNA has been shown to be expressed predominantly in visceral and parietal epithelial cells of kidney of patients with renal fibrosis 25. The present study also demonstrated CTGF mRNA expression in human primary cultured bronchial epithelial cells and alveolar epithelial cell carcinoma cell line A549, suggesting that pulmonary epithelial cells may have the ability to express CTGF. To date, there is little information on the role of CTGF in pulmonary fibrosis, especially in the epithelium. The present results suggest that the expression of CTGF protein and its mRNA in IPF were increased. These data demonstrate that both proliferating type II alveolar epithelial cells and activated fibroblasts may contribute to the pathogenesis of lung fibrosis by producing CTGF.
The present study observed strong expressions of CTGF in proliferating type II alveolar epithelial cells and activated fibroblasts situated in the area of fibrotic interstitium only in the earlier stage of fibrotic changes. In the later stage of fibrotic changes, active fibroblasts are few and lack the expression of CTGF. Therefore, it is speculated that CTGF produced by activated fibroblasts may contribute to active fibrotic process in an early stage of pulmonary fibrosis.
Previous studies have demonstrated that CTGF activation is followed by that of TGF-β 16–18. CTGF may serve as a more specific mediator in processes involving connective tissue formation during wound repair or fibrotic disorders. The present study was not able to investigate CTGF functions as a downstream mediator of TGF-β action on connective tissue cells directly. However, in fibrotic lung tissues, TGF-β expression was observed in type II epithelial cells, fibroblasts and alveolar macrophages by immunohistochemical staining 34, 35. TGF-β shared part of the same immunostaining pattern as CTGF without alveolar macrophages. The present authors suggest that CTGF produced mainly by type II epithelial cells and activated fibroblasts, may also be a mediator of TGF-β action on fibroblasts in IPF.
The present results indicate that not only activated fibroblasts, but also proliferating type II alveolar epithelial cells, are main sources of connective tissue growth factor in the lungs of fibrotic diseases such as idiopathic pulmonary fibrosis. Via both an autocrine and a paracrine route of cytokine action, connective tissue growth factor produced by type II alveolar epithelial cells and activated fibroblasts may have the potential to stimulate fibroblast proliferation and the production of extracellular matrix involved in pulmonary fibrosis.
- Received August 25, 2000.
- Accepted February 7, 2001.
- © ERS Journals Ltd