Type II alveolar epithelial cells and interstitial fibroblasts express connective tissue growth factor in IPF

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.

Cell culture

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⇓).

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Fig. 1.—

Normal lung tissue, immunohistochemical staining (consecutive serial sections): a) Staining for connective tissue growth factor (CTGF) arrow indicates type II epithelial cell which is positive. b) Staining for cytokeratin, arrows indicate type II epithelial cells which are positive c) Staining for CD68, arrows indicate macrophages which are positive.

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).

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Fig. 2.—

Fibrotic lung tissue of IPF. Consecutive serial sections. (a: hematoxylin-eosin staining; bed: immunohistochemical staining). a) Proliferating alveolar macrophages, type II epithelial cells and fibroblasts. b) Staining for connective tissue growth factor (CTGF), arrows indicate proliferating type II epithelial cells which are positive. Small arrows indicate activated fibroblast which is positive. c) Staining for cytokeratin, arrows indicate type II epithelial cells which are positive. d) Staining for CD68, arrows indicate alveolar macrophages which are positive.

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Fig. 3.—

Fibrotic lung tissue of idiopathic pulmonary fibrosis. Consecutive serial sections. a) Staining for connective tissue growth factor (CTGF), arrows indicate proliferating type II epithelial cells which are positive. Small arrows indicate activated fibroblast which is positive. b) Staining for cytokeratin, arrows indicate type II epithelial cells which are positive. c) Staining for alpha smooth muscle actin (a-SMA), arrows indicate activated fibroblasts which are positive.

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⇓).

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Fig. 4.—

Fibrotic lung tissue of idiopathic pulmonary fibrosis. In situ hybridization. Methyl green as counterstain. a) In situ hybridization for connective tissue growth factor (CTGF), arrows indicate type II alveolar epithelial cells which are positive. b) In situ hybridization for CTGF, arrows indicate activated fibroblasts which are positive. c) Control with sense probes, no positive signals are observed.

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.

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Fig. 5.—

Expression of connective tissue growth factor (CTGF) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger ribonucleic acids (mRNAs) detected by reverse transcription-polymerase chain reaction. Ethidium bromide-stained agarose gel showing panel a) (CTGF) and panel b) (GAPDH). 1–2: normal lung tissue; 3–6: Idiopathic pulmonary fibrosis; 7: human foetal lung-1; 8: human primary cultured bronchial epithelial cells; 9: A549. M: molecular markers (phage Hae III fragments). Arrows indicate bands of PCR products of CTGF (329 bp) in panel 9 and GAPDH (983 bp) in panel b.