Targeted injury of type II alveolar epithelial cells induces pulmonary fibrosis

Abstract

Rationale: Ineffective repair of a damaged alveolar epithelium has been postulated to cause pulmonary fibrosis. In support of this theory, epithelial cell abnormalities, including hyperplasia, apoptosis, and persistent denudation of the alveolar basement membrane, are found in the lungs of humans with idiopathic pulmonary fibrosis and in animal models of fibrotic lung disease. Furthermore, mutations in genes that affect regenerative capacity or that cause injury/apoptosis of type II alveolar epithelial cells have been identified in familial forms of pulmonary fibrosis. Although these findings are compelling, there are no studies that demonstrate a direct role for the alveolar epithelium or, more specifically, type II cells in the scarring process.

Objectives: To determine if a targeted injury to type II cells would result in pulmonary fibrosis.

Methods: A transgenic mouse was generated to express the human diphtheria toxin receptor on type II alveolar epithelial cells. Diphtheria toxin was administered to these animals to specifically target the type II epithelium for injury. Lung fibrosis was assessed by histology and hydroxyproline measurement.

Measurements and main results: Transgenic mice treated with diphtheria toxin developed an approximately twofold increase in their lung hydroxyproline content on Days 21 and 28 after diphtheria toxin treatment. The fibrosis developed in conjunction with type II cell injury. Histological evaluation revealed diffuse collagen deposition with patchy areas of more confluent scarring and associated alveolar contraction.

Conclusions: The development of lung fibrosis in the setting of type II cell injury in our model provides evidence for a causal link between the epithelial defects seen in idiopathic pulmonary fibrosis and the corresponding areas of scarring.

Figure 1.

Functional integrity of the murine surfactant protein C promoter and the diphtheria toxin receptor gene (SPC-DTR) expression cassette. MLE-12 cells were grown to 80% confluency in a 96-well plate in 100 μl of media and treated with transfectant reagent alone, with transfectant reagent + the SPC-DTR expression cassette, or with phosphate-buffered saline (PBS) alone for 24 hours. The cells were washed, and subsets from each treatment group were exposed to diphtheria toxin (DT) or an equal volume of PBS for 24 hours. The metabolic activity of the cells was assessed with a 3-(,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Data are reported as the absorbance at 550 nm (mean ± SEM; n = 6 for each treatment condition). Each group is compared using a Student's t test.

Figure 2.

Tissue diphtheria toxin receptor (DTR) mRNA expression. Total RNA was isolated from lung, kidney, spleen, liver, and heart tissue of adult transgene–positive surfactant protein C ([SPC]-DTR+) and transgene-negative (SPC-DTR−) mice. The RNA was analyzed by reverse transcription–polymerase chain reaction (RT-PCR) for expression of the DTR message with primers specific for this gene construct. The resultant products of the RT-PCR were separated on a 1.5% agarose gel and photographed. Each result is from a single mouse from two out of three reproductive founder lines.

Figure 3.

Type II alveolar epithelial cell diphtheria toxin receptor (DTR)mRNA expression. Type II alveolar epithelial cells were isolated from transgenic–positive surfactant protein C ([SPC]-DTR+) and wild-type (WT) (SPC-DTR−) control mice. RNA was extracted from the isolated cells and analyzed by real-time polymerase chain reaction with primers specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), SPC, and DTR. The products of the reverse transcription–polymerase chain reaction were separated on a 2.2% FlashGel cassette and photographed.

Figure 4.

Weight loss associated with diphtheria toxin treatment. Transgenic surfactant protein C diphtheria toxin receptor (SPC-DTR) and wild-type (WT) mice were treated with daily intraperitoneal doses of diphtheria toxin (DT) at 10 μg/kg in 100 μl of phosphate buffered saline (PBS) for 14 days. A control group of mixed transgenic and WT mice (SPC-DTR and WT) received daily intraperitoneal injections of 100 μl PBS for 14 days. The mice were weighed daily during the injections and then for a subsequent 14 days. Data are reported as the change in weight from Day 0 (mean ± SEM; n = 6 mice per group). The weight curves were compared by linear regression analysis. The slopes between SPC-DTR/DT and WT/DT significantly differ (P < 0.001), as do the curves between SPC-DTR/DT and SPC-DTR and WT/PBS (P < 0.001).

Figure 5.

Lung hydroxyproline content after diphtheria toxin treatment. In two separate experiments, transgenic surfactant protein C diphtheria toxin receptor⁺(SPC-DTR⁺) and wild-type (WT) mice (SPC-DTR⁻) were treated with daily intraperitoneal doses of diphtheria toxin⁺ (DT⁺) at 10 μg/kg in 100 μl of phosphate buffered saline for 14 days. A control group of mixed transgenic and WT (SPC-DTR^+/−) mice received daily intraperitoneal injections of 100 μl phosphate buffered saline (DT−) for 14 days. In the first experiment, the right lung from each mouse was excised and analyzed for hydroxyproline content on Day 21 (n = 6–8 per group). In the second experiment, the right lung from each mouse was excised and analyzed for hydroxyproline content on Day 28 (n = 6 per group). Data are reported as the mean hydroxyproline content (μg/ml) ± SEM. The three groups are compared using a one-way analysis of variance with a Bonferroni's post hoc multiple comparison test.

Figure 6.

Lung histology after diphtheria toxin treatment. Transgenic surfactant protein C diphtheria toxin receptor (SPC-DTR) and wild-type (WT) mice were treated with daily intraperitoneal doses of (A and B) diphtheria toxin (DT) at 10 μg/kg or (C) 12.5 μg/kg in 100 μl of phosphate buffered saline for 14 days. A control group of transgenic mice (SPC-DTR) received daily intraperitoneal injections of 100 μl phosphate buffered saline for 14 days. On Day 28, the left lung was fixed at 25 cm H₂O and cut into 8-μm sections. Sections were stained using the Masson's Trichrome method (A and C) or with Picrosirius Red (B). Representative regions of the lung at 10× and 20× magnification are shown.

Figure 7.

Diphtheria toxin receptor (DTR) and surfactant protein C (SPC) expression after diphtheria toxin treatment. Transgenic (SPC-DTR⁺) and WT (SPC-DTR⁻) mice were treated with daily intraperitoneal doses of diphtheria toxin (DT⁺) at 10 μg/kg in 100 μl of PBS for 3, 7, and 14 days. Control groups of transgenic mice (SPC-DTR⁺) received daily intraperitoneal injections of 100 μl phosphate buffered saline (DT⁻) for 3, 7, and 14 days. After the last DT injection, total RNA was isolated from the lungs of each mouse and analyzed by real-time polymerase chain reaction with primers specific (A) for the DTR or (B) for SPC. The expressions of DTR and SPC for each sample were normalized to the corresponding expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The results are reported as the relative expression of DTR and SPC compared with the control group of mice (mean ± SEM; n = 5–9 mice per group). The groups are compared using a one-way analysis of variance with a Bonferroni's post hoc multiple comparison test.

Figure 8.

MTT activity after DT treatment of primary type II alveolar epithelial cells. Type II alveolar epithelial cells were isolated from transgenic surfactant protein C diphtheria toxin receptor⁺ (SPC-DTR+) and wild-type (WT) control mice. The cells were maintained in culture in a 96-well plate for 3 days, and subsets were treated with diphtheria toxin (DT) (1.0 μg/ml) or an equal volume of phosphate buffered saline for 24 hours. The mitochondrial activity of the cells was then assessed with a 3-(,(5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results are reported as the decrease in MTT activity of the DT-treated cells compared with PBS-treated cells (n = 8) ± SEM. The WT and SPC-DTR groups are compared using Student's t test.

Figure 9.

Immunohistochemical staining for SPC and BrdU. Transgenic surfactant protein C diphtheria toxin receptor (SPC-DTR) and wild type (WT) mice were treated with daily intraperitoneal doses of diphtheria toxin (DT) at 10 μg/kg in 100 μl of phosphate buffered saline for 1, 3, and 7 days. A group of WT mice received no treatment and served as the Day 0 control group. One hour before death, each animal received an intraperitoneal injection of bromodeoxyuridine (BrdU). After the last DT injection, the lungs from each animal were fixed by distending at 25 cm H₂O with 10% neutral-buffered formalin, removed en bloc, further fixed in 10% neutral-buffered formalin overnight, and embedded in paraffin. Eight-micron sections were immunostained for SPC and BrdU. The number of SPC-positive (brown stained) and SPC-BrdU double-positive (brown-red stained) cells per 40× high-powered field were counted. Upper panel: Representative images of DT-treated WT and SPC-DTR mice. Arrows point to BrdU-positive cells, and arrowheads point to double-stained cells. Lower panel: Graphic representation of (A) the number of SPC-positive cells and (B) SPC-BrdU double-stained cells per high-powered field. Results are reported as the mean ± SEM (n = 3–4 per group). The WT and SPC-DTR groups are compared using a two-way ANOVA. The effect on genotype on SPC-BrdU is statistically significant (P < 0.05). There is no effect of genotype or day on the number of SPC-positive cells (P = not significant).