Epithelial endoplasmic reticulum stress and apoptosis in sporadic idiopathic pulmonary fibrosis

Abstract

Rationale: The molecular pathomechanisms underlying idiopathic pulmonary fibrosis (IPF) are elusive, but chronic epithelial injury has recently been suggested as key event.

Objectives: We investigated the possible implication of endoplasmic reticulum (ER) stress-mediated apoptosis in sporadic IPF.

Methods: We analyzed peripheral explanted lung tissues from patients with sporadic IPF (n = 24), chronic obstructive pulmonary disease (COPD) (n = 9), and organ donors (n = 12) for expression of major ER stress mediators and apoptosis markers by means of immunoblotting, semiquantitative reverse transcription-polymerase chain reaction, immunohistochemistry, and the TUNEL method.

Measurements and main results: Compared with COPD and donor lungs, protein levels of ER stress mediators, such as processed p50 activating transcription factor (ATF)-6 and ATF-4 and the apoptosis-inductor CHOP (C/EBP-homologous protein), as well as transcript levels of spliced X-box binding protein (XBP)-1, were significantly elevated in lung homogenates and type II alveolar epithelial cells (AECIIs) of IPF lungs. Proapoptotic, oligomeric forms of Bax, which play a key role in ER stress-mediated apoptosis downstream of CHOP induction, as well as caspase-3 cleavage, could be detected in IPF lungs. By means of immunohistochemistry, exclusive induction of active ATF-6, ATF-4, and CHOP in AECIIs was encountered in IPF but not in COPD or donor lungs. Immunoreactivity was most prominent in the epithelium near dense zones of fibrosis and fibroblast foci, where these ER stress markers colocalized with markers of apoptosis (TUNEL, cleaved caspase-3).

Conclusions: Severe ER stress response in the AECIIs of patients with sporadic IPF may underlie the apoptosis of this cell type and development of fibrosis in this disease.

Figure 1.

Induction of the endoplasmic reticulum stress–mediated apoptosis pathway in idiopathic pulmonary fibrosis (IPF). (A) Example immunoblots and (B–F) quantitative immunoblot analysis of peripheral lung tissue from patients with sporadic IPF (IPF_LTX) (n = 20), lung tissue from patients with chronic obstructive pulmonary disease (COPD) (n = 9), and human donor (HD) lungs (n = 12) using specific antibodies against activating transcription factor (ATF)-6α (B), ATF-4 (C), CHOP (D), Bax (E), pro–caspase 3 (F), and β-actin as loading control. Densitometric ratios of the respective protein to β-actin are depicted as a box-and-whisker diagram (box indicates 25th and 75th, horizontal line indicates the 50th percentile [median], and extensions above and below reflect extreme values). Measurements of individual samples were done in duplicate. *P < 0.05; **P < 0.01; ***P < 0.001. ns = not significant.

Figure 2.

X-box binding protein (XBP)-1 activation in idiopathic pulmonary fibrosis (IPF) lungs. Total RNA was isolated from peripheral lung tissue of patients with IPF (IPF_LTX), from lung tissue of patients with chronic obstructive pulmonary disease (COPD), and human donors (HD) and subjected to reverse transcription–polymerase chain reaction analysis with primers spanning the intron of unspliced XBP-1 mRNA. XBP-1(u) denotes unspliced, inactive XBP-1; XBP-1(s) denotes spliced, active XBP-1, which was observed in all 20 IPF_LTX lungs, but in none of the COPD or HD lungs. Representative results are shown. MW = molecular weight.

Figure 3.

Apoptotic endoplasmic reticulum stress response in alveolar epithelial type II cells (AECIIs) in idiopathic pulmonary fibrosis (IPF). (A) Reverse transcription–polymerase chain reaction of human AECIIs isolated from peripheral explanted lung tissue (LTX) of patients with IPF (n = 3) and healthy donors (HD) (n = 3) demonstrated strong induction of C/EBP-homologous protein (CHOP) expression and exclusive splicing of the X-box binding protein (XBP)-1 transcript in IPF; in contrast, expression of surfactant protein (SP)-B, SP-C, thyroid transcription factor 1 (TTF-1) and β-actin was similar in IPF and donor lungs. (B) Immunoblot analysis of human AECIIs isolated from peripheral LTX lung tissue of patients with IPF (n = 2) and HD (n = 3) revealed up-regulated protein expression of Bip, ATF-4, and CHOP in IPF. Loading control: β-actin. MW = molecular weight.

Figure 4.

Up-regulation of activating transcription factor (ATF)-6 in alveolar epithelial type II cells (AECIIs) of idiopathic pulmonary fibrosis (IPF) lungs. Representative immunohistochemistry for pro–surfactant protein (SP)-C in IPF (A, C, E, G) and donor (I, K) lung tissues (red stain indicates AECIIs) and for ATF-6 in serial sections of IPF (B, D, F, H) and donor (J, L) lung tissues. (B, D, F, H) In IPF, localization of ATF-6 was exclusively observed in AECIIs surrounded by fibrotic tissue (B and D, violet stain), in AECIIs of thickened alveolar septae (B and F, arrows), and in AECIIs of histologically normal appearing areas of IPF lung parenchyma (B and H, arrows). AECIIs near dense zones of fibrosis revealed a very strong staining for ATF-6 (B and D). (J, L) No notable staining for ATF6 was observed in any cell of the donor tissues. Original magnification of photomicrographs A and B: ×100 (bar = 200 μm); original magnification of photomicrographs C–H, K, L: ×400 (bar = 50 μm); original magnification of photomicrographs I and J: ×200 (bar = 100 μm).

Figure 5.

Colocalization of pro–surfactant protein (SP)-C, activating transcription factor (ATF)-6, and activated caspase 3 in alveolar epithelial type-II cells (AECIIs) in areas of dense fibrosis in idiopathic pulmonary fibrosis (IPF). Representative immunohistochemistry for pro–SP-C (A–C), ATF-6 (D–F), and cleaved caspase 3 (G–I) in serial sections of IPF lung tissues. AECIIs covering dense areas of fibrosis (A, B) and fibroblast foci (C) showed robust staining for ATF-6 (D–F, arrows) and caspase-3 activation (G–I, arrows). Some pro–SP-C, ATF-6, and caspase-3–positive cells were also detected in the airspaces, presumably representing detached apoptotic AECIIs, prone to the fate of phagocytosis by alveolar macrophages (asterisks in C, F, and J). Original magnification of photomicrographs A, B, D, E, G, and H: ×400 (bar = 50 μm); original magnification of photomicrographs C, F, and I: ×200 (bar = 100 μm).

Figure 6.

Induction of C/EBP-homologous protein (CHOP) in alveolar epithelial type II cells (AECIIs) of idiopathic pulmonary fibrosis (IPF) lungs. Representative immunohistochemistry for pro–surfactant protein (SP)-C in IPF (A–D, I, J) and donor (M, N) lung tissues (red stain, arrows) and for CHOP in serial sections of IPF (E–H, K, L) and donor (O, P) lung tissues (pale pink stain, arrows). In IPF, CHOP stained the nucleus and cytoplasm of hyperplastic AECIIs covering zones of overt fibrosis (E–H) and fibroblastic foci (K, L). Positive staining for CHOP was not seen in any cell and in any of the donor lung tissues (O, P). Original magnification of photomicrographs A, C, E, G, I, K, M, and O: ×200 (bar = 100 μm); original magnification of photomicrographs B, D, F, H, J, L, N, and P: ×400 (bar = 50 μm).

Figure 7.

Induction of C/EBP-homologous protein (CHOP) in alveolar epithelial type II cells (AECIIs) of histologically normal appearing areas of idiopathic pulmonary fibrosis (IPF) lung parenchyma. Representative immunohistochemistry for pro–surfactant protein (SP)-C (A–C, G, I) and for CHOP (D–F, H, J) in serial sections of IPF lung tissue. In AECIIs of histologically normal appearing areas of IPF lung parenchyma, CHOP induction was observed to a lesser extent (D–F; arrows indicate CHOP-positive AECIIs) than in AECIIs lining airspaces near dense fibrotic zones (H, J). Photomicrographs A–C, G, I, D–F, H, and J are from the same IPF lung tissue section. Original magnification of photomicrographs A and D: ×100 (bar = 200 μm); original magnification of photomicrographs B, E, G, and H: ×200 (bar = 100 μm); original magnification of photomicrographs C, F, I, and J: ×400 (bar = 50 μm).

Figure 8.

“Hyperplastic” alveolar epithelial type II cells (AECIIs) show severe endoplasmic reticulum stress and consecutive apoptosis. Colocalization of pro–surfactant protein (SP)-C, activated caspase 3, C/EBP-homologous protein (CHOP), activating transcription factor (ATF)-6, and ATF-4 in AECIIs covering fibroblastic foci. Representative immunohistochemistry for pro–SP-C (A, D), cleaved caspase 3 (B), CHOP (C), ATF-6 (E), and ATF-4 (F) in serial sections of idiopathic pulmonary fibrosis lung tissue. Original magnification of photomicrographs A–F: ×400 (bar = 50 μm).