Extracellular superoxide dismutase has a highly specific localization in idiopathic pulmonary fibrosis/usual interstitial pneumonia

Abstract

Aims: Recent studies suggest the importance of oxidant stress in the progression of pulmonary fibrosis. The aim of this study was to investigate extracellular superoxide dismutase (ECSOD), the major antioxidant enzyme of the extracellular matrix of human lung, in biopsy-proven idiopathic pulmonary fibrosis (IPF) related to usual interstitial pneumonia (UIP).

Methods and results: Fibrotic areas and fibroblastic foci in UIP lungs were notable for absence of ECSOD by immunohistochemistry. Western blotting showed significantly lowered immunoreactivity of ECSOD in fibrotic compared with non-fibrotic areas of the diseased lung. The only cell type that showed intense ECSOD positivity in UIP was the interstitial mast cell. In order to investigate the mechanism for ECSOD depletion in fibrotic areas, alveolar epithelial cells were exposed to tumour necrosis factor-alpha and transforming growth factor (TGF)-beta1; TGF-beta suggested a trend towards decreased synthesis. Patients with UIP were also assessed to determine whether this disease is associated with a naturally occurring mutation in ECSOD (Arg213Gly) which leads to a loss of tissue binding of ECSOD. No significant differences could be found in the allele or genotype frequencies of this polymorphism between 63 UIP patients and 61 control subjects.

Conclusion: Overall, consistent with several other antioxidant enzymes, ECSOD is very low in fibrotic areas of UIP, which may further increase the oxidant burden in this disease.

Figure 1

Extracellular superoxide dismutase (ECSOD) immunoreactivity in usual interstitial pneumonia and double labelling for mast cells and alveolar macrophages. A, Variable expression of ECSOD in type 2 pneumocytes, alveolar macrophages and a few interstitial cells (arrows). B, Strong expression of ECSOD in granular interstitial cells evidently representing mast cells. C, Alveolar macrophages showing staining for both CD68 (indicated by black) and ECSOD (same cells also showing red). Notice that type 2 pneumocytes and a few interstitial cells at the bottom are positive for ECSOD (red). Notice also that the fibroblast foci (asterisks) are negative for ECSOD. D, Mast cells showing staining for both ECSOD (red ) and CD117 (black) (arrows).

Figure 2

Western Blotting of normal alveolar parenchyma and fibrotic lung parenchyma from usual interstitial pneumonia (UIP) specimens. Homogenates of normal (N) and fibrotic (F) regions from four UIP specimens (nos 1–4) were subjected to reducing SDS–PAGE and Western blotting with an antibody specific for extracellular superoxide dismutase (ECSOD). Both the cut and uncut forms of ECSOD are shown. Densitometry was performed and results expressed as a percentage of normal (normal = 100%). *P = 0.021, Student’s paired t-test, two-tailed.

Figure 3

Polymerase chain reaction for the studies of extracellular superoxide dismutase polymorphism in the blood samples of controls and usual interstitial pneumonia patients. Lane 1 contains a size marker with bands of 25, 50, 75 and 100 bp. Lane 2 is negative control. Lane 3 represents a heterozygous for the minor Gly213 allele with Mwo1-digested products of 28, 35 and 63 bp. Lane 4 contains a homozygous sample for the major Arg213 allele with digested products of 28 and 35 bp.

Figure 4

Extracellular superoxide dismutase (ECSOD) immunoreactivity in alveolar epithelial cells exposed to transforming growth factor (TGF)- β (A) and tumour necrosis factor (TNF)- α (B) for 24–76 h; the experiments were carried out in duplicate. The expression was standardized against β-actin as described. A, □, Control; ▪, TGF. B, □, Control; ▪, TNF.