Original Contributions
Localization of Extracellular Superoxide Dismutase in Rat Lung: Neutrophils and Macrophages as Carriers of the Enzyme

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Abstract

Immunohistochemistry (IHC) and in situ hybridization (ISH) was used to localize extracellular superoxide dismutase (EC-SOD) and its mRNA in rat lung before and after a lipopolysaccharide (LPS)- and hyperoxia-induced inflammation. In control rats, EC-SOD mRNA was synthesized in macrophages and in cells of the arterial vessel walls and the alveolar septa. The EC-SOD protein was mainly localized in plasma and on the apical side of the epithelial cells located near bronchus-associated lymphoid tissue (BALT). ISH did not reveal major changes in the distribution of EC-SOD mRNA upon induction of inflammation. In contrast, IHC demonstrated a progressive staining of the epithelium of the larger bronchi for the protein. Neutrophils and macrophages invading the lung showed an intensive staining for the EC-SOD protein concomitantly with a decrease of the enzyme in the plasma. Twenty-four hours after LPS stimulation only a spotty positivity remained on neutrophils in and between the alveolar spaces. In the bronchoalveolar lavage fluid (BALF), only macrophages showed a strong positivity for EC-SOD mRNA while the protein was detected in macrophages and neutrophils. Exposure to hyperoxia did not affect the distribution of EC-SOD mRNA and protein. The presented data demonstrated that in lung tissue the EC-SOD enzyme may have a protective function for activated macrophages, neutrophils, and lympoid tissue-associated epithelial cells.

Introduction

During a normal lifespan, the lung is exposed to high amounts of oxygen. Superoxide radicals are generated by the cellular oxidative metabolism, by inhalation of toxic substances and by hyperoxic exposure.[1]The lung contains several antioxidant enzymes to protect cells against free radical damage including superoxide dismutase (SOD, EC. 1.15.1.1), catalase (EC.1.11.1.6), and glutathione peroxidase (EC. 1.11.1.9). The SODs account for the detoxification of superoxide anion radicals present as pollutants in the inhaled air or produced by inflammatory cells during phagocytosis.

Several forms of SOD have been described. Intracellular MnSOD and Cu,ZnSOD isoenzymes are located in the mitochondrial matrix and in the cytosol, respectively. Their function, properties, and expression have been studied extensively.[2]Extracellular SOD (EC-SOD) is present in all extracellular fluids[3]and has been isolated from human lung[4]and from rat C6 glioma.[5]The human EC-SOD is a tetramer and exists in three subtypes, denominated A, B, and C, with a low, moderate, and high affinity for heparin, respectively.[6]The rat EC-SOD is a dimer that only exists as A and B subtypes.[5]The heparin-binding domain is located in the C-terminal region,7, 8and is conserved between human and rat.[5]The affinity of EC-SOD for heparan sulfate and proteoglycans determines its tissue distribution and explains the differences in localization observed between humans and rats.9, 10In general, EC-SOD is present in high amounts in plasma, kidney, and lung.[10]However, due to the lack of a high affinity EC-SOD C subtype in the rat, a relatively higher concentration of EC-SOD is present in the tissue interstitium, and no binding has been observed to heparan sulfate of endothelial cells.11, 12In the rat lung the relative amount of EC-SOD to the total SOD content is approximately one-tenth of that in humans, while the plasma concentration is substantially higher in rats than in humans.[10]

In the human lung EC-SOD has a specific distribution in the connective tissue matrix around larger vessels and airways.[9]Based on these findings, a function of EC-SOD in the protection of collagen matrix elements against oxidative stress has been suggested. The function of EC-SOD in humans may also be related to modulation of the vascular tone by prevention of NO conversion to peroxynitrite.13, 14This proposition is based on the high level of EC-SOD on the surface of smooth muscle cells and in the extracellular matrix around these cells in human blood vessels.

In vitro studies have shown that the expression of EC-SOD is profoundly influenced by some inflammatory cytokines.[15]In this communication we examined the effect of neutrophil-mediated lung inflammation and hyperoxic exposure on the “in vivo” expression and synthesis of EC-SOD in rat lung tissue. The presented data identify macrophages and neutrophils as carriers of EC-SOD after a lipopolysaccharide (LPS)-induced inflammation, pointing to a function of EC-SOD in the protection of the latter cells against superoxide radical damage.

Section snippets

Animal Protocols, Tissue, and Cell Slide Preparations

Male Wistar rats (200–300 g) were obtained from Iffa Credo (Brussels, Belgium). Intratracheal injections were performed under anaesthesia with pentobarbital (30 mg/kg IP). After dissection of the soft tissue overlaying the trachea, 1 mg LPS (E. coli 026:B6) dissolved in 500 μl saline, was administered via a 27-gauge needle. Control rats received 500 μl saline.

Three and 24 h after LPS injection rats were killed and the lungs removed. The left lung was used for immunohistochemistry (IHC) and in

Localization of EC-SOD in Rat Lung Tissue

To identify the sites of EC-SOD mRNA synthesis and the function of the enzyme in the rat lung, we investigated tissue sections by ISH and IHC before and after a LPS-induced inflammation.

The specificity of the probes used in the ISH experiments was demonstrated by Northern blotting of total rat lung RNA (Fig. 1a) and by ISH on EC-SOD producing C6 cells (Fig. 1b and c). The antisense probe detected a 1.4 kb mRNA on Northern blots and intensively stained the EC-SOD producing cells, while no

Discussion

Studies of human lung have immunolocalized EC-SOD in the connective tissue near epithelial and smooth muscle fibers around vessels and airways.9, 20In the vasculature, the EC-SOD enzyme is present in high concentrations in both pulmonary and systemic arteries and is located underneath the endothelium around smooth muscle cells and throughout the adventia.[13]The observation that rat lung tissue contains less EC-SOD but that its plasma contains a much higher concentration of enzyme than human

Acknowledgements

The authors would like to thank R. Van Den Bossche, H. Fret, and A. Van Hoydonck for their technical assistence. We acknowledge Prof. Dr. G. Slegers of the Pharmaceutical Institute of the University of Ghent for his assistance in the hyperoxic experiments. This work was supported by a grant from the Fund for Medical Scientific Research (Levenslijn 7.0016.94) of the Belgian National Fund for Scientific Research. B.L. and E.V.M. equally contributed to this work.

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