Elsevier

Journal of Hepatology

Volume 30, Issue 1, January 1999, Pages 61-69
Journal of Hepatology

Nitric oxide mediates the lipopolysaccharide dependent upregulation of the heme oxygenase-1 gene expression in cultured rat Kupffer cells

https://doi.org/10.1016/S0168-8278(99)80008-7Get rights and content

Abstract

Background/ Aims: Heme oxygenase catalyzes the rate-limiting enzymatic step of heme degradation. The inducible isoform of heme oxygenase, heme oxygenase-1, is expressed at a low level in most tissues and is upregulated by its substrate heme and various stress stimuli. Kupffer cells which represent the largest population of the body's tissue macrophages serve physiological functions in the defense against various pathogens such as lipopolysaccharide. The goal of the present study was to investigate the heme oxygenase-1 gene expression in Kupffer cells of rat liver and in isolated Kupffer cell cultures during treatment with lipopolysaccharide.

Methods: Cryostat sections of normal rat liver were investigated by immunofluorescence double-staining using specific antibodies for rat heme oxygenase-1 and ED2. Isolation and cell culture of Kupffer cells and primary hepatocytes from rat liver, as well as Northern and Western blot analysis, were performed with standard protocols.

Results: Heme oxygenase-1 protein was highly expressed in large sinusoidal cells of normal rat liver, which were identified as Kupffer cells by staining with the macrophage surface marker ED2. By contrast, no expression of heme oxygenase-1 was detected in liver parenchymal cells. High expression of heme oxygenase-1 was also found in isolated Kupffer cells in culture by immunocytochemical staining as well as by Western and Northern blot analysis. After treatment of Kupffer cells cultures with lipopolysaccharide, heme oxygenase-1 was upregulated on the protein and mRNA level in a time- and dose-dependent manner. This increase in heme oxygenase-1 expression by lipopolysaccharide was prevented by the nitric oxide inhibitor NG-monomethyl-L-arginine which was reversed by an excess of L-arginine. Various nitric oxide donors up-regulated heme oxygenase-1 mRNA expression in Kupffer cells.

Conclusions: The lipopolysaccharide-dependent upregulation of the heme oxygenase-1 gene which is highly expressed in Kupffer cells is mediated by a nitric oxide-dependent mechanism.

Section snippets

Animals

Male Wistar rats (2 months old, body weight 170 to 200 g) were used throughout the study.

Materials

Media M199 and RPMI 1640 were obtained from Gibco BRL (Karlsruhe, Germany), while radioisotopes, nitrocellulose filters and the chemiluminescent detection system for Western blot were from Amersham-Buchler (Braunschweig, Germany). The nucleotide removal kit was from Quiagen (Düsseldorf, Germany), the multiprime labeling kit and restriction endonucleases were from New England Biolabs (Cambridge, MA, USA),

Expression of HO-1 in KC as detected by immunofluorescence double-staining of rat liver sections

It has been shown previously that hepatic parenchymal and sinusoidal cells exhibit differential levels of HO enzyme activity (17). To investigate HO-1 protein expression in rat liver, immunofluorescence studies were performed with cryostat sections of normal rat liver using a specific rat HO-1 antibody. HO-1 was detected in large sinusoidal cells, but not in parenchymal liver cells (Fig. 1A, C). These cells were identified as KC by immunofluorescence double-staining using a

Discussion

The major finding of the present study is that HO-1, which is considered the inducible isoform of the first and rate-limiting enzyme of heme degradation, is constitutively expressed in KC, but not in parenchymal cells of rat liver. The HO-1 gene is upregulated in cultures of isolated KC by treatment with LPS via an NO-dependent mechanism.

KC represent the largest population of tissue macrophages that reside in the sinusoidal space of the liver. KC normally remove particulate and other foreign

Acknowledgements

Supported by Deutsche Forschungsgemeinschaft grants Im 20/2–1 and SFB 402. We wish to thank Dr S. Shibahara, Sendai, Japan, for providing the cDNA of rat HO-1. We also thank Mirjam Borcholt for excellent technical assistance. Part of this work was presented in abstract form at the Annual Meeting of the German Association for the Study of the Liver in Halle, Germany (Z Gastroenterol 1998; 36; 65).

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