HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 16/11/1450 01-0948fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by GLOSLI, H. Articles by PRYDZ, H. Search for Related Content PubMed PubMed Citation Articles by GLOSLI, H. Articles by PRYDZ, H.

Human TNF- in transgenic mice induces differential changes in redox status and glutathione-regulating enzymes¹

HEIDI GLOSLI, KARL JOHAN TRONSTAD^*, HEGE WERGEDAL^*, FREDRIK MÜLLER, ASBJØRN SVARDAL, PÅL AUKRUST^,, ROLF KRISTIAN BERGE^* and HANS PRYDZ²

Biotechnology Centre of Oslo, University of Oslo, PB 1125 Blindern, 0317 Oslo, Norway;
^* Department of Clinical Biochemistry, University of Bergen, Haukeland Hospital, Bergen, Norway;
Research Institute for Internal Medicine, Rikshospitalet, Oslo, Norway;
Section of Clinical Immunology and Infectious Diseases, Medical Department, Rikshospitalet, Oslo, Norway; and
Department of Pharmacology and Toxicology, University of Bergen, Bergen, Norway

²Correspondence: Biotechnology Centre of Oslo, University of Oslo, PB1125, Blindern, 0317 Oslo, Norway. E-mail: hans.prydz{at}biotek.uio.no

SPECIFIC AIM

Various effects of TNF- are mediated by the induction of a cellular state consistent with oxidative stress. Glutathione is the major redox buffer of several eukaryotic cell types and important in the defense against oxidative stress. We hypothesized that persistent TNF- secretion could induce oxidative stress through modulation of glutathione (GSH) metabolism. This hypothesis was examined in a transgenic mouse model with low and persistent expression of human TNF- in the T cell compartment.

PRINCIPAL FINDINGS

1. Transgenic mice expressing hTNF- in T cells
The transgenic mouse line used was established in C57Bl/6 by Probert et al. using standard methods. A CD2-hTNF--ß-globin construct was prepared in the Bluescript vector (Stratagene, La Jolla, CA). A 5 kb BamHI-XbaI fragment from the 3' flanking region of the human CD2 locus, containing the CD2 locus control region, was linked 5' to a 2.8 kb EcoRI fragment containing the promoter and the entire coding region of the hTNF- gene. This was linked 5' to a 0.77 kb EcoRI-SalI fragment of the human ß-globin gene containing its polyadenylation signal in its 3'-untranslated sequence. A BamHI-SalI fragment was used for microinjection. The CD2 locus control region directs the low and persistent expression of hTNF- only to the T cells in the transgenic mice. Tissues were frozen in liquid nitrogen immediately after removal and stored at -80°C until analysis. Isolation of RNA and DNA and Southern and Northern blot analysis were carried out by standard procedures. The results confirmed that there was expression of hTNF- only in the lymphoid compartment. The experimental animals in this study were homozygous for the transgene. Titration showed they harbored two copies of the transgene in their genome, one on each allele. The mice were 45 days of age and of either sex. The controls were preferably sex-matched littermates.

2. Tissue-specific changes in GSH redox status and GSH-regulating enzymes
Glutathione is the major redox buffer of several eukaryotic cell types and an important factor in the cellular defense against oxidative stress. This is due to its own antioxidant capacity and because this thiol is involved in the recycling of other antioxidants. Glutathione homeostasis is regulated by several enzymes such as glutathione reductase (GR), glutathione peroxidase (GPX), and glutathione transferase (GST); it has been suggested that the capacity of the GSH redox cycle, rather than the intracellular levels of reduced GSH, determines the resistance to oxidative stress at least in some cell types. Catalase activity was measured with H₂O₂ as substrate. GPX was measured with t-butyl hydroperoxide as substrate.

We observed marked tissue-specific changes in GSH redox status and GSH-regulating enzymes, with the most pronounced changes in liver (Table 1 ). Reduced levels of catalase and glutathione peroxidase (Table 1) were observed in liver, reflecting impaired redox buffering capacity.

View this table: [in this window] [in a new window]   Table 1. Antioxidant enzymes in hTNF transgenic mice compared with control micea

3. Moderate changes in GSH metabolism in lung and kidney
The metabolism of GSH was also affected in transgenic lungs and kidneys, but these changes were more moderate than in the liver. Despite evidence of oxidative stress, these tissues seemed to be better protected against oxidative stress due to up-regulation of GSH-regulating enzymes.

To gain insight into the regulatory aspects of GSH synthesis and degradation, we also looked at the mRNA levels of GGCS and GGTP. Both levels were reduced by 30% in kidney (P=0.07 for GGCS and P=0.021 for GGTP). The mRNA levels for SOD1 (cytosolic form) and SOD2 (mitochondrial form) showed only minor changes except for SOD2 in liver and lung (increase of 45%, only significant in liver, P<0.02).

4. Tissue-specific decrease of GSH
For quantification of total GSH levels, cells were extracted with 0.3 mL ice-cold 5% sulfosalicylic acid (Merck, Darmstadt, Germany) containing 50 mmol/L dithioerythritol (Sigma). Total GSH (reduced glutathione+glutathione disulfide+soluble glutathione mixed disulfide) and reduced GSH were determined in the acid extract by a chromatographic procedure. The fraction of oxidized GSH (GSSG) (glutathione disulfide+soluble glutathione mixed disulfide) was calculated by subtracting the amount of reduced from the total amount of GSH. Liver, lung, and kidney from transgenic mice had decreased levels of total GSH (Fig. 1 ), whereas the level of total GSH was unchanged in spleen. Splenic CD4⁺ and CD8⁺ T cells had an altered composition of GSH with a marked increase in GSSG and a significant decrease in reduced GSH (Table 2 ). The ratio between reduced GSH and total GSH, a good indicator of oxidative stress, was significantly reduced in spleen.

View larger version (17K): [in this window] [in a new window]   Figure 1. Total glutathione levels (nmol/g of tissue) in liver, lung, and kidney of transgenic (gray) or control (black) mice. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. control mice.

CONCLUSIONS AND SIGNIFICANCE

Using transgenic mice, we have observed a connection between a low constitutive level of hTNF- expression in T cells and differences in GSH metabolism as assessed by GSH redox status and the expression of GSH-regulating enzymes. These changes were highly tissue specific, with the most striking alterations in the liver. The liver in the transgenic mice was characterized by markedly decreased catalase and GPX activity accompanied by low levels of total and reduced GSH. Such a combined depletion of substrate (i.e., reduced GSH) and enzyme (i.e., GPX) could obviously impair the redox buffering capacity of this organ. GSH depletion may enhance the inflammatory response to various stimuli (e.g., TNF-) within the liver, leading to further enhancement of oxidative stress, potentially constituting a vicious circle causing augmented tissue injury. More modest changes were observed in kidneys from the transgenic animals with normal or even up-regulated levels of catalase and GSH-regulating enzymes. Our findings support a relationship between persistent low-grade TNF- expression and enhanced oxidative stress involving marked organ-specific alterations in GSH metabolism.

The activity pattern of the enzymes involved in GSH metabolism in lung and kidney was somewhat different from that in liver, implying tissue specificity in the regulation of these enzymes. In the lung from transgenic mice, the combined up-regulation of GPX and GR provides a more efficient defense against oxidative stress than in the liver. In kidney, only slight differences in the pattern of the transgenics compared to wild-type mice were observed. Nonetheless, the ratio GPX/GR, which is considered more reliable than the separate levels of enzymatic activity of GPX and GR in evaluating oxidative stress, was down-regulated in liver, lung, and kidney, as in the presence of oxidative stress. Accordingly, a pattern of oxidative stress appears to be an important feature in the development of the phenotype observed in hTNF- transgenic mice not only in liver, but also in other organ systems. However, lung and kidney both seem to be better protected against oxidative stress and its deleterious effects than the liver.

Synthesis of GSH seems, in this mouse model, to be most pronounced in lung and kidney. -Glutamylcystene synthethase is unchanged in lung, down-regulated in kidney, and not detectable in liver. Degradation of GSH is only detectable in kidney where -glutamyl transpeptidase is significantly reduced. In this line of transgenic mice, the kidney seems to be the most important organ in the maintenance of GSH levels.

Oxidative stress induced by persistent low-grade exposure to TNF- appears to involve marked organ-specific alterations in GSH redox status and GSH-regulating enzymes, with the most pronounced changes in the liver. These mice constitute a useful model for immunodeficiency syndromes and chronic inflammatory diseases involving pathogenic interaction between TNF- and oxidative stress. A summary of our findings is given in Tables 1 and 2 .

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0948fje; to cite this article, use FASEB J. (July 18, 2002) 10.1096/fj.01-0948fje

This article has been cited by other articles:

				K. P. Tan, M. Yang, and S. Ito Activation of Nuclear Factor (Erythroid-2 Like) Factor 2 by Toxic Bile Acids Provokes Adaptive Defense Responses to Enhance Cell Survival at the Emergence of Oxidative Stress Mol. Pharmacol., November 1, 2007; 72(5): 1380 - 1390. [Abstract] [Full Text] [PDF]

				K. Zhang, L. Shan, M. S. Rahman, H. Unruh, A. J. Halayko, and A. S. Gounni Constitutive and inducible thymic stromal lymphopoietin expression in human airway smooth muscle cells: role in chronic obstructive pulmonary disease Am J Physiol Lung Cell Mol Physiol, August 1, 2007; 293(2): L375 - L382. [Abstract] [Full Text] [PDF]

				I. Talior, M. Yarkoni, N. Bashan, and H. Eldar-Finkelman Increased glucose uptake promotes oxidative stress and PKC-{delta} activation in adipocytes of obese, insulin-resistant mice Am J Physiol Endocrinol Metab, August 1, 2003; 285(2): E295 - E302. [Abstract] [Full Text] [PDF]

				D. Hurlimann, A. Forster, G. Noll, F. Enseleit, R. Chenevard, O. Distler, M. Bechir, L. E. Spieker, M. Neidhart, B. A. Michel, et al. Anti-Tumor Necrosis Factor-{alpha} Treatment Improves Endothelial Function in Patients With Rheumatoid Arthritis Circulation, October 22, 2002; 106(17): 2184 - 2187. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 16/11/1450 01-0948fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by GLOSLI, H. Articles by PRYDZ, H. Search for Related Content PubMed PubMed Citation Articles by GLOSLI, H. Articles by PRYDZ, H.