[39 Chemokine Expression in Transgenic Mice Overproducing Human Glutathione Peroxidases

By NOBUYA ISHIBASHI and OLEG MlROCHNITCHENKO Introduction

Interest in the participation of reactive oxygen species (ROS) in diseases has grown rapidly and has been an area of active research. An issue that remains unresolved is the exact role of ROS in injury process. Although their harmful effects on lipids, proteins, and DNA are more or less understood, the ability of ROS and antioxidants to affect cellular signaling and in this way control gene expression needs additional investigation. As second messengers, ROS are thought to be involved in a number of inflammatory and/or immune-mediated disorders. Although many conditions can elicit inflammation, there are common pathways that contribute to the recruitment of inflammatory cells. A significant amount of data accumulated so far suggests that expression of chemokines, a large family of structurally related chemotactic cytokines produced by a variety of cells, is regulated by oxidative stress. Chemotactic factors not only induce leukocyte movement but also enhance endothelial and leukocyte adhesiveness and endothelial and extracellular matrix permeability. Activation of leukocyte receptors triggers these cells to secrete ROS and other mediators, providing a mechanism to amplify the inflammatory response. The chemokine system is characterized by apparent redundancies of ligands and receptors, which complicates investigation of specific chemokine-regulated events. Four chemokine subfamilies are now described (CXC, CC, CX3C, and C).1 In general, CXC chemokines show chemoatractant activity for neutrophils, whereas CC chemokines are chemotactic for monocytes and lymphocytes, although there is some overlap. Chemokines interact with G protein-coupled seven-transmembrane domain receptors. Five human CXC receptors and nine human CC chemokine receptors have been reported.2

Although studies revealed that ROS are critical regulators of chemokine activation in vitro, few of them have addressed the potential role of these mediators in vivo. One of the ways to approach this issue is to use animals with a genetically changed level of specific antioxidant enzyme. This way it is possible to study chemokine behavior at the level of the whole organism in its natural environment. Another important aspect of this approach is that the level of oxidants/antioxidants changes under certain disease conditions, and the ability of these changes to influence the development of inflammation-related pathological conditions might

2 L. M. Gale and S. R. McColl, Bioassays 21, 17 (1999).

be investigated as well. This chapter focuses on methods for studying chemokine expression in transgenic mice overexpressing human glutathione peroxidases. As a model of pathological conditions involving oxidative stress, kidney ischemia-reperfusion is used. Both animal studies and clinical data strongly implement ROS in ischemia-reperfusion injury, whereas the inflammatory response is crucial for amplification of kidney malfunction.

Oxidative Stress and Chemokine Activation

The best studied among chemokines in relation to oxidative stress-mediated activation is interleukin 8 (IL-8). In lipopolysaccharide (LPS)-stimulated human whole blood and cellular models, scavengers of oxygen and nitrogen intermediates inhibited IL-8 production.3 Addition of exogenous ROS increased IL-8 expression in these cells. H2O2 induced IL-8 expression in epithelial cells, but not in endothelial cells.4 The effect of reduced oxygen pressure in human glioblastoma and of oxidant tone in epithelium infected with respiratory syncytial virus on IL-8 activation was also mediated by ROS.5'6 Intracellular glutathione redox status was an important modulator of the monocyte chemoattractant protein type 1 (MCP-1) level in a model of rat pulmonary granulomatous vasculitis.7 Induction of MCP-1 protein in mesangial cells by tumor necrosis factor a (TNF-a) and IL-l/i was sensitive to antioxidants.8 Oxidative stress is also known to regulate macrophage inflammatory protein 1 a (MIP-la) mRNA expression in alveolar macrophages.9 Analysis of the pathways of activation of chemokines suggested at least two major mechanisms: one at the level of transcription activation and the other involving posttranscriptional stabilization of mRNA. 5'-Regulatory regions of chemokine genes contain several transcription elements, mostly combinations of NF-kB, AP-1, NF-IL-6, and C/EBP DNA-binding sites. These transcription factors are known to be sensitive to changes in the intracellular redox state.10 Differential sensitivity of these transcriptional factors to oxidative stress establishes distinct patterns of cell-type specific and stimulus-specific gene expression. Ox/redox regulation of transcriptional factors occurs at the level of signal transduction pathways (including the activity of protein kinases and phosphatases) or redox status

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of critical cysteine residues, influencing the interaction between subunits or with other factors or DNA.1112 Additional protein factors regulating the redox state of NF-kB and AP-1 were also described (thioredoxin and Ref-1).13

Transgenic Mice Overexpressing Antioxidant Enzymes as Model System to Study Role of Reactive Oxygen Species under Pathological Conditions

Free radical reactions and consequent lipid peroxidation are important regulators of cellular functions. Achieving the proper balance between the generation of ROS and the ability of different type of cells to detoxify or respond to ROS might be critical for a successful outcome under stress conditions. Previous animal studies addressing the role of free radicals and antioxidants have used mainly direct injection or infusion of antioxidants/oxidants. In most cases, this approach leads only to the modulation of the extracellular ox/redox balance, whereas the intracellular level of oxidative stress might be achieved by changing endogenous levels of antioxidant enzymes. When the increasing evidence of the role of ROS, not only as damaging agents, but also as intracellular signaling molecules is taken into account, it is clear that transgenic mice overexpressing antioxidant enzymes, or having these enzymes deleted, represent a new and promising tool for studying the mechanism of injury. For example, transgenic mice overexpressing intracellular and extracellular copper/zinc superoxide dismutase (Cu,ZnSOD), manganese SOD (MnSOD), and intracellular and extracellular glutathione peroxidases (GPs) were developed by us and others.14-16 These animals show an increased resistance to a variety of pathological conditions, such as brain, heart, and kidney ischemia-reperfusion injury, hyperoxia, and doxorubicin (Adriamycin) and paraquat toxicity.17-19 Nevertheless, GP mice showed increased sensitivity to hyperthermia and skin-induced

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17 M. Weisbrot-Lefkowitz, K. Reuhl, B. Perry, P. Chan, M. Inouye, and O. Mirochnitchenko, Mol. Brain Res. 53, 333 (1998).

18 Z. Chen, B. Siu, Y. Ho, R. Vincent, C. C. Chua, R. C. Hamdy, and B. H. Chua, J. Mol. Cell. Cardiol. 30, 2281 (1998).

19 N. Ishibashi, M. Weisbrot-Lefkowitz, K. Reuhl, M. Inouye, and O. Mirochnitchenko, J. Immunol. 163, 5666 (1999).

BamHI BamHI EcoRV

HMGCR hGPxl promoter hGPxP

Fig. 1. The DNA fragments used for generation of transgenic mice overexpressing human GPxl and PGxP. The cross-hatched box represents the coding region for GPxl and GPxP. The promoter region (box with arrow), the first exon, and intron belong to the mouse HMGCR gene.

tumor promotion, whereas Cu,ZnSOD-overexpressing animals possess reduced microbicidal and fungicidal activity.I4'20'21 These results reflect the complex role of ROS generated under stress or disease conditions. In experiments described below, transgenic mice overproducing human extracellular and intracellular glutathione peroxidases (GPxP and GPxl, respectively) are used. GP is a critical antioxidant enzyme for the detoxification of peroxides. Its relatively low substrate specificity and particular kinetics make it an efficient reducer of peroxides, far more so than catalase, which is the other enzyme that detoxifies H2O2.

Construction of Transgenic Mice Overexpressing Human Glutathione Peroxidases

To create animals overexpressing antioxidant enzymes to study the effect of modulation of the level of oxidative stress on the development of pathological processes we use a non-tissue-specific promoter from the mouse hydroxymethyl-glutaryl-coenzyme A reductase gene (HMGCR) in order to avoid tissue-specific expression. It has been demonstrated that the bacterial cat reporter gene under the control of this promoter shows a ubiquitous pattern of expression in transgenic mice.22 Fragments containing cDNA sequences of human GPxl23 and GPxP24 are inserted under the control of the HMGCR promoter (Fig. 1). Other groups reported construction of animals overproducing antioxidant enzymes with its own promoter15 or with a tissue-specific promoter.25

Notl fragments are micronjected into the pronucleus of C57BL/6 x CBA/J hybrid eggs. Newborn babies are analyzed for the presence of transgene and

20 Y.-P. Lu, Y. Lou, P. Yen, H. L. Newmark, O. I. Mirochnitchenko, M. Inouye, and M. T. Huang, Cancer Res. 57, 1468 (1997).

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25 T. D. Oury, Y. Ho, C. A. Pantadosi, and J. D. Crapo, Proc. Natl. Acad. Sci. U.S.A. 89, 9715 (1992).

expression of human glutathione peroxidases. In our experiments, GPxl overexpression was detected in almost all tested tissues of several mouse lines,14 whereas overexpression of GPxP was detected mostly in blood, as expected from its extracellular localization, as well as in kidney, which is known to be the major organ secreting GPxP in mice.26 To obtain nontransgenic and heterozygous transgenic animals for experiments, transgenic founders are bred with (C57BL/6 x CBA/J)F| mice. Transgenic mice should be identified by Southern or slot-blot analysis of tail DNA, using labeled hGPxP and hGPxl probes.27 Nontransgenic littermates are used in experiments as wild-type controls.

Kidney Ischemia-Reperfusion Model

Ischemia-reperfusion injury of the kidney is a common problem with potentially catastrophic ramifications. Reactive oxygen species (ROS) and leukocytes recruited into the tissue have been implicated in the pathogenesis of this process. Insight concerning the mechanisms of leukocyte migration, as well as signal transduction pathways participating in cell activation, indicates that there is an important link between ROS generation and regulation of the inflammatory response. Pathways leading to oxidative stress-induced chemokine activation, including that mediated by NF-kB, are summarized in Fig. 2.

To test a hypothesis that a prooxidant state during kidney ischemia-reperfusion is a critical regulator of chemokine activation, unilateral renal artery occlusion model in mice overexpressing human GPs is used.28 It is a reproducible, well-studied experimental procedure leading to uniform renal cell injury and severe renal impairment. Normal or transgenic males weighing 25-35 g are anesthetized with sodium pentobarbital (25 mg/kg) and xylazine (10 mg/kg) and administered heparin (300 USP units/kg of body weight) subcutaneously before surgery. Unilateral renal ischemia is induced by occluding the left side renal vein and artery with a microaneurysm clamp. Body temperature is maintained at 37° during the whole procedure. After 32 min of ischemia, the left kidney is reperfused by declamping the microaneurysm applicator and right nephrectomy is performed. Sham surgery consists of a surgical procedure that is identical except that the microaneurysm clamp is not applied. Mice are killed at various time points after surgery. Kidneys should be removed immediately after perfusion with cold phosphate-buffered saline (PBS) and rapidly frozen in liquid nitrogen to obtain extracts and perform RNA analysis.

26 N. Avissar, J. C. Whitin, P. Z. Allen, D. D. Wagner, P. Liegey, and H. J. Cohen, J. Biol. Chem. 264, 15850(1989).

27 B. Hogan, R. Beddington, F. Constantini, and E. Lacy, "Manipulating the Mouse Embryo: A Laboratory Manual." Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1994.

28 K. Kelly, W. Williams, R. Colvin, S. Meehan, T. Springer, J. Gutierrez-Ramos, and J. Bonventre, J. Clin. Invest. 97, 1056 (1996).

■PMN-Monocvtes -Platelets cytokines, LTs, PGs

■PMN-Monocvtes -Platelets cytokines, LTs, PGs

Pmn And Ros

FIG. 2. Involvement of ROS in CXC chemokine activation during ischemia-reperfusion. Among the primary sources of ROS are activated leukocytes and endogenous reactions such as xanthine oxidase-mediated conversion of xanthine to hypoxanthine, the mitochondrial electron transport chain, microsomal oxidation, and arachidonic acid metabolism. Cell activation by cytokines, leukotrienes (LTs), prostaglandins (PGs), and so on, also leads to the intracellular generation of ROS. NF-/cB is shown as an example of an oxidative stress-sensitive transcription factor, leading to the induction of chemokine expression. Several proteolytic mechanisms (26S proteasome, caspases, calpains, and lysosomal proteases) able to degrade I-/cB inhibitors in several circumstances are presented. The mechanism functional under ischemia-reperfusion conditions is not known at present.

FIG. 2. Involvement of ROS in CXC chemokine activation during ischemia-reperfusion. Among the primary sources of ROS are activated leukocytes and endogenous reactions such as xanthine oxidase-mediated conversion of xanthine to hypoxanthine, the mitochondrial electron transport chain, microsomal oxidation, and arachidonic acid metabolism. Cell activation by cytokines, leukotrienes (LTs), prostaglandins (PGs), and so on, also leads to the intracellular generation of ROS. NF-/cB is shown as an example of an oxidative stress-sensitive transcription factor, leading to the induction of chemokine expression. Several proteolytic mechanisms (26S proteasome, caspases, calpains, and lysosomal proteases) able to degrade I-/cB inhibitors in several circumstances are presented. The mechanism functional under ischemia-reperfusion conditions is not known at present.

Assessment of Effect of Antioxidant Enzyme Overexpression on Chemokine Activation During Kidney Ischemia-Reperfusion

Several methods may be employed to analyze chemokine expression in kidneys of normal and transgenic mice after ischemia-reperfusion. Level of mRNA, depending on the abundance of the mRNA for a particular chemokine, is characterized by Northern blot analysis, RNase protection assay, or reverse transcription-polymerase chain reaction (RT-PCR). Two of those methods are described below. The use of RT-PCR for assessment of chemokine expression is described in detail elsewhere.29 Primers for detection of several mouse chemokines are commercially available (e.g., from BioSource International, Camarillo, CA). Protein expression

29 T. Standiford, S. L. Kunkel, and R. M. Strieter, Methods Enzymol. 288, 220 (1997).

might be directly assessed by Western blot analysis or enzyme-linked immunosorbent assay (ELISA) in tissue extracts or by immunohistochemistry of the tissue sections, which is less quantitative but will give information regarding cell-specific localization of the protein.

Preparation ofRNA and Northern Blot Analysis

Total RNA is isolated from kidneys, using TRIzol reagent (Life Technologies, Gaithersburg, MD) according to the manufacturer protocol. If necessary, poly (A)+ RNA may be isolated with an oligo(dT) column. The RNA concentration is measured by a spectrophotometer. For Northern blot analysis, RNA samples [20 fig of total RNA or 3 fig of poly (A)+ RNA] are denatured in 1 x morpholinepropanesulfonic acid (MOPS) electrophoresis buffer [0.04 MMOPS (pH 7.0), 0.01 M sodium acetate, 1 ml EDTA], 6.5% (v/v) formaldehyde, and 50% (v/v) formamide for 5 min at 65°. After cooling the samples on ice, a 1/10 volume of loading buffer containing 0.025% (w/v) bromphenol blue and 50% (w/v) glycerol is added and the mixture is loaded on a 1.4% (w/v) agarose gel with 2.2 M formaldehyde. After electrophoresis, RNA should be transferred from the gel to a nylon membrane (GeneScreen; New England Nuclear, Boston, MA). After transfer the membrane is fixed and prehybridized in 50% (v/v) formamide, 5 x SSPE (0.6 M NaCl, 0.04MNaH2P04,5 mMEDTA,pH7.4), 5x Denhardt's solution [0.5% (w/v) polyvinylpyrrolidone, 0.5% (w/v) bovine serum albumin (BSA), 0.5% (w/v) Ficoll 400], 1% (w/v) sodium dodecyl sulfate (SDS), 10% (w/v) dextran sulfate, and carrier DNA (100 /tig/ml) at 42° for at least 1 hr. As a probe DNA fragments labeled with a random primed DNA-labeling kit (Boehringer Mannheim, Indianapolis, IN) are used. Hybridization is performed overnight in prehybridization solution containing labeled probe at 42°. The membrane is washed once with 2x SSPE solution for 15 min at room temperature, twice with 2x SSPE and 2% (w/v) SDS for 30 min at 65°, and once with O.lx SSPE for 15 min at room temperature and autoradiographed. To compare RNA amounts in the bands, the films may be scanned by a densitometer and final values factored relative to the levels of control RNA, for example, /J-actin. As shown in Fig. 3A, the level of KC mRNA assessed by Northern blot analysis was 10 and 4.5 times higher in normal mice compared with those in GPxl and GPxP animals 6 hr after reperfusion, respectively.

RNase Protection Assay

The RNase protection assay is performed with a RiboQuant kit and an mCK-5 multiprobe template set (PharMingen, San Diego, CA) according to the manufacturer protocol. In brief, RNA probes for mouse chemokines [Ltn, RANTES (regulated on activation, normal T cell expressed and secreted), eotaxin, MIP-1/3, MIP-la, MIP-2, interferon-inducible protein 10 (IP-10), MCP-1, and TCA-3 (T-cell activation-3)] as well as for positive controls [L32 and glyceraldehyde-3-phosphate

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