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Regulation of endothelial glutathione by ICAM-1: implications for inflammation

C. G. KEVIL^*, H. PRUITT, T. J. KAVANAGH, J. WILKERSON, F. FARIN, D. MOELLERING, V. M. DARLEY-USMAR^,||, D. C. BULLARD and R. P. PATEL^,||,1

^* Department of Pathology, Louisiana State University, Shreveport, Shreveport, Louisiana;
Department of Pathology, University of Alabama at Birmingham,
Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, Washington, USA; and
Department of Genetics and
^|| Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, Alabama, USA

¹Correspondence: Department of Pathology University of Alabama at Birmingham, 901 19^th Street South, BMR-2, Room 307, Birmingham, AL 35294, USA. E-mail: patel{at}path.uab.edu

SPECIFIC AIMS

Inflammation plays a central role in development of vascular dysfunction associated with atherosclerosis. At the molecular level important determinants of endothelial function during the inflammatory process are redox status of the cell and expression of adhesion molecule-1 (ICAM-1). Glutathione (GSH) is an important intracellular antioxidant that determines sensitivity of redox signaling pathways in the cell. Depletion of GSH increases susceptibility of cells to inflammation-induced apoptosis mediated by oxidized lipids found in atherosclerotic lesions. ICAM-1 is expressed constitutively in the endothelium and is induced during inflammation and so contributes to the progression of vascular dysfunction. In addition to mediating leukocyte adherence ICAM-1 has also been shown to modulate endothelial cell signaling but it is not known whether redox signaling pathways exhibit cross-talk with those controlling GSH levels. In this study a novel signaling pathway is identified involving regulation of endothelial GSH by constitutive ICAM-1.

PRINCIPAL FINDINGS

1. Functional deletion of constitutive ICAM-1 in endothelial cells increases glutathione levels via glutamate cysteine ligase
Aortic endothelial cells from wild-type (WT) and ICAM-1-deficient (ICAM-1–/–) mice (MAEC) were isolated and cultured. Endothelial cells from ICAM-1 deficient mice exhibit a phenotype similar to their wild-type counterparts in culture and show greatly decreased leukocyte adhesion in response to tumor necrosis factor and oxidized lipids. Figure 1 A shows that total GSH (reduced GSH plus oxidized GSH (i.e., GSSG)) is increased almost twofold in ICAM-1–/– cells, relative to WT cells. This was further verified by fluorescence microscopy studies using GSH-reactive probes. The ratio of GSH:GSSG was also increased in ICAM-1–/– cells (Fig. 1B ), suggesting that constitutive ICAM-1 modulates endothelial redox status. Western blotting analysis of 2 subunits (catalytic and modulatory) of glutamate cysteine ligase (GCL), the enzyme that catalyzes the rate-determining step in the GSH biosynthesis pathway, revealed that expression of the catalytic subunit was increased in ICAM-1–/– cells whereas no changes in the modulatory subunit were observed (Fig. 2 B). No differences in GSH efflux rates nor expression of GCLC or GCLM RNA were observed, suggesting that ICAM-1 may be regulating GCLC protein turnover. Inhibiting GCL using buthionine sulfoximine (BSO) decreased GSH concentrations in ICAM-1–/– cells (Fig. 2A ). Inhibition of -glutamyl transpeptidase (-GT) by acivicin in ICAM-1–/– cells had no effect on GSH levels, suggesting that ICAM-1 deficiency does not modulate availability of substrates required for GSH synthesis.

View larger version (15K): [in this window] [in a new window]   Figure 1. Effect of ICAM-1 on GSH metabolism in EC. A) Basal indices of GSH metabolism (i.e., total GSH concentrations, activities of glutathione reductase (GRed), and glutathione peroxidase (GPx)) and Catalase (Cat) activity were measured in WT (clear bars) and ICAM-1–/– (filled bars) MAEC after exposure to MCDB-131 + 0.5%FBS for 16 h. Data shown represent a fold difference in concentrations or activities of ICAM-1–/– cells relative to WT MAEC. *P < 0.0001 (n=17), **P < 0.001, #P < 0.05 (n=3–4). B) GSH:GSSG ratios in WT (clear bar) and ICAM-1 –/– (filled bar) MAEC. *P < 0.01 (n=3).

View larger version (14K): [in this window] [in a new window]   Figure 2. Role of -GCL and -GT in modulating GSH concentrations in ICAM-1–/– MAEC. A) ICAM–/–1-deficient MAEC were exposed to Acivicin (5µM) or BSO (200µM) for 14 h in MCDB-131 + 0.5%FBS and total GSH determined. B) Relative amounts of GCLC and GCLM in WT and ICAM-1–/– MAEC as assessed by Western blot (representative blots shown). Bar graph represents quantitation performed by densitometric analysis. *P < 0.01.

2. Effects of functional deletion of constitutive ICAM-1 on GSH-dependent enzymes and role of reactive oxygen and nitrogen species
The specific activity of glutathione peroxidase (GPx) and glutathione reductase (GRed), two enzymes that utilize and regenerate GSH from GSSG, respectively, to metabolize and safely detoxify oxidants/xenobiotics were also measured. GRed activity increased 30%, whereas that of GPx decreased 30% (Fig. 1A ). A decrease, also 30%, in expression of GPx protein was also detected. No changes in the activity of catalase, a GSH-independent enzyme important in metabolism of peroxides, were observed (Fig. 1A ), suggesting that ICAM-1 is a specific modulator of GSH metabolism. Recent insights suggest that reactive oxygen and nitrogen species (ROS and RNS), together with specific lipid oxidation products control GSH levels in endothelial cells. In ICAM-1–/– cells, neither inhibition of NO production (using nitric oxide synthase inhibitor, L-NAME), scavenging of ROS and RNS using manganese porphyrinic compound, MnTBAP, nor inhibition of lipid peroxidation using butylated hydroxytoluene (BHT) affected increased levels of GSH.

3. Glutathione inhibits expression of constitutive ICAM-1
To investigate the effects of GSH on constitutive ICAM-1 expression, GSH concentrations were increased twofold in WT cells by addition of glutathione ethyl ester (GEE). This resulted in a decrease in expression of constitutive ICAM-1. This suggests that ICAM-1 and GSH are regulated through common and reciprocal pathways.

4. Inducible ICAM-1 and GSH
WT cells were treated with proinflammatory cytokine TNF- to stimulate expression of the inducible isoform of ICAM-1. Under these conditions, no change in GSH was detected, highlighting a functional difference between constitutive and inducible ICAM-1 in the context of regulating cellular redox status. To test effects of GSH on induction of ICAM-1, WT cells were pretreated with GEE to increase cellular GSH and then treated with TNF-. Increasing basal levels of GSH (by twofold) inhibited induction of ICAM-1 by 60%. Supporting an important functional significance for the relationship between cellular redox and ICAM-1 it was found that increasing GSH levels also prevented TNF--dependent monocyte adhesion by 60%.

CONCLUSIONS AND SIGNIFICANCE

This study identifies a novel pathway linking endothelial redox status and inflammation in which constitutive ICAM-1 regulates endothelial GSH. ICAM-1 is expressed both constitutively on vascular endothelial cells and is readily induced in response to proinflammatory stimuli. Induced ICAM-1 modulates adhesion of leukocytes and has been linked to vascular pathologies including atherosclerosis. Little is known about the biological function(s) of constitutive ICAM-1. In this study we reveal a cellular cross-talk between constitutive expression of ICAM-1 and control of cellular redox status through both control of GSH synthesis and GSH:GSSG ratio.

Glutathione is a ubiquitous cellular reductant/antioxidant playing diverse roles from xenobiotic metabolism to preventing tissue injury during oxidative stress and controlling redox cell signaling. In the context of inflammation, the prevailing view is that GSH is antiinflammatory. Consistent with this concept, GSH protects against oxidative stress and its concentrations in the vascular wall are decreased in hypercholesterolemia. However, little is known of how cellular GSH is controlled during inflammation. In this study a mechanism involving ICAM-1 for regulating GSH is identified. Using a model system of endothelial cells deficient in functional ICAM-1, we show an increase in GSH levels via up-regulation of the catalytic subunit of GCL, the rate limiting enzyme in GSH biosynthesis. Signaling pathways that mediate this function of ICAM-1 are not yet known, but do not appear to involve altered reactive oxygen and nitrogen species metabolism. Our data suggest that constitutive ICAM-1 suppresses GSH synthesis under basal conditions and thereby regulates the basal redox state in the endothelium.

These data support the concept that adhesion molecules have functions beyond physical tethering of one cell type to another, and include modulation of cell signaling. Roles have previously been described for induced ICAM-1 in regulating specific signaling pathways involving (for example) MAP kinases, Rho proteins, and the cytoskeleton. To our knowledge, our data demonstrate for the first time a role for constitutively expressed ICAM-1 in modulating endothelial function via redox cell signaling. How ICAM-1 regulates pathways that control GSH metabolism is unknown. The small cytoplasmic domain of ICAM-1 lacks any obvious signaling consensus motifs, although a weak immunoreceptor tyrosine-based inhibitory modif (ITIM) has been proposed. The specific signaling pathways through which ICAM-1 modulates GSH biosynthesis are under investigation.

Increasing GSH decreased constitutive ICAM-1, whereas deleting ICAM-1 increased GSH levels. This suggests that under basal conditions, a dynamic and coordinated relationship between the cellular redox state and ICAM-1 exists. However, inducing ICAM-1 in endothelial cells with TNF- had no effect on GSH levels and revealed a functional difference between constitutive and induced ICAM-1. In contrast, GSH inhibited induction of ICAM-1 and was associated with inhibition of monocyte adhesion. Taken together these data suggest that constitutive ICAM-1 can regulate endothelial responses to inflammation via GSH. These concepts are illustrated in Fig. 3 .

View larger version (21K): [in this window] [in a new window]   Figure 3. Scheme illustrating proposed model of interplay between constitutive ICAM-1, GSH, and subsequent inflammation dependent induction of ICAM-1. Data presented in this study shows that deletion of constitutive ICAM-1 results in an increase in endothelial GSH via up-regulation of the catalytic subunit of GCL (Step 1). In turn this suggests that constitutive ICAM-1 acts as a tonic repressor of GSH synthesis (Step 2), revealing a novel signaling function for this adhesion molecule. Regulation of GSH by constitutive ICAM-1 is reciprocal (i.e., increasing GSH concentrations decrease constitutive ICAM-1 expression, Step 3). This suggests that the ICAM-1-GSH pathway is self-regulating. GSH also inhibits induction of ICAM-1 (and subsequent leukocyte adhesion) stimulated by TNF- (Step 4).

This study raises several interesting questions that may impact understanding of how endothelial responses to inflammation are controlled and the role of constitutive ICAM-1. It is hypothesized that lower levels of constitutive ICAM-1 could inhibit inflammation via increased GSH. In this model, constitutive ICAM-1, via its effects on GSH metabolism, will modulate inflammatory response providing a novel framework that links mechanistically, cellular redox state and responses to inflammation. These data reveal a previously unsuspected cross-talk linking redox signaling in the endothelial cell with leukocyte adhesion, a key early step in the inflammatory process.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1401fje; doi: 10.1096/fj.03-1401fje

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