Alpha-lipoic acid modulates NF-κB activity in human monocytic cells by direct interaction with DNA

Experimental Gerontology 37 (2002) 401±410

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Alpha-lipoic acid modulates NF-kB activity in human monocytic cells by direct interaction with DNA Heather A. Lee 1, David A. Hughes* Immunology Group, Nutrition and Consumer Science Division, Institute of Food Research, Norwich Research Park, Colney, Norwich, Norfolk NR4 7UA, UK Received 1 June 2001; accepted 1 September 2001

Abstract The constitutive activity of the redox-sensitive transcription factor, NF-kB, which regulates the production of many in¯ammatory cytokines and adhesion molecules, appears to be up-regulated in an age-associated manner and it is thought this might contribute to the increased incidence of chronic in¯ammatory conditions observed with increasing age. As some antioxidants have demonstrated protective effects against rheumatoid arthritis, we are investigating the effects of vitamin E, vitamin C and alpha-lipoic acid (ALA) on NF-kB activity and on the expression of intracellular adhesion molecule (ICAM)-1. MonoMac6 cells (a human monocytic cell line) stimulated with tumour necrosis factor-a (TNF-a) were treated with antioxidants at physiological achievable levels and ICAM-1 mRNA levels investigated. Both vitamin E and vitamin C had no effect on ICAM-1 expression at the doses used, but ALA reduced the TNF-a-stimulated ICAM-1 expression in a dose-dependent manner, to levels observed in unstimulated cells. Alpha-lipoic acid also reduced NF-kB activity in these cells in a dosedependent manner. Addition of ALA to the binding reaction of nuclear extract with DNA prior to gel-shift analysis showed that it caused inhibition at this level. These initial results suggest that antioxidant modulation of monocyte activity might have potential bene®ts in inhibiting the dysregulated activity of redox-sensitive transcription factors that occurs with increasing age. q 2002 Elsevier Science Inc. All rights reserved. Keywords: Monocytic cells; NF-kB; Alpha-lipoic acid; Antioxidant; Direct DNA interaction

1. Introduction It is now known that the transcription factor, NFkB, serves as a critical regulator of the inducible expression of many genes. It is triggered by a wide variety of stimuli and plays a pivotal role in many cellular responses to environmental changes, e.g. * Corresponding author. Tel.: 144-1603-255345; fax: 144-1603507723. E-mail address: [email protected] (D.A. Hughes). 1 Present address: School of Life and Environmental Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK.

stress, injury and in¯ammation. NF-kB is a family of ®ve proteins all containing the conserved Rel homology domain (p50, p52, p65, c-Rel and RelB), which exists in the cytoplasm of almost every type of cell, bound to its inhibitor, IkB (Baeuerle and Baltimore, 1988). Stimulation of the cell by, for example, TNF-a or interleukin (IL)-1 in chronic in¯ammation (Baeuerle and Henkel, 1994), initiates a signalling cascade where IkB is phosphorylated, tagged by ubiquinone and degraded by proteosomes. This results in the release of NF-kB from the complex and its rapid translocation into the nucleus where it binds to the promoter regions of many genes. Active NF-kB is a

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dimer, usually composed of p50 and p65 in humans, although many other heterodimers and homodimers have been described, which bind to slightly different DNA sequences (Parry and Mackman, 1994). Homodimers of the p50 subunit (which lacks a transactivation domain) have been shown to be present constitutively in monocytes (Frankenberger et al., 1994). There is evidence to suggest that the deleterious changes to the immune system that occur with increasing age are associated with a decreased ability to effectively handle oxidative stress (Poynter and Daynes, 1998). In support of this, an age-associated up-regulation of constitutive NF-kB expression has been observed in several animal models (Spencer et al., 1997; Helenius et al., 2001), together with an increased production of pro-in¯ammatory cytokines (e.g. TNF-a, IL-1, IL-6) and an elevated expression of ICAM-1 by stimulated cells (Yan et al., 1999). Indeed, it is thought that the pathophysiology of many disease states seen in the elderly is linked to the dysregulated production of cytokines. In chronic in¯ammatory diseases such as rheumatoid arthritis, psoriasis, and asthma, cytokines are involved, along with chemokines, in the recruitment of activated immune cells to the site of lesions. These cells also produce cytokines and other mediators of in¯ammation and so the in¯ammatory state is ampli®ed and perpetuated (Brennan et al., 1995). The pro-in¯ammatory cytokines TNF-a and IL-1 both activate and are produced by NF-kB (Barnes and Karin, 1997). ICAM-1 is expressed on a wide variety of cells (Rothlein et al., 1986) and as well as being vital for lymphocyte adhesion to many cell types including epithelial cells (facilitating migration to sites of in¯ammation), ICAM-1 plays a pivotal role in antigen presentation to T cells (Springer, 1990). Its induction is largely regulated at the mRNA level with surface expression occurring within 4 h after stimulation (Simmons et al., 1988). Rheumatoid arthritis patients have elevated expression of ICAM-1 and MHC class II molecules, indicating continuous antigen presentation, in chronically in¯amed joints (Wicks et al., 1992). Thus, control of ICAM-1 levels could be very important in in¯ammatory diseases. To explain how NF-kB can be stimulated by a wide variety of factors (cytokines, viral and bacterial products, UV light, hydrogen peroxide, cigarette

smoke) it was suggested that all these factors increased intracellular hydrogen peroxide levels, and therefore NF-kB was activated by oxidative stress (Schreck et al., 1992). This was based on a number of observations; direct addition of hydrogen peroxide to cultures activated NF-kB in some cells; intracellular levels of reactive oxygen species (ROS) increased in response to agents that activated NF-kB; antioxidant compounds (e.g. PDTC) could inhibit the pathway to NF-kB activation; and inhibition or overexpression of enzymes that affect the levels of intracellular ROS could modulate NF-kB activation (Bowie and O'Neill, 2000a). We have previously shown that ICAM-1 expression in human monocytes (which mature in tissues to become macrophages that can present antigen) stimulated in vitro with IFN-g can be reduced with …n 2 3† polyunsaturated fatty acids (Hughes et al., 1996b), and that dietary supplementation of …n 2 3† PUFAs in ®sh oil reduced surface ICAM-1 levels on both stimulated and unstimulated monocytes (Hughes et al., 1996a). Also it has been shown that a diet rich in carotenoids, vitamins C and E is bene®cial to rheumatoid disease patients (Hanninen et al., 2000) decreasing joint stiffness and pain. In view of these ®ndings and the oxidative stress model of NF-kB activation, we decided to investigate the effects of the dietary antioxidants vitamin C, vitamin E and ALA on NF-kB activity in TNF-a stimulated monocytic cells (MonoMac6), and on subsequent ICAM-1 expression. In this paper, we present our initial ®ndings, which suggest that ALA has a suppressive effect, and present data supporting the suggestion that this is achieved via a direct interaction with NF-kB±DNA binding.

2. Materials and methods 2.1. Cell cultures The human monocyte cell line MonoMac6 (ZeiglerHeitbrock et al., 1988) was obtained from the German Collection of Microorganisms and Cell Cultures. It was grown in RPMI medium containing 10% fetal calf serum (FCS), sodium pyruvate (1 mmol/l), glutamine (2 mmol/l), gentamycin (50 mg/ml), non-essential amino acids (diluted 1/100) and insulin (10 mg/ml)

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and maintained between 3 £ 10 and 10 £ 10 cells/ ml. TNF-a (BD Biosciences, Cowley, UK) was dissolved in phosphate-buffered saline (PBS) containing bovine serum albumin (BSA, 1.0%) at a concentration of 10 4 U/ml, and stored at 2208C. It was added to the cell culture at a concentration of 10 3 U/ml. All antioxidants were purchased from Sigma-Aldrich, (Poole, UK). Vitamin C was dissolved in water to make a stock solution of 100 mmol/l and added to the cell culture as required. Vitamin E, as (1/2)-atocopherol, was dissolved in ethanol to give a stock solution of 200 mmol/l, ¯ushed with nitrogen gas, and stored at 2808C. To treat the cells, the required amount was ®rst added to FCS (0.5 ml), incubated at 378C for 15 min, and then added to the culture ¯ask. ALA was added to water to give a stock solution of 200 mmol/l and NaOH (3 mol/l) added until the ALA dissolved. It was added directly to the cell culture ¯ask to give the required concentration. Pyrrolidine dithiocarbamate (PDTC) was dissolved in water as a stock solution of 200 mmol/l, and added directly to the cell culture medium. For each experimental condition, approximately 10 7 cells in 20 ml culture were used. 2.2. RNA extraction and electrophoresis Cells were pretreated with antioxidant for 2 h, stimulated with TNF-a for 2 h, and then harvested. After centrifuging at 500g for 10 min, the cells were transferred to an eppendorf tube and spun again at 3000g for 20 s to remove all culture supernatant. Total RNA was extracted using an RNeasy Mini Kit (Qiagen, Crawley, UK), eluted with 2 £ 30 ml of RNAase free water and stored at 2808C. RNA concentration was estimated from the absorbance at 260 nm. Samples (5±10 mg) were heated at 658C with RNA sample loading buffer (Sigma-Aldrich, Poole, UK) before being electrophoresed on a 1% agarose formaldehyde gel in MOPS buffer overnight at 20 V at room temperature. The gel was washed with DEPCtreated water, 50 mmol/l NaOH and 5 £ SSC buffer (20 £ SSC is 3 mol/l NaCl, 0.2 mol/l sodium citrate) before being blotted onto a Hybond-N 1 membrane (Amersham Pharmacia Biotech, Amersham, UK) using an Appligene vacuum blotter. The blot was washed in 50 mmol/l NaOH and water and ®xed

403

under a UV crosslinker (Stratagene, Amsterdam, The Netherlands). 2.3. Northern blotting Two probes were prepared. One, as a 532 base pair fragment, ampli®ed by PCR, from a plasmid containing the ICAM-1 sequence (Simmons et al., 1988), and the other, glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1500 bp), a housekeeping gene, was digested from a plasmid using PstI (Dugaiczyk et al., 1983). The probes were labelled using the Megaprime DNA Labelling System (Amersham Pharmacia Biotech, Amersham, UK) and [a- 32P]dATP (370 MBq/ml; 1.1 MBq). Occasionally, the probes were puri®ed before use with ElutipD columns (Schleicher and Schuell, London, UK) using Low Salt buffer (0.4 mol/l NaCl, 20 mmol/l Tris/HCl, 1.0 mmol/l EDTA, pH 7.4) and High Salt buffer (2.0 mol/l NaCl, 20 mmol/l Tris/HCl, 1.0 mmol/l EDTA, pH 7.4). Probes were used immediately and added to the blot that had prehybridised for 1 h at 658C in UltraHyb solution (Flowgen, Ashby de la Zouch, UK). After hybridising for 16±24 h at 658C, the membrane was rinsed twice and washed once for 10 min in Low Stringency buffer (1 £ SSC, 0.1% SDS) then washed three times for 15 min with High Stringency buffer (0.2 £ SSC, 0.1% SDS). Lastly, the blot was dried, wrapped in Saran wrap and exposed to a phosphorimager plate overnight before being imaged on a Fuji ®lm BAS-1500 imager using MacBas V2.2 software. Band densities were measured and values for ICAM-1 expressed as the density of the ICAM-1 band divided by the density of the GAPDH band. 2.4. Nuclear protein extraction The cells were pretreated with antioxidant for 2 h, then stimulated with TNF-a for 0.5±2 h before being harvested. Nuclear protein extracts were prepared according to the method of Wang et al. (1995). Cells were washed in ice-cold PBS, then resuspended in cold PBS (1 ml) and transferred to an eppendorf tube. After being centrifuged at 2000g for 5 min, they were resuspended in Buffer A (10 mmol/l Hepes-NaOH, 15 mmol/l KCl, 0.1 mmol/l EDTA, 2 mmol/l MgCl2, 1 mmol/l DTT, 1 mmol/l PMSF, pH 7.9), left on ice for 10 min (tapped once during

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this time), 10 ml of 10% IGEPAL was added and the cells left for a further 5 min. The nuclei were pelleted at 13 000g for 30 s and the supernatant removed. The pellet was then resuspended in 20 ml of Buffer B (20 mmol/l Hepes-NaOH, 1.5 mmol/l MgCl2, 0.42 mmol/l NaCl, 0.2 mmol/l EDTA, 25% glycerol, 0.5 mmol/l DTT and 0.5 mmol/l PMSF, pH 7.9) and incubated on ice for 30 min. After centrifuging again at 13 000g for 30 s, the supernatant, containing the nuclear protein, was transferred to a tube containing 20 ml of Buffer C (20 mmol/l Hepes-NaOH, 50 mmol/ l KCl, 0.2 mmol/l EDTA, 0.5 mmol/l DTT, 0.5 mmol/ l PMSF, pH 7.9). The extracts were divided into 10 ml aliquots and stored at 2808C. 2.5. Microtitre plate assay to determine protein concentration A stock of BSA standard (10 mg/ml), stored at 2208C, was diluted to give a range of standards from 2 to 20 mg/ml in water. All nuclear protein extracts were diluted 1/400 in water. Bio-Rad Protein Assay Reagent (30 ml) was added to the wells of a microtitre plate. Samples, standards and water for a blank were added in triplicate (120 ml), the plate tapped and left at room temperature for 30 min. The well absorbances were read at 610 nm (the optimal wavelength is 595 nm) and a standard curve constructed. Absorbance versus dose is linear in this range, and the extract concentrations were calculated from the curve. 2.6. NF-k B gel-shift assay NF-kB activity was determined using either an oligonucleotide from Santa Cruz (Autogen Bioclear, Calne, UK) or the Promega Gel Shift Assay System (Southampton, UK) containing the same consensus sequence: 5 0 AGT TGA GGG GAC TTT CCC AGG C 5 0 3 0 TCA ACT CCC CTG AAA GGG TCC G 3 0 The 32P-labelled probe was prepared by incubating oligonucleotide (10 pmol), 10 £ kinase buffer, [g- 32P] ATP (370 MBq/ml; 1.1 MBq) and T4 polynucleotide kinase (1.0 ml) for 60 min at 378C in a total volume of 20 ml or following the instructions for the Promega Kit. The probe was subsequently puri®ed

using ElutipD columns. The column was presoaked with Low Salt buffer (20 mmol/l NaCl, 20 mmol/l Tris/HCl, 1.0 mmol/l EDTA, pH 7.4), then the probe, diluted to 1 ml in Low Salt buffer, was pushed through the column using a syringe. After washing with 5 ml of Low Salt buffer, the probe was eluted with 0.2 ml of High Salt buffer (1.0 mol/l NaCl, 20 mmol/l Tris/HCl, 1.0 mmol/l EDTA, pH 7.4) and stored in four aliquots at 2208C. Before adding the probe (,50 000 cpm/reaction) nuclear extract (10 mg) was preincubated at room temperature for 15 min in Gel Shift Binding buffer (1 mmol/l MgCl2, 0.5 mmol/l EDTA, 50 mmol/l NaCl, 10 mmol/l Tris/HCl, 4% glycerol) with DTT (2.5 mmol/l) and poly(dI±dC).poly(dI±dC) (2± 4 mg). If required, unlabelled probe, or competitor (unlabelled NF-kB oligonucleotide; 100 ng), or antioxidant (0.5±2.0 mmol/l) was also added. If Supershift Antibody (Autogen Bioclear, Calne, UK; 2± 4 mg) was added, the extract was preincubated for 30 min at room temperature. Once the probe was added, the extract was incubated for a further 30 min. The samples were then analysed on a 6% polyacrylamide gel (Novex TBE or DNA Retardation gels, Invitrogen, Groningen, The Netherlands), run in 0.5 £ TBE buffer for 45 min at room temperature. The gels were dried for 1 h, wrapped in Saran wrap and exposed overnight to a phosphorimager plate before being analysed on a Fiji ®lm BAS-1500 imager using MacBas V2.2. Band densities were measured to compare NF-kB binding activity between tracks. 2.7. Statistical analysis For the comparison of effects of different antioxidants on ICAM-1 mRNA expression, one-way analysis of variance was used to identify signi®cant differences between treatments. Where there was a signi®cant difference, means were compared using the Scheffe post hoc test, to reduce the possibility of false positives. 3. Results 3.1. ICAM-1 mRNA expression Levels of ICAM-1 mRNA in MonoMac6 cells were upregulated when the cells were stimulated

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Table 1 The effect of different doses of vitamin C, vitamin E and alpha-lipoic acid separately on ICAM-1 mRNA in MonoMac6 cells stimulated with TNF-a (10 3 U/ml) (The data points are the ratio of the density (measured in arbitrary units) of the ICAM-1 bands and the corresponding GAPDH bands from Northern blots, expressed as a percentage of the value obtained when the cells were stimulated in the absence of antioxidant.) Dose (mmol/l) Unstimulated (no TNF-a)

0

Stimulated

0 0.01 0.5 0.125 0.25 0.50 1.00 2.00

Vitamin C 6.1 100 ± 96 98 85 100 97 ±

with TNF-a for 2 h. Pre-incubation with either vitamin E or vitamin C for 2 h prior to stimulation did not decrease the upregulated levels of ICAM-1 mRNA at any of the concentrations tested. Vitamin E acetate was also tested and no response was seen (data not shown). However, pre-incubation with ALA decreased the mRNA levels in a dose-dependent manner to the same level as in unstimulated cells with 1 mmol/l of the antioxidant (Table 1 and Fig. 1). Alpha-lipoic acid, or most probably its reduced form, dihydrolipoic acid (DHLA), is able to regenerate other antioxidants such as vitamin C and (indirectly) vitamin E (Scholich et al., 1989; Podda et al., 1994). Therefore, it was decided to examine the effect of ALA with vitamin E and vitamin C, both separately and together, on stimulated ICAM-1 mRNA levels. The inhibitory effect of ALA (p , 0:01 compared with control) was not altered by the presence of either of the vitamins alone, but the combination of all three antioxidants further reduced the production of ICAM-1 mRNA compared with control values …p , 0:001† (Table 2 and Fig. 2).

Antioxidant vitamin E 13.7 100 90 ± 97 ± 80 ±

a-Lipoic acid 26 100 ± ± 67 58 41 28 15

3.2. NF-k B binding activity The gel-shift assay for NF-kB was validated by adding anti-p50 and anti-p65 antibodies or unlabelled oligonucleotide to the binding reaction before the probe was added. Fig. 3 shows that both of the NF-kB subunit antibodies caused a supershift in the gel-shift assay, and competition with excess NF-kB oligonucleotide con®rmed the position of the NF-kB±oligonucleotide complex. Additionally, an anti-c fos antibody did not cause a supershift and there was no competition with an excess of AP-1 oligonucleotide. Addition of ALA and PDTC to the culture medium 2 h before the cells were stimulated with TNF-a for 0.5 h (Fig. 4) shows that both of these compounds reduce the upregulated NF-kB activity. Alphalipoic acid reduced the binding activity in a dose-dependant manner up to 60.6% of the control activity with 1 mmol/l, and PDTC (200 mmol/l) reduced the activity to 1.5%.

Fig. 1. Northern blot of ICAM-1 mRNA from MonoMac6 cells pre-incubated with increasing concentrations of alpha-lipoic acid (ALA) and stimulated with TNF-a (10 3 U/ml; 1).

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Table 2 The combined effect of vitamin C, vitamin E and alpha-lipoic acid (ALA) on ICAM-1 mRNA in TNF-a stimulated (10 3 U/ml) MonoMac6 cells (The density is the ratio of the ICAM-1 band to the corresponding GAPDH band from Northern blots expressed as a percentage of the control. Concentrations of antioxidants were vitamin C, 100 mmol/l; vitamin E, 100 mmol/l; alpha-lipoic acid, 250 mmol/l.) Antioxidant

Density (mean, n ˆ 4)

Statistically different from control

None (control) Vitamin C Vitamin E ALA Vitamin C 1 vitamin E Vitamin C 1 ALA Vitamin E 1 ALA Vitamin C 1 vitamin E 1 ALA

100 105.4 113.3 66.4 97.3 70.5 67.6 58.5

± ns ns P , 0:01 ns P , 0:01 P , 0:01 P , 0:001

3.3. Direct effect on DNA binding To see if the effect of ALA was upstream in the NFkB regulatory pathway, or if it was directly interfering with the binding of NF-kB to the DNA consensus sequence, ALA (and PDTC as a control) was added to the binding reactions before the gel was run. We found that PDTC had no effect on NF-kB activity (Table 3) as expected, but that ALA had a marked effect that was variable depending on the age of the a-lipoic acid solution. If the solution was prepared and stored frozen for 1 week, as little as 0.2 mmol/l, a-lipoic acid could reduce NF-kB activity to nothing, but if the solution was prepared and used immediately, the reduction was minimal as shown in Table 3.

4. Discussion Reports on the ability of vitamin E to lower NF-kB activity in different cells with different stimuli are variable and confusing. The water-soluble vitamin E acetate has been shown to decrease NF-kB activity in Jurkat T cells stimulated with TNF-a, whereas the

lipid-soluble vitamin E had no effect in these cells (Suzuki and Packer, 1993). Vitamin E also had no effect on an astrocyte cell line stimulated with TNFa (Hirano et al., 1998), on THP-1 monocytes stimulated with lipopolysaccharide (LPS) (Nakamura et al., 1998), or on the macrophage cell line J774 stimulated with LPS (Abate et al., 2000). However, it did decrease NF-kB activity in U937 monocytes stimulated with LPS (Islam et al., 1998) and ®broblasts stimulated with polyunsaturated fatty acids (Maziere et al., 1999). Vitamin C alone has either been shown to have no effect on NF-kB activity in TNF-a stimulated astrocyte cells (Hirano et al., 1998), to increase the binding of NF-kB to DNA in TNF-a stimulated Jurkat cells (Munoz et al., 1997), or to inhibit activation of NF-kB by TNF-a in endothelial cells (Bowie and O'Neill, 2000b). The inhibition required millimolar concentrations of vitamin C, more than was used in our study, and was shown to occur via blocking of IkBa phosphorylation. Hirano et al. (1998) have shown that although neither vitamin E nor vitamin C alone had any effect, a compound (EPC-K1) composed of the two vitamins linked together through a phosphate diester bond, could inhibit TNF-a

Fig. 2. Northern blot of ICAM-1 mRNA from MonoMac6 cells pre-incubated with combinations of antioxidants and stimulated with TNF-a (10 3 U/ml) Lanes: (1) no antioxidant; (2) vitamin C; (3) vitamin E; (4) ALA; (5) vitamin C 1 vitamin E; (6) vitamin C 1 ALA; (7) vitamin E 1 ALA; (8) vitamin C 1 vitamin E 1 ALA. Concentrations used: vitamin C, 100 mmol/l; vitamin E, 100 mmol/l; ALA, 250 mmol/l.

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Fig. 3. NF-kB binding activity for nuclear extracts (10 mg) from MonoMac6 cells stimulated with TNF-a, evaluated by gel-shift assay. Lanes: (1) control; (2) 1 anti-p65 monoclonal antibody (Mab); (3) 1 anti-p50 Mab; (4) 1 anti-c fos Mab; (5) 1 NF-kB oligonucleotide; (6) 1 AP1 oligonucleotide.

induced NF-kB activity in astrocytes. They suggest that in this form vitamin E can switch vitamin C from a pro-oxidant to an antioxidant (Wefers and Seis, 1988), and that their compound is amphipathic compared to the lipophilic vitamin E and so can reach the mitochondria where it scavenges radical intermediates and reduces the production of ROS. We did not see any effect with either vitamin alone or together on ICAM-1 expression, which is under the control of NF-kB, in TNF-a stimulated MonoMac6 monocytes (Table 1 and Fig. 2), although in our case the two vitamins were not chemically linked. There are no reports, to our knowledge, of the effects of vitamin E or vitamin C on ICAM-1 expression in human monocytes. Alpha-lipoic acid is a potent thiol antioxidant in both fat- and water-soluble media (Packer, 1998), which can be synthesised by humans and functions as a cofactor in the oxidative decarboxylation of aketo acids. It is also found in potatoes, carrots, yams, sweet potatoes, and red meat (Ley, 1996) and is easily absorbed, taken up by cells and reduced to dihydrolipoic acid (DHLA, Handelman et al., 1994). Alphalipoic acid has potential as a therapeutic agent. In

407

Fig. 4. Reduction of NF-kB activity in MonoMac6 cells with increasing doses of alpha-lipoic acid (ALA) and pyrroline dithiocarbamate (PDTC) added to the cell culture 2 h before the cells were stimulated with TNF-a for 0.5 h. US ˆ extract from unstimulated cells. The density of the bands was measured in arbitrary units and expressed as a percentage of the band from the TNF-a-stimulated cell extract with no antioxidants added.

animal models, it has been shown to be useful in diabetes, atherosclerosis, cataracts, ischemia-reperfusion injury, HIV activation, neurodegeneration and radiation damage; pathologies in which ROS have been implicated (Packer et al., 1995). In Europe, it has been successfully used in clinical trials to treat diabetic polyneuropathy (Ziegler et al., 1999), a condition where cell adhesion molecules are implicated in both development and progression (Jude et al., 1998). Investigations of the effect of ALA and DHLA on Table 3 NF-kB activities of nuclear extracts from MonoMac6 cells stimulated with TNF-a (10 3 U/ml), with pyrrolidine dithiocarbamate (PDTC) or alpha-lipoic acid (ALA), either fresh or a 1-week old solution, added to the binding reaction (The results are the densities of the NF-kB bands in the gel-shift assay expressed as a percentage of the bands obtained when no antioxidant is added to the binding reaction.) Concentration (mmol/l)

PDTC

ALA 1-week old

ALA fresh

0.0 0.2 0.5 1.0

100

100 1.0 0 1.2

100 99 99 93

124 145

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NF-kB activity have shown that there are two steps which can be in¯uenced by thiol antioxidants. The early stages of the signalling pathway where NF-kB is released from its inhibitory protein IkB, which are believed to be under redox control, and the actual binding of NF-kB to DNA. This involves cysteine residues, particularly Cys62 in the p50 subunit, with reduced cysteine apparently enhancing binding (Matthews et al., 1992). Because of this, the effects of thiol-containing antioxidants can be complex. Inhibition of NF-kB by a-lipoic acid has been reported for Jurkat cells stimulated by TNF-a or phorbol myristate 13-acetate (PMA) (Suzuki et al., 1992). The inhibition was dose-dependent, as seen in our studies, with 4 mmol/l required for complete inhibition. We saw approximately 40% inhibition with 1 mmol/l, but did not increase the dose to obtain complete inhibition. More recently, ALA has been shown to reduce NF-kB activity by 38% in peripheral blood mononuclear cells isolated from patients with diabetic nephropathy who have been given 600 mg of the antioxidant for 3 days (Hofmann et al., 1999). Another study has shown reduction in expression of the adhesion molecules ICAM-1 and VCAM-1 by alpha-lipoate in stimulated Jurkat T cells (Roy et al., 1998). The effect was signi®cant with a dose of 100 Umol/l, which is similar to the concentrations we found caused a decrease in ICAM-1 expression (Table 1). We also found that ICAM-1 levels could be reduced to unstimulated levels with 1 mmol/l ALA whereas this amount only reduced NF-kB levels by approximately 40%, with background levels being 17%. These authors also found that the effect was enhanced when a combination of alpha-lipoate and vitamin E was used. Although we did not observe this, we did see an enhancement of the effect of ALA when added in combination with both vitamin E and vitamin C (Table 2). Further work by Suzuki et al. (1995) have also shown that the redox regulation of NF-kB by ALA could be as a result of its inhibitory action on DNA. In addition, in contrast to our own study, they showed that DHLA enhanced binding. In our study, we showed that ALA inhibited DNA±NF-kB binding, but that this effect was very dependent on the age of the solution, with fresh ALA solution causing no inhibition and a 1-week old solution causing almost complete inhibition (Table 3). If we assume that

ALA in solution is reduced to DHLA, then the effect we see is the opposite of that observed by Suzuki et al. (1995). An explanation for this could be that they eliminated dithiothreitol (DTT) from the binding reaction. When DTT is removed, no NF-kB±DNA binding is observed because DTT keeps NF-kB in a reduced state that is necessary for DNA binding. Addition of DHLA, a strong reductant (the DHLA/ ALA couple has a redox potential of 20.32 V; Packer, 1998), restores the NF-kB to its reduced state. It could be argued that in our case, in the presence of DTT, NF-kB was already reduced, and the added DHLA had an effect on the redox balance. Alpha-lipoic acid is not such a strong reductant as DHLA and had no effect on the system. In the cell, most protein thiol groups are strongly `buffered' against oxidation by the highly reduced environment inside the cell and only accessible thiol groups, with high thiol-disulphide oxidation potentials, are redox sensitive (Roy et al., 1998). It would seem that our system, where DTT is included in the binding reaction, more closely mimics the normal environment of the cell. There is now much evidence to show that the effect of antioxidants on NF-kB activation is stimulus-speci®c as well as cell-speci®c (Bowie and O'Neill, 2000a,b). As a comparison to ALA, PDTC was tested in our system. This antioxidant is well known as an inhibitor of NF-kB (Schreck et al., 1992), and has been shown to reduce lipid peroxides (Somers et al., 2000) that could be intermediates in the signalling pathway generated by TNF-a. In MonoMac6 cells, PDTC was a strong inhibitor of NF-kB activity when added to the cell culture medium (Fig. 4), but showed no activity when added to the binding reaction (Table 3). This result indicated that TNF-a-stimulated NF-kB in MonoMac6 cells could be inhibited by an antioxidant via upstream signalling, and that although ALA was effective, its mechanism of action could be at the DNA binding level rather than at one of the multiple pathways involving a range of protein kinases that now appear to regulate NF-kB (Bowie and O'Neill, 2000a). In conclusion, we have shown that the antioxidant ALA, which is found in food and is available as supplements, can reduce TNF-a-stimulated NF-kB in a human monocyte cell line. Of course, this does not necessarily mean that monocytes and macrophages in vivo will respond in the same manner. We

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hope to undertake further work on peripheral blood monocytes, obtained from young and elderly individuals, to assess the in¯uence of ALA on stimulated NF-kB activity in vitro, before undertaking a dietary intervention study in these age groups. Alpha-lipoic acid has proved useful in treating in¯ammatory conditions such as diabetes and probably lowers expression of adhesion molecules. Therefore, ALA could be a useful compound in controlling dysregulated NF-kB activity in monocytes in elderly individuals and in treating in¯ammation in autoimmune conditions such as rheumatoid arthritis and psoriasis. Acknowledgements This work was funded by a Competitive Strategic Grant from the Biotechnology and Biological Sciences Research Council, UK. We thank Dr Kamal Ivory for commenting on the manuscript and Anthony Wright for the statistical analysis. References Abate, A., Yang, G., Dennery, P.A., Oberle, S., Schroder, H., 2000. Synergistic inhibition of cyclooxygenase-2 expression by vitamin E and aspirin. Free Rad. Biol. Med. 29, 1135±1142. Baeuerle, P., Baltimore, D., 1988. IkB: a speci®c inhibitor of the NF-kB transcription factor. Science 242, 540±546. Baeuerle, P.A., Henkel, T., 1994. Function and activation of NF-kB in the immune system. Ann. Rev. Immunol. 12, 141±179. Barnes, P.J., Karin, M., 1997. Nuclear factor-kB: a pivotal transcription factor in chronic in¯ammatory diseases. N. Engl. J. Med. 336, 1066±1071. Bowie, A., O'Neill, L.A.J., 2000a. Oxidative stress and nuclear factor-kB activation. A reassessment of the evidence in the light of recent discoveries. Biochem. Pharmacol. 59, 13±23. Bowie, A.G., O'Neill, L.A., 2000b. Vitamin C inhibits NF-kappa B activation by TNF via the activation of p38 mitogen-activated protein kinase. J. Immunol. 15, 7180±7188. Brennan, F.M., Maine, R.N., Feldmann, M., 1995. Cytokine expression in chronic in¯ammatory disease. Br. Med. Bull. 51, 368± 384. Dugaiczyk, A., Haron, J.A., Stone, E.M., Dennison, O.E., Rothblum, K.N., Schwart, R.J., 1983. Cloning and sequence of a deoxyribonucleic acid copy of glyceraldehyde 3-phosphate dehydrogenase messenger ribonucleic acid isolated from chicken muscle. Biochemistry 22, 1605±1613. Frankenberger, M., Pforte, A., Sternsdorf, T., Passlick, B., Baeuerle, P.A., Zeigler-Heitbrock, H.W.L., 1994. Constitutive nucler NFkB in cells of the monocyte lineage. Biochem. J. 304, 87±94. Handelman, G.J., Han, D., Tritschler, H., Packer, L., 1994. a-Lipoic

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