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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:1125-1133.)
© 1999 American Heart Association, Inc.

Original Contributions

-Tocopherol Decreases Interleukin-1ß Release From Activated Human Monocytes by Inhibition of 5-Lipoxygenase

Sridevi Devaraj; Ishwarlal Jialal

From the Center for Human Nutrition (S.D., I.I.) and Departments of Pathology (S.D., I.J.) and Internal Medicine (I.J.), University of Texas Southwestern Medical Center, Dallas, Tex.

Correspondence to I. Jialal, MD, PhD, Department of Internal Medicine and Pathology, The University of Texas Southwestern Medical Center at Dallas Dallas, TX 75235-9073. E-mail jialal.i{at}pathology.swmed.edu

	Abstract

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Abstract—Cardiovascular disease is the leading cause of morbidity and mortality in westernized populations. Low levels of -tocopherol (AT) are associated with increased incidence of atherosclerosis and increased intakes appear to be protective. Recently, we showed that supplementation with AT resulted in significant decreases in monocyte superoxide anion release, lipid oxidation, interleukin-1ß (IL-1ß) release, and adhesion to endothelium. The reduction in superoxide and lipid oxidation by AT seemed to be mediated by inhibition of protein kinase C. The aim of this study was to investigate the mechanism(s) by which AT inhibits IL-1ß release. Potential mechanisms examined included its effect as an antioxidant and its inhibitory effects on protein kinase C and the cyclooxygenase-lipoxygenase pathways. Although AT decreased superoxide release from activated monocytes, superoxide dismutase and catalase had no effect on IL-1ß release. Also, a similar antioxidant, ß-tocopherol, had no effect on IL-1ß release. The protein kinase C inhibitor, bisindolylmaleimide, did not inhibit IL-1ß release from activated monocytes, in spite of AT decreasing protein kinase C activity. Leukotriene B₄, a major product of 5-lipoxygenase, has been shown to augment IL-1ß release. In the presence of AT, a significant reduction in leukotriene B₄ and IL-1ß levels was observed, which was reversed by the addition of leukotriene B₄. Similar observations were seen with specific inhibitors of 5-lipoxygenase. The product of cyclooxygenase, prostaglandin E₂, has been shown to inhibit IL-1ß activity in some systems. However, AT had no significant effect on prostaglandin E₂ levels in activated monocytes. In the presence of indomethacin, a cyclooxygenase inhibitor, AT inhibited IL-1ß activity. Also, AT had no effect on IL-1ß mRNA levels or stability, suggesting a posttranscriptional effect. Thus, in activated human monocytes, AT exerts a novel biological effect of inhibiting the release of the proinflammatory cytokine, IL-1ß, via inhibition of the 5-lipoxygenase pathway.

Key Words: vitamin E • cytokine • interleukins • protein kinase C • leukotriene

	Introduction

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Although low levels of -tocopherol (AT) are associated with increased risk for cardiovascular disease, increased intakes appear to be protective.^{1 2 3 4 5 6} In addition to AT decreasing the oxidative susceptibility of LDL,^{7 8 9} it could have effects on other crucial cells in atherogenesis, such as monocytes, that could prove beneficial. We previously showed that in vivo supplementation with AT significantly decreases monocyte superoxide anion release, lipid oxidation, interleukin-1ß (IL-1ß) release, and adhesion to endothelial cells.¹⁰ In that study it appeared that the inhibition of superoxide anion release and lipid oxidation was mediated by inhibition of protein kinase C (PKC) by AT. However, we did not investigate the mechanism(s) by which AT inhibits IL-1ß release and monocyte–endothelial cell adhesion. Recently, we showed that AT enrichment of monocytes decreased agonist-induced adhesion to human endothelium by decreasing the expression of CD11b and VLA-4 on the monocytes.¹¹

Several lines of evidence suggest that the proinflammatory cytokine, IL-1ß, is proatherogenic.^{12 13 14 15 16} IL-1ß appears to have procoagulant activity, stimulates monocyte–endothelial cell adhesion and cholesterol esterification in macrophages, and appears to stimulate smooth muscle cell proliferation by platelet-derived growth factor.^{13 14 15 16} Oxidized LDL has been shown to augment IL-1ß release.¹⁷ Also, IL-1ß has been demonstrated in the atherosclerotic lesion with a 640-fold increase in mRNA for IL-1ß.¹⁸ Increased IL-1ß protein has been demonstrated in coronary arteries of patients with ischemic heart disease compared with nonischemic cardiomyopathy and has been shown to correlate with the severity of atherosclerosis.¹⁹ Hence, in this study, we examined the mechanisms by which AT could inhibit IL-1ß release from LPS-activated human monocytes. The potential mechanisms that were examined included its effect as a general antioxidant, its inhibitory effect on PKC activity, and its effect on the cyclooxygenase-lipoxygenase pathways, inasmuch as it has previously been shown that AT can inhibit both cyclooxygenase (COX) and lipoxygenase (LO) in certain systems.^{20 21 22 23} Also, the effect of AT on mRNA for IL-1ß was studied.

	Methods

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Isolation of Human Peripheral Blood Monocytes
Human monocytes were isolated from blood obtained from fasting, healthy volunteers by Ficoll Hypaque gradient as described previously.¹⁰ Twenty milliliters of blood (anticoagulated with 10 U/mL heparin) was layered carefully on 15 mL of Ficoll-Hypaque gradient (Sigma Immunochemicals) and centrifuged at 500g without brakes at room temperature for 30 minutes. The mixed mononuclear band was aspirated, and the cells were washed 3 times with RPMI 1640 containing 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mmol/L glutamine and suspended in a known volume. Leukocyte counts were performed on a Coulter counter, and cells were plated (5 to 7x10⁶ cells) in 6-well Primaria plates in RPMI 1640 medium. Incubation was performed at 37°C for 2 hours in 5% CO₂/95% air, after which nonadherent cells were removed by washing 3 times with RPMI 1640 medium. Nonspecific esterase staining revealed that 88% of cells isolated in this manner were monocytes.¹⁰ All incubations were performed immediately after isolation of the monocytes. All reagents used were tested for endotoxin contamination by Limulus endotoxin assay¹⁰ and concentrations were <12.5 pg/mL. The viability of the monocytes was found to be 94% by Trypan blue exclusion.¹⁰ LPS (10 µg/mL) was used to activate monocytes because we used it previously in our in vivo supplementation study.

Release of IL-1ß
The release of IL-1ß was measured in the culture supernatants with ELISA using a human immunoassay kit as described previously, which is specific for human IL-1ß and does not cross-react with other IL-1s.^{10 24} The assay is based on a solid-phase ELISA, which uses an antibody for human IL-1ß bound to the wells of a microtiter plate together with a biotinylated antibody to human IL-1ß and Amdex amplification reagent. Pooled blood from different normal healthy volunteers, not on antioxidant supplements or anti-inflammatory drugs, were used to isolate monocytes for the different experiments. The variability in the level of IL-1ß released from LPS-activated monocytes ranged from 2143 to 4954 pmol/mg cell protein, with a mean of 3803±932 pmol/mg cell protein; % coefficient of variation, 24%). All experiments reported were conducted on at least 3 different pools in duplicate.

Determination of PKC Activity
PKC activity in human monocytes in the presence and absence of 50 and 100 µmol/L AT was performed with RIA techniques using reagents from Amersham Corp.²⁵ The system is based on the PKC-catalyzed transfer of the -phosphate group of ATP to a peptide that is specific for PKC.

Quantitation of Leukotriene B₄ and Prostaglandin E₂ Levels
Leukotriene B₄ (LTB₄) levels were measured in the cell culture supernatants from monocytes activated with LPS in the absence and presence of 5-LO inhibitors and AT and assayed with an enzyme immunoassay (Amersham Corp).²⁶ This assay is based on the competition between unlabeled LTB₄ and a fixed quantity of peroxidase-labeled LTB₄ for a limited amount of LTB₄-specific antibody. The peroxidase ligand that is bound to the antibody is immobilized on precoated wells. The amount of labeled LTB₄ is determined using a stabilized substrate. The concentration of unlabeled LTB₄ in the sample is determined by interpolation from a standard curve. Prostaglandin E₂ (PGE₂) levels were determined using an enzyme immunoassay identical to that described for LTB₄.²⁷

Dose-Response of AT on IL-1ß Release
Mononuclear cells were incubated with AT (25, 50, and 100 µmol/L) during the 2-hour adherence incubation and for 30 minutes after the 3 washes with RPMI 1640. Thereafter the monocytes were activated with LPS and incubated at 37°C for 18 hours. The supernatants were then collected, and IL-1ß secretion was assayed by ELISA. The cells were dissolved with 0.1N NaOH, protein content was measured by the method of Lowry et al¹⁰ as reported previously, and IL-1ß activity was expressed as picomoles of IL-1ß per milligram cell protein. In some experiments, cell-associated IL-1ß was measured by subjecting the cells to 3 cycles of freeze-thawing and measuring IL-1ß in the supernatant after centrifugation. Also, AT content of the monocytes was assayed after enrichment with AT and was increased 2-fold.

Potential mechanisms by which AT could inhibit IL-1ß release include (1) inhibition of PKC; (2) inhibition of reactive oxygen species (ROS); (3) acting as a chain-breaking antioxidant; (4) inhibition of the 5-LO pathway; and (5) inhibition of the COX pathway.

Role of PKC
Mononuclear cells were incubated with PKC inhibitors, calphostin C (250 and 500 nmol/L) or with bisindolylmaleimide (BIM; 0.1 and 1 µmol/L) during the 2-hour incubation for adherence of monocytes. Thereafter, the cells were washed 3 times with RPMI-1640 medium and monocytes were activated with LPS. After an 18-hour incubation, IL-1ß activity was measured in the supernatants. The concentrations of the inhibitors used did not show any antioxidant activity as determined by assaying the lag phase of copper-catalyzed LDL oxidation (data not shown). Superoxide anion release and lipid oxidation of the monocytes was determined as described previously.¹⁰

Role of ROS
The effect of the antioxidant enzymes, superoxide dismutase (SOD, 125 µg/mL) and catalase (CAT, 100 µg/mL), alone and in combination, was tested on human monocytes by adding them during the 2-hour adherence incubation of the mononuclear cells before addition of LPS. IL-1ß activity was measured after an 18-hour incubation. Because SOD may not be able to get into the cells readily, we also tested the effect of encapsulated SOD on IL-1ß release. Liposome-encapsulated SOD and empty liposomes (which served as a control) were obtained as a gift from Dr. M. Tarpey (University of Alabama, Birmingham). After incubation of the monocytes with encapsulated SOD, cytosolic SOD activity was measured after cell lysis by the method of McCord and Fridovich.²⁸

To test whether inhibition of IL-1ß release by AT was purely an antioxidant property, human monocytes were also incubated in the presence of another antioxidant, ß-tocopherol (50 and 100 µmol/L), which is not an inhibitor of PKC.²⁹

Role of 5-LO
To test the hypothesis that AT could inhibit IL-1ß release by inhibition of 5-LO, we measured LTB₄ levels, a major product of 5-LO activity, in the presence and absence of AT. Also, we tested the effect of 2 inhibitors of 5-LO, MK886 and REV5901, on IL-1ß release and LTB₄ levels. Human monocytes were incubated with 1 µmol/L of MK886 and 10 µmol/L REV5901 for 30 minutes before addition of LPS and incubated for 18 hours at 37°C. Both IL-1ß and LTB₄ levels were measured in the supernates.

Role of COX
Monocytes were incubated in the presence of the COX inhibitor, indomethacin (10 µmol/L), and in the presence of 100 µmol/L AT for 2 hours before addition of LPS. PGE₂ release (as a measure of COX activity) and IL-1ß levels were measured in the cell culture supernatants after an 18-hour incubation.

Effect of AT on mRNA for IL-1ß
Monocytes were incubated with AT (100 µmol/L) during the 2-hour adherence incubation and for 30 minutes after 3 washes with RPM1 1640 medium. Thereafter, the monocytes were incubated with LPS for 4 hours at 37°C. RNA was isolated using Trizol reagents from Gibco BRL.³⁰ Message for IL-1ß was quantified using RNase protection assay, using reagents from Pharmingen, with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as control.³¹ For mRNA stability experiments, after activation with LPS for 4 hours, cells were incubated with actinomycin D (10 µg/mL) for 2 hours to stop all further RNA synthesis.³² Total RNA was thereafter isolated every 20 minutes for 6 hours, and message for IL-1ß was quantified using RNase protection assay.

Statistical Analysis
Data were expressed as the mean±SE of at least 3 experiments. Statistical analysis was performed by Student's paired t test to determine differences. Significance was defined at the 5% level.

	Results

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The effect of AT on IL-1ß release from LPS-activated monocytes is shown in Figure 1A. There was a dose-dependent inhibition of IL-1ß release from LPS-activated monocytes (45% and 66% inhibition with 50 and 100 µmol/L of AT, respectively; P<0.001). There was no significant decrease in IL-1ß levels with 25 µmol/L of AT (P=0.21). Cell-associated IL-1ß was also decreased in the presence of AT (100 µmol/L: LPS, 1157±319 pmol/mg protein; LPS+AT, 559±163.2 pmol/mg protein; P<0.02, n=4 experiments).

View larger version (17K): [in this window] [in a new window]   Figure 1. A, Dose-response effect of AT on IL-1ß release. Human peripheral blood mononuclear cells were isolated by Ficoll Hypaque gradient and incubated for 2 hours at 37°C in 5% C02/95% air in the presence and absence of AT, after which nonadherent cells were removed by 3 washes with RPMI-1640 medium. Thereafter the cells were incubated for an additional 30 minutes in the presence and absence of AT. Monocytes were then activated with LPS, and IL-1ß activity was measured after an 18-hour incubation at 37°C by sandwich ELISA as described in Methods. Data are mean±SE of 4 separate experiments performed in duplicate. P<0.001 compared with LPS-activated cells. B, Effect of AT on PKC activity in human monocytes. Monocytes were enriched with AT as described in A. PKC activity in LPS-activated human monocytes in the presence and absence of AT (50 and 100 µmol/L) was measured using RIA. Data are mean±SE of 3 separate experiments performed in duplicate. P<0.01 compared with LPS-activated cells.

Because AT has been shown to inhibit PKC activity in smooth muscle cells and platelets,^{33 34} we tested the effect of AT enrichment of monocytes on PKC activity; the results are shown in Figure 1B. Incubation with AT (50 and 100 µmol/L) resulted in significant inhibition of PKC activity when compared with LPS-activated monocytes (34% and 39%, respectively; P<0.01). In a recent study¹⁰ we showed that calphostin C, an inhibitor of the regulatory domain of PKC, when used at concentrations that did not exhibit antioxidant activity, significantly reduces superoxide anion release and lipid oxidation from activated human monocytes but had no effect on IL-1ß release. In the present study, we also tested another PKC inhibitor, BIM, which is a catalytic site inhibitor of PKC. Similar results as for calphostin C were also obtained with BIM (Figure 2A and 2B). Although BIM (0.1 and 1 µmol/L) inhibited superoxide anion release and lipid oxidation, it had no effect on IL-1ß release from LPS-activated monocytes (Figure 2B).

View larger version (25K): [in this window] [in a new window]   Figure 2. Effect of BIM on monocyte superoxide anion release and lipid oxidation (A) and IL-1ß release by monocytes (B). Mononuclear cells were incubated with BIM (0.1 and 1.0 µmol/L) during the 2-hour adherence incubation as described in Figure 1. Thereafter the cells were stimulated with LPS. Superoxide anion release and lipid oxidation was assayed as described previously10 and IL-1ß was measured as described in Methods. Data are mean±SE of 3 experiments. P<0.001 for superoxide anion release compared with control; P<0.05 for thiobarbiturate-reactive substance thiobarbituric acid reactive substances release when compared with control.

Kasama et al³⁵ have shown that superoxide stimulates IL-1-like factor release from human monocytes. Because AT is a known chain-breaking antioxidant that preserves membrane integrity,³⁶ it could prevent induction of IL-1ß release by decreasing ROS. Hence, we tested the effect of scavengers of ROS, namely, SOD and CAT, on IL-1ß release from LPS-activated human monocytes. Incubation of monocytes with SOD and CAT, either alone or in combination, did not produce any significant reduction in IL-1ß release from LPS-activated cells (Figure 3). To rule out the possibility that SOD had no effect because it failed to enter the cells, we tested the effect of encapsulated SOD on IL-1ß release. Monocytes were incubated with either vehicle control or liposome-encapsulated SOD for 3 hours before activation with LPS. Cytosolic SOD activity in the presence of encapsulated SOD was enhanced approximately 6.0-fold when compared with control (1036±437 U/mg protein compared with 174±22 U/mg protein, respectively). Incubation with encapsulated SOD also did not result in inhibition of IL-1ß release from activated monocytes (LPS, 3335±440 pmol/mg cell protein; LPS+SOD-liposomes, 3279±55.5 pmol/mg cell protein, n=3 experiments).

View larger version (13K): [in this window] [in a new window]   Figure 3. Effect of SOD and CAT on IL-1ß release from LPS-activated monocytes. Monocytes were incubated with either SOD (125 µg/mL) or both SOD and CAT (100 µg/mL) during the 2-hour adherence incubation as described in Figure 1. Thereafter the cells were activated with LPS. IL-1ß activity was measured after an 18-hour incubation at 37°C. Data are mean±SE of 4 experiments.

To test whether the inhibition of IL-1ß release by AT was purely an antioxidant function, we tested the effect of another similar antioxidant, ß-tocopherol, on IL-1ß release from human monocytes. Incubation with ß-tocopherol (50 and 100 µmol/L) produced only a slight, insignificant reduction in IL-1ß release from activated monocytes (6% and 9%, respectively) whereas AT produced a 39% and 45% reduction in IL-1ß release at similar concentrations (Figure 4).

View larger version (23K): [in this window] [in a new window]   Figure 4. Effect of AT and ß-tocopherol on IL-1ß release. Human monocytes were incubated along with AT or ß-tocopherol (50 and 100 µmol/L, respectively) during the 2-hour adherence incubation and for an additional 30 minutes as described in Figure 1. After activation with LPS, monocytes were incubated overnight, and IL-1ß activity was measured in the supernatants with ELISA as described in Methods. Data are mean±S.E of 3 experiments. P<0.02 for LPS+AT 50 µmol/L and P<0.01 for LPS+AT 100 µmol/L compared with LPS alone.

LTB₄, a product of 5-LO activity, has been shown to increase IL- 1ß release from monocytes,^{37 38} and AT has been shown in certain systems to inhibit 5-LO.^{20 21 22 23} Hence, we measured LTB₄ levels in monocytes treated with AT (100 µmol/L). AT significantly decreased LTB₄ release from LPS-activated cells (LPS, 98.2±5.9 versus 44.0±6.02 pmol/mg protein with AT, 55% inhibition; P<0.005). However, ß-tocopherol had no significant effect on LTB₄ release (LPS, 82.4±22 pmol/mg protein; LPS+ß-tocopherol, 77.1±24.7 pmol/mg protein; n=4 experiments). As shown in Figure 5, AT (100 µmol/L) also decreased IL-1ß release from LPS-activated monocytes. Because AT decreased LTB₄ levels and IL-1ß release from activated monocytes, it could possibly act by inhibition of 5-LO. To test this hypothesis, we added LTB₄ (100 nmol/L) to monocytes incubated with AT before adding LPS to activate the cells. When LTB₄ was added along with AT, there was a reversal of the inhibition of IL-1ß release from activated monocytes incubated with AT (Figure 5). Although LTB₄ alone significantly increased IL-1ß release from monocytes (Figure 5), the addition of LTB₄ to LPS did not have any significant effect on IL-1ß release.

View larger version (13K): [in this window] [in a new window]   Figure 5. Effect of AT on 5-LO and IL-1ß release from human monocytes. Mononuclear cells were incubated in the presence and absence of AT (100 µmol/L) during the 2-hour adherence incubation and then for an additional 30 minutes as described in Figure 1. Thereafter, the cells were activated with LPS or LTB4 (10-7 mol/L). Also, LTB4 was added to one set of monocytes that had been preincubated with AT before activation with LPS. Data are mean±SE of 4 experiments performed in duplicate. P<0.001 for LPS+AT100 µmol/L compared with either LPS alone, LTB4 alone, or LPS+AT+LTB4.

To further determine the role of LTB₄ on IL-1ß release from activated monocytes, we tested the effect of 2 specific inhibitors of 5-LO, MK886 and REV5901, on IL-1ß release and LTB₄ levels from activated monocytes. The 5-LO inhibitors, MK886 and REV5901, significantly inhibited LTB₄ levels in LPS-activated monocytes (Figure 6A). Both inhibitors were also able to inhibit IL-1ß release from activated human monocytes (58% and 68% inhibition, respectively). When LTB₄ was added to either system (ie, LPS+MK886 or LPS+REV5901), there was resurrection of IL-1ß activity (Figure 6B). Although both AT and MK886 alone significantly inhibited IL-1ß release (LPS, 4755±416 pmol/mg protein; LPS+AT, 1670±560 pmol/mg protein; LPS+MK886, 1221±198 pmol/mg protein; P<0.05 versus LPS alone), there was no additive effect on IL-1ß release when monocytes were incubated with -tocopherol alone or in combination with MK886 (LPS+AT, 1670±560 pmol/mg cell protein versus LPS+AT+MK886, 1211±201 pmol/mg cell protein; n=3 ex-periments, P=0.11) (Table 1).

View larger version (21K): [in this window] [in a new window]   Figure 6. Effect of 5-LO inhibitors on LTB4 levels (A) and IL-1ß release (B) from LPS-activated monocytes. Adherent monocytes were incubated with the 5-LO inhibitors, MK886 (1 µmol/L) and REV5901 (10 µmol/L), for 30 minutes before addition of LPS. LTB4 (10-7 mol/L) was added to one set of monocytes that had been preincubated with the 5-LO inhibitors. After an 18-hour incubation, LTB4 and IL-1ß release were measured in the supernatants as described in Methods. Data are mean±SE of 4 experiments performed in duplicate. P<0.005 for LPS+MK886 or LPS+REV 5901 compared with LPS alone, LPS+MK886+LTB4, or LPS+REV5901+LTB4.

View this table: [in this window] [in a new window]   Table 1. Effect of MK886, AT, and Indomethacin on IL-1ß Release From Activated Human Monocytes

To examine the possibility that AT could act through the COX pathway, we measured the release of a major product of COX, PGE₂, from cells incubated in the presence of AT or indomethacin, a known inhibitor of COX. Although indomethacin decreased PGE₂ release from activated cells by 94% (P<0.001), AT produced an insignificant (12%) decrease in PGE₂ release (LPS, 456±94 pg/well; LPS+AT 100 µmol/L, 401±68 pg/well; LPS+indomethacin 10 µmol/L, 29±5 pg/well; n=3 experiments). However, indomethacin did not have a significant effect on IL-1ß release from activated monocytes (LPS, 2871±309; LPS+indomethacin, 3456±308 pmol/mg protein; P=0.76, n=3 experiments). Furthermore, when MK886 was incubated alone or in combination with indomethacin, there was a significant reduction in IL-1ß release from activated monocytes (Table 1). Also, in the presence and absence of indomethacin, AT significantly decreased IL-1ß release from activated cells (Table 1).

To examine whether AT had any effect on IL-1ß synthesis, mRNA for IL-1ß was measured. As shown in Figure 7, AT (100 µmol/L), did not have any significant effect on mRNA levels for IL-1ß from LPS-activated monocytes as measured by the intensity of the protected probes compared with GAPDH control in the RNase protection assay. Also, AT did not have an effect on the stability of mRNA, assessed by RNase protection assay in activated cells during a 6-hour time course after treatment with actinomycin D to stop further RNA synthesis (data not shown, n=3 experiments).

View larger version (71K): [in this window] [in a new window]   Figure 7. Effect of AT on mRNA for IL-1ß from activated human monocytes. Monocytes were incubated with AT (100 µmol/L) during the 2-hour adherence incubation and for an additional 30 minutes at 37°C as described in Figure 1. Thereafter, the cells were activated with LPS for 4 hours at 37°C. RNA was isolated, and mRNA for IL-1ß was quantified by RNase protection assay as described in Methods.

	Discussion

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Much data support the concept that atherosclerosis is an inflammatory process.³⁹ Several studies^{13 14 15 16 17 18 19} indicate a causal role for IL-1ß in atherosclerosis and suggest that modalities that can decrease IL-1ß could possibly be beneficial. In the CHAOS study,⁴⁰ AT supplementation for a duration of only 17 months (400 or 800 IU/d) resulted in a 77% reduction in recurrent myocardial infarction. It is not unreasonable to ascribe this benefit to an anti-inflammatory effect of AT. Akeson et al⁴¹ have shown in vitro that THP-1 cells respond to phorbol ester activation by induction of IL-1ß mRNA. In this in vitro system, probucol and AT were able to inhibit PMA-induced IL-1ß secretion. Cannon et al⁴² have shown that vitamin E supplementation (800 IU/d) prevented the increase in IL-1ß release from LPS-activated mononuclear cells after exercise. However, studies were not directed at elucidating the mechanism of inhibition. Recently, we have shown that AT supplementation (1200 IU/d) to human volunteers for 8 weeks resulted in a significant decrease in superoxide anion release, lipid oxidation, IL-1ß release, and monocyte–endothelial cell adhesion.¹⁰ The inhibition in superoxide anion release and lipid oxidation by monocytes after AT supplementation could be attributed to an inhibition of PKC activity rather than a general antioxidant effect. It has been previously shown that superoxide anion release and lipid oxidation is mediated through PKC.⁴³ However, the exact mechanism by which IL-1 release from monocytes is modulated is not well understood. In this study, we show that in vitro enrichment of human monocytes with AT decreases IL-1ß release from activated monocytes as shown previously after in vivo supplementation.¹⁰ The concentrations of AT used can be attained after supplementation of human volunteers.^{44 45} Thus, we investigated potential mechanisms through which AT could effect this decrease in IL-1ß.

We first investigated the effect of PKC inhibition by AT on IL-1ß release. PMA and other phorbol esters are thought to induce IL-1 activity through activation of cAMP and PKC.⁴⁶ Shapira et al⁴⁷ have shown that LPS-induced IL-1ß production from human monocytes involves both PKC and protein tyrosine kinase. AT has been shown to inhibit PKC activity in vascular smooth muscle cells and human platelets.^{33 34} This appears to be the mechanism by which AT inhibits smooth muscle cell proliferation and platelet aggregation.^{33 34} Also, it appears to be the mechanism by which AT ameliorates diabetic microvascular complications.^{48 49} In the present study, we also showed that AT was able to inhibit PKC activity in activated human monocytes. However, no appreciable decrease in IL-1ß release from activated monocytes was observed in our earlier studies using the regulatory subunit inhibitor of PKC, calphostin C. We also studied the effect of another inhibitor, BIM, on IL-1ß release from activated monocytes. BIM is a catalytic site inhibitor of PKC.⁵⁰ However, no significant reduction in IL-1ß release from activated monocytes was observed with BIM. Thus, AT does not appear to inhibit IL-1ß release from activated human monocytes through inhibition of PKC.

ROS have been shown to act as positive immunomodulators and cause induction of IL-1.⁵¹ Because AT is a known chain-breaking antioxidant that provides membrane integrity, it could prevent induction of IL-1ß release by decreasing ROS. Kasama et al³⁵ have shown that superoxide stimulates IL-1-like factor release from human monocytes and that both SOD and vitamin E inhibit IL-1-like factor release from human monocytes. We found that both SOD and CAT had no effect on IL-1ß release from human monocytes. To eliminate the possibility that SOD did not produce an effect because it could not enter the cells, we incubated the activated monocytes with liposome-encapsulated SOD. Even in the presence of encapsulated SOD, there was no significant change in IL-1ß release from human monocytes despite a substantial increase in cytosolic SOD activity. To further delineate the role of AT on IL-1ß release, ie, to differentiate its general antioxidant effect from other intracellular effects, we studied the effect of ß-tocopherol, another antioxidant, which does not inhibit PKC.²⁹ Also, their mode of uptake by cells is very similar and they do not compete with each other for transport.⁵² However, ß-tocopherol had no significant effect on IL-1ß release from activated monocytes. From these experiments, it can be concluded that in activated human monocytes, AT does not appear to act through a classic chain-breaking antioxidant mechanism to inhibit IL-1ß release.

LTs are a family of potent proinflammatory compounds derived from the metabolism of arachidonic acid by the 5-LO pathway. The major product of the 5-LO pathway in human monocytes is LTB₄.⁵³ LTB₄, at concentrations ranging from 10^-8 to 10^-7 mol/L, has been shown to enhance IL-1 production from resting and LPS-activated monocytes 2- to 3-fold.^{37 38} Among the LT products of 5-LO, LTB₄ appears to be more potent than LTC₄ and LTD₄ in augmenting IL-1ß release.^{37 54} Also, 5-LO inhibitors have been shown to inhibit IL-1 production.^{55 56 57 58} AT has been shown to inhibit 5-LO activity in cell-free systems and neutrophils.^{20 21 22 23} AT inhibits 5-LO purified from potato tubers, and this effect appears to be unrelated to its antioxidant function.²¹ The inhibition was found to be irreversible and noncompetitive with respect to arachidonic acid. Also, radiolabeled AT was found to bind strongly to 5-LO.²¹ Hence, we tested the effect of AT on LTB₄ levels in activated human monocytes. AT significantly inhibited LTB₄ and IL-1ß release from activated monocytes. When LTB₄ was added to the system containing AT and LPS, there was a reversal of the inhibition of IL-1ß release from activated monocytes. Although LTB₄ alone augmented IL-1ß release from human monocytes to the extent seen with LPS, there was no significant increase in IL-1ß release with the combination of LPS and LTB₄. Thus, AT appears to inhibit IL-1ß release from LPS-activated monocytes by inhibition of 5-LO. To test this hypothesis further we also studied specific inhibitors of 5-LO, such as MK886 and REV5901, on IL-1ß release. MK886 has been shown to irreversibly block LT synthesis in human leukocytes without affecting COX, 12-LO, and 15-LO activity. MK886 binds 5-LO-activating protein and blocks the membrane translocation of 5-LO and its subsequent activation.^{57 58} REV5901 (PF5901) specifically inhibits 5-LO by virtue of its structural similarity to 15-hydroxyeicosatetraenoic acid, a known inhibitor of 5-LO.⁵⁹ It is inactive against COX and 12-LO. REV5901 acts as a peptido-LT receptor antagonist and does not appear to have antioxidant properties.⁵⁹ In the present study, both inhibitors decreased IL-1ß release from activated monocytes to the same extent as that seen with AT. Also, in both systems, addition of LTB₄ reversed the inhibition of IL-1ß release from activated monocytes. Dinarello et al⁵⁵ have earlier shown that pretreatment with BW755C (an LO inhibitor) also resulted in decreased IL-1 activity in mononuclear cells treated with endotoxin. Further support for LTs being involved in IL-1 release comes from the work of Rola-Pleszczynski and Lemaire³⁷ and Tatsuno et al,³⁸ who have clearly shown that LTB₄ significantly enhances IL-1 production from LPS-activated monocytes. Also, Gagnon et al⁶⁰ have shown that LTB₄ is a potent activator of IL-1 release from human monocytes. With regard to inhibitors, the results of studies using stimulated monocyte populations have yielded variable results. Rainsford et al⁶¹ have examined the effects of some 5-LO inhibitors, MK886, L656,224, PF5901, and tepoxalin, on IL-1 production and have shown that all these inhibitors were able to decrease IL-1 release from human synovial tissue explants whereas IL-1 synthesis inhibitors such as tenidap did not have any effect. However, Hoffman et al⁶² failed to see any inhibition of IL-1ß or tumor necrosis factor- production from LPS-activated human monocytes treated with the 5-LO inhibitor, MK886. The only difference between their system and ours was that they did not preincubate their cells with MK886 before addition of LPS. If AT worked primarily by inhibition of LT synthesis, one would anticipate no additive effect when incubated with a 5-LO inhibitor. In our study, when monocytes were incubated with AT and the specific 5-LO inhibitor, MK886, there was no additive effect on IL-1ß release when compared with AT alone. Thus, our data demonstrate that AT inhibits IL-1ß release from LPS-activated human monocytes by inhibition of 5-LO. Although previous studies have shown that AT inhibits 5-LO, future mechanistic studies should be directed at elucidating the effect of AT on 5-LO-activating protein and 5-LO translocation to the membrane.

COX-derived arachidonate metabolites, such as PGE₂, have been shown to decrease LPS-induced IL-1 activity from murine macrophages and blood monocytes.⁶³ Knudsen et al⁶⁴ have shown that PGs posttranscriptionally decreased IL-1 activity from monocytes by decreasing intracellular cAMP. Correspondingly, nonsteroidal anti-inflammatory compounds that effectively suppress COX activity demonstrated a dose-dependent augmentation of IL-1 release.⁶⁵ AT, in certain systems such as rat peritoneal and avian macrophages, has been shown to decrease COX activity and would thus be expected to increase IL-1ß levels.⁶⁶ However, in the present report, AT had no significant effect on PGE₂ levels from LPS-activated monocytes. Our findings are supported by the work of Sakamoto et al,⁶⁵ who also showed that AT failed to inhibit PGE₂ production in LPS-stimulated rat macrophages. In addition, we show that indomethacin, a COX inhibitor, although significantly decreasing PGE₂ levels in LPS-activated monocytes, did not produce any significant increase in IL-1ß release from activated monocytes. This is supported by the work of other investigators using the COX-2 inhibitor, NS-398, who also failed to show an augmentation of IL-1 release.^{67 68 69} Furthermore, when AT was added along with indomethacin, there was inhibition of IL-1ß release from activated monocytes. This suggests that the mild inhibition of the COX pathway (12%) seems to be overridden by a significant inhibition of 5-LO (55%) in the presence of AT. The apparently discrepant findings with respect to COX inhibition and IL-1 release could be because of the different agonists used generating different effector pathways, the different cell types (monocytes, macrophages, cell lines), and the different assay systems used to measure IL-1, including bioactivity, immunoassays, mRNA, used by different investigators.

To examine whether AT had an effect on IL-1ß synthesis, mRNA for IL-1ß was measured. There was no change in mRNA expression for IL-1ß in the presence of AT. Similar findings have been reported previously with dexamethasone.³² Also, there was no change in mRNA for IL-1 receptor antagonist (data not shown). Stability of mRNA also did not seem to be altered in the presence of AT. Kern et al³² have shown that IL-1ß production in human monocytes is also inhibited by dexamethasone, and its effect appears to be at the posttranscriptional level. Dexamethasone treatment resulted in an increase in IL-1ß mRNA half-life, which they suggested was probably biologically insignificant. Thus, one can propose several posttranscriptional mechanisms by which AT decreases IL-1ß secretion in human monocytes, and these possibilities will be explored in future studies.

To date, it appears that most of the biological effects of AT that are potentially antiatherogenic appear to be mediated either by a classic antioxidant effect (decreasing LDL oxidation) or by its inhibition of PKC (inhibiting smooth muscle cell proliferation and platelet aggregation and ameliorating diabetic microvascular complications). The findings in this report continue to add to the biological effects of AT on pivotal cells in atherogenesis. In this manuscript, we point to a novel mechanism by which AT inhibits the release of the proinflammatory, proatherogenic cytokine, IL-1ß, in activated human monocytes, by inhibition of 5-LO. Thus, in addition to its well-documented effect on decreasing the oxidation of LDL, AT has inhibitory effects on important enzymes, such as PKC and 5-LO, in critical cells in atherogenesis, which could also be antiatherogenic.⁷⁰

	Acknowledgments

 
This work was supported in part by research grants from the American Diabetes Association, the Henkel Corporation, and General Clinical Research Center grant No. M01-RR00633. We thank Beverly Adams-Huet for statistical expertise and Ron Tankersley for manuscript preparation.

Received August 20, 1998; accepted October 28, 1998.

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