Monocyte suppressing action of fenofibrate

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Monocyte suppressing action of fenofibrate

Bogus³aw Okopieñ, Jan Kowalski, Robert Krysiak, Krzysztof £abuzek, Aldona Stachura-Ku³ach, Andrzej Ku³ach, Marek Zieliñski,

Zbigniew S. Herman

Department of Clinical Pharmacology, Medical University of Silesia, Medykow 18, PL 40-752 Katowice, Poland Correspondence:Bogus³aw Okopieñ, e-mail: mbkdokop@mp.pl

Abstract:

Since atherosclerosis has been proven to be an inflammatory disease, it is obvious that the proper treatment for dyslipidemia should not only correct lipid parameters but also inhibit inflammation. Monocytes and monocyte-derived proinflammatory cytokines are widely known to be involved in the formation and rupture of the atherosclerotic plaque. The aim of our study was to assess the effect of fenofibrate, a commonly used hypolipidemic drug, on the release of interleukin 1b (IL-1b), interleukin 6 (IL-6) and monocyte chemoattractant protein 1 (MCP-1) by monocytes from patients with combined hyperlipidemia.

Fourteen patients with biochemically confirmed type IIb dyslipidemia who did not respond to a low-fat diet were treated with micronized fenofibrate for 1 month. The control group included 12 healthy, normolipidemic, age-matched subjects. To accurately evaluate the levels of the inflammatory cytokines, we excluded patients with any inflammatory disease. Monocytes were isolated from peripheral blood before and after the treatment. IL-1b, IL-6 and MCP-1 release was measured by enzyme-linked immunosorbent assay (ELISA) after lipopolysaccharide stimulation.

IL-1b, IL-6 and MCP-1 levels were significantly higher in hyperlipidemic patients compared to the control (143.9 ± 6.5 vs. 74.4 ± 4.4 pg/ml;

8212 ± 285vs. 6110 ± 170 pg/ml; 19.6 ± 0.9 vs. 12.3 ± 0.6 ng/ml, respectively). Thirty-day fenofibrate treatment decreased the release of IL-1b by 43% (143.9 ± 6.5 vs. 86.2 ± 5.9 pg/ml), of IL-6 by 22% (8212 ± 285 vs. 6330 ± 234 pg/ml), and of MCP-1 by 29%

(19.6 ± 0.9vs. 14.0 ± 0.8 ng/ml).

The evaluated cytokines were markedly elevated in patients with type IIb dyslipidemia. Effective fenofibrate therapy had a significant inhibitory effect on the release of monocyte-derived inflammatory cytokines.

Key words:

dyslipidemia, atherosclerosis, fibrate, proinflammatory cytokines, monocyte

Abbreviations: IL-6 – interleukin 6; IL-1b – interleukin 1b;

MCP-1 – monocyte chemoattractant protein 1

Introduction

Atherosclerotic cardiovascular disease is the principal cause of death and disability nowadays. Recent years have witnessed many studies indicating that some in-

flammatory factors play a significant role in the initiation and growth of the atherosclerotic plaque. Such immune cells as monocytes, macrophages and T lymphocytes belong to the most important constituents of the atherosclerotic plaque [24]. Activated immune cells produce and secrete such cytokines as monocyte chemoattractant protein 1 (MCP-1), interleukin 1b (IL-1b) and interleukin 6 (IL-6). It has been proved that the migration of monocytes into the subendothelial space and the release of proinflammatory cytokines initiate atherosclerotic injury to the arterial wall [19].

Pharmacological Reports 2005, 57, 367`372 ISSN 1734-1140

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smooth muscle cells and is responsible for the altera- tions in the structure of the arterial wall [22]. IL-1b affects coagulation and fibrinolysis processes by in- creasing fibrinogen and thromboxan production and by decreasing tissue plasminogen activator (tPA) levels [8, 21]. Moreover, IL-1b stimulates the production of other proinflammatory cytokines such as IL-6 and MCP-1.

IL-6 is produced by monocytes in response to IL-1b, tumor necrosis factor-a (TNF-a) and others. IL-6 has a pleiotropic effect on immune cells (it activates B and T-lymphocytes), stimulates the liver to produce acute phase protein and is thought to be one of the most important markers of inflammation. The elevated IL-6 levels in patients with unstable angina support the hy- pothesis that IL-6 participates in atherogenesis [20].

MCP-1 is the most important chemotactic factor involved in the migration of monocytes into the subintimal space. MCP-1 production is stimulated by oxidized low density lipoprotein and such cytokines as IL-1b, TNF-a and INF-g [28, 29]. The role of MCP-1 in atherogenesis has been confirmed in trans- genic mice knocked out of the MCP-1 gene (or the MCP receptor gene) in which, despite high cholesterol levels, the lack of MCP-1 or its receptor inhibited monocyte migration into the arterial wall [5, 11].

While the injury-related migration of monocyte into the subendothelial space is a physiological process, the persistence and acceleration of this phenomenon leads to atherosclerotic plaque formation.

The treatment of atherosclerosis focuses mainly on lipid disturbances. However, recent trends have turned attention to the potential anti-inflammatory action of drugs commonly used in the treatment of lipid metabolism disorders. Fibrates are first-line drugs for the treatment of type IIb dyslipidemia [2]. Their effect is mediated by peroxisome proliferator activated receptors (PPARs), which transfer signals triggered by fat- soluble compounds to the nucleus. Fibrates are artifi- cial activators of PPAR-a [27]. The activation of PPARs leads to the inhibition of various nuclear factors, such as NF-kB, AP-1 and STAT, which eventu- ally modulate proinflammatory gene expression [6].

PPAR activation has been reported to produce varied effects. In adipocytes, it intensifies synthesis of lipoprotein lipase (LPL). In hepatocytes PPAR-a is re- sponsible for enhanced fatty acid uptake andb-oxidation and diminished apolipoprotein CIII production.

HDL [25]. Recent data show that PPAR-a activators play an important role not only in lipid metabolism, but also in coagulation and inflammatory processes. It has been proven, that PPAR-a activators decrease in vitro and in vivo both plasminogen activator inhibitor (PAI) and fibrinogen levels [3, 23]. However, the effect of fibrates on inflammatory parameters is still being studied. There are some studies showing the effect of fibrates on plasma concentrations of proinflammatory cytokines [17, 18]. Contrary, there are just a few experiments evaluating fibrate influence on cytokines secreted by cultured monocytes.

The aim of our study was to evaluate the effect of micronized fenofibrate on the release of IL-1b, IL-6 and MCP-1 by monocytes in patients with combined hyperlipidemia.

Materials and Methods

Patients

The study was carried out in 14 patients with biochemically confirmed combined hyperlipidemia (type IIb dyslipidemia according to the Friedrickson classi- fication). The patients had to meet the following in- clusion criteria: TC > 200 mg/dl, LDL-C > 135 mg/dl and TG > 200 mg/dl. In each patient, red blood cell count (RBC), white blood cell count (WBC), leuko- cyte differentiation, liver enzyme marker tests were conducted, HBs antigen, HCV antibodies, protein electrophoresis and urinalysis were performed.

Patients with secondary dyslipidemias as well as those with obesity (BMI > 30 kg/m²), myocardial in- farction, stroke or any serious disease within 6 months before the study start-up were excluded. Subjects re- ceiving drugs affecting inflammatory processes in the arterial wall (angiotensin converting enzyme inhibi- tors, calcium channel antagonists and non-steroidal anti-inflammatory drugs) and drugs affecting lipid metabolism were also excluded. Eligible patients were treated with micronized fenofibrate (Lipanthyl, Fournier) at a daily dose of 267 mg for 30 days.

The control group included 12 age-matched subjects with normal lipidograms.

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Lipid parameters

Lipid profile including TC, HDL and TG was determined with bioMerieux enzymatic kits 12 h after the last meal.

Isolation of peripheral blood mononuclear cells

The 5 ml of blood was collected between 8.00 and 10.00 a.m. to avoid circadian fluctuations of the studied parameters. Peripheral blood mononuclear cells (PBMC) were separated by Histopaque (Sigma, USA) density gradient centrifugation. Monocytes were isolated from PBMC by negative immunomagnetic sepa- ration using Pan-T and Pan-B Dynabeads (Dynal) [10].

The isolated cells were labeled with monoclonal anti- body (Daco) against the monocyte-specific antigen CD14. The procedure gave 90% of CD14-positive cells in the isolated fraction. The viability of the im- munomagnetically isolated cells was more than 98%

as assessed by trypan blue exclusion test.

Monocyte cultures

The isolated monocytes, 1 × 10⁶monocytes per 1 ml, were placed in a plastic 24-well microliter plate (Bec- ton, Dickinson) and left for 2 h to let them adhere to the bottom. Afterwards, the medium composed of RPMI 1640, 10% of FCS (low in endotoxin) (Gibco), 2 mM glutamine, 100 U/ml of penicilin, 100mg/ml of streptomycin and 10mg/ml of fungizone (Gibco) were added, and the cultures were incubated for 24 h. Con- sequently, the medium was replaced with medium of similar composition, supplemented with a submaxi- mal dose of LPS (Sigma; 1mg/ml) and incubated for 48 h. The supernatant was collected, centrifuged and frozen at –80°C until assayed for IL-1b, IL-6 and MCP-1 levels.

Measurement of concentrations of cytokines by ELISA

Cytokines were estimated using commercial ELISA kits (R&D) according to the manufacturer’s instruc- tions. All serum supernatant samples were stored at –80°C and were assayed at the same time by the same ELISA to avoid variation of assay conditions. The detection limits of IL-1b, IL-6 and MCP-1 were 1 pg/ml, 0.7 pg/ml and 5 pg/ml, respectively. The intra-assay coefficients of variation for IL-1b, IL-6 and MCP-1 were 6.9%, 5.2% and 10.1%, respectively.

Statistical analysis

All values were expressed as the means ± standard error (AVG ± SE). Statistical analysis of data was performed using one-way ANOVA. If the ANOVA re- vealed significance, differences between the means were evaluated using thepost-hoc Bonferroni test. To compare pre- and post-treatment values the Wilcoxon test was performed. Differences were considered to be significant if p < 0.05. All statistical calculations were performed using the GraphPad Prism 2.01 software (GPA-26576-117).

The study was accepted by the Ethics Committee of the Medical University of Silesia. The investiga- tion conforms to the principles outlined in the Decla- ration of Helsinki.

Results

Baseline characteristics

Baseline characteristics of participants of the study are listed in Table 1. There were no marked differences between the two groups in demographic characteristics and such risk factors as BMI and smoking.

CHD and hypertension were more often observed in hyperlipidemic patients.

Lipidogram

Baseline and post-treatment lipid parameters are shown in Table 2. Dyslipidemic patients showed sig-

Monocyte suppressing action of fenofibrate

Bogus³aw Okopieñ et al.

Tab. 1.Baseline characteristics of participants

Parameter Fenofibrate

group (n = 14)

Control group (n = 12)

Sex (F/M) 6/8 6/6

Age (years)* 51.2 ± 3.1 50.3 ± 3.0

Range of age 37–64 36–62

Mild or moderate hypertension (n) 5 3

Coronary heart disease (n) 7 1

BMI (kg/m)* 28 ± 0.7 27 ± 0.5

Smokers (n) 3 3

* values are the mean ± standard error (SE)

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nificant lipid disturbances compared to the control.

Fenofibrate treatment markedly decreased TC, TG and LDL-C levels. Moreover, fenofibrate increased HDL levels, though this effect was not statistically significant.

Inflammatory cytokines

In dyslipidemic patients IL-1b, IL-6 and MCP-1 were significantly higher than those in control (143.9 ± 6.5 pg/ml vs. 74.4 ± 4.4 pg/ml, p < 0.001; 8212 ± 285 pg/mlvs. 6110 ± 170 pg/ml, p < 0.001 and 19.6 ± 0.9 ng/mlvs. 12.3 ± 0.6 ng/ml, p < 0.001, respectively).

After fenofibrate treatment, the cytokines’ levels were as follows (Fig. 1):

– IL-1b 143.9 ± 6.5 pg/ml vs. 86.2 ± 5.9 pg/ml, p < 0.001; reduction by 43.3%

– IL-6 8212 ± 285 pg/ml vs. 6330 ± 234 pg/ml, p < 0.001; reduction by 22.5%

– MCP-1 19.6 ± 0.9 ng/ml vs. 14.0 ± 0.8 ng/ml, p < 0.001; reduction by 28.5%

Discussion

This study has demonstrated that 1-month micronized fenofibrate treatment 1) normalizes lipid parameters i.e. it decreases TC, LDL-C and TG plasma levels and increases HDL-C, and 2) suppresses inflammatory processes by inhibiting monocyte activity in patients with combined hyperlipidemia.

As expected, the effect of fenofibrate on TG levels was the most pronounced. The marked reduction of LDL-C (29%) is also important in the light of recent

thought to modify the structure of the LDL particle rather than to decrease its concentration. The expected increase in HDL-C level did not reach statistical significance in this study.

There are many studies [1, 14] showing that IL-1b, a primary proinflammatory cytokine, and IL-6, a secondary proinflammatory cytokine, are elevated in patients with combined hyperlipidemia. Similarly, numerous studies show that MCP-1 plasma levels are increased in this group [16, 18]. In contrast, a few studies [17, 30] focus on the source of the cytokines.

The present study shows not only that monocytes are one of the sources of proinflammatory cytokines, but also that fenofibrate affects the proinflammatory action of monocytes.

Since it has been proved that macrophages involved in the formation of atherosclerotic plaque originate from blood monocytes [13], one can conclude that the inhibited cytokine release by cultured monocytes reflects the processes operating within the atherosclerotic plaque.

The effect of fibrates on monocyte activity seems to be multidirectional. The oxidized LDL particles are known to activate monocytes to produce cytokines and, therefore, it is highly probable that the elevated plasma LDL level was responsible for the increased cytokine release. As fibrates decrease LDL-C plasma levels and inhibit oxidation processes, it is tempting to conclude that this is one of important mechanisms contributing to the decreased cytokine release.

The direct effect of fibrates on monocytic cytokine synthesis is still being evaluated. Fibrates, due to PPAR-a activation, inhibit nuclear factor-k-B (NF-kB), a transcription factor, which is involved in the gene expression of numerous cytokines. Inhibition of MCP-1 gene expression was observed in mice treated with fibrates [9]. There are very few data on the influence of PPAR-a activators on IL-1b and IL-6 gene expression. As expected, they inhibit IL-1b-induced IL-6 and CRP mRNA expression [7, 15]. Similarly, the IL-1b-dependent expression of CRP, cyclooxyge- nase and NO synthase genes is disturbed by PPAR-a agonists [12].

While IL-1b and IL-6 participate rather in the formation and rupture of atherosclerotic plaque, MCP-1 takes part in recruiting monocytes from blood and in their migration into the subintimal space. Conse- Baseline After treatment

TC (mg/dl) 277.2 ± 12.3^^ 217.8 ± 8.4* 197.3 ± 3.6 TG (mg/dl) 316.7 ± 27.7^^^ 220.6 ± 21.8* 135.8 ± 18.4 LDL-C (mg/dl) 183.6 ± 12.7^ 129.4 ± 5.2* 130.4 ± 11.7 HDL-C (mg/dl) 35.1 ± 3.7^^ 41.9 ± 3.2 52.0 ± 2.6

TC total cholesterol, TG triglycerides, LDL-C low-density lipoprotein, HDL-C high-density lipoprotein * p < 0.05, ** p < 0.01,

*** p < 0.001 statistical significance vs. baseline results. ^ p < 0.05,

^^ p < 0.01, ^^^ p < 0.001 statistical significance vs. control group

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quently, fibrates are expected to prevent these processes.

The results of our study have not explained whether fenofibrate suppresses monocytes directly or through its lipid-lowering action or by other mechanisms. Kowalski et al. [17] showed that hypolipidemic drugs (fenofibrate and statins) decrease MCP-1 release in the hyperlipidemic patients to levels below the control (diet treatment) value, which suggests both lipid and extralipid mechanisms of their action.

To elucidate this issue, we undertook experiments to analyze both secretion of IL-1b, IL-6 and MCP-1 and their mRNA expression in monocyte culture treated with fenofibrate.

The anti-monocyte action of fibrates has many potential advantages. Important agents that affect atherosclerotic plaque stability are metalloproteinases, the enzymes produced by activated monocytes [12] and said to be crucial in atherosclerotic plaque rupture. It has been proven that IL-1b and IL-6 increase metalloproteinase production and secretion [3]. It is, therefore, reasonable to suppose that fibrates can stabilize the plaque by inhibiting metalloproteinase production.

This conclusion is supported by Shu et al. [26], who showed that the release of matrix metalloproteinase 9 (MMP-9) by human monocytes was inhibited by fibrates.

Depending on the stage of atherosclerosis, fibrates can either inhibit the early phase of vascular changes or stabilize advanced atherosclerotic plaques. The ability of fibrates to prevent formation and rupture of the atherosclerotic plaque may not only slow the course of atherosclerosis, but also plays a key role in the prevention of sudden coronary and cerebral epi- sodes.

In conclusion, a monocyte activation coexists with lipid disturbances in patients with combined hyperlipidemia. We have shown that fibrate treatment not only corrects lipid parameters, but also markedly reduces monocyte activity.

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Monocyte suppressing action of fenofibrate

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x1000

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Fig. 1.Effects of micronized fenofibrate on monocytic release of proinflammatory cytokines

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Received:

October 8, 2004; in revised form: March 7, 2005.