This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Dunzendorfer, S. Articles by Wiedermann, C. J. Search for Related Content PubMed PubMed Citation Articles by Dunzendorfer, S. Articles by Wiedermann, C. J.

(Circulation Research. 1997;81:963-969.)
© 1997 American Heart Association, Inc.

Articles

Mevalonate-Dependent Inhibition of Transendothelial Migration and Chemotaxis of Human Peripheral Blood Neutrophils by Pravastatin

Stefan Dunzendorfer, Dorothea Rothbucher, Peter Schratzberger, Norbert Reinisch, Christian M. Kähler, , Christian J. Wiedermann

From the Department of Internal Medicine, University of Innsbruck (Austria).

Correspondence to Dr Christian J. Wiedermann, Department of Internal Medicine, Anichstrasse 35, A-6020 Innsbruck, Austria. E-mail Christian.Wiedermann{at}uibk.ac.at

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract Pravastatin, a hydrophilic inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, has been reported to beneficially affect atherogenesis, plaque stability, and transient myocardial ischemia in significant coronary artery disease by influencing lipid metabolism and by intracellular signaling via mevalonate pathway products other than cholesterol. Leukocytes are implicated to play a pathophysiological role in these events. We were interested in finding out whether pravastatin could affect transendothelial migration (TEM), chemotaxis, and respiratory burst activity of the neutrophil ex vivo. In addition, effects on monocyte and T-lymphocyte chemotaxis were tested. For TEM assays, monolayers of human umbilical vein endothelial cells (HUVECs) were grown to confluence on polycarbonate filters bearing 5-µm pores in Transwell (Costar) culture plate inserts. Chemotaxis experiments were performed using modified Boyden chambers with cellulose nitrate micropore filters. Respiratory burst activity was measured fluorometrically. Treatment of neutrophils and monocytes with pravastatin at 2 to 200 µmol/L and 10 to 1000 µmol/L, respectively, significantly decreased chemotaxis triggered by fMet-Leu-Phe. This effect was abolished in the presence of mevalonic acid (500 µmol/L); no effect of pravastatin was seen on T-lymphocyte chemotaxis triggered by interleukin-8. Preincubation of neutrophils with pravastatin (200 µmol/L) also resulted in a significant reduction in the number of neutrophils that transmigrated a tumor necrosis factor–stimulated or lipopolysaccharide-stimulated HUVEC monolayer. At none of the concentrations tested (2 pmol/L to 200 µmol/L) did pravastatin affect neutrophil respiratory burst activity. We conclude that pravastatin may alter monocyte chemotaxis and neutrophil-endothelial interactions in migratory responses at concentrations obtained in vivo with cholesterol-lowering doses.

Key Words: atherosclerosis • neutrophil • 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Intense activation of neutrophils in unstable angina and acute myocardial infarction has been described as being related to ongoing in vivo cellular activation.¹ In ischemic injury, neutrophils rapidly accumulate in the areas of injury,² where they discharge oxygen-derived free radicals, proteolytic enzymes, and mediators of inflammation.³ The increased neutrophil chemotactic activity and arachidonic acid metabolite generation may be markers of angina pectoris.¹ Mechanisms of neutrophil activation in coronary heart disease are largely unknown and may involve priming with hormones and proinflammatory mediators.⁴ The activation of an increased number of neutrophils may be associated with increased risk of acute myocardial infarction,^5–8 its recurrence,⁹ and the incidence of ventricular fibrillation in the postinfarction period.¹⁰ Neutrophils may occasionally be found in disrupted plaques beneath coronary thrombi and may also migrate into the arterial wall shortly after reperfusion of occluded arteries in response to ischemia/reperfusion.¹¹

Mevalonate is the product of HMG CoA reductase (E.C. 1.1.1.34), a major regulatory enzyme of cholesterol biosynthesis. The mevalonate pathway produces isoprenoids that are vital for diverse cellular functions, ranging from cholesterol synthesis to growth control.^12,13 A nonsterol isoprenoid product has been found to be required for natural killer cell chemotaxis,¹⁴ and inhibition of mevalonate biosynthesis was associated with a reduction in the chemotactic migration of monocytes.¹⁵ Treatment of peripheral blood neutrophils with compactin, an HMG CoA reductase inhibitor, produced no effect on the ability of these cells to support respiratory burst activity in response to fMLP, whose initial product is superoxide anion generated by an NADPH oxidase.¹⁶ Treatment of neutrophils during differentiation from HL-60 cells, however, inhibited superoxide anion formation due to a blockage of isoprenoid synthesis, suggesting that an isoprenoid pathway intermediate is necessary for the activation of the neutrophil NADPH oxidase during differentiation of the cells.¹⁶ Thus, in part, the beneficial effects of HMG CoA reductase inhibitors in the prevention of cardiovascular events could be due to the manipulation of the regulatory system of the mevalonate pathway in leukocytes unrelated to cholesterol biosynthesis.¹³

Neutrophil-endothelial interactions and neutrophil migration into the subendothelial or interstitial space are critical events in the pathophysiology of inflammatory diseases, including acute coronary syndromes.^11,17 The effects of HMG CoA reductase inhibition on neutrophil-endothelial interaction and migration are unknown. Thus, the present study was undertaken to determine the effects of PN, a hydrophilic HMG CoA reductase inhibitor,¹⁸ on (1) neutrophils and endothelium for enhanced transmigration, (2) concentration gradient–dependent chemotaxis, and (3) respiratory burst activity of the leukocytes. Furthermore, the effects of PN on human peripheral blood monocytes and T-lymphocyte chemotaxis were tested.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
RPMI 1640 with or without phenol red and FCS were from Biological Industries, Kibbutz Beit Haemek, Israel. BSA was from Behring Werke AG. ECGM with low serum contents was from Promo Cell, Bioscience Alive. Human fibronectin (ECAF) was from Biomedica. Collagenase was from Seromed, Biochrom KG. Transwell culture plate inserts (6.5-mm diameter, 5-µm pore size, Transwell 3421) were from Costar. FITC-labeled BSA and MTT were from Sigma Chemical Co. PN (Bristol-Myers Squibb) was dissolved in HBSS to a stock solution of 4 mmol/L. TNF- and endotoxin (LPS, from Escherichia coli serotype 026:B6) (both from Sigma) were diluted to stock solutions in 0.2N acetic acid. DCFH-DA (Molecular Probes) was dissolved in N-N-dimethylformamide to a stock solution of 10 mmol/L. MACS magnetic microbeads were from Miltenyi Biotec. All stock solutions, except PN (4°C), were stored at -20°C before use. All other reagents not further specified were from Sigma.

Neutrophil Isolation
Neutrophils were obtained from the peripheral blood of healthy volunteers (anticoagulated with EDTA) or from buffy coat residues (mixed with normal saline in a ratio of 3:1) after discontinuous density gradient centrifugation on Percoll by dextran sedimentation and centrifugation through a layer of Ficoll-Hypaque, followed by hypotonic lysis of contaminating erythrocytes using sodium chloride solution.⁴ Cell preparations yielded >95% neutrophils (by morphology in Giemsa stains) and >99% viability (by trypan dye exclusion). Chemotaxis experiments were performed in RPMI 1640/0.5% BSA.

MNC Isolation
MNCs were isolated from buffy coats by Ficoll-Paque density gradient centrifugation and washed in HBSS as described previously.¹⁹ The MNC preparations (>98% viability by trypan blue exclusion) were suspended at 1x10⁶ cells/mL in RPMI 1640 medium containing 0.5% BSA.

T-Lymphocyte and Monocyte Isolation
The MNC preparations (>98% viability by trypan dye exclusion) were resuspended in RPMI 1640 containing 20% FCS (both from Biological Industries) and put into tissue culture dishes (1x10⁶ cells/mL). After 30 minutes of incubation at 37°C, PBLs were put into new tissue culture dishes and incubated for another 90 minutes. PBLs were then washed and resuspended in RPMI 1640 containing 0.5% BSA (Behringwerke AG). Negative selection of T lymphocytes (CD19^- cells) and positive selection of monocytes (CD14⁺ cells) was performed by adding MACS colloidal superparamagnetic microbeads conjugated with monoclonal anti-human CD19 antibodies (Miltenyi Biotec) to a cooled, freshly prepared PBL suspension in MACS buffer (PBS with 5 mmol/L EDTA and 0.5% BSA) or by adding monoclonal anti-human CD14 antibodies to MNC preparations, according to the manufacturer's instructions. Cells and microbeads were incubated for 15 minutes at 6°C with occasional gentle mixing. Concurrently, the separation column was washed, incubated with MACS buffer, and positioned in the MACS strong magnetic field at room temperature. The column was flushed with ice-cold MACS buffer before the separation procedure. Recommended volumes of ice-cold MACS buffer were added to the cell/microbead mixture, and cells were gently resuspended. The cell suspension was loaded onto the top of the separation column. CD19⁺ lymphocytes or CD14⁺ cells from MNC preparations were trapped and remained in the separation column. The effluent containing CD19^- lymphocytes was collected, washed, and resuspended in RPMI 1640 containing 0.5% BSA. For monocyte isolation, the remaining cells were washed out of the column after removal of the magnet and were collected as a positive fraction. As for lymphocytes, monocytes were also washed and resuspended in RPMI 1640/0.5% BSA. To increase sensitivity, the separation procedures were repeated for a second time. Purity of sorted CD19^- and CD14⁺ cells was >95%, as determined by fluorescence automated cell sorting.

Chemotaxis Experiments
Chemotaxis of neutrophils, monocytes, or T lymphocytes into cellulose nitrate to gradients of soluble attractants was measured using a 48-well microchemotaxis chamber (Neuroprobe) in which a 5-µm pore-sized filter (Sartorius) separates the upper and lower chamber. Cells were incubated for 30 minutes with various concentrations of PN (0.2 nmol/L to 200 µmol/L) or remained untreated and were washed before testing for chemotaxis. To investigate the effect of mevalonic acid on PN-affected neutrophil chemotaxis, cells received PN and mevalonic acid simultaneously for 30 minutes before they were washed and placed into the upper chamber (5x10⁴ per well in RPMI 1640/BSA 0.5%). Neutrophils and monocytes were allowed to migrate toward the soluble attractants in the lower chambers for 35 minutes and 90 minutes, respectively, at 37°C in a humidified atmosphere (5% CO₂). Incubation time for T-lymphocyte chemotaxis was 120 minutes. After this incubation time, the nitrocellulose filters were dehydrated, fixed, and stained with hematoxylin-eosin. Migration depth of cells into the filter was quantified by microscopy, measuring the distance (µm) from the surface of the filter to the leading front of three cells. Data are expressed as chemotaxis index, which is the ratio between the distances of directed and undirected migration of neutrophils into the nitrocellulose filters.

Respiratory Burst of Neutrophils
Respiratory burst activity of neutrophils was detected by an assay using DCFH-DA. This assay is based on the oxidation of nonfluorescent DCFH-DA to highly fluorescent 2',7'-dichlorofluorescein both intracellularly and extracellularly.²⁰ Neutrophils were primed for 30 minutes at 37°C (5% CO₂ atmosphere) with various concentrations of PN (0.2 nmol/L to 200 µmol/L), washed twice, and resuspended in HBSS. Then 100 µL/well (96-well plate, Falcon 3072) of 2x10⁵ neutrophils were immersed at 37°C in a 1x10^-5 mol/L solution of DCFH-DA in phenol red–free HBSS containing 1 µmol/L of fMLP, as a triggering agent, or medium. The plates were covered with lids and placed in a humidified incubator (95% air/5% CO₂) for various time intervals. Fluorescence activity was determined at 485±20-nm excitation and 530±25-nm emission wavelengths using the CytoFluor 2350 fluorescence measurement system (Millipore Corp). Since the cells in the plates were not disturbed by the measuring procedure, the plates could be incubated for various time periods and reread as desired. Readings were taken every 10 minutes for 1 hour.

Endothelial Cell Culture
HUVECs from fresh placental cords were isolated and grown to confluence at 37°C in 5% CO₂.²¹ The growth medium was a complete HUVEC growth medium (ECGM) supplemented with 10% FCS. Tissue culture flasks were coated with 1 µg/cm² human fibronectin (ECAF) before the seeding of HUVECs. Cells were passaged by treatment for 3 minutes with collagenase. HUVECs used for experiments were from passages 2 to 4.²¹

Culture of HUVECs on Transwell Tissue Culture Plates
At confluence, HUVECs were detached as described and seeded out on polyvinylpyrrolidone-free polycarbonate filters bearing 5-µm pores in Transwell culture plate inserts. The filters were prepared by coating with ECAF, followed by removal of excess fluid and air drying. HUVECs at 1.0x10⁵ cells per culture plate insert were added to the cups above the filter in 0.1 mL ECGM, and 0.6 mL of the same medium was added to the lower compartment beneath the filter. HUVECs formed a tight permeability barrier within 4 to 6 days. Medium was exchanged for fresh medium the day after seeding and 2 days before the monolayers were used for transendothelial migration assays.²¹

Test of Endothelial Cell Monolayer Permeability
To evaluate the functional integrity of the HUVEC monolayer, medium was removed, and 100 µL of colorless RPMI 1640, containing 100 µg/mL FITC-labeled BSA, was added to the upper compartment above the HUVEC monolayers on the filters. The lower compartment contained colorless RPMI 1640 without BSA and FITC; 10 minutes to 4 hours later, 50-µL samples were taken from the upper compartment and from the lower chamber beneath the HUVEC monolayer growing on the polycarbonate filter, and fluorescent activity was measured using the CytoFluor 2350 fluorescence measurement system (Millipore Corp). The ratio of fluorescence activity between the two samples was calculated. Without HUVEC monolayers, 25% of equilibrium developed within 10 minutes, which increased to roughly 50% after 4 hours. In contrast, when HUVECs were cultured to confluence on the micropore inserts, <1% and 5% of equilibrium was measured after 10 minutes and 4 hours, respectively, indicating that a tight barrier was formed.

Culture of HUVECs on 96-Well Tissue Culture Plates
For cell survival experiments, HUVECs were cultured on ECAF-treated 96-well tissue culture plates according to the methods described above. HUVECs were passaged and seeded on the plates (10⁵ cells in 100 µL/well), where they grew to confluence within 5 days.

Assay of Cytotoxicity and Cell Survival
For detection of cytotoxicity and cell survival, we used the MTT test. This colorimetric assay is based on the conversion of the tetrazolium salt MTT to formazan crystals. It detects living but not dead cells, and the absorbance generated is directly proportional to the number of cells.²² MTT was dissolved in Dulbecco's PBS without Ca²⁺ and Mg²⁺ to a final concentration of 5 mg/mL.

When HUVEC monolayers in 96-well plates had grown to confluence, cells were washed twice with PBS and incubated for 60 minutes with various concentrations of PN. Thereafter, monolayers were washed, and 20 µL/well of MTT solution and 30 µL/well of colorless RPMI 1640 were added for 6 hours. During this period of time, MTT was converted to formazan crystals, which were subsequently dissolved by the addition of 150 µL dimethyl sulfoxide/well. The plates were agitated for 5 minutes on a plate shaker, and the optical density was then read with an ELISA reader at 550 nm (Labsystems Multiskan).

Cytotoxicity and cell survival of neutrophils and MNCs were measured in an identical fashion, except that nonadherent cells were pelleted by centrifugation before and after washing.

Assay of Neutrophil Transmigration of HUVEC Monolayers
For transmigration experiments,^21,23 confluent HUVEC monolayers grown on Transwell filters were washed twice with PBS, incubated with PN (200 mmol/L) alone or in combination with TNF (10 ng/mL) or LPS (10 µg/mL) for 4 hours, and then washed again twice for the removal of all agents. RPMI 1640/0.5% BSA, which was added to the upper compartment, was used to detect random migration. Incubation of the monolayers with the test substances was followed by washing of the upper and lower surfaces of the Transwell filter cups with PBS and then transferring them to new clean wells (lower compartments). There, neutrophil suspensions (0.1 mL, unstimulated) were added to the upper compartments (cups), and neutrophils were allowed to migrate across the HUVEC monolayers for 45 minutes. In another set of experiments, HUVEC monolayers were stimulated with TNF (10 ng/mL) or LPS (10 µg/mL) as described above. Neutrophils were incubated with PN (200 mmol/L) for 30 minutes, washed twice, and then added to the upper compartment for a transmigration period of 45 minutes.

Migration of neutrophils was stopped by washing and thereby removing excess neutrophils from the upper surfaces of the filter cups. Thereafter, the tissue culture plates bearing the Transwell filters were centrifuged in a Beckman GPR centrifuge (2000 rpm, 10 minutes) in order to pellet migrated neutrophils on the bottom of the lower compartments. Finally, pelleted neutrophils were counted in three microscopic fields per cup at a magnification of x100 (Olympus CK2). The number of neutrophils that had migrated across unstimulated HUVECs was 50 cells per microscopic field. Counting results were verified by a sensitive fluorometric assay based on the fluorescence enhancement of propidium iodide on binding to double-stranded nucleic acids.²⁴ Data are expressed as a "transmigration index," which represents the ratio of neutrophil migration across stimulated and unstimulated HUVEC monolayers.

Statistical Methods
Data are expressed as mean±SEM. Means were compared by the Mann-Whitney U test and Kruskal-Wallis ANOVA. Statistical analyses were performed using the StatView software package (Abacus Concepts).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
PN Cytotoxicity
After exposure of HUVEC monolayers and neutrophils or MNCs to PN at concentrations ranging from 0.02 µmol/L to 200 mmol/L for 4 hours and 60 minutes, respectively, viability of the cells was measured using the MTT test. Significant negative effects on HUVEC survival were seen at concentrations of 20 mmol/L (Mann-Whitney U test, P<.01). Thus, in all experiments with HUVECs, the highest concentration of PN tested was 200 µmol/L, which is 100-fold less than the observed toxic concentration for HUVECs. Cytotoxicity of PN for neutrophils and MNCs was tested at concentrations ranging from 0.002 µmol/L to 200 mmol/L, and adverse effects on cell survival were obtained at concentrations of 2 mmol/L (Table 1).

View this table: [in this window] [in a new window]   Table 1. Cytotoxicity of Pravastatin for MNC and PMNL

Monocyte Chemotaxis
Random migration of monocytes into nitrocellulose micropores remained unaffected after preincubation (30 minutes) with PN (0.1 µmol/L to 1 mmol/L) followed by washing of the cells. Directed migration toward fMLP (10 nmol/L) was significantly diminished by incubation of monocytes with 1 µmol/L (Mann-Whitney U test, P<.05) concentrations of PN tested (Mann-Whitney U test, P<.01) (Fig 1).

View larger version (68K): [in this window] [in a new window]   Figure 1. Migration of human monocytes into nitrocellulose micropore filters (5-µm pore size). Cells were incubated at various concentrations of PN (0.1 µmol/L to 1 mmol/L) for 30 minutes at 37°C in a humidified atmosphere, washed twice, and then allowed to migrate for 90 minutes. Data are expressed as mean±SEM (n=4) of the chemotaxis index, which is the distance of stimulated migration divided by that of random migration. Statistical analysis was by Mann-Whitney U test after the Kruskal-Wallis test (P<.01). *P<.05; **P<.01.

T-Lymphocyte Chemotaxis
Neither random migration nor directed migration of T lymphocytes to interleukin-8 (1 nmol/L) was affected by preincubation (30 minutes) with PN (0.02 to 200 µmol/L) (Table 2).

View this table: [in this window] [in a new window]   Table 2. Effects of Pravastatin on T-Lymphocyte Chemotaxis

Neutrophil Chemotaxis
Random migration of neutrophils into nitrocellulose micropores remained unaffected after preincubation (30 minutes) with PN (2 pmol/L to 200 µmol/L) followed by washing of the cells. Continuous presence of PN (2 pmol/L to 200 µmol/L) during neutrophil migration also failed to have any significant effects. Directed migration toward fMLP (10 nmol/L) was significantly diminished after incubation of neutrophils with PN in a dose-dependent manner (tested at 2 pmol/L to 200 µmol/L) (Fig 2). There was no difference in the degree of migration inhibition between cells that were washed after exposure to PN (Fig 2, right panel) and that in which PN was present during the whole migration period (Fig 2, left panel). Addition of 500 µmol/L DL-mevalonic acid lactone to the cells during the incubation period with PN (30 minutes), followed by two washing steps, completely restored the inhibition by PN of directed migration (Fig 3).

View larger version (44K): [in this window] [in a new window]   Figure 2. Migration of human neutrophils into nitrocellulose micropore filters (5-µm pore size). Neutrophils were incubated at various concentrations of PN (2 pmol/L to 200 µmol/L) for 30 minutes at 37°C in a humidified atmosphere, washed twice, and then allowed to migrate for 35 minutes (right panel). There was a continuous presence of PN (2 pmol/L to 200 µmol/L) during the neutrophil migration period of 35 minutes (left panel). Data are expressed as mean±SEM (n=9) of the chemotaxis index, which is the distance of stimulated migration divided by that of random migration. Statistical analysis comparing the highest concentrations of PN tested for preincubation and coincubation revealed a value of P=.121. Statistical analysis was by Mann-Whitney U test after the Kruskal-Wallis test (P<.01). **P<.01.

View larger version (88K): [in this window] [in a new window]   Figure 3. Influence of mevalonic acid on PN-inhibited neutrophil chemotaxis. Human neutrophils were incubated simultaneously with PN (0.2 nmol/L to 200 µmol/L) and DL-mevalonic acid lactone for 30 minutes, followed by two washing steps. The subsequent period of migration into nitrocellulose filters (5 µm) was 35 minutes. Data are expressed as mean±SEM (n=7) of the chemotaxis index, which is the distance of stimulated migration divided by that of random migration. Statistical analysis was by Mann-Whitney U test after the Kruskal-Wallis test (P<.01). *P<.05; **P<.01.

Respiratory Burst of Neutrophils
Cells were washed twice after a 30-minute incubation of neutrophils with PN at various concentrations (2 pmol/L to 200 µmol/L); thereafter, the oxidative burst was triggered with fMLP (10 nmol/L). At all concentrations tested, PN failed to exert significant effects on neutrophil basal and fMLP-triggered respiratory burst activity (Table 3).

View this table: [in this window] [in a new window]   Table 3. Effects of Pravastatin on Neutrophil Respiratory Burst Activity

Effect of PN on HUVECs for TNF- and LPS-Induced Activation of Neutrophil Transmigration
Incubation of endothelial cells with TNF (10 ng/mL) led to a >5-fold increase in the number of unstimulated neutrophils that passaged the monolayer. Similar results were obtained after stimulation of the monolayer with LPS (10 µg/mL). When HUVECs were treated with TNF (10 ng/mL) or LPS (10 µg/mL) for 4 hours in the presence of 200 µM PN and then washed, PN failed to show any effect on TNF- or LPS-induced activation of HUVECs for neutrophil transmigration (Fig 4, left panel).

View larger version (36K): [in this window] [in a new window]   Figure 4. Effect of PN on transendothelial migration of neutrophils (PMNLs). HUVEC monolayers were treated for 4 hours with TNF (10 ng/mL), LPS (10 µg/mL), or medium (RPMI/BSA, 0.5%) with and without PN (200 µmol/L). After two washing steps, unstimulated PMNLs were allowed to transmigrate monolayers (left panel). PMNLs were incubated for 30 minutes with PN (200 µmol/L) or medium, washed twice, and added to unstimulated (medium) or stimulated (TNF [10 ng/mL] or LPS [10 µg/mL] for 4 hours) monolayers (right panel) and allowed to transmigrate. Data are given as mean±SEM (n=3) of the transmigration index. Transmigration index represents the ratio of PMNL migration across stimulated and unstimulated HUVEC monolayers. Statistical analysis was by Mann-Whitney U test after the Kruskal-Wallis test (P<.01). *P<.05.

Effect of PN on Neutrophils for TNF- and LPS-Induced Activation of Transendothelial Migration
Neutrophils were incubated with PN (200 µM) or medium for 30 minutes. After two washing steps, neutrophils were allowed to transmigrate unstimulated or stimulated HUVEC monolayers. Basal migration remained unaffected by treatment of neutrophils with PN (200 µM), whereas neutrophil transmigration across TNF (10 ng/mL)- or LPS (10 µg/mL)- stimulated HUVEC monolayers was significantly reduced (Mann-Whitney U test, P<.05) (Fig 4, right panel).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The data presented here demonstrate that incubation of neutrophils and monocytes with PN inhibits functional parameters of migration resulting from inhibition of mevalonate synthesis. No effect of PN on lymphocyte migration was seen. Although studies in lymphocytes suggested that HMG CoA reductase inhibitors induce alteration in receptor function,²⁵ the inhibition of triggered transendothelial migration and chemotaxis in neutrophils by PN, its reversal by exogenous mevalonate, and at the same time a lack of effect of PN on respiratory burst suggest that a postreceptor event is involved. Previous study has demonstrated that p21^ras is isoprenylated and that this isoprenylation is required for functional association of p21^ras with the membrane.^26,27 If other guanine nucleotide binding proteins (G proteins) present in neutrophils are modified in this way, then inhibition of isoprenoid synthesis by PN could suppress G-protein function.^28,29 G proteins are present in neutrophils and are involved in transducing chemotactic signals.³⁰

Studies in human monocytes have demonstrated that treatment of the monocytes with PN for incubation periods of 12 hours resulted in inhibition of both cholesterol synthesis and chemotaxis.¹⁵ Cholesterol synthesis in monocytes normally requires several hours of incubation to become detectable in in vitro assays.¹⁵ Previously, in a monocytic cell line, HMG CoA reductase inhibition resulted in suppressed respiratory burst activation and cytokine production by altering isoprenoid synthesis.³¹ In order to be absolutely sure that the effects observed in monocyte chemotaxis were indeed cholesterol independent, we repeated the chemotaxis experiments, allowing PN to exert effects on a purified population of monocytes only for 30 minutes, a time period too short to significantly affect cholesterol synthesis. Our result show that short-term incubation with PN also inhibited monocyte chemotaxis in a dose-dependent manner, as it did in neutrophils. It is well known that neutrophils are unable to produce cholesterol in significant amounts.³² This fact along with only a 30-minute incubation of neutrophils with PN supports our conclusion that the inhibitory effect of PN on migration is independent of cholesterol synthesis.

At concentrations of PN that significantly inhibited monocyte and neutrophil chemotaxis, interleukin-8–directed migration of T lymphocytes was unaffected, supporting the conclusion that effects on monocytes and neutrophils are unrelated to cytotoxicity.

At doses of PN that are at least 10 times lower than cytotoxic concentrations, no significant effects on physiologically (fMLP) triggered respiratory bursts of intact neutrophils were observed. In previous publications on the effects of PN on NADPH-related oxidative bursts, inhibition was seen in membrane preparations,³³ a monocytic cell line,³¹ or a myeloid cell line that differentiated to mature neutrophils in the presense of HMG CoA reductase inhibitors.¹⁶ Our failure to find effects of PN on oxidative bursts may be due to the completely different assay systems used. Since we used freshly prepared human peripheral blood neutrophils and triggered respiratory burst in a surface receptor–dependent manner, data are novel and question the in vivo significance of mevalonate-dependent respiratory burst inhibition.^16,31,33

Despite similarities in the chemical structure of HMG CoA reductase inhibitors, pharmacological differences have been observed. PN is more hydrophilic than simvastatin or lovastatin.^18,34 Probably because of this property, they have different inhibitory effects on different cell types. The same inhibitory action of lovastatin, simvastatin, and PN on rat liver sterol synthesis has been observed both in vivo and ex vivo.³⁵ However, in several human extrahepatic cells, including HUVECs, PN was much less efficient in inhibiting sterol synthesis than was lovastatin or simvastatin.³⁶ In the present study, inhibition of transendothelial migration of neutrophils was seen when neutrophils but not HUVECs were pretreated with PN. Lack of sufficient uptake of PN in HUVECs was observed in labeling studies using ¹⁴C-PN,³⁷ which may explain our observation of cell type–dependent inhibition of transmigration.

Recent studies of dose-response relationships of PN and lipophilic HMG CoA reductase inhibitors, such as fluvastatin, in smooth muscle cell proliferation and in monocyte or hepatocyte cholesterol synthesis revealed that PN exerts significant effects on monocytes and hepatocytes at concentrations reached by an in vivo treatment.¹⁸ In contrast, compared with fluvastatin, PN showed no effect at therapeutic doses on smooth muscle cell proliferation.³⁸ Since effects of PN on neutrophils occurred at concentrations lower than necessary for monocyte effects, we suggest that neutrophils may be affected at plasma levels obtained with therapeutic doses of 20 to 40 mg PN daily (50 to 100 µmol/L)¹⁸; hence, our observations may have clinical relevance. Since monocyte invasion of the arterial wall is important in the generation and progression of atherosclerosis, the clinical effects of HMG CoA reductase inhibitors may be due in part to direct alteration of monocyte function by manipulation of isoprenylation. In addition, inhibition of neutrophil transendothelial migration and chemotaxis without affecting endothelial cell functions may be of relevance in neutrophil-dependent effects in acute coronary syndromes, including effects on coagulation, vessel wall inflammation, plaque rupture, and ischemia/reperfusion injury.

	Selected Abbreviations and Acronyms

 

DCFH-DA = 2',7'-dichlorofluorescein diacetate ECAF = endothelial cell attachment factor ECGM = endothelial cell growth medium fMLP = fMet-Leu-Phe HMG CoA = 3-hydroxy-3-methylglutaryl coenzyme A HUVEC = human umbilical vein endothelial cell LPS = lipopolysaccharide MNC = mononuclear leukocyte MTT = 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide PBL = nonadherent mononuclear leukocyte PN = pravastatin TNF = tumor necrosis factor

	Acknowledgments

 
This study was supported by the Austrian Science Funds grant No. 09977 to Dr Wiedermann and by Bristol-Myers-Squibb, Vienna, Austria.

Received March 24, 1997; accepted September 11, 1997.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Mehta J, Dinerman JW, Mehta P, Saldeen TGP, Lawson D, Donelly WH, Wallin R. Neutrophil function in ischemic heart disease. Circulation. 1989;79:549–556.[Abstract/Free Full Text]

2. Robbins SL, Cotran RS, Kumar N. Inflammation, and Repair in Pathologic Basis of Disease. Philadelphia, Pa: WB Saunders; 1984:40–49.

3. Fantone JL, Ward PA. Role of oxygen derived free radicals and metabolites in leukocyte dependent inflammatory reactions. Am J Pathol. 1982;107:394–418.

4. Wiedermann CJ, Niedermühlbichler M, Braunsteiner H. Priming of polymorphonuclear neutrophils by atrial natriuretic peptide in vitro. J Clin Invest. 1992;89:1580–1586.

5. Friedman GD, Klatsky AL, Siegelaub AB. The leukocyte count as a predictor of myocardial infarction. N Engl J Med. 1974;290:1275–1278.

6. Zalokar JB, Richard JL, Claude VR. Leukocyte count, smoking and myocardial infarction. N Engl J Med. 1981;304:465–468.[Medline] [Order article via Infotrieve]

7. Schlant RC, Forman S, Stamler V, Canner PL. The natural history of coronary heart disease: prognostic factors after recovery from myocardial infarction in 2789 men. Circulation. 1982;66:401–408.[Free Full Text]

8. Prentice RL, Shuimizu Y, Lin CH, Petersen AV, Kato H, Mason MW, Szatrowski TP. Leukocyte counts and coronary heart disease in a Japanese cohort. Am J Epidemiol. 1982;116:496–506.[Abstract/Free Full Text]

9. Lowe GD, Machado SG, Krol WF, Barton BA, Forbes CD. White blood cell count and haematocrit as predictors of coronary recurrence after myocardial infarction. Thromb Haemost. 1985;54:700–703.[Medline] [Order article via Infotrieve]

10. Maisel AS, Gilpin EA, LeWinter M, Henning H, Ross J Jr, Engler R. Initial leukocyte count during acute myocardial infarction independently predicts early ventricular fibrillation. Circulation. 1985;72(suppl III):III-414. Abstract.

11. Falk E, Shah PK, Fuster V. Coronary plaque disruption. Circulation. 1995;92:657–671.[Free Full Text]

12. Goldstein JL, Braun MS. Regulation of the mevalonate pathway. Nature. 1990;343:425–430.[Medline] [Order article via Infotrieve]

13. Vaugham CJ, Murphy MB, Buckley BM. Statins do more than just lower cholesterol. Lancet. 1996;348:1079–1082.[Medline] [Order article via Infotrieve]

14. Cutts JL, Bankhurst AD. Reversal of lovastatin-mediated inhibition of natural killer cell cytotoxicity by interleukin-2. J Cell Physiol. 1990;145:244–252.[Medline] [Order article via Infotrieve]

15. Kreuzer J, Bader J, Jahn L, Hautmann M, Kubler W, von Hodenberg E. Chemotaxis of the monocyte cell line U937: dependence on cholesterol and early mevalonate pathway products. Atherosclerosis. 1991;90:203–915.[Medline] [Order article via Infotrieve]

16. Bokoch GM, Prossnitz V. Isoprenoid metabolism is required for stimulation of the respiratory burst oxidase of HL-60 cells. J Clin Invest. 1992;89:402–408.

17. Adams DH, Shaw S. Leukocyte-endothelial interactions and regulation of leukocyte migration. Lancet. 1994;343:831–836.[Medline] [Order article via Infotrieve]

18. Haria M, McTavish D. Pravastatin: a reappraisal of its pharmacological properties and clinical effectiveness in the management of coronary heart disease. Drugs. 1997;53:299–336.[Medline] [Order article via Infotrieve]

19. Wiedermann CJ, Reinisch N, Braunsteiner H. Stimulation of monocyte chemotaxis by human growth hormone and its deactivation by somatostatin. Blood. 1993;82:954–960.[Abstract/Free Full Text]

20. Wan CP, Myung E, Lau BH. An automated micro-fluorometric assay for monitoring oxidative burst activity of phagocytes. J Immunol Methods. 1993;159:131–138.[Medline] [Order article via Infotrieve]

21. Wiedermann CJ, Schratzberger P, Kähler CM. Migration of neutrophils across endothelial monolayers is stimulated by treatment of the monolayer with ß-endorphin. Brain Behav Immun. 1994;8:270–277.[Medline] [Order article via Infotrieve]

22. Mosman T. Rapid colorimetric assay for cell growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65:55–63.[Medline] [Order article via Infotrieve]

23. Moser R, Schleiffenbaum B, Groscurth P. Interleukin 1 and tumor necrosis factor stimulate human vascular endothelial cells to promote transendothelial neutrophil passage. J Clin Invest. 1989;83:444–455.

24. Wan CP, Sigh RV, Lau BH. A simple fluorometric assay for the determination of cell numbers. J Immunol Methods. 1994;173:265–272.[Medline] [Order article via Infotrieve]

25. Cutts JL, Scallen TJ, Watson J, Bankhurst AD. The role of mevalonic acid in the regulation of natural killer cell cytotoxicity. J Cell Physiol. 1989;139:550–557.[Medline] [Order article via Infotrieve]

26. Hancock JF, Magee AJ, Childs JE, Marshall CJ. All ras proteins are polyisoprenylated but only some are palmitoylated. Cell. 1989;57:1167–1177.[Medline] [Order article via Infotrieve]

27. Schafer WR, Kim R, Sterne LR, Thorner J, Kin S-H, Rine J. Genetic and pharmacological suppression of oncogenic mutations in ras genes of yeast and humans. Science. 1989;245:379–385.[Abstract/Free Full Text]

28. Scheer A, Gierschik P. Farnesylcysteine analogues inhibit chemotactic peptide receptor-mediated G-protein activation in human HL-60 granulocyte membranes. FEBS Lett. 1993;319:110–114.[Medline] [Order article via Infotrieve]

29. Chiloeches A, Usera F, Lasa M, Ropero S, Montes A, Toro MJ. Effect of mevalonate availability on the association of G-protein -subunits with the plasma membrane in GH₄C₁ cells. FEBS Lett. 1997;401:68–72.[Medline] [Order article via Infotrieve]

30. Snyderman R, Pike MC, Edge S, Lane B. A chemoattractant receptor on macrophages exists in two affinity states regulated by guanine nucleotides. J Cell Biol. 1984;98:444–448.[Abstract/Free Full Text]

31. Terkeltaub R, Solan J, Barry M Jr, Santoro D, Bokoch GM. Role of the mevalonate pathway of isoprenoid synthesis in IL-8 generation by activated monocytic cells. J Leukoc Biol. 1994;55:749–755.[Abstract]

32. Yachnin S, Toub DB, Mannickarottu V. Divergence in cholesterol biosynthetic rates and 3-hydroxy-3-methylglutaryl-CoA reductase activity as a consequence of granulocyte versus monocyte-macrophages differentiation in HL-60 cells. Proc Natl Acad Sci U S A. 1984;81:894–897.[Abstract/Free Full Text]

33. Heyworth PG, Knaus UG, Xu X, Uhlinger DJ, Conroy L, Bokoch GM, Curnutte JT. Requirement for postranslational processing of Rac GTP-binding proteins for activation of human neutrophil NADPH oxidase. Mol Biol Cell. 1993;4:261–269.[Abstract]

34. Serajuddin AT, Ranadive SA, Mahoney EM. Relative lipophilicities, solubilities, and structure-pharmacological considerations of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors pravastatin, lovastatin, mevastatin, and simvastatin. J Pharmacol Sci. 1991;80:830–834.[Medline] [Order article via Infotrieve]

35. Koga T, Fukuda K, Shimada Y, Fukami M, Koike H, Tsujita Y. Tissue selectivity of pravastatin sodium, lovastatin and simvastatin: the relationship between inhibition of de novo sterol synthesis and active drug concentrations in the liver, spleen and testis in rat. Eur J Biochem. 1992;209:315–319.[Medline] [Order article via Infotrieve]

36. Tsujita Y, Kuroda M, Shimada Y, Tanzawa K, Arai M, Kaneko I, Tanaka M, Masuda H, Tarumi C, Watanabe Y, Fujii S. CS-514, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase: tissue-selective inhibition of sterol synthesis and hypolipidemic effect on various animal species. Biochim Biophys Acta. 1986;877:50–60.[Medline] [Order article via Infotrieve]

37. Van Vliet AK, van Thiel GCF, Huisman RH, Moshage H, Yap SH, Cohen LH. Different effects of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors on sterol synthesis in various human cell types. Biochim Biophys Acta. 1995;1254:105–111.[Medline] [Order article via Infotrieve]

38. Corsini A, Pazzucconi F, Pfister P, Paoletti R, Sirtori CR. Inhibitor of proliferation of arterial smooth-muscle cells by fluvastatin. Lancet. 1996;348:1584. Letter.[Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				S. Dublin, M. L Jackson, J. C Nelson, N. S Weiss, E. B Larson, and L. A Jackson Statin use and risk of community acquired pneumonia in older people: population based case-control study BMJ, June 16, 2009; 338(jun16_1): b2137 - b2137. [Abstract] [Full Text] [PDF]

				B. M. Maher, T. N. Dhonnchu, J. P. Burke, A. Soo, A. E. Wood, and R. W. G. Watson Statins alter neutrophil migration by modulating cellular Rho activity--a potential mechanism for statins-mediated pleotropic effects? J. Leukoc. Biol., January 1, 2009; 85(1): 186 - 193. [Abstract] [Full Text] [PDF]

				S. Nolan, R. Dixon, K. Norman, P. Hellewell, and V. Ridger Nitric Oxide Regulates Neutrophil Migration through Microparticle Formation Am. J. Pathol., January 1, 2008; 172(1): 265 - 273. [Abstract] [Full Text] [PDF]

				M. B. Fessler, S. K. Young, S. Jeyaseelan, J. G. Lieber, P. G. Arndt, J. A. Nick, and G. S. Worthen A Role for Hydroxy-Methylglutaryl Coenzyme A Reductase in Pulmonary Inflammation and Host Defense Am. J. Respir. Crit. Care Med., March 15, 2005; 171(6): 606 - 615. [Abstract] [Full Text] [PDF]

				V. M. Conraads, J. M. Bosmans, A. J. Schuerwegh, L. S. De Clerck, C. H. Bridts, F. L. Wuyts, W. J. Stevens, and C. J. Vrints Association of lipoproteins with cytokines and cytokine receptors in heart failure patients: Differences between ischaemic versus idiopathic cardiomyopathy Eur. Heart J., December 2, 2003; 24(24): 2221 - 2226. [Abstract] [Full Text] [PDF]

				C. P. Tiefenbacher, J. Kapitza, V. Dietz, C.-H. Lee, and F. Niroomand Reduction of myocardial infarct size by fluvastatin Am J Physiol Heart Circ Physiol, June 5, 2003; 285(1): H59 - H64. [Abstract] [Full Text] [PDF]

				Z. T. Bloomgarden Inflammation and Insulin Resistance Diabetes Care, June 1, 2003; 26(6): 1922 - 1926. [Full Text] [PDF]

				M. Sasaki, S. Bharwani, P. Jordan, T. Joh, K. Manas, A. Warren, H. Harada, P. Carter, J. W. Elrod, M. Wolcott, et al. The 3-Hydroxy-3-methylglutaryl-CoA Reductase Inhibitor Pravastatin Reduces Disease Activity and Inflammation in Dextran-Sulfate Induced Colitis J. Pharmacol. Exp. Ther., April 1, 2003; 305(1): 78 - 85. [Abstract] [Full Text]

				N. C. Kaneider, P. Egger, S. Dunzendorfer, P. Noris, C. L. Balduini, D. Gritti, G. Ricevuti, and C. J. Wiedermann Reversal of Thrombin-Induced Deactivation of CD39/ATPDase in Endothelial Cells by HMG-CoA Reductase Inhibition: Effects on Rho-GTPase and Adenosine Nucleotide Metabolism Arterioscler Thromb Vasc Biol, June 1, 2002; 22(6): 894 - 900. [Abstract] [Full Text] [PDF]

				N. C. Kaneider, P. Egger, S. Dunzendorfer, and C. J. Wiedermann Rho-GTPase-Dependent Platelet-Neutrophil Interaction Affected by HMG-CoA Reductase Inhibition With Altered Adenosine Nucleotide Release and Function Arterioscler Thromb Vasc Biol, June 1, 2002; 22(6): 1029 - 1035. [Abstract] [Full Text] [PDF]

				S. Wassmann, U. Laufs, A. T. Baumer, K. Muller, K. Ahlbory, W. Linz, G. Itter, R. Rosen, M. Bohm, and G. Nickenig HMG-CoA Reductase Inhibitors Improve Endothelial Dysfunction in Normocholesterolemic Hypertension via Reduced Production of Reactive Oxygen Species Hypertension, June 1, 2001; 37(6): 1450 - 1457. [Abstract] [Full Text] [PDF]

				O. Feron, C. Dessy, J.-P. Desager, and J.-L. Balligand Hydroxy-Methylglutaryl-Coenzyme A Reductase Inhibition Promotes Endothelial Nitric Oxide Synthase Activation Through a Decrease in Caveolin Abundance Circulation, January 2, 2001; 103(1): 113 - 118. [Abstract] [Full Text] [PDF]

				J. G. Wagner and R. A. Roth Neutrophil Migration Mechanisms, with an Emphasis on the Pulmonary Vasculature Pharmacol. Rev., September 1, 2000; 52(3): 349 - 374. [Abstract] [Full Text] [PDF]

				D. C. Hess, A. M. Demchuk, L. M. Brass, and F. M. Yatsu HMG-CoA reductase inhibitors (statins): A promising approach to stroke prevention Neurology, February 22, 2000; 54(4): 790 - 796. [Abstract] [Full Text] [PDF]

				A. Kaser, S. Dunzendorfer, F. A. Offner, T. Ryan, A. Schwabegger, W. W. Cruikshank, C. J. Wiedermann, and H. Tilg A Role for IL-16 in the Cross-Talk Between Dendritic Cells and T Cells J. Immunol., September 15, 1999; 163(6): 3232 - 3238. [Abstract] [Full Text] [PDF]

				A. M. Lefer, B. Campbell, Y.-K. Shin, R. Scalia, R. Hayward, and D. J. Lefer Simvastatin Preserves the Ischemic-Reperfused Myocardium in Normocholesterolemic Rat Hearts Circulation, July 13, 1999; 100(2): 178 - 184. [Abstract] [Full Text] [PDF]

				P. Schratzberger, S. Dunzendorfer, N. Reinisch, C. M Kahler, M. Herold, and C. J Wiedermann Release of chemoattractants for human monocytes from endothelial cells by interaction with neutrophils Cardiovasc Res, May 1, 1998; 38(2): 516 - 521. [Abstract] [Full Text] [PDF]

This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Dunzendorfer, S. Articles by Wiedermann, C. J. Search for Related Content PubMed PubMed Citation Articles by Dunzendorfer, S. Articles by Wiedermann, C. J.