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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3557-3564.)
© 1997 American Heart Association, Inc.

Articles

Adhesiveness of Mononuclear Cells in Hypercholesterolemic Humans Is Normalized by Dietary L-Arginine

Gregor Theilmeier; Jason R. Chan; Christoff Zalpour; Barbara Anderson; Bing-yin Wang; Andreas Wolf; Philip S. Tsao; ; John P. Cooke

From the Section of Vascular Medicine. Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, Calif.

Correspondence to John P. Cooke, MD, PhD, Division of Cardiovascular Medicine, CVRC, Stanford University School of Medicine, Stanford, CA 94305-5246.

	Abstract

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Abstract Hypercholesterolemia reduces vascular nitric oxide (NO) activity. This dysfunction may promote endothelial monocyte interaction, as NO is a potent inhibitor of cell adhesion. We have previously shown that in hypercholesterolemic (HC) rabbits, chronic oral supplementation of L-arginine (Arg) restores NO activity and inhibits monocyte–endothelial cell interaction, in association with a reduction in atherogenesis. We hypothesized that enhancement of endothelial NO activity in HC humans would reduce monocyte adhesiveness. We used a functional binding assay to assess the adhesiveness of human mononuclear cells (MNCs) ex vivo to determine the effects of hypercholesterolemia and L-arginine administration. MNCs from HC subjects adhered in greater numbers (50% more cells per high-power field; P<.0001) than cells derived from normocholesterolemic (NC) subjects. To determine whether enhancement of endogenous NO activity could inhibit mononuclear cell adhesiveness, in a double-blinded placebo-controlled study, oral arginine HCl (8.4 g/d) was administered to HC subjects. Over a course of 2 weeks, this treatment abolished the increased adhesiveness of HC MNCs (160±11% versus 104±5%; before and after 2 weeks of Arg treatment; results expressed as a percentage of the binding values obtained using cells derived from paired NC individuals). By contrast, MNC adhesion remained significantly elevated in placebo-treated HC subjects. To examine whether endothelium-derived NO could act as a paracrine modulator of monocyte behavior, monocytes were exposed to NO donors or cocultured in the presence of endothelial cells exposed to antagonists of NO synthase in the presence or absence of L-arginine. NO donors inhibited monocyte adhesiveness. Furthermore, the adhesiveness of monocytes cocultured with endothelial cells was increased by antagonists of NO synthase; this effect was reversed by L-arginine. This study shows that the adhesiveness of human MNCs is increased by hypercholesterolemia. The increase in adhesiveness was reversed in vivo by administration of the NO precursor L-arginine. NO donors or endothelium-derived NO inhibits the adhesiveness of monocytes in vitro, supporting the hypothesis that the effects of L-arginine are mediated by NO.

Key Words: endothelium • atherosclerosis • nitric oxide • monocytes • adhesion

	Introduction

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Hypercholesterolemia enhances the adherence and infiltration of monocytes and T lymphocytes into the vessel wall, and evidence suggests that this process is mediated by the induced expression of specific adhesion molecules and chemotactic proteins.^{1 2 3} Hypercholesterolemia also impairs endothelial vasodilator function, largely due to reduced activity of endothelium-derived NO.^{4 5 6} This endothelial impairment may promote the interaction of platelets and MNCs with the vessel wall because NO is not only a vasodilator but also a potent inhibitor of platelet and leukocyte adhesion.^{7 8 9 10 11 12 13} NO elaborated by ECs in vivo alters the behavior of platelets, inhibiting their reactivity.^{11 12 13} This effect of endothelium-derived NO on circulating blood elements may be mediated by its elevation of intracellular cGMP, which may phosphorylate proteins, modulating adhesion signaling and/or intracellular calcium levels.^{11 12}

There is abundant evidence that the reduction of NO activity by hypercholesterolemia can be reversed by administration of the NO precursor L-arginine. We and others have found that intravenous administration of L-arginine restores endothelial vasodilator function in the peripheral and coronary circulation of HC animals and humans.^{14 15 16 17} Chronic administration of L-arginine to HC rabbits enhances the synthesis of endothelium-derived NO, reduces endothelial elaboration of superoxide anion, inhibits monocyte-EC interaction, and reduces or reverses progression of intimal lesions.^{18 19 20 21}

Accordingly, we hypothesized that chronic oral administration of L-arginine to HC humans would enhance the in vivo generation of endothelium-derived NO and thereby alter the behavior of circulating MNCs. In this investigation, we have developed a reproducible ex vivo assay for the binding of human MNCs to cultured ECs. We have used this functional binding assay to examine the effect of hypercholesterolemia on mononuclear adhesiveness. Furthermore, we have observed the effect of arginine treatment (to restore vascular NO activity in vivo) on the adhesiveness of circulating MNCs.

	Methods

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Subjects
Demographics
The control subject population in this study included 12 normal volunteers, comprising 10 males and 2 females, with an average age of 37±2 years. Normality was determined by a careful history, physical examination, and laboratory analysis to exclude individuals with hematologic, renal, or hepatic dysfunction or clinically evident atherosclerosis. There were 20 patients with hypercholesterolemia, as defined by a total plasma cholesterol >240 mg/dL and an LDL cholesterol level >160 mg/dL. These individuals had an average age of 51±2 years and included 10 males and 10 females. None of the patients had historical evidence of atherosclerosis, as determined by the absence of symptoms of angina, claudication, or cerebrovascular ischemia, nor clinical evidence of arterial occlusive disease, as would be suggested by decreased pulses, asymmetrical blood pressure, or bruits. In addition, no patient had hypertension, diabetes mellitus, or congestive heart failure. None of the subjects were taking diuretics, vasoactive medications, or antiplatelet or hypolipidemic medications. This study was approved by the Stanford University Administrative Panel on Human Subjects in Medical Research, and each subject gave written informed consent before entry into the study. (Because of the age difference between the NC and HC groups, we performed a subset analysis, comparing data derived from the five oldest NC subjects (45±6 years) and the five youngest HC subjects (40±6 years).

To confirm our findings that MNCs derived from HC subjects are more adhesive than those of NC individuals, we performed confirmatory adhesion studies (see below) in 14 NC and 22 HC individuals using a different binding assay.

Hematology and Biochemistry
Venous blood was processed for plasma lipid analysis (total cholesterol, LDL, and triglyceride), as determined by enzymatic colorimetric methods. LDL cholesterol was calculated using the Friedewald equation. A number of standard biochemical parameters were assessed, including measures of hepatic function (SGOT, SGPT, alkaline phosphatase, and albumin), measures of renal function and electrolyte balance, (blood urea nitrogen, creatinine, sodium, potassium, and chloride), hematologic function (lactate dehydrogenase, hematocrit, hemoglobin, complete blood count, differential white blood count), and glucose. These studies were performed in the Stanford University Hospital Laboratory using standard clinical laboratory methods. Citrated plasma was frozen at -20°C for determination of nitrogen oxides by chemiluminescence and arginine using an automated amino acid analyzer (model 6300, Beckman Instruments Inc).

Nitrogen Oxide Measurements
Nitrogen oxide levels were determined as described previously.¹⁸ Briefly, plasma (200 µL) samples were deproteinated by 1:2 incubation with 100% ethanol for 20 minutes at 4°C, then pelleted at 10 000g, and the supernatant was aspirated. Nitrogen oxides were measured in the supernatant with a commercially available chemiluminescence apparatus (model 2108, Dasibi) after reduction of the samples in boiling acidic vanadium (III) chloride at 98°C. Boiling acidic vanadium quantitatively reduces the oxidative metabolites of NO (NO₂^- and NO₃^-) to NO, which is quantified by the chemiluminescence detector after reaction with ozone. Signals from the detector were analyzed by a computerized integrator and recorded as areas under the curve. Standard curves for NO₂/NO₃ were linear over the range of 50 pmol to 10 nmol.

Cell Culture and Functional Binding Assays
MNC Isolation
Human MNCs were isolated from peripheral venous blood, using density ultracentrifugation. Briefly, citrated peripheral venous blood was centrifuged at 500g for 10 minutes. Platelet-rich plasma was removed and the volume was replaced with 0.5 mmol/L EDTA/HBSS, which was free of calcium and magnesium. Blood was centrifuged again at 1200g for 10 minutes and the buffy coat removed and resuspended to 6 mL with HBSS. The suspension was then carefully layered onto a cushion of Histopaque-1077 (Sigma) and centrifuged at 650g for 30 minutes at room temperature. After centrifugation, the MNCs were aspirated from the opaque interface. Cells were then washed with EDTA/HBSS three times at 1000g and subsequently resuspended in HBSS containing 2 mmol/L Ca²⁺, 2 mmol/L Mg²⁺, and 20 mmol/L HEPES, adjusting the cell number to 6x10⁶ per milliliter for binding studies. Viability of the cells was assessed using the trypan blue exclusion test. Viability was >95% in all samples used in these experiments. The isolation procedure, as well as the experiments, was carried out at room temperature. Isolation of MNCs was begun on blood samples from all subjects studied within 2 hours of the venipuncture.

EC Culture
In our initial binding assay, we used the transformed EC line, bEnd3 (mouse brain-derived polyoma middle T-antigen–transformed ECs)²² kindly provided to us by Dr Werner Risau. The BEnd3 cells express a number of EC-specific antigens, including von Willebrand factor, and take up acetylated LDL. The BEnd3 cells express endothelial adhesion molecules and bind monocytes in a cytokine-inducible fashion, with kinetics similar to those observed with human umbilical vein endothelium. Cells were used at passages 27 to 32 and were grown to confluence in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Cells were split into 35-mm-diameter wells on six-well plates (Falcon) 3 days before the adhesion assays.

In a confirmatory study, we used the transformed human umbilical vein EC line ECV304 (American Type Culture Collection) as the binding surface. This cell line retains many endothelial characteristics, including the cytokine-regulated expression of endothelial adhesion molecules, including intercellular adhesion molecule-1, lymphocyte function-associated antigen-3, and vascular cell adhesion molecule-1.^{23 24} These cells were cultured in medium M199 (Applied Scientific, Inc) containing 10% fetal calf serum (GIBCO-BRL). Two days before adhesion studies, cells were passaged into 35-mm wells on six-well culture plates (Nunc, Inc). Confluence was confirmed before binding of all wells. Sixty minutes before adhesion assays, the medium was removed from the wells and replaced by HBSS containing 5 mmol/L HEPES and 2 mmol/L Mg²⁺ and Ca²⁺ (binding buffer).

Adhesion Assay
MNC suspension (1 mL) was added to the wells containing the endothelial monolayers to reach a final cell number of 3x10⁶ per milliliter. The six-well plates were transferred to a rocking platform (Research Products International Corp) and rocked for 30 minutes at room temperature. Parenthetically, we found that the use of the larger six-well plates was critical in obtaining reproducible results. Use of the smaller microwell plates led to higher and more variable cell counts per hpf. The six-well plates were turned 90 degrees at 15 minutes. After 30 minutes, the cell suspension was aspirated from each well and wells were then carefully rinsed twice with 2 mL binding buffer to remove nonadherent MNCs. Plates were rocked for an additional 5 minutes and the binding buffer was aspirated and replaced by HEPES/HBSS containing 2% glutaraldehyde. Wells were kept in this fixing solution until they were counted by videomicroscopy. Videomicroscopic counting of adherent cells was performed using a computer-aided image analysis system (Image Analyst, Automatix Corp). To quantitate the functional binding assay, we counted the adherent cells in 50 hpfs (250x) in each of two duplicate wells, to obtain a mean value of bound cells per hpf for each subject.

Confirmatory Adhesion Studies
In a separate series of studies, we confirmed our results by repeating the binding assays with an internal control, as well as with a human EC line, using additional samples derived from 36 human subjects. These adhesion studies used the transformed human umbilical vein EC line ECV304.

We modified the adhesion assay described above as follows. Peripheral blood MNCs (2x10⁶) were added to confluent 35-mm culture dishes in triplicate. As an internal control, WEHI 78.24 monocytoid cells (2x10⁵) were labeled with TRITC (3 mg/mL) and simultaneously added to each well to control for interassay and intra-assay variability. The suspension of WEHI cells and PBMCs were allowed to incubate with the endothelial monolayer at room temperature for 30 minutes on a rocking platform (Research Products International Corp), as previously described.

Adherent cells were removed by incubation with HBSS supplemented with 5 mmol/L EDTA for 5 minutes, resuspended in 200 mL HBSS, and then fixed with 1% paraformaldehyde. Samples were then analyzed by using a flow cytometer (FACScan, Becton Dickinson). The ratio of PBMC events to TRITC-labeled WEHI 78.24 events was determined and compared between NC (n=14) and HC (n=22) individuals.

Drugs
HBSS was obtained from Applied Scientific. L-Arginine and placebo capsules were obtained from Tyson Inc. fMLP, L-arginine, and sodium nitroprusside for the in vitro studies, as well as all other chemicals used, were obtained from Sigma Chemical Co. Agonists were dissolved in distilled water, except for fMLP, which was dissolved in glacial acetic acid (14.7 mol/L) and then diluted in distilled water 1:100.

Experimental Protocols
Blood was drawn from each subject in the postabsorptive state. Alcohol, caffeine, and tobacco consumption were prohibited 12 hours before the venipuncture. All subjects participated in at least one of the following three protocols

Effect of Hypercholesterolemia on Adhesiveness of Peripheral Blood MNCs
In the first study protocol, we tested the hypothesis that MNCs derived from the peripheral blood of HC volunteers would have greater adhesiveness for ECs in culture than MNCs derived from NC subjects. After resting quietly for at least 20 minutes, peripheral venous blood (30 cc) was obtained, with the subjects in a seated position. Whole-blood samples were collected into a syringe containing sodium citrate at a final concentration of 3.8% and processed as described below for biochemical studies and MNC isolation. HC subjects (n=20) and NC individuals (n=12) were studied in experimental sets of three to five individuals. Each experimental set consisted of one to two NC and two to four HC individuals. Blood samples from subjects in each experimental set were obtained and processed in parallel, and the adhesion assays were performed in parallel. This parallel processing was implemented to minimize differences between the HC and NC groups due to day-to-day variability of the binding assay.

Open-Label L-Arginine Treatment: Effect on Adhesion
In a second protocol, we tested the hypothesis that oral administration of L-arginine would normalize the increased adhesiveness of MNCs from HC individuals. L-Arginine was administered orally as the hydrochloride salt in 700-mg capsules, three capsules four times daily (for a total daily dose of 8.4 g arginine hydrochloride, which is equivalent to 7.0 g L-arginine), for 2 weeks to HC subjects (n=3). The average American consumes approximately 5.4 g arginine daily.²⁵ Therefore, in this study arginine intake was increased approximately 230% above normal dietary levels. Venous blood was collected and processed as described above before administration of L-arginine and after completing the 2-week course of open-label treatment.

Double-Blinded Placebo-Controlled Randomized Study of Oral Arginine: Effect on Adhesion
Finally, to confirm the hypothesis that oral arginine supplementation reduces MNC adhesiveness, we conducted a double-blind, placebo-controlled study with seven HC subjects randomized to L-arginine (at the above dosage) and three HC subjects randomized to placebo for a 2-week course. Six NC subjects not receiving any study drug were also studied in parallel to control for any variability of the functional adhesion assay over time. Venous blood was collected and processed as described below before administration of the study drug, after completing the 2-week course and again after a 2-week washout period.

Role of NO: In Vitro Studies
To delineate the mechanisms for the observations made during the human studies, the following in vitro investigations were undertaken. To determine whether NO could be responsible for the effects of L-arginine administration, in some studies MNCs were exposed in vitro for 30 minutes to various agents (sodium nitroprusside, L-arginine, L-nitro-arginine, or fMLP) or vehicle control. They were then centrifuged at 1000g for 10 minutes and the pellet was resuspended in fresh binding buffer before the binding assay. The cultured ECs were not exposed to any of these agonists.

To determine whether endothelium-derived NO could affect the behavior of circulating monocytes, the following coculture experiment was done. BAECs, passage 9 to 11, transformed BEnd3, and monocytoid cells (WEHI 78.24) were cultured separately in Dulbecco's modified Eagle's medium HI glucose (Applied Scientific, Ltd), supplemented with 10% fetal calf serum and streptomycin (100 µ/mL penicillin-G; 100 ng/mL streptomycin, Applied Scientific, Ltd). Immediately before coculture, BAECs were placed in serum-free medium. Monocytoid cells (2x10⁶ cells per milliliter) were placed into contact with medium conditioned by the BAECs, using coculture inserts (Falcon). The cell-culture insert has a semipermeable membrane that permits the monocytoid cells to be exposed to small molecules elaborated by the ECs but prevents direct contact of the monocytoid cells with the endothelium. The coculture system was placed on a rotating platform (at 120 rpm) for 24 hours. We have previously demonstrated that the fluid shear stress impacted by these conditions stimulates endothelial release of NO.²⁶ In some experiments, antagonists of NOS (L-NAME, 5 mmol/L; or L-nitro-arginine, 5 mmol/L), or NOS antagonists in the presence of L-arginine (10 mmol/L) were added to the medium. In some experiments, monocytoid cells were placed into the same conditions in the absence of ECs to allow observation of the direct effects of the NOS inhibitors and L-arginine on monocytoid cell adhesiveness.

After 24 hours of coculture, monocytoid cells were removed from the coculture, washed in binding buffer, and resuspended to a final concentration of 5x10⁵ cells per milliliter. A functional binding assay was then performed to assess monocytoid adhesiveness. Monocytoid cells were incubated with BEnd3 ECs at room temperature on a rocking platform for 30 minutes, rotating the samples 90° every 15 minutes to ensure equal distribution. ECs were then washed twice in binding buffer and placed again on the rocking platform for 5 minutes to remove nonadherent cells. Samples were then fixed in 0.25% glutaraldehyde and counted by microscopy as described below.

Data Analysis
Adhesiveness of the MNCs from HC or NC individuals was assessed by determining the number of bound cells per hpf at a magnification of 250x. The value for each individual represents the average of 50 hpfs in each of two duplicate wells and is expressed as the mean±SEM.

Results from the different groups were compared using the unpaired Student's t test. The effect on monocyte binding of in vitro treatments versus vehicle controls was compared by the paired Student's t test. Differences were considered to be significant at P<.05.

	Results

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Biochemistry and Hematology
There were no differences between the NC (n=13) and HC (n=20) groups in the measures of hepatic, hematologic, and renal function, electrolyte balance, and glucose, with the exception of a slightly higher alkaline phosphatase value in HC individuals, which was still in the range of normality (77±5 versus 57±5; HC versus NC group; P<.05). The lipid profiles were significantly different, with higher total cholesterol and LDL cholesterol values in the HC group (P<.01 [Table 1]). HDL cholesterol and triglyceride values were not different between the two groups.

View this table: [in this window] [in a new window]   Table 1. Biochemical and Hematological Values

Baseline plasma L-arginine values were not different between the NC and HC groups (84±7 versus 79±10 µmol/L, respectively; P=NS). Administration of L-arginine to a subset of the HC group increased plasma arginine values by 60% (from 79±10 to 128±12 µmol/L; n=7), whereas L-arginine values in the placebo-treated (n=3) and NC (n=6) groups remained unchanged. The administration of oral L-arginine had no effect on any of the biochemical or hematologic parameters (data not shown). Oral L-arginine did not lower total cholesterol or LDL cholesterol (Table 2).

View this table: [in this window] [in a new window]   Table 2. Lipid Profiles Before, During, and After Treatment With Study Drug

NOx measured in citrated plasma was 24±8 nmol/L (n=10) for the NC group and 28±5 nmol/L for the HC group (n=10; P=NS). After oral L-arginine supplementation plasma, NOx in the HC group was unchanged (24±3 nmol/L; n=10; P=NS, in comparison with values obtained before L-arginine therapy).

Effect of Hypercholesterolemia on Adhesiveness of Peripheral Blood MNCs
The composition of the MNC suspension was assessed by differential white blood cell count and revealed that the cell suspensions consisted of <1% granulocytes, basophils, and reactive lymphocytes. The MNCs were comprised of lymphocytes (93±0.8%) and monocytes (7±0.7%). The composition of the cell suspension was not altered by hypercholesterolemia nor by the administration of L-arginine or placebo. Analysis of the MNC suspension by fluorescence-activated cell sorter revealed no differences between HC and NC subjects in the percentages of B cells, T cells, and monocytes (Table 3).

View this table: [in this window] [in a new window]   Table 3. Percentage Composition of Isolated Mononuclear Cells

The results of the adhesion assays were highly reproducible. When aliquots of MNC suspension from the same individual were studied in parallel, the numbers of bound cells per hpf in the duplicate wells correlated highly (r=.9 and R²=.9, P=.001, n=44). There was somewhat less correlation between binding values for the same individual on different experimental days: MNCs were isolated from the same individuals on experimental days 2 weeks apart, and the values at these different time points for the same individual correlated modestly (r=.8 and R²=.7, P=.05, n=7). The correlation was weakened by significant day-to-day variability in the number of cells binding to the endothelial monolayer, likely due to sources of biological variability such as EC passage and manipulation of MNCs during isolation. There was a range of 11 to 64 bound cells per hpf for cells derived from NC volunteers and 12 to 98 bound cells per hpf for cells derived from HC volunteers. The average number of bound cells per hpf was 33±5 versus 51±6 (NC [n=12] versus HC [n=20] volunteers; P=.06). Despite the daily variability in bound cells per hpf, on each experimental day, MNCs obtained from HC patients consistently bound in greater numbers per hpf than cells derived from NC subjects. Therefore, on each experimental day, the number of cells per hpf obtained from the NC subjects was averaged and set to 100. The HC values were then expressed as a percentage of these control values. Expressed in this way, MNCs derived from HC individuals demonstrated a 50±8% increase in bound cells per hpf (P<.0001; Fig 1). The degree of adhesiveness was correlated to the plasma levels of LDL cholesterol (r=.7, n=33; P<.0001; Fig 2).

View larger version (14K): [in this window] [in a new window]   Figure 1. Adherence of human MNCs to ECs in culture. MNCs were isolated from the peripheral blood of NC and HC volunteers and incubated in parallel in binding buffer with EC monolayers. The number of bound cells per hpf are expressed as a percentage of bound cells per hpf obtained using cells from NC control subjects studied in parallel.

View larger version (20K): [in this window] [in a new window]   Figure 2. Correlation of cell binding with the level of LDL. There is a correlation between the number of bound cells per hpf and the level of LDL. Cont indicates control.

Because the mean age of the NC group was significantly less than that of the HC group (37±2 versus 51±2 years, P=.002), it is possible that age-related changes in the adhesiveness of MNCs could contribute to the difference between these groups. To examine this issue, we compared the adhesion responses of the five oldest NC individuals (45±6 years) versus the five youngest HC individuals (40±6 years; P=NS). MNCs from the HC individuals exhibited 34±14% more bound cells per hpf than the age-matched NC group (P<.05).

Confirmatory Adhesion Studies
To confirm the finding that MNCs derived from HC subjects were more adhesive than those from NC subjects, we repeated the binding studies using a human EC line and an internal control (WEHI 78.24 monocytoid cells). The number of adherent MNCs per hpf was expressed in terms of the number of adherent WEHI cells per hpf.

MNCs isolated from HC patients were more adhesive than those isolated from NC subjects (4.0±0.7 PBMC/WEHI, n=22; versus 2.0±0.4 PBMC/WEHI, n=14; P=.05). Therefore, using an alternative methodology, we confirmed that hypercholesterolemia alters the adhesiveness of circulating MNCs.

Open-Label L-Arginine Treatment: Effect on Adhesion
In the open-label study, five HC individuals were treated with oral L-arginine supplementation for 2 weeks and paired with NC controls not receiving arginine. Two of the HC individuals did not complete the course of treatment; one because of reluctance to take the pills, and one due to reactivation of herpes simplex during the study. In the remaining three HC individuals, arginine treatment resulted in a 38% decrease in monocyte adhesiveness; indeed after arginine treatment, the number of bound cells per hpf was only 93±4% of the values for paired NC controls.

Double-Blinded Placebo-Controlled Randomized Study of Oral L-Arginine: Effect on Adhesion
To confirm this effect of L-arginine treatment and to control for any experimental bias, a double-blinded placebo-controlled randomized study was performed. Ten HC subjects were randomized(1:2) to placebo or L-arginine treatment; six NC individuals were studied in parallel to control for variation over time in the binding assay. At baseline, the adhesion of MNCs from both HC groups was increased in comparison with the NC individuals (P<.001; Fig 3). After 2 weeks of L-arginine administration, there was an absolute reduction of 53% in MNC binding (n=7, P<.005, baseline versus 2 weeks; Fig 3). By contrast, there was no significant change in the adhesiveness of MNCs isolated from HC individuals treated with placebo (Fig 3). Two weeks after discontinuation of the L-arginine treatment, the adhesiveness of the MNCs isolated from HC individuals had significantly increased compared with the NC individuals (34±9% increase in bound cells per hpf; P<.05) and was also significantly increased in comparison with the binding obtained after 2 weeks of L-arginine therapy (an increase of 30±9%, P<.05; Fig 3). The adhesiveness of placebo-treated HC MNCs did not change significantly during the washout period (Fig 3).

View larger version (30K): [in this window] [in a new window]   Figure 3. Reduction of MNC adhesiveness by dietary arginine supplementation. HC individuals were randomized to arginine supplementation or placebo. At baseline (before the initiation of study drug), MNCs from both HC groups exhibited greater adhesiveness than monocytes derived from NC individuals. After 2 weeks of arginine supplementation, monocytes derived from these HC individuals demonstrated a significant reduction in adhesiveness. At the 4-week time point (after discontinuing the study drugs for 2 weeks), the adhesiveness of MNCs derived from HC individuals previously treated with dietary arginine had returned to levels significantly greater than that of NC individuals. The number in the bars refers to the number of individuals participating at each time point. Cont indicates control (NC) subjects; Arg, arginine; and plac, placebo. *Significantly different from baseline value.

Effect of NO on Monocyte Adhesion
In some studies, MNCs were exposed to fMLP (100 nmol/L) or vehicle control for 30 minutes in vitro. This agent is a known activator of leukocytes and induces chemotaxis and adhesion. This agent increased cell adhesion significantly in both the HC group (by 25%; n=13, P<.005) and the NC group (by 69%; n=8, P<.005).

In vitro incubation of MNCs from HC subjects with L-nitro-arginine (10^-4 mol/L), an irreversible NOS inhibitor, did not affect their binding to the endothelium (141±2% versus 140±3%, vehicle treatment versus L-nitro-arginine; n=5; values expressed as a percentage of the value for NC cells exposed to vehicle). Preincubation of MNCs from HC individuals with L-arginine (3 mmol/L) also did not affect binding (151±11% versus 148±10%; n=5, P=NS; values expressed as a percent of the NC controls exposed to vehicle). By contrast, preincubation of the cells from HC individuals with the NO donor sodium nitroprusside (10^-5 mol/L) markedly reduced binding (164±9% versus 98±7%, vehicle versus sodium nitroprusside; n=7, P<.0005; values expressed as a percent of the NC control exposed to vehicle; Fig 4). The binding of NC monocytes was unaffected by L-arginine (3 mmol/L) or sodium nitroprusside (10^-5 mol/L; n=5), whereas L-nitro-arginine treatment led to a slight increase in adhesion that did not reach significance (112±5% versus 100±7%, n=5, P=NS).

View larger version (27K): [in this window] [in a new window]   Figure 4. Effect of NO donor on monocyte adhesiveness. MNCs from NC (Cont) and HC (Chol) individuals were exposed to vehicle control or sodium nitroprusside (SNP; 10-7 mol/L). SNP had no effect on the binding of cells derived from NC individuals. However, SNP induced a significant reduction in binding of cells derived from HC individuals.

To determine whether endothelium-derived NO could affect circulating blood elements, monocytoid cells were exposed to the conditioned medium of ECs, in the presence or absence of NOS antagonists. Exposure to ECs in the presence of the NOS antagonist L-NAME (5 mmol/L) markedly enhanced the adhesiveness of monocytoid cells (Fig 5A). This effect of L-NAME was abolished by the addition of arginine (10 mmol/L) to the medium. In the absence of ECs, L-NAME tended to slightly increase the adhesiveness of monocytoid cells, although this effect did not achieve significance. The addition of arginine abolished this direct effect of L-NAME on the monocytoid cells. Arginine alone had no effect on the adhesiveness of monocytoid cells (Fig 5B).

View larger version (22K): [in this window] [in a new window]   Figure 5. A, Effect of conditioned medium from ECs on adhesiveness of monocytoid cells. WEHI 78.24 monocytoid cells were exposed for 24 hours to the conditioned medium of BAECs. When the NOS antagonist L-NAME (5 mmol/L) was added to the coculture, the adhesiveness of the cocultured monocytes was increased by 105±24%, as assessed by a functional binding assay. This enhanced adhesiveness was abrogated by the addition of 10 mmol/L L-arginine to the coculture. B, Direct effect of NOS antagonist or NO precursor on monocytoid adhesiveness. Monocytoid cells were exposed for 24 hours to 5 mmol/L L-NAME and/or 10 mmol/L L-arginine in the absence of conditioned medium from ECs. Subsequently the adhesiveness of monocytoid cells was assessed using a functional binding assay. Cont indicates control; NAME, L-NAME; and Arg, arginine.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The salient findings of this investigation are that (1) hypercholesterolemia enhances the adhesiveness of MNCs for ECs, (2) dietary arginine supplementation reverses the increase in adhesiveness of MNCs from HC individuals, (3) the effect of dietary arginine is mimicked in vitro by exposure of the MNCs from HC individuals to sodium nitroprusside, an NO donor, and (4) ECs stimulated to release NO inhibit the adhesiveness of coincubated monocytes; this effect is reversed by L-nitro-arginine.

We propose that the effects of dietary arginine on the adhesiveness of MNCs are mediated by its conversion to NO by vascular endothelium in vivo. We and others have previously shown that the intravenous infusion of L-arginine restores NO-dependent vasodilation in HC animals and humans.^{14 15 16} The salutary effects of L-arginine are not due to its physicochemical properties, since they are not mimicked by D-arginine.¹⁵ Moreover, L-arginine administration specifically improves endothelium-dependent relaxation without altering responses to endothelium-independent agonists.^{14 15 16} More recently, we have demonstrated that chronic oral administration of L-arginine to HC rabbits enhances the release of endothelium-derived NO from the thoracic aorta (as detected by chemiluminescence) and inhibits endothelial-monocyte binding ex vivo.¹⁸ The NO-associated alteration in monocyte binding may contribute to the effect of dietary arginine to markedly inhibit atherogenesis in this animal model.^{19 20 21} This hypothesis is bolstered by the observations that inhibition of NO elaboration by oral administration of NOS antagonists enhances endothelial-monocyte binding and accelerates atherogenesis.^{18 27}

The effect of exogenous L-arginine to enhance endothelial elaboration of NO may reflect its competition with an endogenous inhibitor. ADMA is a competitive antagonist of NOS. Its levels are increased in HC rabbits^{28 29} and HC humans.^29A Plasma levels of ADMA are positively correlated with the severity of peripheral arterial disease.³⁰ Administration of L-arginine to patients with peripheral arterial disease increases limb blood flow, in association with an increase in urinary nitrate excretion.³¹ Finally, isolated vascular rings contract in response to inhibition of dimethylarginine dimethylaminohydrolase (the enzyme that degrades ADMA); the contraction is reversed by administration of L-arginine.³² These studies indicate that ADMA is an endogenous regulator of NOS, which may be abnormally elevated in disease states.

Bath and colleagues¹⁰ have previously demonstrated that the in vitro binding of human MNCs to ECs in culture is inhibited by adding NO donors to the media in the presence of superoxide dismutase. Similarly, in the present study, we found that the enhanced binding of MNCs from HC individuals is reversed by exposure of the MNCs to the NO donor sodium nitroprusside. The mechanism by which NO inhibits adhesion is not known, although the effect is associated with increases in intracellular cGMP. This second messenger may act by phosphorylating proteins that reduce intracellular calcium levels or that interfere with signal transduction of adhesion receptors.^{11 12}

In this study, the effect of dietary arginine supplementation was most likely due to its known effect to enhance NO elaboration by the endothelium, rather than due to a direct effect on the MNCs. Indeed, exposure of MNCs (from untreated HC individuals) to arginine in vitro had no effect on adherence. The antagonist of NOS, L-nitro-arginine, also had little effect in vitro on the behavior of MNCs from HC individuals. Therefore, it is less likely that autocrine elaboration of NO by MNCs is responsible for the effect of dietary L-arginine. Our previous studies and those of others^{14 15 16 17 18} would suggest that the effect of oral arginine was to increase the synthesis of endothelium-derived NO. Endothelium-derived NO is known to affect circulating blood elements. Vascular NO increases cyclic GMP in platelets passing through the coronary microvasculature and inhibits their ability to aggregate.^{11 12} Endothelium-derived NO may circulate in plasma in the form of nitrosoalbumin or other nitrosothiols, in concentrations that have been demonstrated to alter the reactivity of platelets and leukocytes.³³ The available evidence supports our hypothesis that the known effect of arginine to enhance the endothelial elaboration of NO in HC individuals is responsible for its effect on MNC adhesion. However, this study does not exclude the possibility that administration of L-arginine influenced monocyte behavior independently of an effect on NO synthesis.

We did not observe a systemic increase in circulating plasma nitrogen oxides. This may be due to the fact that plasma nitrogen oxide values are significantly influenced by dietary nitrite and nitrate,³⁴ which were not controlled in this study. In addition, many other cell types are capable of generating NO and likely contribute to the circulating pool of plasma nitrogen oxide. Furthermore, the chemiluminescence technique used in this study for detection of nitrogen oxides does not differentiate between vasoactive NO and its one-electron oxidation products NO₂^- and NO₃^-. Evidence suggests that in comparison with endothelium from NC animals, the endothelium of HC animals releases as much or more NO, as measured by chemiluminescence.^{18 35} However, much of this NO is in an oxidized form, which lacks vasodilator potency.^{35 36} Therefore, more direct methods to detect vasoactive endothelial-derived NO in vivo need to be used in future studies of therapies directed at restoring endothelial function.

The mechanisms by which hypercholesterolemia augments the adhesiveness of MNCs for the endothelium are poorly defined. However, an alteration in the oxidative state of the endothelium may be involved. Under conditions of hypercholesterolemia (or direct inhibition of NO production by NOS antagonists), the endothelium begins to generate oxygen-derived free radicals.^{37 38} Under these conditions, superoxide anion or lipid peroxidation products elaborated by the endothelium could directly enhance the adhesiveness of circulating leukocytes.^{39 40}

To conclude, we have developed a reproducible assay for the interaction of human MNCs with the endothelium. We have observed a consistent increase in the adhesiveness of MNCs from HC individuals, which is correlated with the level of LDL. The effect of hypercholesterolemia on the adhesiveness of MNCs is abrogated by dietary arginine and in vitro exposure to an NO donor or to endothelium-derived NO. We propose that the effect of dietary arginine is mediated by an enhanced elaboration of endothelium-derived NO, which may directly influence cell adhesiveness and/or reduce the endothelial generation of oxygen-derived free radicals and lipid peroxidation products that activate circulating MNCs. These effects of dietary arginine have been associated with an inhibition of atherogenesis in animal models. It remains to be seen whether supplements of the NO precursor will inhibit atherogenesis in humans.

	Selected Abbreviations and Acronyms

 

ADMA = asymmetric dimethylarginine BAEC = bovine aortic EC EC = endothelial cell fMLP = N-formyl-methionyl-leucyl-phenylalanine HC = hypercholesterolemic hpf = high-power field L-NAME = NG-nitro-L-arginine methyl ester MNC = mononuclear cell NC = normocholesterolemic NO = nitric oxide PBMC = peripheral blood MNC

	Acknowledgments

 
This work was supported in part by grants from the National Heart, Lung, and Blood Institute (1P01-HL-48638 to 01); the University of California Tobacco Related Disease Program IRT 215; and the Peninsula Community Foundation and was done during the tenure of a Grant-in-Aid Award from the American Heart Association and Sanofi Winthrop. Dr Theilmeier is the recipient of scholarships from the German Scholarship Program and the Gottlieb Daimler and Karl Benz Foundation. Dr Wolf is the recipient of a Stanford University Dean's Postdoctoral Fellowship. Dr Cooke is a recipient of the Vascular Academic Award from the National Heart, Lung, and Blood Institute (1K07-HC-02660) and is an Established Investigator of the American Heart Association. The transformed EC line BEnd3 was kindly provided to us by Dr Werner Risau. We thank Drs Leslie McEvoy and Eugene Butcher for advice on adhesion assays and cell-labeling methods and for development of the WEHI internal standard.

Received May 9, 1995; accepted April 15, 1997.

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