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(Circulation. 1996;93:1488-1492.)
© 1996 American Heart Association, Inc.

Articles

Increased Adhesiveness of Isolated Monocytes to Endothelium Is Prevented by Vitamin C Intake in Smokers

Presented in part at the 64th Meeting of the European Atherosclerosis Society, Utrecht, the Netherlands, June 10-13, 1995, and published in abstract form in Atherosclerosis (1995;115:S59).

Christian Weber, MD; Wolfgang Erl, MSc; Kim Weber, MBBS; Peter C. Weber, MD

From the Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Ludwig-Maximilians-Universität, München, Germany, and Brigham and Women's Hospital (K.W.), Boston, Mass.

Correspondence to Christian Weber, MD, Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Pettenkoferstrasse 9, D-80336 München, FRG.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background To explore pathophysiological mechanisms of cigarette smoking involved in atherogenesis, we compared adhesiveness of isolated blood monocytes with endothelium and plasma levels of the aqueous phase antioxidant vitamin C in nonsmokers and smokers before and after supplementation, using a novel monocyte adhesion assay with fixed human endothelial cells.

Methods and Results Monocyte adhesion to unstimulated human umbilical vein endothelial cells ranged from 0.17% to 0.51% in the nonsmoker group (0.37±0.09%, mean±SD, n=13). In smokers with a 1 to 2 packs per day consumption, monocyte adhesion was increased to 0.71±0.17% (mean±SD, n=10, P<.001), ranging from 0.46% to 0.99%. Increased adhesiveness was mediated by the integrin CD11b/CD18, as shown by inhibition with a monoclonal antibody to CD11b but not associated with altered CD11b surface expression. Plasma vitamin C levels were reduced in smokers (48.2±14.1 µmol/L) versus nonsmokers (67.7±17.6 µmol/L; P<.025), while no significant differences were found in retinol, vitamin E, or ß-carotene levels. This confirms that the radical scavenger vitamin C reacts sensitively to oxidative stress induced by cigarette smoke in human plasma. Consistently, dietary supplementation with vitamin C (2 g per day) for 10 days raised plasma levels to 82.6±11.0 µmol/L (n=10, P<.001) in smokers and decreased monocyte adhesion to values found in nonsmokers (0.38±0.18%, P<.001). In contrast, vitamin C intake did not affect monocyte adhesiveness in nonsmokers (0.37±0.14%, n=6) despite increasing plasma levels to 82.9±11.8 µmol/L.

Conclusions Our data show that cigarette smoking increases CD11b-dependent monocyte adhesiveness in humans. Restoring reduced plasma vitamin C concentrations in smokers by oral supplementation decreased monocyte adhesion to values found in nonsmokers.

Key Words: smoking • antioxidants • endothelium

	Introduction

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Smoking has been recognized as a major risk factor in atherogenesis.¹ Cigarette smoke elicits leukocyte adhesion to endothelium in animal models,² and potentiated adherence of monocytes to endothelial cells may be an initial event in the pathogenesis of a cigarette smoke–induced inflammatory response in the vessel wall involved in atherosclerosis.

While cigarette smoke has been shown to introduce reactive oxygen intermediates (ROI) in the circulation,³ ascorbate (vitamin C) is an outstanding antioxidant in human plasma⁴ that acts by scavenging aqueous phase–ROI and preventing initiation of lipid peroxidation.^{5 6} In an animal model, cigarette smoke–induced leukocyte adhesion was inhibited by pretreatment with superoxide dismutase² or with vitamin C but not with vitamin E,⁷ implying aqueous phase–ROI as mediators. It has been speculated that vitamin C interferes with P-selectin translocation to the endothelial cell surface in response to radicals.^{7 8} In human endothelial cells stimulated to generate radicals, water-soluble antioxidants recently have been shown to inhibit vascular cell adhesion molecule-1 induction and monocyte adhesion.⁹

In the present study, we determined the adhesiveness of isolated monocytes to fixed human umbilical vein endothelial cells (HUVECs) and measured plasma vitamin C levels in smokers and nonsmokers. We also investigated the effect of vitamin C supplementation on these parameters in both groups.

	Methods

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Protocol and Monocyte Isolation
Venous blood (40 mL) was collected from consenting male volunteers (age, 20 to 30 years) who fasted and refrained from smoking for at least 8 hours until blood samples were drawn. Nonsmokers were clinically healthy, while smokers (average consumption of 1 to 2 packs per day) had a chronic cough as the only known symptom or pathology. Pairs of nonsmokers and smokers were evaluated in side-by-side experiments. In all smokers who maintained their smoking habits and in 6 nonsmokers, blood sampling was repeated after supplementing 2 g per day of ascorbate powder (freshly solubilized in water before intake, University Pharmacy Munich) for 10 days. The study was approved by the ethical committee of the University of Munich. Peripheral blood mononuclear cells were separated by Ficoll density gradient centrifugation. Monocytes were isolated by magnetic cell separation (Miltenyi Biotec) with Fe-dextran–labeled anti-CD14 monoclonal antibody (mAb), yielding purities 95%, as judged by immunofluorescence. In some experiments, monocytes were stimulated with f-methionyl-leucyl-phenylalanine (fMLP, 10 nmol/L) for 10 minutes before immunofluorescence or adhesion assays were performed. Ethidium bromide/acridin orange fluorescence showed viability 97% under all conditions.

The surface expression of L-selectin, which is shed from monocytes upon activation,¹⁰ was compared in monocytes isolated by magnetic cell separation versus monocytes in whole blood collected from the same donor and in whole blood monocytes from nonsmokers and smokers. For whole blood samples, red cells were lysed in 180 mmol/L NH₄Cl, 9 mmol/L KHCO₃, 0.1 mmol/L EDTA, pH 7.4, for 5 minutes at 25°C. Remaining cells were pelleted at 300g and resuspended in PBS at 4°C. Surface expression of L-selectin did not differ between isolated monocytes (219±13 channels) and monocytes in whole blood from nonsmokers (224±15 channels) but was slightly reduced on monocytes in whole blood from smokers (193±12 channels, mean±SD, n=3, P<.050 versus nonsmokers).

Endothelial Cell Culture
HUVECs obtained from umbilical cords by digestion with chymotrypsin were cultured in T-75 flasks (Falcon Primaria, Becton Dickinson) precoated with collagen using endothelial cell growth medium (PromoCell) in 5% CO₂/air at 37°C, as described previously.⁹ Purity was assessed by morphology and factor VIII staining. In brief, HUVECs were detached by 0.01% trypsin/EDTA, antagonized by fetal calf serum. Confluent monolayers from the same preparation (passage 2) grown in 48-well plates were washed twice with PBS and fixed with 2% paraformaldehyde for 15 minutes; the fixing solution was decanted, and cells were dried in air and stored at 4°C, desiccated, and protected from light.

Adhesion Assay
Isolated monocytes were resuspended in medium 199 (Life Technologies) with 10 mmol/L HEPES buffer to a concentration of 1.5 to 2.0x10⁵/mL. HUVECs were washed with medium 199 (10 mmol/L HEPES) before addition of monocytes (3 to 5x10⁴ per well) and coincubated at 37°C, 5% CO₂/air, and 90% humidity for 1 hour. For optimal stimulation with fMLP, monocytes were coincubated with HUVECs for 15 minutes. Some monocyte samples were pretreated with a blocking mAb to CD11b (clone 44) or IgG₁ isotype control (clone B-C14, both Serotec, 10 µg/mL) for 15 minutes and washed. Monocyte suspensions were withdrawn, HUVECs were washed with PBS, and inverted plates were centrifuged (50g, 1 minute).¹¹ Cells were treated with formalin/ethanol and stained with Coomassie/Giemsa solution. The number of adherent monocytes was counted in 20 separate areas per well and adhesion was expressed as percentage of monocytes added. The intra-assay and interassay variations for control monocytes isolated from one donor were 11% and 16% (n=4), respectively. All experiments were performed in triplicate.

Immunofluorescence
Monocytes were reacted with phycoerythrin-conjugated Mo2 mAb and fluorescein isothiocyanate–conjugated IgM isotype control mAb or with phycoerythrin-IgM isotype control mAb and fluorescein isothiocyanate–Mo1 mAb (Coulter) in PBS with 0.5% bovine serum albumin for 30 minutes on ice.¹² Cells were also reacted with Dreg-56 mAb (Biocol) or IgG₁ isotype control and stained with fluorescein isothiocyanate–conjugated goat anti-mouse IgG₁ (Serotec). After gating, monocytes were analyzed by a Becton Dickinson fluorescence activated cell sorter. Mo1 is directed to CD11b (Mac-1 -chain), Mo2 to a CD14 epitope, expressed on mature monocytes, and Dreg-56 to L-selectin. After correction for unspecific binding, data were presented as specific mean fluorescence intensity in channels on a log₁₀ scale, as described.¹¹

Determination of Antioxidants
Serum was collected by centrifugation. Total serum lipids were extracted with hexane. Lipophilic antioxidants were separated on C18 reverse phase columns (Ultrasphere ODS, 5x150 mm, Beckman) with acetonitrile/Cl2CH2/CH3OH/NH4 acetate (70:20:10:0.01) as a mobile phase (flow rate, 1.5 mL/min) and detected on a Waters 490E programmable multiwavelength detector. Vitamin C levels were measured by high performance liquid chromatography with isoluminal chemiluminescence detection.¹³ All other reagents were from Sigma Chemical Co.

Statistics
Adhesion data and plasma vitamin C levels were expressed as mean±SD. Differences were analyzed by two-sided unpaired and paired Student's t test comparing (1) nonsmokers with smokers and (2) smokers and nonsmokers before and after vitamin C intake, respectively. Values of P<.05 were considered significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Influence of Cigarette Smoking on Monocyte Adhesion to Endothelium
Monocyte counts determined after isolation in smokers (3.9±1.6x10⁵/mL, mean±SD, n=10) were found to be significantly increased compared with nonsmokers (1.8±0.6x10⁵/mL, n=13), consistent with previous findings.¹⁴ The range of monocyte adhesion to HUVECs was 0.17% to 0.51% in the nonsmoker group (n=13) with a mean±SD of 0.37±0.09% (Fig 1A). The adherence of isolated monocytes was increased by about 90% to a mean±SD of 0.71±0.17% in the smoker group (P<.001, n=10), ranging from 0.46% to 0.99% (Fig 1A). The fixation process with formaldehyde enabled us to use confluent HUVECs from the same preparation and passage, thus minimizing interassay variations. Because differences in monocyte adhesion between smokers and nonsmokers were not detected using cytokine-stimulated HUVECs (data not shown), we used unstimulated HUVECs, which express intercellular adhesion molecule-1 (ICAM-1) but not vascular cell adhesion molecule-1 or E-selectin.⁹

View larger version (27K): [in this window] [in a new window]   Figure 1. Monocyte adhesion and plasma vitamin C levels in nonsmokers and smokers. Monocytes from male volunteers were isolated by magnetic cell separation and coincubated with fixed HUVECs (37°C, 1 hour). Cells were treated with formalin/ethanol and stained with Coomassie/Giemsa solution. Number of adherent monocytes was counted in 20 separate areas in triplicate wells and expressed as percentage of monocytes added. Plasma vitamin C levels were measured by high-performance liquid chromatography with isoluminal chemiluminescence detection. Dot blots show monocyte adhesion values (A) and plasma vitamin C levels (B) of nonsmokers () and smokers () before vitamin C intake. Mean±SD values of nonsmokers (n=13) and smokers (n=10) are displayed separately for each population. Differences in monocyte adhesion (A) and vitamin C levels (B) were statistically significant (P<.001 and P<.025, respectively). Also shown are plasma vitamin C levels (C) and monocyte adhesion values (D) of smokers before () and after () vitamin C intake (2 g per day) for 10 days. The mean±SD values (n=10) before and after intake of vitamin C are displayed as open or closed squares, respectively. Differences in vitamin C (C) and monocyte adhesion (D) levels were statistically significant (P<.001).

Mechanism of Increased Adhesiveness in Monocytes From Smokers
Since cigarette smoke condensate has been shown to upregulate CD11b,¹⁵ we determined CD11b surface expression in monocytes. However, we found no differences between smokers and nonsmokers (330±16 versus 327±24; mean±SD, n=5). In contrast, fMLP (10 nmol/L) stimulated CD11b expression in monocytes isolated from nonsmokers (372±31 versus 316±19, n=3, P<.050). In accordance with previous findings of transient neutrophil adhesion,¹⁶ fMLP increased adhesion of monocytes from nonsmokers and smokers from 0.35±0.08% and 0.66±0.15% to 1.01±0.21% and 1.08±0.25% (mean±SD, n=5, P<.010 and P<.025), respectively, after coincubation with HUVECs for 15 minutes but not for 60 minutes (data not shown). This finding indicates that additional stimulation of monocytes overrides differences in adhesiveness detectable in our assay. Adhesiveness of the integrin Mac-1 (CD11b/CD18) does not require increased surface expression but activation of a CD11b subpopulation.^{17 18 19} To assess a role of the ICAM-1 receptor Mac-1 for monocyte adhesion to HUVECs, we performed inhibition studies with a blocking mAb to CD11b. This mAb did not alter adhesion of monocytes from nonsmokers (0.39±0.13% versus 0.47±0.06% with isotype control mAb, n=5) but reduced adhesion of monocytes from smokers from 0.91±0.16% with isotype control mAb to 0.59±0.11% (n=5, P<.010). Thus, increased adhesiveness of monocytes from smokers may be mediated by activation of Mac-1, independent of surface expression.

Effect of Cigarette Smoking on Antioxidant Vitamin Levels
To elucidate whether our results were related to a cigarette smoke–induced decrease in antioxidative vitamins, we measured plasma levels of hydrophilic ascorbate (vitamin C) and lipophilic retinol, -tocopherol (vitamin E), and ß-carotene. While plasma retinol (smokers, 1.8±0.3 µmol/L; nonsmokers, 1.9±0.3 µmol/L) and vitamin E levels (smokers, 15.6±2.1 µmol/L; nonsmokers, 17.6±4.4 µmol/L) were not affected, plasma ß-carotene levels were lower in smokers (0.31±0.11 µmol/L) versus nonsmokers (0.42±0.15 µmol/L) but not significantly (P<.100). In contrast, vitamin C levels were significantly (P<.025) reduced by about 40% in smokers (48.2±14.1 µmol/L) versus nonsmokers (67.7±17.6 µmol/L, Fig 1B). This indicates that vitamin C is a reagible antioxidant counteracting oxidative stress produced by cigarette smoke in human serum.

Effect of Vitamin C Intake on Plasma Levels and Monocyte Adhesiveness
Since plasma vitamin C levels were reduced in smokers and vitamin C prevents cigarette smoke–induced leukocyte adhesion to endothelium in an animal model,⁷ we studied whether oral intake of vitamin C (2 g per day) would reverse increased monocyte adhesion to HUVECs ex vivo in smokers. Supplementation with vitamin C for 10 days increased plasma levels from 48.2±14.1 to 82.6±11.0 µmol/L (n=10, P<.001, Fig 1C). Importantly, vitamin C intake reduced adhesion of isolated monocytes to values found in nonsmokers (0.38±0.18%, n=10, P<.001). An inhibition was evident in all smokers tested (Fig 1D), with an average reduction to 52.1±16.9% of values before vitamin C intake. In contrast, vitamin C intake for 10 days slightly increased plasma levels in nonsmokers from 67.7±17.6 to 82.9±11.8 µmol/L while not significantly lowering monocyte adhesiveness (0.37±0.14%, n=6, Fig 2A and 2B; closed circles). The mean±SD values of both parameters for all groups are shown in Fig 2A and demonstrate that smokers before vitamin C intake form a clearly distinguishable population, while the other groups reveal overlapping distributions. All individual data points (n=39) with arrows connecting corresponding data pairs before and after vitamin C intake are shown in Fig 2B. Increasing plasma vitamin C levels reduced monocyte adhesiveness in all smokers with varying responsiveness but showed no consistent effect in nonsmokers.

View larger version (19K): [in this window] [in a new window]   Figure 2. Dot blots show plasma vitamin C levels (x axis) versus percent monocyte adhesion (y axis) for nonsmokers (), smokers (), nonsmokers after vitamin C intake (), and smokers after vitamin C intake (). Data were obtained as described in Fig 1. Mean±SD values of the four groups (A) and all individual data points (n=39) (B) are displayed. Smokers before vitamin C intake form a distinct population, while the other groups are overlapping (A). Arrows connecting corresponding data pairs before and after vitamin C supplementation demonstrate a reduction of monocyte adhesion values after vitamin C intake in all smokers but no consistent effect in nonsmokers (B).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
We have found that chronic cigarette smoking was associated with a CD11b-dependent increase in adhesiveness to endothelium of isolated blood monocytes in healthy 20- to 30-year-old men. Surface expression of CD11b was not altered on monocytes from smokers. This may be due to the reversibility of CD11b upregulation after acute cigarette smoke exposure.¹⁵ Increased surface expression of the ICAM-1 receptor Mac-1 is not sufficient to mediate adhesiveness,^{17 18} which requires the activation of a competent subset of CD11b molecules.¹⁹ Indeed, inhibition studies with an mAb to CD11b indicated that increased adhesiveness to endothelium of monocytes isolated from smokers was due to activation of Mac-1 independent of surface expression. Similarly, activation of CD11b has been involved in enhanced monocyte adhesiveness stimulated by modified LDL.²⁰ The surface expression of L-selectin was not affected by the isolation procedure but was slightly reduced in whole blood monocytes from smokers, consistent with a moderate activation of monocytes by cigarette smoking. However, L-selectin cannot account for increased adhesiveness of monocytes in our assay, since it contributes to nonstatic rather than static monocyte adhesion to endothelium, and no evidence has been provided for a functional significance of L-selectin ligands on unstimulated HUVECs.¹⁰

In this study we have also demonstrated that chronic smoking was associated with reduced plasma levels of vitamin C but not of vitamin E. Oral supplementation of vitamin C for 10 days restored plasma levels and decreased monocyte adhesiveness in smokers. Vitamin C but not lipid-soluble antioxidants recently was shown to prevent cigarette smoke–induced leukocyte adhesion to endothelium in an animal model.⁷ Furthermore, vitamin C has been reported to act as the initially reagible, hydrophilic antioxidant in human plasma,^{4 6} and smoking substantially increases vitamin C consumption and requirements.²¹ Vitamin C but not the lipophilic antioxidant vitamin E completely prevents lipid peroxidation in human plasma exposed to cigarette smoke.⁴ Hence, vitamin C may protect against cigarette smoke damage by interfering with aqueous phase–ROI originating from cigarette smoke rather than by inhibiting subsequent lipid peroxidation induced by ROI. Oxidized LDL (oxLDL) has been shown to enhance adhesion of human monocytes to endothelial cells.²⁰ Since vitamin C reacts with cigarette smoke–derived, aqueous phase–ROI before the initiation of cigarette smoke–induced LDL oxidation,^{6 22} it may prevent oxLDL-stimulated monocyte adhesion. Thus, oxLDL may mediate the cigarette smoke–induced increase in monocyte adhesiveness.

The increase in monocyte adhesiveness found in smokers can be reversed by restoring their plasma vitamin C levels above the estimated threshold plasma concentration of 50 to 60 µmol/L for effective protection from cardiovascular disease.^{23 24} Interestingly, monocyte adhesion was not correlated with plasma vitamin C levels in nonsmokers, which already exceeded the protective threshold of 60 µmol/L before supplementation. Intake of vitamin C for 10 days raised plasma concentrations both in smokers and nonsmokers to the level of tissue saturation achieved at plasma concentrations 80 µmol/L.²⁵

An inverse correlation of vitamin C as well as of carotene levels with coronary heart disease has been demonstrated,²³ and low plasma ß-carotene levels in smokers have been associated with an increased risk of myocardial infarction.²⁶ In our study, we could not find a significant reduction of ß-carotene levels in smokers. This may be attributed to the smaller sample size compared with epidemiological studies. Despite low absolute concentrations, however, carotenes may contribute to the antioxidative balance. In this context, it is notable that vitamin C can compensate for reduced levels of lipophilic antioxidants.^{26 27}

Monocyte adhesion to endothelium is crucially involved in atherogenesis.¹ In patients with atherosclerosis, monocyte adhesiveness is not related to the severity of atherosclerosis but to the presence of risk factors such as smoking.²⁴ Enhanced monocyte adhesiveness in smokers has been suggested to depend on cigarette consumption.²⁸ Our finding that the CD11b-dependent increase in monocyte adhesiveness in smokers can effectively be prevented by vitamin C intake infers that it is mediated by increased formation of radicals, which can be scavenged by ascorbate. In this context, the adhesion assay may be useful to assess possible protective effects of other micronutrients or drugs and to screen for altered monocyte adhesiveness in other conditions or disease states.

	Acknowledgments

 
This work was supported by grants from Bundesministerium für Forschung und Technologie (BMFT) and August-Lenz Stiftung. It partially fulfills requirements for the doctoral thesis of W. Erl. We thank Dr T. Schulz and Prof R. Lorenz for helpful discussions and B. Zimmer and Dr N. Hrboticky for help with lipophilic antioxidant determination.

Received January 23, 1996; accepted February 7, 1996.

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