(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).
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.
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Abstract |
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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
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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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.
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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.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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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
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) 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 (
) 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.
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), and smokers after vitamin C intake ( |
Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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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.
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Acknowledgments |
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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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P. Libby and M. Aikawa Vitamin C, Collagen, and Cracks in the Plaque Circulation, March 26, 2002; 105(12): 1396 - 1398. [Full Text] [PDF]
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C. M Loria, M. J Klag, L. E Caulfield, and P. K Whelton Vitamin C status and mortality in US adults Am. J. Clinical Nutrition, July 1, 2000; 72(1): 139 - 145. [Abstract] [Full Text] [PDF]
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R. A Riemersma, K. F Carruthers, R. A Elton, and K. A. Fox Vitamin C and the risk of acute myocardial infarction Am. J. Clinical Nutrition, May 1, 2000; 71(5): 1181 - 1186. [Abstract] [Full Text] [PDF]
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A. C Carr and B. Frei Toward a new recommended dietary allowance for vitamin C based on antioxidant and health effects in humans Am. J. Clinical Nutrition, June 1, 1999; 69(6): 1086 - 1107. [Abstract] [Full Text] [PDF]
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K. N. Islam, S. Devaraj, and I. Jialal {alpha}-Tocopherol Enrichment of Monocytes Decreases Agonist-Induced Adhesion to Human Endothelial Cells Circulation, November 24, 1998; 98(21): 2255 - 2261. [Abstract] [Full Text] [PDF]
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S. Thorne, M. J. Mullen, P. Clarkson, A. E. Donald, and J. E. Deanfield Early endothelial dysfunction in adults at risk from atherosclerosis: different responses to L-arginine J. Am. Coll. Cardiol., July 1, 1998; 32(1): 110 - 116. [Abstract] [Full Text] [PDF]
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