Circulating monocytes in patients with diabetes mellitus, arterial disease, and increased CD14 expression

Circulating Monocytes in Patients With Diabetes Mellitus, Arterial Disease, and Increased CD14 Expression Ramo´n Patin ˜o, MD, Jose´ Ibarra, MD, Antonia Rodriguez, RN, Margarita Ruiz-Yagu¨e, MD, Emilio Pintor, MD, Arturo Fernandez-Cruz, Angeles Figueredo, MD

MD,

and

Low serum concentrations of high-density lipoprotein (HDL) cholesterol and elevated levels of acute-phase reactans are frequently found in patients with non–insulindependent diabetes mellitus (NIDDM) and cardiovascular disease. Changes in the phenotype of circulating monocytes have been reported with both of these circumstances in nondiabetic subjects. In the present study, we explored the possibility that similar changes may occur in circulating monocytes of patients with NIDDM and arterial disease. Two groups of subjects with NIDDM were studied: patients with cardiovascular disease (n ⴝ 25) were compared with a group without cardiovascular disease (n ⴝ 26); both groups were ageand sex-matched, had the same length of diabetes duration, and degree of glycemic control. Healthy nondiabetic volunteers of comparable age and sex (n ⴝ 35) formed the control group. There was no significant difference in the numbers of the CD14ⴙ/CD16ⴙ monocyte subpopulations between the 3 groups. However, a sig-

nificant graded increase of the mCD14 intensity expression values was observed among the groups, with the highest levels in patients with NIDDM patients and the lowest in nondiabetic subjects. The serum C-reactive protein concentrations were significantly higher in the group with arterial disease compared with those without arterial disease or healthy controls. In the group of patients as a whole, relative mCD14 intensity expression was significantly correlated with HDL cholesterol levels (inversely) and with serum concentrations of C-reactive protein. Serum HDL cholesterol levels and the C-reactive protein concentrations were also significantly correlated. We concluded that the increased mCD14 intensity expression on circulating monocytes may be an important contributor to the increased inflammatory response observed in patients with NIDDM and arterial disease, and eventually, to atherogenesis. 䊚2000 by Excerpta Medica, Inc. (Am J Cardiol 2000;85:1288 –1291)

levated levels of systemic inflammatory markers have been reported in patients with atherosclerotic E vascular disease who are with or without diabetes.

tory and/or infectious diseases, such as sepsis and acquired immune deficiency syndrome,6,7 and in individuals with low high-density lipoprotein (HDL) cholesterol levels.8 The initial goal of the present study was to measure the percentage of this subpopulation of monocytes in patients and normal control subjects, and to correlate the results to the presence of cardiovascular disease, lipid profile, and serum level of C-reactive protein.

1,2

The causes of this phenomenon are unknown. Adhesion of monocytes to endothelium and their subsequent extravasation into the vessel wall is one of the prominent features in the development of atherosclerotic plaque.3 Recent studies have shown that it is possible to define different monocyte phenotypes by flow cytometry, on the basis of the expression of surface antigens. The expression of the 55-kDa lipopolysaccharide receptor (CD14) and of the Fc ␥ receptor III (CD16) can be used to define 2 subpopulations of circulating monocytes with different functional characteristics.4,5 Increased numbers of the circulating proinflammatory CD14⫹ (dim)/CD16⫹ subset have been observed in patients with inflammaFrom the Service of Internal Medicine III, Cardiovascular Preventive Area, and Service of Immunology, Hospital Clı`nico Universitario de San Carlos, Medicina III, Madrid, Spain. This study was supported in part by the Department of Medicine, Faculty of Medicine, Universidad Complutense de Madrid, Madrid, Spain. E. Pintor was supported by a grant from the Fundacio´n Fernandez-Cruz, Madrid, Spain. Manuscript received July 30, 1999; revised manuscript received and accepted December 20, 1999. Address for reprints: Ramo´n Patin˜o, MD, Hospital Clı´nico De San Carlos. Medicina Interna III, Martin Lagos s/n 28040, Madrid, Spain.

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©2000 by Excerpta Medica, Inc. All rights reserved. The American Journal of Cardiology Vol. 85 June 1, 2000

METHODS The patients were selected from our outpatient diabetes clinic. Twenty-five patients with cardiovascular disease (see the following) and 26 without, with similar age and sex distribution, duration of diabetes, and level of glycemic control constituted the study groups. Thirty-five healthy volunteers of comparable age and sex served as controls. All patients with cardiovascular disease had clinical and/or angiographic evidence of coronary disease, Doppler ultrasound and/or angiographic signs of carotid artery disease, and/or peripheral vascular disease. The cardiovascular status was considered to be normal when a patient scored negative on the World Health Organization cardiovascular questionnaire9 and had no evidence of myocardial ischemia on a 12-lead electrocardiogram analyzed according to the Minnesota code. Hypertension was defined as systolic 0002-9149/00/$–see front matter PII S0002-9149(00)00757-8

TABLE I Clinical and Biochemical Characteristics of Patients and Control Subjects

Men/women Age (yrs) Diabetes duration (yr) BMI (kg/m2) HbA1c (%) Hypertensive (%) Microalbuminuric (%) Smoking (%) Insulin treatment (%) HMG-CoA inhibitors (%) Oral agents (%) Aspirin (%) Total triglyceride (mg/dl) Total cholesterol (mg/dl) LDL cholesterol (mg/dl) HDL cholesterol (mg/dl)

Normal Controls (n ⫽ 35)

NIDDM Without CVD (n ⫽ 26)

NIDDM With CVD (n ⫽ 25)

15/20 61 ⫾ 7.7 — 25 ⫾ 2 — 0 0 20 — — — — 98 (78–133) 228 ⫾ 19 151 ⫾ 19 57 ⫾ 12

11/15 64 ⫾ 5 15 ⫾ 6 29 ⫾ 4* 7.3 ⫾ 0.8 35 19 23 68 27 46 19 115 (80–160) 221 ⫾ 29 147 ⫾ 31 51 ⫾ 16

9/16 65 ⫾ 9 14 ⫾ 7 28.7 ⫾ 4 7.1 ⫾ 1 44 60† 23 65 32 35 68† 142 (98–204) 225 ⫾ 31 156 ⫾ 35 40.6 ⫾ 10†

*p ⬍0.01 patients with NIDDM without CVD versus controls; †p ⬍0.01 patients with NIDDM with CVD versus NIDDM patients without CVD. Data are mean ⫾ SD, or median (range) for values with skewed distribution. CVD ⫽ cardiovascular disease.

blood pressure of ⱖ140 mm Hg and/or a diastolic pressure of ⱖ90 mm Hg in subjects not taking antihypertensive medication. Target blood pressures during antihypertensive treatment were ⱕ140 mm Hg systolic and ⱕ85 mm Hg diastolic. Patients with macroalbuminuria (albumin excretion rate ⬎200 ␮g/min) were not included. The study was approved by our Hospital’s Ethics Committee. Circulating monocytes from whole blood were collected from patients and controls at the same time in tubes containing ethylenediaminetetraacetic acid and phenotyped by flow cytometry (FACScan; EPICS XL, Coulter, Miami, Florida). After incubation with antibodies (phycoerythrin-conjugated [R-PE] anti-CD14 antibody, fluorescein-conjugated [FITC] anti-CD16 antibody, and their respective isotype conjugated controls [Caltag Labs, Burlingame, California]) at 4°C and under saturating conditions, monocytes were gated by their forward- and side-scatter properties and analyzed by 2-color immunofluorescence. The mean fluorescence intensities (MFI) of CD14 receptors in the CD14⫹⫹(bright) monocyte subset were recorded. After subtracting for the nonspecific binding, the specific antibody binding in each patient was expressed as percent change from the corresponding paired control value measured in the same day and under identical experimental conditions.10 C-reactive protein levels were measured by nephelometry (Array 360 System, Beckman, La Brea, California) with a detection limit of 1 mg/L. In the statistical calculation, all values ⬍1 mg/L were treated as 0.9 mg/L. The urinary albumin concentration was measured by radioimmunoassay (Pharmacia, Uppsala, Sweden). Total and HDL cholesterol and triglyceride levels were assayed by enzymatic methods (Boehringer Mannheim, Mannheim, Germany). Glycosylated hemoglobin was measured by automated liquid chromatogra-

phy (Hitachi L-9100, Tokyo, Japan). The low-density lipoprotein (LDL) cholesterol fraction was calculated according to the Friedewald formula. Statistical analysis: Differences between groups in nominal variables were initially tested by analyses of variance for multiple comparisons (Scheffe´ or Kruskal-Wallis tests). Differences between 2 groups were analyzed by the Mann-Whitney U statistic test or the unpaired t test. The chi-square test was used for comparing proportions. Differences in MFI CD14⫹⫹ expression between patient and control pairs were evaluated using the Wilcoxon matched-pairs signed-rank test. The correlation between variables was assessed by the Spearman’s rank correlation test.

RESULTS

The clinical and laboratory data of the subjects are shown in Table I. No significant differences were found either in the total number of circulating monocytes or in the percentage subpopulations expressing both CD14⫹ (dim) and CD16 among the 3 groups. CD14⫹/CD16⫹ cells (median [range]) accounted for 9% (7 to 13) of the total monocytes in healthy controls versus 10% (8 to 12) in patients with uncomplicated diabetes and 11% (8 to 14) in those with cardiovascular disease (Figure 1). CD14 intensity expression in the CD14⫹⫹ (bright)/ CD16- monocyte subset showed significantly higher results in both groups of patients compared with their corresponding paired healthy controls (Figure 2). The comparison of CD14 intensities, expressed as percent of normal, between both groups of patients showed a significantly higher value in patients with cardiovascular disease (Figure 3). The serum C-reactive protein levels were higher in the group of patients with arterial disease compared with those without arterial disease and normal controls (median [95% confidence intervals (CI) range]), 3.2 mg/L (1.3 to 5) versus 1.3 mg/L (0.9 to 2.5) and 1.1 mg/dl (0.9 to 1.6), respectively. Bivariate correlations between relative CD14 expression and relevant biochemical and clinical features in the whole group of patients with non–insulindependent diabetes mellitus (NIDDM) revealed that only the HDL cholesterol (inversely) and the C-reactive protein levels correlated with CD14 relative intensity expression (Table II). A significant inverse relation was observed between the HDL cholesterol and C-reactive protein levels (r ⫽ 0.31, p ⫽ 0.027).

DISCUSSION We did not find any significant difference in the percentage of CD14⫹/CD16⫹ monocytes in patients with NIDDM either with or without vascular compli-

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and/or HMG-CoA reductase inhibitors (that have been shown to downregulate the membrane expression of CD14 in all circulating monocytes from hypercholesterolemic patients).11 This was a rather unexpected finding for which we have no clear-cut explanation. The monocyte CD14 is a multifunctional innate immune receptor for “infectious nonself” components,15 which may be involved in monocyte integrin CD11b/CD18 adhesion to cytokine-activated endothelium16 and may interact with “apoptotic self” components.17 Typically, binding of surface CD14 induces the production of inflammatory cytokines and reactive oxygen species, increased procoagulant acFIGURE 1. Percentage of the CD14ⴙ/CD16ⴙ monocyte cell subset in normal controls tivity, and clinical symptoms in huand patients with NIDDM who have or do not have cardiovascular disease (CVD). Difmans. The important point in relaferences are not significant. Columns are median ⴞ interquartile range. tion to the main finding in this study, however, is that CD14-mediated events are critically dependent on CD14 intensity expression.18 Therefore, patients with increased CD14 expression in their monocytes may be supposed to be prone to an augmented and/or unbalanced inflammatory response to endotoxin challenge, which may contribute to the development of atherosclerosis.19 This hypothesis is supported by the present study and by other studies, which provide evidence that patients with NIDDM show an augmented acute-phase response.1,20 In addition, it has been very recently demonstrated that genetic polymorphisms of the lipopolysaccharide receptor that lead to increased CD14 density in monocytes represent a risk factor for myocardial infarction.21,22 We do not know why the mCD14 expression was upregulated in the patients studied. High endotoxin blood levels were unlikely in our patients23; all of FIGURE 2. Comparison of CD14 MFI expression of patients with NIDDM who have or do not have cardiovascular disease (CVD) them were studied while they were under stable conwith their corresponding paired control group. Columns are meditions, without clinical evidence of ongoing infecdian ⴞ interquartile range. *p <0.01; ** p <0.001. tion. Increased plasma HDL has been shown to induce mCD14 downregulation in humans.24 Whether low HDL levels have the opposite effect is unknown, but cations compared with healthy-matched control sub- our results suggest that HDL is a parameter that injects. Increased numbers of this subset of circulating versely influences CD14 expression. Complement acmonocytes have been observed in nondiabetic hyper- tivation and higher blood levels of platelet-derived cholesterolemic patients with coronary heart dis- microparticles known to occur in patients with ease,11 a type of dyslipidemia basically absent in our NIDDM and arterial disease25,26 have been shown to patients, as is the rule in most of the diabetic popula- upregulate mCD14 expression and to induce cytokine tion in Spain.12 However, the expression of mem- production in monocytes,27,28 which together with the brane-bound CD14 in the largest subset of circulating cytokines originating in adipose tissue,29 might further monocytes, the CD14⫹⫹/CD16- subpopulation, was contribute to the observed increase in mCD14 density. significantly increased in both groups of patients comThe genetic origin of the phenotypical alteration pared with the healthy controls, the highest values described here cannot be wholly excluded; however, being observed in the group with cardiovascular dis- the previously mentioned genetic change that was ease. The degree of the abnormality may even be associated with increased CD14 expression was not greater, because a substantial number of the patients related to any classic vascular risk factor, including studied, in particular those with evidence of arterial diabetes.21,22 disease, were treated with aspirin (which may moduWhatever the reason, the increased mCD14 expreslate cytokine-dependent CD14 gene expression)13,14 sion that we have found in our patients could influence 1290 THE AMERICAN JOURNAL OF CARDIOLOGY姞

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FIGURE 3. Relative MFI CD14 expression in patients NIDDM who have or do not have cardiovascular disease (CVD). Columns are mean ⴞ SD. *p <0.001.

TABLE II Correlation of Relative MFI CD14 Expression With Clinical and Biochemical Risk Factors Variable Hypertension (yes vs no) Microalbuminuric (yes vs no) Smoking (yes vs no) HbA1c Triglyceride Total cholesterol HDL cholesterol LDL cholesterol C-reactive protein

r

p Value

⫺0.007 0.18 0.17 ⫺0.261 0.05 0.16 ⫺0.37 0.17 0.3

0.6 0.19 0.45 0.064 0.6 0.25 0.006 0.2 0.03

Spearman’s rank test.

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