INCREASED SERUM HIGH-DENSITY LIPOPROTEIN CHOLESTEROL LEVEL IS ASSOCIATED WITH A REDUCTION IN PERIPHERAL MONOCYTE COUNT

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1 INCREASED SERUM HIGH-DENSITY LIPOPROTEIN CHOLESTEROL LEVEL IS ASSOCIATED WITH A REDUCTION IN PERIPHERAL MONOCYTE COUNT IN NORMAL INDIVIDUALS Zei-Shung Huang, 1 Kwan-Lih Hsu, 1 Kuo-Liong Chien, 1 Tien-Chun Chang, 1 and Chiu-Hwa Wang 2 Background and Purpose: Monocytes and high-density lipoprotein cholesterol (HDL-C) both play important roles in the process of atherosclerosis. This retrospective study investigated whether an increase in serum HDL-C level would be followed by a reduction in monocyte count. Methods: A total of 781 participants in a health check program in 1996 who had received a second health check in 1997, 1998, or 1999 were included. Based on the change in HDL-C at the second health check, the subjects were divided into the following 3 groups: 1) increase in HDL-C of 5% (n = 426); 2) change of HDL-C of < 5% (n = 162); and 3) decrease of HDL-C of 5% (n = 193). The relations between the change in HDL-C and the change in monocyte count were analyzed. Results: A significant inverse relation between the change in HDL-C level and the change in monocyte count was found among the 3 study groups by 1-way analysis of variance (p = 0.002). Subjects with increased HDL-C had significantly decreased monocyte count at the second check while subjects with decreased HDL-C had increased monocyte count. Multivariate regression analysis of data from all subjects revealed that the change of HDL-C was independently associated with a significant inverse change in monocyte count (p = 0.007). Conclusions: In view of the documented inflammatory nature of atherosclerosis, the inverse relation between the change of HDL-C level and the change of monocyte count may partly explain why a higher serum HDL-C level can protect arteries against atherosclerosis. Key words: Arteriosclerosis; Leukocyte count; Lipoproteins, HDL cholesterol; Monocytes J Formos Med Assoc 2005;104:231-5 Monocytes and the derived macrophages play important roles in atherosclerosis. 1 3 A higher peripheral monocyte count, usually reflecting an increased immunoinflammatory activity within the body, has been found to be associated with atherosclerosis 4,5 and its risk factors such as smoking, 6 8 hypertension, 9,10 hypertriglyceridemia, 8,10 and obesity. 8 A unique exception is that hypercholesterolemia has been found to be associated with a lower monocyte count, 8,10 especially in purely hypercholesterolemic subjects without coexisting hypertriglyceridemia. 8 This may be due to the following 2 reasons: 1) purely hypercholesterolemic subjects usually have a significantly higher serum highdensity lipoprotein cholesterol (HDL-C) level than normocholesterolemic subjects; 8 and 2) there is a significant inverse relationship between serum HDL-C level and peripheral monocyte count by single blood measurement. 8,11 14 In view of the immunoinflammatory nature of atherosclerosis, 3,17 the established roles of monocytes in both the inflammatory and the atherosclerotic process, 1 5 the positive associations between monocyte count and atherosclerosis 4,5 and most of atherosclerotic risk factors, 6 10 and an inverse relation between monocyte count and serum HDL-C level, 8,11 14 it seems reasonable to assume that the well-known protective effect of a higher serum HDL-C level against atherosclerosis 18,19 may be partially due to its association with a lower monocyte count. If this assumption is true, we would expect that, on serial measurements, an elevation of serum HDL-C level would be followed by a reduction in the peripheral monocyte count. This Departments of 1 Internal Medicine and 2 Laboratory Medicine, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan. Received: 10 June 2004 Revised: 19 July 2004 Accepted: 5 October 2004 Reprint requests and correspondence to: Dr. Zei-Shung Huang, Department of Internal Medicine, National Taiwan University Hospital, College of Medicine, National Taiwan University, No. 7, Chung-Shan South Rd., Taipei, Taiwan. J Formos Med Assoc 2005 Vol 104 No 4 231

2 Z.S. Huang, K.L. Hsu, K.L. Chien, et al retrospective study examined this relationship using data from subjects who underwent serial health checks. Methods Study subjects Study subjects were selected from participants in a health check program at our hospital in The enrollment conditions for this study included: 1) participants of the health check program in 1996 who had received a second health check in 1997, 1998, or We used chart numbers in 1996 to search computer data files to identify those who met this enrollment condition; 2) peripheral leukocyte analysis was checked with the same blood cell counter (Sysmex Cell Counter NE-8000; TOA Medical Electronics Co., Ltd, Kobe, Japan); and 3) serum HDL-C level (mg/dl) was checked with the same biochemical analyzer (Automatic Multichannel Biochemical Analyzer) [Hitachi-7450; Hitachi, Japan]. The purpose of the second and third conditions was to increase the accuracy and reliability of laboratory data. A total of 781 subjects met the inclusion criteria. Among them, 447 subjects received their second health checks in 1997, 234 subjects received their second health checks in 1998, and 100 subjects received their second health checks in The ages of the 781 subjects in 1996 ranged from 30 to 96 years, with a median of 56 and a mean of There were 629 men and 152 women. The age of the men ranged from 33 to 96 years, with a median of 57 and a mean of The age of the women ranged from 30 to 80 years, with a median of 54 and a mean of Data collection Data on age, gender, serum lipid profiles including HDL-C, total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and triglyceride (TG) levels, and the results of leukocyte analysis in the 2 health checks were obtained by chart review. The leukocyte analysis included total leukocyte count and differential percentages of neutrophils, lymphocytes, monocytes, eosinophils, and basophils. The differential percentages of these 5 kinds of leukocytes added up to 100%. The absolute count of a leukocyte subtype was calculated as the product of its respective differential percentage and total leukocyte count. Statistical methods Based on the change in HDL-C at the second health check, the 781 subjects were divided into the following 3 groups: 1) the increased HDL-C group included subjects with HDL-C increase by 5% or more (n = 426); 2) the stable HDL-C group included subjects with change in HDL-C of less than 5% (n = 162); and 3) the decreased HDL-C group included subjects whose HDL- C level decreased by 5% or more (n = 193). The means of changes in differential leukocyte counts/percentages between the 2 health checks were compared among the 3 groups by 1-way analysis of variance (ANOVA) [Table 1]. The increased HDL-C group was further divided into the following 3 subgroups: HDL-C increased 5% (n = 426), 10% (n = 337), and 20% (n = 211), Table 1. Change in differential leukocyte count and percentages at the second health check among increased high-density lipoprotein cholesterol (HDL-C), stable HDL-C, and decreased HDL-C groups analyzed by 1-way analysis of variance. Change in leukocyte count (10 9 /L)* All leukocytes Neutrophils Lymphocytes Monocytes Eosinophils Basophils Increased HDL-C group ± ± ± (0.009 ± 0.006) ± (0.005 ± 0.001) (n = 426) Stable HDL-C group ± ± ± ± ± (0.007 ± 0.002) (n = 162) Decreased HDL-C group (0.011 ± 0.086) (0.090 ± 0.068) ± ± ± (0.005 ± 0.002) (n = 193) p value Change in leukocyte percent (%)* Increased HDL-C group (n = 426) ± ± (0.250 ± 0.083) ± (0.101 ± 0.019) Stable HDL-C group (n = 162) ± (0.883 ± 0.571) (0.067 ± 0.141) ± (0.131 ± 0.033) Decreased HDL-C group (n = 193) (1.127 ± 0.519) ± ± ± (0.068 ± 0.029) p value < * Data are equal to [(value at the second check) (value at the first check)] and expressed as mean ± standard error of the mean. For monocyte count (percent), the significant p value indicates an inverse trend relation between the change in monocyte count (percent) and the change in serum HDL-C level among the studied 3 groups. For neutrophil percent, a significant difference was found only between the stable HDL-C and the decreased HDL-C groups. 232 J Formos Med Assoc 2005 Vol 104 No 4

3 Table 2. Change in monocyte count and percentage at the second health check in different study subgroups by paired t test and sign rank test. Increased HDL-C subgroups Decreased HDL-C subgroups 5% 10% 20% 5% 10% 20% (n = 426) (n = 337) (n = 211) (n = 193) (n = 127) (n = 48) Change in monocyte count (10 9 /L) p value by paired t test 0.054* * p value by sign rank test Change in monocyte percent (%) p value by paired t test 0.001* * p value by sign rank test * The marker or + before a probability value indicates a decrease or an increase, respectively, of monocyte count (percent) in the 2 consecutive checks. HDL-C = high-density lipoprotein cholesterol. respectively. The decreased HDL-C group was also further divided into 3 subgroups: HDL-C decreased 5% (n = 193), 10% (n = 127), and 20% (n = 48), respectively. Analyses of changes in monocyte count/ percent between the 2 health checks in these subgroups were analyzed by paired t test and sign rank test (Table 2). Results Table 1 shows the results of comparison of the mean change in differential leukocyte counts at the second health check among the increased HDL-C, stable HDL- C, and decreased HDL-C groups by 1-way ANOVA. A significant inverse relationship was found between the change in serum HDL-C level and the change in monocyte count in the 3 groups (p = 0.002), indicating that an increased serum HDL-C level was associated with a reduction of monocyte count, and decreased serum HDL-C level was associated with an increased monocyte count. There was no significant relationship between change of serum HDL-C level and other leukocyte counts. Comparison of changes in differential leukocyte percents also revealed a significant inverse relation between the change in serum HDL-C level and the change in monocyte percent in the 3 groups (p < 0.001). The differences in the changes in other leukocyte counts/percents and total leukocyte count between the 2 health checks were all insignificant except for the neutrophil percent that was significantly different between the stable HDL-C and the decreased HDL-C groups (p = 0.047, Table 1). We analyzed the relation between the extent of change in serum HDL-C level (%) and the mean of change in monocyte count (10 9 /L, mean ± SE) between the 2 health checks by 1-way ANOVA. Six levels of the extent of change in serum HDL-C level were defined as follows: decrease > 20.0%, decrease 19.9~10.0%, decrease 9.9~0.0%, increase 0.0~9.9%, increase 10.0~ 19.9%, increase > 20.0%. The results showed a trend of inverse relationship between the change in monocyte count and the percent change in HDL-C level of borderline significance (Fig., p = 0.074), suggesting an association between increase in HDL-C level and decrease in monocyte count. In the subgroup of subjects with HDL-C increase 10% (n = 337), analyses of changes in monocyte count between the 2 health checks by paired t test and sign rank test revealed a significant reduction of monocyte count in the second check (p = and 0.006, respectively; Table 2). In the subgroups of HDL- C increase 5% (n = 426) and HDL-C increase 20% (n = 211), the reduction of monocyte count in the second check was only borderline significant by paired t test (p = and 0.065, respectively) but was significant by sign rank test (both p = 0.008). The same analyses in the 3 decreased HDL-C subgroups revealed all had significant elevation of monocyte Change in monocyte count (10 9 /L) (n = 48) (n = 79) (n = 161) (n = 156) One-way ANOVA p = (n = 126) Percent change in serum HDL-C level 20 (n = 211) Fig. Relation between the extent of change in serum highdensity lipoprotein cholesterol (HDL-C) level (%, x axis) and the mean of change in monocyte count (mean ± standard error, y axis) at the second health check in all 781 study subjects analyzed by 1-way analysis of variance (ANOVA). A trend toward an inverse relationship was found between these 2 parameters with borderline significance (p = 0.074). J Formos Med Assoc 2005 Vol 104 No 4 233

4 Z.S. Huang, K.L. Hsu, K.L. Chien, et al Table 3. Multivariate regression analysis of the relationship between the change in monocyte count at the second health check and age, gender, and the changes of serum high-density lipoprotein cholesterol (HDL-C), total cholesterol (TC), lowdensity lipoprotein cholesterol (LDL-C), and triglyceride (TG) levels in all study subjects (n = 781). Change of monocyte count (10 9 /L) Regression coefficient p value (standard error)* Age (in 1996) ( ) Gender ( ) Change in HDL-C level (mg/dl) ( ) Change in TC level (mg/dl) ( ) Change in LDL-C level (mg/dl) ( ) Change in TG level (mg/dl) ( ) * Regression coefficients were estimated by ordinary least squares method and probability values were calculated by t test. For the analysis of LDL-C level, 26 cases with TG level 450 mg/dl were excluded because of a profound influence of such a high TG level on the laboratory measurement of LDL-C level. count (Table 2). Analyses of mococyte percent revealed similar results as those for monocyte count, but all p values obtained in analyses of monocyte percent were more significant than those for monocyte count (Table 2). As shown in Table 3, multivariate regression analysis of the change of monocyte count on age, gender, and the changes of serum HDL-C, TC, LDL- C, and TG levels revealed that the change of HDL-C level was independently associated with a significant inverse change in monocyte count (p = 0.007) and the change of LDL-C level was associated with a marginally significant inverse change in monocyte count (p = 0.050). There was no significant association between the change of monocyte count and the change of TC level or the change of TG level (p = and 0.209, respectively; Table 3). Discussion Although several previous studies have found a significant inverse association between serum HDL-C level and peripheral monocyte count on single blood measurement, 8,11 14 this study is the first study examining this relationship in serial blood measurements. Our results indicate that increased serum HDL-C level was associated with a reduction in peripheral monocyte count. This finding together with the inflammatory nature of atherosclerosis 3,17 and the established roles of monocytes in both the inflammatory activity and the atherosclerotic process, 1 5 may provide some insight into the mechanism responsible for the antiatherogenic effect of a higher serum HDL-C level. Monocytes and derived macrophages are known to play important roles in atherosclerosis. 1 5 A higher peripheral monocyte count usually reflects an elevated immunoinflammatory activity within the body and thus may be able to promote the atherosclerotic process due to its chronic immunoinflammatory nature. 3,17 Several clinical studies have found positive associations between peripheral monocyte count and risk of both atherosclerosis 4,5 and atherosclerosis-related diseases such as coronary artery disease Therefore, a reduction in monocyte count associated with an elevation in serum HDL-C level, as found in the study, may partly contribute to the protective action of a higher serum HDL-C level against atherosclerosis and atherosclerosisrelated diseases. 18,19 Possible reasons for the association of increased serum HDL-C level with a reduction in peripheral monocyte count might include: 1) increased serum HDL-C level indicated improved reverse cholesterol transport by HDL particles and thus reduced need for monocytes and macrophages to remove cholesterol deposits from arterial walls 19,23,24 ; 2) more HDL particles in the blood might reduce monocyte recruitment by either inhibiting vascular intercellular adhesion molecule production 19,25,26 or preventing lipid peroxidation and LDL oxidation via HDL particle-associated paraoxonases 19,27 29 ; and 3) the HDL particles may possess an anti-inflammatory property of being able to bind on certain inflammatory mediators, neutralize their leukocyte chemotactic activities, 15,16 and thus suppress monocyte recruitment. In summary, a lower serum HDL-C level and a higher peripheral monocyte count are both associated with atherosclerosis and atherosclerosisrelated diseases such as coronary artery disease. The association of a reduction in peripheral monocyte count with an increase in serum HDL-C level in serial blood measurements suggests the role and contribution of this relationship in the well known protective effect of a higher serum HDL-C level against atherosclerosis and related diseases. ACKNOWLEDGMENT: This study was financially supported by the grant Management and Prevention of Stroke of National Taiwan University Hospital. References 1. Gerrity RG: The role of the monocyte in atherosclerosis. I. Transition of blood-borne monocytes into foam cells in fatty lesions. Am J Pathol 1981;103: Joris I, Zand T, Nunnari JJ, et al: Studies on the pathogenesis of atherosclerosis. I. Adhesion and emigration of mononuclear cells in the aorta of hypercholesterolemic rats. 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