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Lymphocyte T subset counts in children with elevated low-density lipoprotein cholesterol levels
ATHEROSCLEROSIS Atherosclerosis117(I 995) 119- 123
Lymphocyte T subset counts in children with elevated low-density lipoprotein cholesterol levels A. Sarria*a,
L.A. Moreno”, M. Mu-“, A. LBzaro”, M.P. Lasierrab, L. Roda”, A. Ginerc, L. Larradb, M. Buenos
“Departamento de Pediatria, Hospital Chico Universitario ‘Lozano Bless’, Ava’a. San Juan Bosco 15, 50009 Zaragoza, Spain bServicio de Inmunologia, Hospital Clinic0 Universitario ’ Lozano Bless’, Au&. San Juan Bosco 15, 50009 Zaragoza, Spain ‘Departamento de Bioquimica, Hospital Clinic0 Universitario ‘Lozano Bless’, Avda. San Juan Bosco 15, 50009 Zaragoza, Spain
Received5 August 1994;revision received3 February 1995;acceptedIO March 1995
Abstract
The aim of this study was to determine blood lymphocyte T subset counts in children with elevated levels of low-density lipoprotein cholesterol. We studied 107 children, ages 2.0 to 15.9 years, from 79 families who were referred to our Lipid Research Clinic becausetotal cholesterol serum levels higher than 200 mg/dl had been detected in at least one child. At the time of diagnosis we analyzed serum lipoprotein profile and blood lymphocyte T subsets (CD3, CD4 and CD8). Children were classified according to LDL-C levels into three groups: (1) normal, if levels were between the 5th and 75th percentiles (50 and 125 mg/dl, respectively); (2) at moderate risk, if levels were between the 75th and 95th percentiles (125 and 150 mg/dl, respectively); and (3) at high risk, if levels were above the 95th percentile (150 mg/dl). In children aged 2.0 to 6.9 years, all lymphocyte T subset counts were higher in the high risk group than in the normal group (P < 0.05 and P < 0.01). In children aged 11.Oto 15.9 years, the CD4 subset count was also significantly higher in the high risk group than in the other two groups (P < 0.05 and P < 0.01). These results are in agreement with pathologic findings in the atheromatous plaque. Keywords: T lymphocyte subsets; Low-density lipoprotein cholesterol; Atherosclerosis
1. Introduction Recently, it has been suggestedthat the inflammatory process and specific immunological mechanisms are associated with atherogenesis [ 1,2]. * Correspondingauthor. Fax: + 34 76 563 154.
Immunohistochemical analysis using monoclonal antibodies for specific cell types has shown that the lipid-rich core region of advanced human atherosclerotic plaques is dominated by macrophages [3,4]. The fibrous cap that surrounds the lipid core is dominated by vascular smooth muscle cells, but it also contains substantial numbers of both T lymphocytes and macrophages
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[3,5]. Other studies showed that monocyte-derived macrophages and T lymphocytes are present from a very early stage of atherosclerosis and are detectable in the fatty streak [5,6]. In healthy normolipidemic boys, significant correlations between low-density lipoprotein cholesterol and CD3 lymphocytes were found [7]. The aim of this study was to determine blood lymphocyte T subset counts in children with elevated levels of low-density lipoprotein cholesterol. 2. Patients and methods 2.1. Patients We studied 107 children of 79 families who came to our clinic between January 1990 and December 1992 because at least one child had cholesterol serum levels higher than 200 mg/dl. Before inclusion in the study all the participants were submitted to an interview and a medical examination to ascertain the possible presence of the following excluding factors: immunological diseases, use of corticosteroids or immunosuppressive drugs in the previous month and/or hepato-biliary diseases. When a child had an infectious diseaseor fever, the laboratory determinations were done 2 months after the end of the clinical and biological manifestations. At this time we analyzed serum lipoprotein profile and blood lymphocyte T subsets (CD3, CD4 and CD8). 2.2. Laboratory procedures Blood was obtained by vein puncture between 08:OOand 09:30 h, after overnight fasting. Total cholesterol was determined by an enzymatic method (cholesterol oxidase-peroxidase, Technicon, Basingstoke, UK) [8]. Total triglycerides were determined by the triglyceride-phosphateoxidase method in a Hitachi 705 autoanalyser [9]. High density lipoprotein-cholesterol (HDL-C) was determined after precipitation of lipoproteins linked to apolipoprotein B, with phosphotungstic acid and magnesium chloride [lo]. The low-density lipoprotein cholesterol (LDL-C) values were calculated according to Friedewald et al. [l 11. Lymphocyte T (CD3, CD4, CD8) subsets were measured using a FACScan flow cytometer, which had been calibrated using CaliBRITE Beads and
117 (1995) 119-123
AutoCOMP software. Flow cytometric two parameter dot plots and quadrant statistics were generated by SimulSET software (all from Becton Dickinson Immunocytometry Systems, San Jose, CA) [12]. After quality control assessment,flow cytometric measurements were electronically transferred from results files generated by SimulSET to a computer database, and quadrant statistics were analyzed. A complete blood cell count was also performed with a Technicon Counter. Absolute subset counts were obtained as the product of the absolute lymphocyte count and the percentage of the lymphocyte subset population of interest, as determined by flow cytometry [13,14]. 2.3. Statistical analysis All the variables studied, except triglycerides, had a Gaussian distribution. Thus for statistical analysis triglyceride serum levels were transformed logarithmically. For the analysis, children were divided into three age groups: 2.0 to 6.9 years, 7.0 to 10.9 years and 11.0 to 15.9 years; these groups were those used in studies concerning lymphocyte subpopulations in healthy children. Children were also divided by different LDL-C serum levels, according with values obtained in a healthy Spanish population [ 15,161:(1) normal, if levels were between the 5th and 75th percentiles (50 and 125 mg/dl, respectively); (2) at moderate risk, if levels were between the 75th and 95th percentiles (125 and 150 mg/dl, respectively); and (3) at high risk, if levels were above the 95th percentile (150 mg/dl). We have applied a twoway ANOVA model, with sex and age groups as group variables. There were significant effects only for age groups concerning blood lymphocyte T subsets (CD3, CD4 and CD8) counts. There was not significant interaction taking account of sex and age groups. Comparison between LDL-C groups were made using one-way ANOVA test (Fisher test); post-hoc testing has been carried out using Scheffe’s S-test. We have also calculated Spearman correlations with lipids and lipoproteins and lymphocyte T subset counts. Statistical analyses were performed using SPSS (Statistical Package for the Social Sciences).
A. Sarria et al. 1 Atherosclerosis
121
I I7 (1995) I19- 123
Table I Lipids and lipoproteins in children with different levels of LDL-C (mean f SD.) Normal
Moderate risk
High risk
15 14 12
9 11 8
14 17 7
TC (wldl) 2.0-6.9 years 7.0- 10.9 years 11.O- 15.9 years
170.53+ 24.25 183.00k 18.39 176.92k 21.01
210.11 +21.09b 215.36+ 14.81 214.38+ 11.83’
251.93 & 35.50=,e 270.53f 60.04”~’ 289.14-+ 56.55”,d
TG (mgldl) 2.0-6.9 years 7.0- 10.9 years ll.O-15.Y years
56.33+ 15.72 56.21 + 15.44 63.75 + 25.83
64.44 f 17.16 63.18 f 20.20 67.88 _+9.66
67.07 + 22.92 68.65 + 23.81 82.29& 35.66
58.60 + 13.92 63.79 & 13.89 60.25 + 13.05
60.22 f 19.63 67.73 k 14.44 67.00 + 10.45
54.07 + 11.61 64.59 f. 14.29 54.14 + 10.02’
100.67+ 15.04 108.07+ 9.36 104.00f 14.25
137.00k 5.72” 135.00& 5.80 133.88It 7.22
N 2.0-6.9 years 7.0- 10.9 years I1 .O- 15.9 years
HDL-C (mgldl) 2.0-6.9 years
7.0- 10.9 years 11.0-15.9 years LDL-C
(mg/dl)
2.0-6.9 years 7.0- 10.4 years 11.0-15.5, years
184.50f 34.45a,d 192.06-+ 60.15a.d 218.57-+ 62.42”.d
“-‘Significantly different from normal: “P < 0.001; bP < 0.01; “P < 0.05. d-fSignificantly different from moderate-risk group: dP < 0.001: “P < 0.01: ‘P < 0.05.
3. Results Table 1 gives lipids and lipoproteins in children divided by age and different levels of LDL-C. For all age groups, total and LDL cholesterol serum levels were significantly higher in the two risk groups than in the normal one. From 11.Oto 15.9 years, HDL-C serum concentration was significantly lower in the high risk group than in the moderate risk one. In children aged 2.0-6.9 years all the lymphocyte subset counts studied were significantly higher in the high risk group compared with the mean lymphocyte count in the normal group. In children aged 11.O- 15.9 years, lymphocyte CD4 count was higher in the high risk group compared with counts in the other two groups (Table 2). In children aged 2.0-6.9 years, Spearman correlations between total and LDL cholesterol vs. CD3 and CD8 lymphocyte counts were statistically significant. In children aged 1l.O- 15.9 years, triglyceride serum levels showed significant correlations
with all the lymphocyte subset counts studied. In children aged 7.0-10.9 years, triglyceride serum levels showed significant correlation with CD3 lymphocyte subset count; and in children aged 2.0-6.9 years, serum triglyceride levels also showed significant correlation with CD4 lymphocyte subset count (Table 3). 4. Discussion It has been suggested that monitoring of immune responses could be useful for the diagnosis and evaluation of the atherosclerotic process [2]. To assessatherosclerosis risk we have taken into account serum levels of LDL-C, because of its relation with the presenceof aortic fatty streaks in children [17]. In our study, serum levels of lipids and lipoproteins are in agreement with groups established by different levels of LDL-C. In the normal group, serum levels of all lipids and lipoproteins studied were similar to those observed in healthy Spanish children [ 15,161.
122
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117 (1995) 119- 123
Table 2 Lymphocyte T subsets in children with different levels of LDL-C (mean + SD.) Normal
Moderate risk
High risk
2.70 k 0.61 2.10 f 0.48 1.95* 0.52
3.04 + 0.86 2.32 + 0.40 2.02 + 0.45
3.68 &- 1.13” 2.37 f 0.50 2.54 f 0.89
1.41 + 0.35 1.Ol f 0.23 0.88 f 0.20
1.55+ 0.34 1.15_+0.32 0.99 * 0.28
1.75* 0.55b l.lO&O.27 I .32 -+ 0.33”,’
0.91 f 0.23 0.72 i 0.24 0.70 + 0.23
1.13* 0.39 0.84_+0.15 0.76 + 0.22
1.34* 0.51” 0.83 k 0.27 0.86 + 0.32
CD3 (IO’/!,
2.0-6.9 years 7.0-10.9 years 11.0-15.9 years CD4 (1 O’jl)
2.0-6.9 years 7.0- 10.9 years 11.0-15.9 years CD8 (109/l)
2.0-6.9 years 7.0- 10.9 years 1l.O- 15.9 years
a~bSignificantlydifferent from normal: “P < 0.01; bP < 0.05. “Significantly different from moderate-risk group: P < 0.05.
In children aged 2.0-6.9 years, all the T lymphocyte subset counts showed significant differences between the high risk group and the normal one. In children aged 1l.O- 15.9 years the CD4 lymphocyte subset count showed significant differences between the high risk group and the other two groups. These findings are parallel to those observed at the atherosclerotic plaque level. Immunocytochemical studies have shown that, in addition to macrophages being present in atherosclerotic plaques, about 20% of the cell population in the fibrous cap expressed the CD3 antigen, indicating that they were T lymphocytes [3]: CD8 lymphocytes are found mostly in early plaques [18] and fatty streaks [6], while CD4 lymphocytes are seen mostly in complicated arteriosclerotic lesions [3]. Significant correlations between total and LDL-C on the one hand, and some lymphocyte subset counts on the other hand, agree with results in Table 2. However, we do not have any explanation for the significant correlation between HDL-C and CD8 lymphocyte count. Significant correlation of lymphocyte counts and triglycerides also confirms some of our previous findings in healthy normolipidemic children [7].
To the best of our knowledge, there are no data about peripheral blood T lymphocyte subset counts in patients with coronary heart disease, nor in individuals with atherosclerosis risk or different levels of LDL-C. However, there are ever increasing data confirming the involvement of the immune system in the atherosclerotic process that begins in childhood. Recently, we have also described, in boys, a relation between C4 serum levels and the risk of atherosclerosis, taking into account serum levels of LDL-C [19]. In conclusion, we think that monitoring the immune responses, as other factors related with the risk of atherosclerosis [20], could be useful for the diagnosis and evaluation of the atherosclerotic process. Now, we must investigate the precise relationship between peripheral T lymphocyte subset counts and the pathogenic sequence of the atherosclerotic plaque formation that begins in childhood. Acknowledgement This work was supported by grant P CM-5/92 from Diputacion General de Aragon, Zaragoza, Spain.
A. Sarria
et al. / Atherosclerosi.~
I I7 (1995)
123
119- 123
Table 3 Spearman correlations between lymphocyte T subsets and lipids and lipoproteins ~~ HDL-C TG CT
LDL-C
CD.?
2.0-6.9 years 7.0- 10.9 years 11.0&159 years
0.442” 0.158 0.215
0.312 0.383b 0.541”
0.234 -0.127 - 0.209
0.371h 0.173 0.120
0.286 0.031 0.399
0.482” 0.118 0.498”
-0.055
-0.215 - 0.228
0.279 0.102 0.337
0.465” 0.124 0.236
0.141 0.266 0.454b
0.358h - 0.065 -0.012
0.363b 0.146 0.132
CD4
2.0-6.9 years 7.0-10 9 years 11.0.-159 years CD8
2.0-6.9 years 7.0-10.9 years 11.0-15.9 years “P
< 0.01; bP < 0.05.
References [I] Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s.Nature 1993;362:801. [2] Hansson GK. Immune and inflammatory mechanisms in the development of atherosclerosis.Br Heart J 1993;69:S38. [3] Jonasson L, Holm J, Skalli 0, Bondjers G, Hansson GK. Regional accumulation of T cells, macrophages and smooth muscle cells in the human atherosclerotic plaque. Arteriosclerosis 1986;6:I3 I. [4] Gown AM, Tsukada T, Ross R. Human atherosclerosis. II. Immunocytochemical analysis of the cellular composition of human atherosclerotic lesions. Am J Pathol 1986;125:191. [5] Van der Wal AC, Das PK, Van de Berg DB, Van der Loos CM, Becker AE. Atherosclerotic lesions in humans. In situ immunophenotypic analysis suggesting an immune mediated response. Lab Invest 1992;61:166. Fl Munro JM, Van Der Walt JD, Munro CS, Chalmers JAC, Cox E1L.An immunohistochemical analysis of human aortic fatty streaks. Hum Pathol 1987;18:375. 171Sarria A, Moreno L, Fleta J, Lasierra MP, Roda L, Giner A, Larraz L, Bueno M. Immunoglobulins, complement components and lymphocyte subsetsare related to plasma lipoproteins in healthy children. Atherosclerosis 1992; 93:157. PI Allain CC, Poon LS, Chan CSG, Richmond W. Enzymatic determination of total serum cholesterol. Clin Chem 1974;20:470. [91 Fossati P, Prencipe L. Serum triglycerides determined calorimetrically with an enzyme that produces hydrogen peroxide. Clin Chem 1982;28:2077. UOINguyen T, Albers A. Comparison of improved precipitation methods for quantification of high-density lipoprotein cholesterol. Clin Chem 1985;31:217. v11 Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in
plasma, without use of preparative ultracentrifuge. Clin Chem 1972;18:499. [I21 Loken MR, Brosnan J, Bach B, Ault KA. Establishing optimal lymphocyte gates for immunophenotyping by flow cvtometrv. Cvtometrv 1990:11:453. [I31 Erkeller-Yuksel FM: Deneys V, Yuksel B, Hannet I, Hulstaert F, Hamilton C, Mackinnon H, Turner Stokes L, Munhyeshuli V, Vanlangendonck F, De Bruyere M, Bach BA, Lydyard PM. Age-related changes in human blood lymphocyte subpopulations. J Pediatr 1992;120:216. 1141Hulstaert F, Hannet I, Deneys V, Munhyeshuli V, Reichert T, De Bruyere M, Strauss K. Age-related changesin human blood lymphocyte subpopulations. Ii. Varying kinetics of percentage and absolute count measurements. Clin Immunol Immunopathol 1994;70:152. [I51 Sarria A, Moreno LA, Mur M. Lbaro A, Bueno M. Usefulness of serum apolipoprotein B levels for screening children with primary dyslipoproteinemias. Am J Dis Child 1992;146:1230. [If31Plaza I, Grupo de Expertos de las SociedadesEspafiolas de Arteriosclerosis. Cardiologia, Pediatria, Nut&ion y Medicina Preventiva, Informe sobre el colesterol en nifios y adolescentes espanoles. Clin Invest Arteriosclerosis 1991;3:47. iI71 Newman NP, Freedman DS, Voors AN, Gard PD, Srinivasan SR, Cresanta JL, Williamson GD, Webber LS, Berenson GS. Relation of serum lipoprotein levels and systolic blood pressure to early atherosclerosis: The Bogalusa Heart Study. N Engl J Med 1986;314:138. [I81 Emeson EE, Robertson AL. T lymphocytes in aortic and coronary intimas. Their potential role in atherogenesis.Am J Pathol 1988;130:369. iI91 Moreno LA, Sarria A, LLaro A, Bueno M, Larrad L. Serum C4 concentration and risk of atherosclerosis.Br Med J 1994;309:1087. PO1Moreno LA, Sarria A, Mur M, Lbaro A, Roda L, Giner A, Bueno M. Serum bilirubin concentrations in children at risk for atherosclerosis. Clin Chem 1994;40:1791.
Lymphocyte T subset counts in children with elevated low-density lipoprotein cholesterol levels A. Sarria*a,
L.A. Moreno”, M. Mu-“, A. LBzaro”, M.P. Lasierrab, L. Roda”, A. Ginerc, L. Larradb, M. Buenos
“Departamento de Pediatria, Hospital Chico Universitario ‘Lozano Bless’, Ava’a. San Juan Bosco 15, 50009 Zaragoza, Spain bServicio de Inmunologia, Hospital Clinic0 Universitario ’ Lozano Bless’, Au&. San Juan Bosco 15, 50009 Zaragoza, Spain ‘Departamento de Bioquimica, Hospital Clinic0 Universitario ‘Lozano Bless’, Avda. San Juan Bosco 15, 50009 Zaragoza, Spain
Received5 August 1994;revision received3 February 1995;acceptedIO March 1995
Abstract
The aim of this study was to determine blood lymphocyte T subset counts in children with elevated levels of low-density lipoprotein cholesterol. We studied 107 children, ages 2.0 to 15.9 years, from 79 families who were referred to our Lipid Research Clinic becausetotal cholesterol serum levels higher than 200 mg/dl had been detected in at least one child. At the time of diagnosis we analyzed serum lipoprotein profile and blood lymphocyte T subsets (CD3, CD4 and CD8). Children were classified according to LDL-C levels into three groups: (1) normal, if levels were between the 5th and 75th percentiles (50 and 125 mg/dl, respectively); (2) at moderate risk, if levels were between the 75th and 95th percentiles (125 and 150 mg/dl, respectively); and (3) at high risk, if levels were above the 95th percentile (150 mg/dl). In children aged 2.0 to 6.9 years, all lymphocyte T subset counts were higher in the high risk group than in the normal group (P < 0.05 and P < 0.01). In children aged 11.Oto 15.9 years, the CD4 subset count was also significantly higher in the high risk group than in the other two groups (P < 0.05 and P < 0.01). These results are in agreement with pathologic findings in the atheromatous plaque. Keywords: T lymphocyte subsets; Low-density lipoprotein cholesterol; Atherosclerosis
1. Introduction Recently, it has been suggestedthat the inflammatory process and specific immunological mechanisms are associated with atherogenesis [ 1,2]. * Correspondingauthor. Fax: + 34 76 563 154.
Immunohistochemical analysis using monoclonal antibodies for specific cell types has shown that the lipid-rich core region of advanced human atherosclerotic plaques is dominated by macrophages [3,4]. The fibrous cap that surrounds the lipid core is dominated by vascular smooth muscle cells, but it also contains substantial numbers of both T lymphocytes and macrophages
0021-9150/95/%09.50 0 1995ElsevierScienceIreland Ltd. All rights reserved SSDZ 002 l-91 50(95)05567-G
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A. Sarria et al. 1 Atherosclerosis
[3,5]. Other studies showed that monocyte-derived macrophages and T lymphocytes are present from a very early stage of atherosclerosis and are detectable in the fatty streak [5,6]. In healthy normolipidemic boys, significant correlations between low-density lipoprotein cholesterol and CD3 lymphocytes were found [7]. The aim of this study was to determine blood lymphocyte T subset counts in children with elevated levels of low-density lipoprotein cholesterol. 2. Patients and methods 2.1. Patients We studied 107 children of 79 families who came to our clinic between January 1990 and December 1992 because at least one child had cholesterol serum levels higher than 200 mg/dl. Before inclusion in the study all the participants were submitted to an interview and a medical examination to ascertain the possible presence of the following excluding factors: immunological diseases, use of corticosteroids or immunosuppressive drugs in the previous month and/or hepato-biliary diseases. When a child had an infectious diseaseor fever, the laboratory determinations were done 2 months after the end of the clinical and biological manifestations. At this time we analyzed serum lipoprotein profile and blood lymphocyte T subsets (CD3, CD4 and CD8). 2.2. Laboratory procedures Blood was obtained by vein puncture between 08:OOand 09:30 h, after overnight fasting. Total cholesterol was determined by an enzymatic method (cholesterol oxidase-peroxidase, Technicon, Basingstoke, UK) [8]. Total triglycerides were determined by the triglyceride-phosphateoxidase method in a Hitachi 705 autoanalyser [9]. High density lipoprotein-cholesterol (HDL-C) was determined after precipitation of lipoproteins linked to apolipoprotein B, with phosphotungstic acid and magnesium chloride [lo]. The low-density lipoprotein cholesterol (LDL-C) values were calculated according to Friedewald et al. [l 11. Lymphocyte T (CD3, CD4, CD8) subsets were measured using a FACScan flow cytometer, which had been calibrated using CaliBRITE Beads and
117 (1995) 119-123
AutoCOMP software. Flow cytometric two parameter dot plots and quadrant statistics were generated by SimulSET software (all from Becton Dickinson Immunocytometry Systems, San Jose, CA) [12]. After quality control assessment,flow cytometric measurements were electronically transferred from results files generated by SimulSET to a computer database, and quadrant statistics were analyzed. A complete blood cell count was also performed with a Technicon Counter. Absolute subset counts were obtained as the product of the absolute lymphocyte count and the percentage of the lymphocyte subset population of interest, as determined by flow cytometry [13,14]. 2.3. Statistical analysis All the variables studied, except triglycerides, had a Gaussian distribution. Thus for statistical analysis triglyceride serum levels were transformed logarithmically. For the analysis, children were divided into three age groups: 2.0 to 6.9 years, 7.0 to 10.9 years and 11.0 to 15.9 years; these groups were those used in studies concerning lymphocyte subpopulations in healthy children. Children were also divided by different LDL-C serum levels, according with values obtained in a healthy Spanish population [ 15,161:(1) normal, if levels were between the 5th and 75th percentiles (50 and 125 mg/dl, respectively); (2) at moderate risk, if levels were between the 75th and 95th percentiles (125 and 150 mg/dl, respectively); and (3) at high risk, if levels were above the 95th percentile (150 mg/dl). We have applied a twoway ANOVA model, with sex and age groups as group variables. There were significant effects only for age groups concerning blood lymphocyte T subsets (CD3, CD4 and CD8) counts. There was not significant interaction taking account of sex and age groups. Comparison between LDL-C groups were made using one-way ANOVA test (Fisher test); post-hoc testing has been carried out using Scheffe’s S-test. We have also calculated Spearman correlations with lipids and lipoproteins and lymphocyte T subset counts. Statistical analyses were performed using SPSS (Statistical Package for the Social Sciences).
A. Sarria et al. 1 Atherosclerosis
121
I I7 (1995) I19- 123
Table I Lipids and lipoproteins in children with different levels of LDL-C (mean f SD.) Normal
Moderate risk
High risk
15 14 12
9 11 8
14 17 7
TC (wldl) 2.0-6.9 years 7.0- 10.9 years 11.O- 15.9 years
170.53+ 24.25 183.00k 18.39 176.92k 21.01
210.11 +21.09b 215.36+ 14.81 214.38+ 11.83’
251.93 & 35.50=,e 270.53f 60.04”~’ 289.14-+ 56.55”,d
TG (mgldl) 2.0-6.9 years 7.0- 10.9 years ll.O-15.Y years
56.33+ 15.72 56.21 + 15.44 63.75 + 25.83
64.44 f 17.16 63.18 f 20.20 67.88 _+9.66
67.07 + 22.92 68.65 + 23.81 82.29& 35.66
58.60 + 13.92 63.79 & 13.89 60.25 + 13.05
60.22 f 19.63 67.73 k 14.44 67.00 + 10.45
54.07 + 11.61 64.59 f. 14.29 54.14 + 10.02’
100.67+ 15.04 108.07+ 9.36 104.00f 14.25
137.00k 5.72” 135.00& 5.80 133.88It 7.22
N 2.0-6.9 years 7.0- 10.9 years I1 .O- 15.9 years
HDL-C (mgldl) 2.0-6.9 years
7.0- 10.9 years 11.0-15.9 years LDL-C
(mg/dl)
2.0-6.9 years 7.0- 10.4 years 11.0-15.5, years
184.50f 34.45a,d 192.06-+ 60.15a.d 218.57-+ 62.42”.d
“-‘Significantly different from normal: “P < 0.001; bP < 0.01; “P < 0.05. d-fSignificantly different from moderate-risk group: dP < 0.001: “P < 0.01: ‘P < 0.05.
3. Results Table 1 gives lipids and lipoproteins in children divided by age and different levels of LDL-C. For all age groups, total and LDL cholesterol serum levels were significantly higher in the two risk groups than in the normal one. From 11.Oto 15.9 years, HDL-C serum concentration was significantly lower in the high risk group than in the moderate risk one. In children aged 2.0-6.9 years all the lymphocyte subset counts studied were significantly higher in the high risk group compared with the mean lymphocyte count in the normal group. In children aged 11.O- 15.9 years, lymphocyte CD4 count was higher in the high risk group compared with counts in the other two groups (Table 2). In children aged 2.0-6.9 years, Spearman correlations between total and LDL cholesterol vs. CD3 and CD8 lymphocyte counts were statistically significant. In children aged 1l.O- 15.9 years, triglyceride serum levels showed significant correlations
with all the lymphocyte subset counts studied. In children aged 7.0-10.9 years, triglyceride serum levels showed significant correlation with CD3 lymphocyte subset count; and in children aged 2.0-6.9 years, serum triglyceride levels also showed significant correlation with CD4 lymphocyte subset count (Table 3). 4. Discussion It has been suggested that monitoring of immune responses could be useful for the diagnosis and evaluation of the atherosclerotic process [2]. To assessatherosclerosis risk we have taken into account serum levels of LDL-C, because of its relation with the presenceof aortic fatty streaks in children [17]. In our study, serum levels of lipids and lipoproteins are in agreement with groups established by different levels of LDL-C. In the normal group, serum levels of all lipids and lipoproteins studied were similar to those observed in healthy Spanish children [ 15,161.
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117 (1995) 119- 123
Table 2 Lymphocyte T subsets in children with different levels of LDL-C (mean + SD.) Normal
Moderate risk
High risk
2.70 k 0.61 2.10 f 0.48 1.95* 0.52
3.04 + 0.86 2.32 + 0.40 2.02 + 0.45
3.68 &- 1.13” 2.37 f 0.50 2.54 f 0.89
1.41 + 0.35 1.Ol f 0.23 0.88 f 0.20
1.55+ 0.34 1.15_+0.32 0.99 * 0.28
1.75* 0.55b l.lO&O.27 I .32 -+ 0.33”,’
0.91 f 0.23 0.72 i 0.24 0.70 + 0.23
1.13* 0.39 0.84_+0.15 0.76 + 0.22
1.34* 0.51” 0.83 k 0.27 0.86 + 0.32
CD3 (IO’/!,
2.0-6.9 years 7.0-10.9 years 11.0-15.9 years CD4 (1 O’jl)
2.0-6.9 years 7.0- 10.9 years 11.0-15.9 years CD8 (109/l)
2.0-6.9 years 7.0- 10.9 years 1l.O- 15.9 years
a~bSignificantlydifferent from normal: “P < 0.01; bP < 0.05. “Significantly different from moderate-risk group: P < 0.05.
In children aged 2.0-6.9 years, all the T lymphocyte subset counts showed significant differences between the high risk group and the normal one. In children aged 1l.O- 15.9 years the CD4 lymphocyte subset count showed significant differences between the high risk group and the other two groups. These findings are parallel to those observed at the atherosclerotic plaque level. Immunocytochemical studies have shown that, in addition to macrophages being present in atherosclerotic plaques, about 20% of the cell population in the fibrous cap expressed the CD3 antigen, indicating that they were T lymphocytes [3]: CD8 lymphocytes are found mostly in early plaques [18] and fatty streaks [6], while CD4 lymphocytes are seen mostly in complicated arteriosclerotic lesions [3]. Significant correlations between total and LDL-C on the one hand, and some lymphocyte subset counts on the other hand, agree with results in Table 2. However, we do not have any explanation for the significant correlation between HDL-C and CD8 lymphocyte count. Significant correlation of lymphocyte counts and triglycerides also confirms some of our previous findings in healthy normolipidemic children [7].
To the best of our knowledge, there are no data about peripheral blood T lymphocyte subset counts in patients with coronary heart disease, nor in individuals with atherosclerosis risk or different levels of LDL-C. However, there are ever increasing data confirming the involvement of the immune system in the atherosclerotic process that begins in childhood. Recently, we have also described, in boys, a relation between C4 serum levels and the risk of atherosclerosis, taking into account serum levels of LDL-C [19]. In conclusion, we think that monitoring the immune responses, as other factors related with the risk of atherosclerosis [20], could be useful for the diagnosis and evaluation of the atherosclerotic process. Now, we must investigate the precise relationship between peripheral T lymphocyte subset counts and the pathogenic sequence of the atherosclerotic plaque formation that begins in childhood. Acknowledgement This work was supported by grant P CM-5/92 from Diputacion General de Aragon, Zaragoza, Spain.
A. Sarria
et al. / Atherosclerosi.~
I I7 (1995)
123
119- 123
Table 3 Spearman correlations between lymphocyte T subsets and lipids and lipoproteins ~~ HDL-C TG CT
LDL-C
CD.?
2.0-6.9 years 7.0- 10.9 years 11.0&159 years
0.442” 0.158 0.215
0.312 0.383b 0.541”
0.234 -0.127 - 0.209
0.371h 0.173 0.120
0.286 0.031 0.399
0.482” 0.118 0.498”
-0.055
-0.215 - 0.228
0.279 0.102 0.337
0.465” 0.124 0.236
0.141 0.266 0.454b
0.358h - 0.065 -0.012
0.363b 0.146 0.132
CD4
2.0-6.9 years 7.0-10 9 years 11.0.-159 years CD8
2.0-6.9 years 7.0-10.9 years 11.0-15.9 years “P
< 0.01; bP < 0.05.
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