© Borgis - Postępy Nauk Medycznych 11/2015, s. 760-765
Anna Niemirska1, Jacek Michałkiewicz2, *Mieczysław Litwin1
Przyspieszone dojrzewanie limfocytów krwi obwodowej u dzieci z pierwotnym nadciśnieniem tętniczym
Accelerated maturation of peripheral blood lymphocytes in children with primary hypertension**
1Department of Nephrology and Arterial Hypertension, The Children’s Memorial Health Institute, Warszawa
2Department of Microbiology and Immunology, The Children’s Memorial Health Institute, Warszawa
Streszczenie
Wstęp. Nadciśnienie tętnicze pierwotne (NTP) związane jest ze zmianami fenotypu i funkcji limfocytów T. Status limfocytów T można określić poprzez ocenę ekspresji izoform powszechnego antygenu leukocytarnego (CD45 Ag) w limfocytach T CD4+ lub CD8+. Limfocyty T CD45RA+ to tzw. komórki „naiwne”, a limfocyty T CD45RO+ i z ekspresją cząsteczek adhezyjnych to komórki „pamięci immunologicznej”.
Cel pracy. Wykazanie czy u dzieci z NTP dochodzi do zmian subpopulacji limfocytów T i czy koreluje to z wysokością ciśnienia tętniczego i uszkodzeniem narządowym.
Materiał i metody. 68 dzieci z nieleczonym NTP (śr. 15,6 ± 2 lat; 59 chłopców) i 26 dzieci z prawidłowym ciśnieniem tętniczym (śr. 14,8 ± 3,7 roku; 19 chłopców). Izoformy CD45RA+ i CD45RO+ na limfocytach T oceniono techniką cytometrii przepływowej.
Wyniki. Dzieci z NTP miały większą intensywność i więcej limfocytów CD4 i CD8 CD45RO+ w porównaniu do grupy kontrolnej. Stosunek RA+/RO+ na limfocytach CD4 i CD8 był mniejszy w porównaniu z grupą kontrolną. Ekspresja RA+ i RO+ nie korelowała z grubością kompleksu błona środkowa – błona wewnętrzna t. szyjnej wspólnej, ale pacjenci z NTP i przerostem lewej komory serca mieli mniej limfocytów CD4 CD45RA+, więcej limfocytów CD45RO+ i mniejszy stosunek RA+/RO+ w porównaniu do pacjentów z NTP i prawidłową masą lewej komory serca. Odsetek limfocytów CD8 CD45RA+ zmniejszał się ze zmianą statusu ciśnienia tętniczego od stanu przednadciśnieniowego, przez ambulatoryjne nadciśnienie tętnicze do ciężkiego nadciśnienia ambulatoryjnego.
Wnioski. Pacjenci z NTP mieli więcej dojrzałych limfocytów T CD4 i CD8 (o fenotypie komórek pamięci) oraz mniej limfocytów nieaktywnych (naiwnych). Te zaburzenia były związane z ciężkością nadciśnienia i uszkodzeniem narządowym.
Summary
Introduction. Primary hypertension (PH) is associated with immune activation. The isoforms of leucocyte common antigen (CD45) in the CD4 and CD8 T cell subsets are markers of T cells status. Naive cells express the RA+ isoform and memory cells express the RO+ isoform. The profile of CD45 isoforms may serve as the marker of T cells functional status and senescence.
Aim. Analysis of distribution of T lymphocytes bearing CD45RA+ (naive) and CD45RO+ (memory) markers and their relationship with hypertension severity and target organ damage (TOD).
Material and methods. 68 children with PH (15.6 ± 2 years, 59 boys) and control group of 26 (14.8 ± 3.7 years, 19 boys) children. The expression of CD45RA+ and CD45RO+ isoforms in the CD4 and CD8 T cell subset was evaluated by flow cytometry technique.
Results. Hypertensive children had greater intensity and percentage of CD45RO+CD4 and CD8 T lymphocytes and lower RA+/RO+ ratio of CD4 and CD8 lymphocytes than controls. T lymphocytes markers expression did not correlate with carotid intima-media-thickness. Children with left ventricular hypertrophy had less CD45RA+CD4 T cells, more CD45RO+ CD4 T cells and lower ratio of RA+/RO+CD4 cells than PH children with normal left ventricular mass index. The percentage of CD45RA+CD8 T cells decreased with increasing blood pressure status from prehypertension, ambulatory hypertension to severe ambulatory hypertension.
Conclusions. PH children had less naive T lymphocytes and more T cells with ‘memory phenotype’. These alterations correlated with hypertension severity and TOD.
Introduction
There is an increasing amount of data indicating that primary hypertension (PH) is not only a hemodynamic phenomenon but also a complex syndrome involving abnormal fat tissue distribution, over-activity of the sympathetic nervous system, metabolic abnormalities and activation of the immune system. It has been also reported that accelerated ageing of the immune system may play a role in the pathogenesis of hypertension and atherosclerosis in adults, with the special role of the innate and adaptive immune responses (1, 2). Immune system maturation and ageing is associated with increased populations of memory lymphocytes with highly diverse repertoire of antigenic specificity that enables their wide reactivity against both foreign and auto-antigens possibly including the vascular ones. The role of these cells in the PH pathogenesis is still obscure, especially in humans.
Aim
The aim of our study was to find out if the PH children differ from controls in terms of their CD4 and CD8 T cells ‘naive’ and ‘memory’ subsets distribution and if these changes correlate with target organ damage (TOD) or hypertension severity.
Material and methods
The study was performed according to the Declaration of Helsinki and with the approval of the Children’s Memorial Health Institute Ethics Committee. All patients (pts) and parents gave consent to participate in the study.
68 pts (mean age: 15.6 ± 2 years; 59 boys) with newly diagnosed and untreated PH, who underwent full diagnostic approach to exclude secondary hypertension, were included to the study. The exclusion criteria were: the presence of any significant chronic disease (except for PH) including diabetes mellitus, chronic kidney disease, chronic inflammatory disorders, any acute illness including infections in the 6 weeks preceding enrolment, and incomplete data. The control group consisted of 26 (19 boys) normotensive children in mean age 14.8 ± 3.7.
PH was diagnosed according to The Fourth Task Force Report and European Society of Hypertension guidelines and confirmed by 24-hour ambulatory blood pressure monitoring (ABPM) (3-5). Blood pressure status was defined according to the ABPM classification (5, 6). Hypertensive TOD (intima-media thickness of carotid arteries and left ventricular hypertrophy) and metabolic risk profile were assessed in PH group.
ABPM measurements
All ABPM measurements were assessed oscillometrically using SpaceLabs Monitor 90207, and the most appropriate cuff was applied on the non-dominant arm. Readings were taken every 20 minutes during daytime and every 30 minutes at night. Recordings lasting ≥ 20 hours with ≥ 80% of readings were considered as valid and were included to the analysis. We used a recently published classification system based on ABPM to classify patients as having normal blood pressure, ambulatory hypertension and severe ambulatory hypertension (5, 6).
Echocardiography
All echocardiography examinations were performed by 1 examiner who knew the clinical diagnosis, but was not aware of the severity of hypertension and the effectiveness of treatment. Echocardiography measurements were performed according to the guidelines of the American Society of Echocardiography (7). To standardize the left ventricular mass to height, left ventricular mass index (LVMI) was calculated according to the de Simone formula (8). Left ventricular hypertrophy (LVH) was defined as a LVMI value above the 95th percentile for age- and sex-based on reference data (9).
Carotid-intima media thickness (cIMT) measurements
cIMT was evaluated by ultrasound, and SD of normal values for cIMT was obtained according to the methodology described previously (10, 11).
Laboratory investigations
The following metabolic cardiovascular risk factors were assessed at diagnosis: plasma glucose level, lipid profile and serum uric acid. Blood samples were taken after 12 hours of fasting.
Evaluation of lymphocyte subsets by flow cytometry
The distribution of CD45RA+ and CD45RO+ isoforms of CD45 common leukocyte antigen in the CD4 and CD8 T cell subsets was determined by conventional three-color direct immuno-fluorescence. The samples of heparinized whole blood (50 ul) were stained for 30 min at room temperature with fluorescein (FITC), phycoerythrin (PE) and R phycoerythrycyanin 5.1 (PC5) conjugated mouse-anti-human monoclonal antibodies (mAbs). After staining, the samples were exposed to lysing solution (OptiLyse C, Beckman-Coulter), then washed and resuspended in PBS containing 2% of FCS and 0.1% sodium azide. The lymphocyte population was gated according to the forward-and side scatter light profile. Fluorescence was measured with a Beckman-Coulter FC-500 flow cytometer. Measurements were made on the FL1-channel (FITC-conjugated mAbs), the Fl-2 channel (PE-conjugated mAbs) and the PC5 mAbs (Fl-4 channel). The gates were adjusted into the appropriate negative control quadrant. A total of 10.000 events was collected. The FITC, PE and PC-5 conjugated mAbs were used in the following combinations, respectively: 1) CD4/CD8/CD3 and CD3/CD56/CD19 (for estimation of the basic lymphocyte subsets distribution), 2) CD45RA+CD4 or CD8, and CD45RO+CD4 or CD8, in combination with CD3 (for evaluation of CD45RA+ and CD45RO+ isoforms expression in the CD4 and CD8 T cell subsets). Simultest Leuco-Gate (CD45-FITC/CD14-PE) as well as gamma-FITC, gamma-PE and gamma-PC5 (Simultest control) were included in each staining panel. The fluorescence intensity (FI) was calculated as a relative mean channel fluorescence (RFI) for each surface molecule as described previously (12, 13).
Statistical analysis
The homogeneity of variance was checked with the Shapiro-Wilk test. Continuous variables with a normal distribution were compared using the Student t-test for independent variables. Continuous values with abnormal distribution were compared using the Wilcoxon test. Variables with normal distribution were presented as mean and SD values, whereas variables with abnormal distribution were presented as median and range values between the 5th and 95th percentiles. The correlation analysis was performed using Spearman test for abnormal distribution. Variables with significant correlation were included in the step-wise multiple regression analysis. P values < 0.05 were considered statistically significant, and values between 0.05 and 0.1 were considered as demonstrating trend toward significance.
Results
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