Chemokine receptors on peripheral blood T lymphocytes in children on peritoneal dialysis

Abstract

Background:

Immune cell dysfunction is listed among complications resulting from chronic kidney disease (CKD). It could be associated with T-cells, which play a role in the lymphocytic migration and infiltration. However, the data on chemokine receptors expression on T-cells in patients with CKD particularly treated with peritoneal dialysis (PD) are still limited.

Methods:

The study aimed at multiparameter flow-cytometric analysis of the absolute numbers and percentage of T-cell subsets with surface chemokine receptors (CCR4, CCR5, CCR7, CXCR3, and CXCR4) or receptors’ combinations in 47 children treated with PD.

Results:

We found lower absolute numbers of total T lymphocytes, lymphocytes with surface CCR5, CXCR4⁺CCR5, CXCR3⁺CCR5 antigens and T-cells with CCR4, CCR4⁺CD4, CXCR3, CXCR3⁺CD4, and CD8 receptors. Lymphocytes T with CD4, CCR7, CD28⁺CCR7, CXCR3⁺CD8 antigens showed higher percentage in children on PD as compared to healthy children and opposite percentage values of CCR4⁺, CCR4⁺CD4⁺, CXCR3⁺ T lymphocytes were diminished. Mean fluorescent intensity for CCR7⁺, CCR7⁺CD45RO⁺, CCR7⁺CD28⁺, CXCR4⁺CD4⁺, CCR5⁺CD4⁺, CCR4⁺, CCR4⁺CD4⁺ T-cells was lower in the PD group than in healthy children. The analysis of correlation between T lymphocyte subpopulations with chemokine receptors and other parameters revealed positive correlation of CCR7⁺ and CCR7⁺CD28⁺ T-cells and weekly creatinine clearance, negative correlation between the percentage of CD45RO⁺CCR7 antigen positive T-cells and KT/V_urea.

Summary:

In conclusion, we could not confirm the phenomenon of earlier senescence of T-cells in children with CKD on PD treatment. This still requires further investigation. The higher percentage of T-cells with CCR7 surface receptor could be responsible for the increase of proliferation activity in this group of children.

Keywords

Chemokine receptors children with CKD peritoneal dialysis T lymphocytes

Introduction

The chemokine-receptor system plays the crucial role in the regulation of both innate and adaptive immunity, therefore it provides appropriate immune response in healthy individuals. Chemokines, described as low molecular-weight proteins, are responsible for chemoattraction. Binding to the corresponding receptors, chemokines activate signaling pathways, that results in cell migration, differentiation, and proliferation, in physiological, as well as in pathological conditions.¹ Chemokine receptors are structurally seven-transmembrane proteins, also known as G protein-coupled receptors.² These molecules have been divided into subfamilies such as CCR, CXCR, CX3CR, or XCR, numbered in accordance with an order of their discovery.^3
–5

Human CC chemokine receptor type 5 (CCR5) has been identified as the receptor for chemokines such as macrophage inflammatory protein 1α (CCL3), macrophage inflammatory protein-1β (CCL4), and regulated on activation, normal T-cell expressed and secreted (RANTES; CCL5),⁶ which also acts as a co-receptor that allows R5-HIV-1 entry into cells, when co-expressed with CD4.⁷

Immune cell dysfunction is listed among complications resulting from chronic kidney disease (CKD). It is believed that the process might be associated with T-cells, due to CCR7 receptor expression on their surface and an influence on the lymphocyte migration and infiltration. It was previously documented that the uremic milieu induces premature aging of T-cells.⁸ However, the data on chemokine receptors expression on T-cells in patients with CKD particularly treated with peritoneal dialysis (PD) are still limited^6,7

Miao et al. demonstrated that AMG487 (small-molecule CXCR3 antagonist) combined with cyclosporine A has effectively reduced the prevalence of graft versus host disease (GvHD) and increased the survival time. This compound has also remarkably inhibited the infiltration of inflammatory cells in GvHD target tissues in a murine aGvHD model, as well as inhibited the production and differentiation of splenic donor-derived T-cells.⁹ Paust et al. suggested that specific targeting of CXCR3 on T_H1 cells might be effective in T_H1-driven inflammatory diseases including crescentic glomerulonephritis.¹⁰

The CXCR4 protein (chemokine CXC motif receptor 4, also called the CD184 antigen or LESTR antigen, a leukocyte-derived seven-transmembrane domain receptor) is a receptor for stromal 1 cell factor (SDF-1; chemokine CXC motif ligand 12 (CXCL12)). CXCR4 performs its main functions together with its ligand both in the mobilization of the stem cells into the bloodstream and during homing, hence the colonization of the marrow niches after the cells transplantation.¹¹ Increased expression of the CXCR4 receptor is a vital element in the diagnosis and prognostic assessment of the rhabdomyosarcoma.¹²

A significant therapeutic potential of these co-receptors is also described in several other processes, such as tissue repair or regeneration and inflammation.¹³ Maluf et al. showed that CXCR4 has been upregulated in fibrotic lesions in renal biopsy samples.¹⁴ During experimental study on animals, Zou et al. found out that blocking the CXCR4 resulted in improvement of the renal interstitial fibrosis in surviving allografts by downregulation of transforming growth factor β1 concentration.¹⁵

In the inflammatory process, there is a high concentration of chemokines that cause the attraction of multiple chemokine receptor-expressing lymphocytes. The studies on animal models in lupus nephritis highlighted the role of chemokines in autoimmune systemic reactions in various tissues, including renal tissues, and proved their responsibility for local renal inflammation with direct effects and subsequent damage eventually leading to irreversible CKD.¹⁶ Current investigations target their efforts to design treatment limiting inflammation. Such agents, presumably consisting of small molecules alongside with monoclonal antibodies, would aim to block the interactions between chemokines and chemokine receptors by neutralizing either one of them. Therefore, the research on chemokine receptors in children undergoing PD might bring to light some new strategies in preventing an enhanced inflammatory response during stage 5D CKD and after kidney transplantation.

Aims

The study aims at multiparameter flow-cytometric analysis of the absolute numbers and percentage of T-cell subsets with selected surface chemokine receptors or receptors’ combinations in children treated with PD.

Materials and methods

The study involved 47 children and young adults with CKD (22 girls and 25 boys) treated with PD at the Department of Dialysis of Children at the Chair and Clinic of Pediatrics in Zabrze, Medical University of Silesia in Katowice. The characteristics of the group are presented in Table 1. In the PD group, the study was performed during routine checkups. There were no signs of infection in the examined group of children. Determination of hemoglobin, erythrocytes, leukocytosis, and lymphocytosis was performed with the MicroDiffII hematology analyzer (Becton Coulter, Fullerton, CA, USA). The concentration of hemoglobin was expressed in g/dl, the number of erythrocytes was expressed as 1 × 10¹²/l, the number of leukocytes and platelets as 1 × 10⁹/l, hematocrit was expressed as a percentage. The concentration of creatinine and urea in the blood serum (Kodak Ektachem 700; Kodak, Rochester, NY, USA) were evaluated in the biochemical laboratory tests. Creatinine concentration was expressed in µmol/l, urea concentration in mmol/l.

Table 1.

Baseline clinical characteristics and selected laboratory tests result in studied children.^a

Analyzed parameter	PD group (n = 47)	Control group (n = 41)	p Value
Mean age (years)	11.9 ± 5.4	12.5 ± 4.1	0.57
Sex (F/M)	22/25	16/25	0.40
Dialysis duration (years)	1.2 ± 1.1
CKD duration (years)	4.6 ± 3.4
Weight (kg)	34.9 ± 18.3	46.7 ± 17.8	<0.01
Height (cm)	134.3 ± 28.5	157.3 ± 23.6	<0.002
BMI (kg/m²)	17.8 ± 3.8	19.8 ± 3.5	<0.04
MAP (mmHg)	81.0 ± 12.0	76.7 ± 13.6	<0.006
Hemoglobin (g/dl)	10.3 ± 1.3	13.6 ± 1.4	<0.0001
Hematocrit (%)	30.1 ± 3.8	39.5 ± 4.7	<0.0001
Erythrocytes (1 × 10¹²/l)	3.3 ± 0.4	4.7 ± 0.4	<0.001
Thrombocytes (1 × 10⁹/l)	245.8 ± 89.2	308.5 ± 89.6	<0.001
Leukocytes (1 × 10⁹/l)	6.3 ± 1.9	7.7 ± 1.9	<0.0005
Creatinine (µmol/l)	664.7 ± 308.0	66.5 ± 16.9	<0.0001
Urea nitrogen (mmol/l)	19.7 ± 8.0	4.3 ± 1.21	<0.001
GFR (ml/min)		124.2 ± 23.2
Kt/V_urea	2.7 ± 0.9
WCC (L/week)	77.1 ± 29.6

PD: peritoneal dialysis, BMI: body mass index, MAP: mean arterial pressure, WCC: weekly creatinine clearance.

^a Absolute values are shown as mean ± standard deviation. p < 0.05: significant PD versus control group.

The mean duration of dialysis was estimated, and the Kt/V_urea dialysis ratio and weekly creatinine clearance (WCC) were calculated in children on PD. WCC in automatic PD was considered adequate for values ≥70 l. Kt/V_urea, as for children with PD values ≥2.1, indicated an adequate dialysis.

All children underwent blood pressure (BP) measurement and mean arterial pressure was calculated (MAP = 1/3 difference in systolic and diastolic pressures + diastolic pressure), body surface area and body mass index (BMI; body mass divided by the square of the body height, kg/m²) were also estimated (Table 1). BP values, expressed in mmHg, were considered abnormal if they exceeded the 95th percentile for age, sex, and height. BMI values were adjusted for sex and classified as lower than 5 percentile: malnutrition; 5–85: normal weight; above 85 percentile: —overweight; and above 95 percentile: obesity.

The control group (ZD) was represented by 41 healthy children (16 girls, 25 boys). These patients were admitted to the hospital due to nocturnal enuresis or for elective surgery (day surgery). BP values and renal function indices in this group were within the normal range for sex and age. Estimated glomerular filtration rate was calculated using the Schwartz formula [ml/min/1.73 m²].¹⁷

Methods of the cytometric parameters determination

Each patient had 2 ml of blood withdrew by puncture of a peripheral vein in order to perform flow cytometry. Blood samples were collected using anticoagulant (unfractionated heparin) and then analyzed within 4 h. Immunophenotyping of each sample was performed by six-color flow cytometry on a BD FACSCanto II flow cytometer (Becton Dickinson, Biosciences, San Jose, California, USA). The whole blood labeling method followed by the lysis of erythrocytes was used. A panel of 11 combinations of monoclonal antibodies was chosen to determine individual lymphocyte populations in accordance with a standard staining protocol. Combinations of antibodies from the diagnostic panel are presented in Table 2.

Table 2.

The panel of monoclonal antibodies for lymphocyte subpopulations flow cytometry.

No	FITC	PE	PerCP	PE-Cy7	APC	APC-Cy7
1	CD4BD	—	CD3BD	CCR4BD Pharmingen	—	CD8BD
2	CD45RODako	CD197 = CCR7BD Pharmingen	CD3BD	CD19BD	CD28BD Pharmingen	CD8BD
3	CD4BD	CD183 = CXCR3BD Pharmingen	CD3BD	CD195 = CCR5BD Pharmingen	CD184 = CXCR4BD Pharmingen	CD8BD

BD: Becton Dickinson; BD Pharmingen: Becton Dickinson Pharmingen-Biosciences; Dako: Dakopatts, Glostrup, Denmark; FITC: fluorescein isothiocyanate; PE: phycoerythrin; PerCP: peridinin-chlorophyll; PE-Cy7: phycoerythrin-cyanin 7; APC: allophycocyanin; APC-Cy7 allophycocyanin-cyanin 7.

After the proper time of incubation, the erythrocytes were lysed with FACS Lysing Solution (Becton Dickinson Biosciences). Subsequently, the samples were rinsed in PBS (NaCl, Na₂HPO₄, NaH₂PO₄, NaN₃, pH (25°C) 7.2 ± 0.1) and then loaded into the flow cytometer.

Data were recorded and then analyzed using a Diva computer program (Becton Dickinson, Immunocytometry Systems, San Jose, California, USA). The FACSCanto II flow cytometer was subjected to daily quality control using the Rainbow Beads microspheres (Spherotech Inc., Greek Oaks, Illinois, USA).

Statistical analysis of the results

The data were analyzed using the STATISTICA 12.0 PL program (StatSoft, Inc. 2014). For the parameters tested, basic descriptive statistics were made and the mean value, median, minimum, and maximum values were determined. For all parameters, their distribution was checked for compatibility with the normal distribution using the Shapiro–Wilk test. The homogeneity of variance was tested using the Levene test. For a comparison of variables with a normal distribution, parametric tests with a separate variance assessment were performed. For comparative analysis of variables with distribution deviated from the normal distribution, nonparametric Mann–Whitney U test was used. The Pearson linear correlation test or Spearman rank correlation enabled to determine the correlation between parameters. The test results were presented graphically in the form of tables. The values fulfilling the condition p < 0.05 were considered statistically significant.

Results

Table 1 summarizes the baseline characteristics of the studied population. Children on PD were described with lower body weight, height, BMI, and higher MAP values. Regarding complete blood count in this group, the red blood cell count, white blood cell count, and platelet count, hemoglobin concentration and hematocrit values were significantly lower in comparison with the control group. Serum creatinine level and blood urea concentration were increased as in stage 5D CKD.

Tables 3 and 4 present the comparison of absolute values and percentage of T lymphocytes with selected chemokine receptors in the group of children on PD and healthy controls. The outcome revealed decreased absolute numbers of total T lymphocytes, lymphocytes with surface CCR5, CXCR4⁺CCR5, CXCR3⁺CCR5 antigens and T-cells with CCR4, CCR4⁺CD4, CXCR3, CXCR3⁺CD4 and CD8 receptors. Lymphocytes T with CD4, CCR7, CD28⁺CCR7, CXCR3⁺CD8 antigens percentage was more numerous in children on PD as compared to healthy children and opposite percentage values of CCR4⁺, CCR4⁺CD4⁺, CXCR3⁺ T lymphocytes were diminished. Mean fluorescent intensity for CCR7⁺, CCR7⁺CD45RO⁺, CCR7⁺CD28⁺, CXCR4⁺CD4⁺, CCR5⁺CD4⁺, CCR4⁺, CCR4⁺CD4⁺ T-cells was lower in the PD group than in healthy children (Table 5).

Table 3.

Absolute values of T-cell subpopulations, including populations with expression of receptors for selected chemokines in examined children and healthy controls.^a

Lymphocyte subpopulations (G/l)	PD group	Control group	p Value
T Lymphocytes	1.63 (0.7–6.8)	1.8 (1.4–4.6)	<0.03
CD4⁺ T-cells	0.98 (0.2–2.6)	0.94 (0.2–2.2)	0.83
CD8⁺ T-cells	0.59 ± 0.32	0.62 ± 0.3	0.67
CCR7⁺ T-cells	0.39 (0.10–1.6)	0.06 (0.005–1.8)	0.39
CD45RO⁺CCR7⁺	0.04 (0.01–0.11)	0.02 (0.01–0.58)	0.19
CD28⁺CCR7⁺ T-cells	0.45 ± 0.36	0.30 ± 0.43	0.18
CXCR4⁺ T-cells	0.34 (0.08–0.82)	0.21 (0.06–2.0)	0.17
CXCR4⁺CD4⁺ T-cells	0.13 (0.02– 0.4)	0.23 (0.00001–0.9)	0.11
CXCR4⁺CD8⁺ T-cells	0.3 (0.04–0.9)	0.2 (0.02–1.1)	0.61
CXCR4⁺CCR5⁺ T-cells	0.04 (0.00004–0.2)	0.13 (0.00001–0.2)	<0.048
CCR5⁺ T-cells	0.26 (0.02–0.63)	0.41 (0.07–1.69)	<0.03
CCR5⁺CD4⁺ T-cells	0.07 (0.00001–0.2)	0.1 (0.014–0.28)	0.13
CCR5⁺CD8⁺ T-cells	0.2 (0.05–0.4)	0.24 (0.02–0.6)	0.20
CCR4⁺ T-cells	0.10 (0.04–0.24)	0.27 (0.02–1.18)	<0.0002
CCR4⁺CD4⁺ T-cells	0.06 (0.03–0.2)	0.14 (0–0.4)	<0.0001
CCR4⁺CD8⁺ T-cells	0.02 (0.00002–0.8)	0.03 (0.00001–0.9)	0.9535
CXCR3⁺ T-cells	0.59 ± 0.32	0.84 ± 0.33	<0.003
CXCR3⁺CD4⁺ T-cells	0.18 ± 0.07	0.25 ± 0.12	<0.03
CXCR3⁺CD8⁺ T-cells	0.32 ± 0.19	0.46 ± 0.21	<0.006
CXCR3⁺CCR5⁺ T-cells	0.19 (0.02–0.54)	0.30 (0.05–1.12)	<0.04
CXCR3⁺CXCR4⁺ T-cells	0.15 (0.01–0.43)	0.25 (0.02–0.96)	0.15

PD: peritoneal dialysis.

^a Absolute values are shown as mean ± standard deviation for Gaussian distribution, otherwise as median (minimum–maximum). p < 0.05: significant PD versus control group.

Table 4.

Percentage values of T-cell subpopulations, including populations with expression of receptors for selected chemokines in examined children and healthy controls.^a

Lymphocyte subpopulations (%)	PD group	Control group	p Value
T Lymphocytes	67.8 (41.7–85.6)	69.3 (36.7–91.9)	0.52
CD4⁺ T-cells	41.0 (22.2–53.6)	34.2 (11.4–58.0)	<0.005
CD8⁺ T-cells	25.8 (12–42.4)	22.2 (8.4–42.1)	0.13
CCR7⁺ T-cells	19.9 (2.4–32.5)	2.4 (0.3–49.6)	<0.005
CD45RO⁺CCR7⁺	1.8 (0.3–4.5)	0.9 (0.3 – 21.2)	0.06
CD28⁺CCR7⁺ T-cells	16.5 (4.9–29.5)	2.6 (0.3–48.5)	<0.001
CXCR4⁺ T-cells	13.6 (3.1–45.8)	14.4 (2.9– 60.1)	0.26
CXCR4⁺CD4⁺ T-cells	6.5 (0.3–24.3)	5.5 (0.0004–38.7)	0.32
CXCR4⁺CD8⁺ T-cells	15.0 (3.1– 29.1)	7.0 (1.1–32.0)	0.78
CXCR4⁺CCR5⁺ T-cells	1.4 (0.003–8.5)	2.5 (0.0002–26.0)	0.09
CCR5⁺ T-cells	10.8 (1.4–31.6)	12.8 (2.2–53.0)	0.14
CCR5⁺CD4⁺ T-cells	3.4 (1.4–5.6)	3.4 (0.6–9.5)	0.38
CCR5⁺CD8⁺ T-cells	9.3 (2.6–18.7)	5.8 (1.1–41.3)	0.51
CCR4⁺ T-cells	4.2 (1.4–9.1)	7.5 (1.0–31.6)	<0.002
CCR4⁺CD4⁺ T-cells	4.5 (1.7–12.3)	5.7 (0–15.9)	<0.0004
CCR4⁺CD8⁺ T-cells	1.1 (0.001– 34.3)	0.9 (0.0002–24.7)	0.40
CXCR3⁺ T-cells	23.4 (11.3–48.8)	31.6 (14.6–50.4)	<0.0005
CXCR3⁺CD4⁺ T-cells	10.0 (4.5–19.3)	9.3 (1.7–17.0)	0.09
CXCR3⁺CD8⁺ T-cells	17.1 (8.9–26.7)	16.5 (7.2–27.8)	<0.005
CXCR3⁺CCR5⁺ T-cells	6.1 (1.2–28.0)	9.2 (2.2–35.1)	0.18
CXCR3⁺CXCR4⁺ T-cells	5.9 (1.0–15.9)	6.9 (1.3–24.1)	0.23

PD: peritoneal dialysis.

^a Percentage values are shown as median value (minimum–maximum). p < 0.05: significant PD versus control group.

Table 5.

Significant differences of MFI values of T-cell subpopulations with expression of receptors for selected chemokines between examined children and healthy controls.^a

Lymphocyte subpopulations (MFI)	PD group	Control group	p Value
CCR7⁺ T-cells	94.9 (52–203)	125 (76–243)	<0.002
CD45RO⁺CCR7⁺	39 (17-69)	50.2 (29–79)	<0.01
CD28⁺CCR7⁺ T-cells	115.1 (60– 240)	144.6 (97–282)	<0.04
CXCR4⁺CD4⁺ T-cells	290.3 (148–745)	395.7 (151–845)	<0.005
CCR5⁺CD4⁺ T-cells	117.8 (58–295)	163 (76–389)	<0.006
CCR4⁺ T-cells	301.3 (161–646)	422.2 (157–893)	<0.02
CCR4⁺CD4⁺ T-cells	271.6 (102–636)	398.3 (129–953)	<0.01

PD: peritoneal dialysis; MFI: mean fluorescent intensity.

^a Values are shown as median value (minimum–maximum). p < 0.05: significant PD versus control group.

The results of the analysis of correlation between T lymphocyte subpopulations with chemokine receptors and other parameters, presented in Table 6, revealed positive correlation of CCR7⁺ and CCR7⁺CD28⁺ T-cells and WCC, negative correlation between the percentage of CD45RO⁺CCR7 antigens positive T-cells and KT/V_urea. Total number of CD45RO⁺CD28 antigens positive T-cells correlated positively with CKD duration (R = 0.4474, p < 0.05).

Table 6.

Significant correlations between T lymphocyte subpopulations with chemokine receptors and other parameters in children with chronic kidney disease on peritoneal dialysis.

Parameter	Lymphocyte T subpopulation	Significance
Age (years)	The absolute number of CD3⁺CCR7⁺ lymphocytes	R = −0.4093^a
Platelet number (1 × 10⁹/l)	The absolute number of CD3⁺CCR5⁺ lymphocytes	R = 0.4412^a
Platelet number (1 × 10⁹/l)	The absolute number of CD3⁺CXCR3⁺ lymphocytes	R = 0.4690^a
BMI (kg/m²)	The percentage of CD3⁺CCR5⁺CXCR4⁺ lymphocytes	R = 0.5555^a
BMI (kg/m²)	The absolute number of CD3⁺CCR5⁺CXCR4⁺ lymphocytes	R = 0.5098^a
BMI (kg/m²)	The percentage of CD3⁺CXCR3⁺CXCR4⁺ lymphocytes	R = 0.4230^a
Kt/V _urea	The percentage of CD3⁺CD45RO⁺CCR7⁺ lymphocytes	R = −0.7039^a
WCC (l/week)	The absolute number of CD3⁺CCR7⁺ lymphocytes	R = 0.4989^a
WCC (l/week)	The absolute number of CD3⁺CCR7⁺CD28⁺ lymphocytes	R = 0.4999^a
Urea concentration (mmol/l)	The percentage of CD3⁺CXCR4⁺ lymphocytes	R = 0.4618^a

WCC: weekly creatinine clearance.

^a p < 0.05.

Discussion

The magnitude of the immune response dependent on T lymphocytes, its potential and phenotype cannot be assessed on the basis of a single parameter. Hence the great significance of multicolor flow cytometry. This method allows to assess the proliferation of lymphocytes and the induction of other cells’ proliferation, as well as the mechanisms of the immune response and the evaluation of the effectors’ functions. The more the specific markers are discovered, the better the assessment of disorders in individual cell subpopulations can be performed.¹⁸ This study has documented a significantly higher percentage of T-cell bearing surface CCR7 receptor in children with CKD undergoing PD, although the MFI of this receptor was lowered. Moreover, it presents the influence of PD on their percentage value showing negative correlation with Kt/V_urea (CD45RO⁺CCR7 T-cells) and on the other hand, positive correlation of CCR7⁺CD28⁺ T-cells and WCC. The additional effect of PD on cellular immunity, apart from conservatively managed CKD, was acknowledged. According to Freitas et al., aging alongside with CKD have an additive effect on decreasing T- and B-cells number.¹⁹ Study by Betjes et al. suggested that CKD adult patients’ T-cells are 20–30 years older than healthy individuals. Such correlation was more prominent in older patient.²⁰ Chung et al. documented that peripheral blood central memory and effector memory T-cells count was increased in patients with uremia comparing to healthy adults.²¹ Wurm et al. conducted a research on animal models, which concluded, that extra- and intraglomerular mesangial cells in healthy rats were CCR7-negative and furthermore, significant CCR7 expression during mesangial proliferation in the course of glomerulonephritis was revealed.²² CCR7⁺CD8⁺ T-cells are believed to regulate the effector T-cells, involved in T-cell-mediated rejection in an in vitro and an ex vivo transplant model.²³ It is likely to assume that the configuration we reported displays action promoting proliferation in children on PD. Crépin et al. observed an early replicative aging in young patients with CKD linked to reduced thymic output.²⁴

Among the CD45RO⁺ memory T-cells, two subpopulations can be distinguished: first, central lymphocytes (TCMs, CM T-cells) expressing the CCR7 molecule, taking part in lymphoid tissue colonization, but not directly fulfilling the effector functions and second, effector memory cells (TEMs) that do not express this receptor. The co-receptor CD28 was often applied to define fractions of memory cells. Historically, downregulation of CD28 was usually associated with loss of functionality, especially described as weakened proliferative ability and loss of telomeres.^25,26 George et al. proved CD28 negative T-cells and CD57 positive T-cells increase, suggesting that it might take part in premature senescence.²⁷ However, in this research such T-cells phenomenon in children on PD could not be estimated, as higher percentage of CM T-cells, with CD28 and CCR7 receptors bearing on their surface, were detected and similar absolute number of such cells occurred in healthy controls. Therefore, it should be clarified in further investigations.

In the literature, there is no consensus on frequency of memory cells in CKD patients. Such ambiguities might originate from the fact that researchers tend to analyze various, selected subpopulations of lymphocytes which are structurally and functionally distinct. Moreover, in pediatric studies, the number of patients is usually limited. One should also take into account that CKD duration in children is shorter than in adults, uremia is acting on developing immune system and children have less comorbidities than older patients.

Biology of the receptors for chemokines is a wide field of study and many discoveries are yet to be unraveled, as the chemokines are known for 25 years so far. Interestingly, it was proved that there may be a parallel expression of several receptors for chemokines on the same cell, which activated would bring different results. Some cells of the lymphoid system appearing at the site of infection tend to present the receptors for chemokines with opposite action, that is why the accurate assessment of their function might be difficult.⁶

The literature underlines the role of CCR5 in various diseases such as rheumatoid arthritis, multiple sclerosis, transplant rejection processes, diabetes mellitus, myeloma, and gastrointestinal disorders. CCR5 facilitates the intracellular migration of HIV-1. Szalai et al. pointed out that CCR5 dysfunction (gene polymorphism), due to the 32 base-pair deletion mutation (CCR5Δ32), is usually associated with better prognosis in atherosclerotic disease.²⁸ The CCR5Δ32 genotype is also correlated with longer survival in type 2 diabetes mellitus.²⁹ The relevance of CCR5 and CXCR4 receptors in allergic disorders in the lungs and remodeling (fibrosis) of lung tissue has been described.³⁰ Moreover, CCR5 affects proteinuria-related renal injury, as albumin and other filtered urinary proteins upregulate its ligand RANTES. Its expression was associated with the presence of interstitial CCR5-positive mononuclear cells including T lymphocytes and fibrosis in patients with acute interstitial nephritis.³¹ Still, Moreno et al. concluded that anti-chemokines or anti-chemokine receptors compounds administering needs to be explored more and their interactions with conventional anti-inflammatory or anti-proteinuric treatment ought to be investigated.³² According to Yuan et al., CXCR4 present in the tubular cell compartment in CKDs, particularly obstructive uropathy, promotes the secretion of cytokines, and influences macrophage trafficking and activation state, resulting in kidney fibrosis.³³

In all clinical areas, including inflammatory diseases (together with acute and CKD), the use of chemokine receptor antagonists is currently being explored. Blocking chemokine receptors might induce selective inhibition of monocyte pro-inflammatory phenotype and not affect immunoregulatory or anti-inflammatory monocytes. Studies on the efficacy of the CCR5 receptor antagonist in the treatment of AIDS remain in the phase of clinical trials. As for kidney diseases, the role of chemokines in the interaction between cytokines, vasoactive substances, and the corresponding target cells in the progression of chronic interstitial fibrosis has been underlined. Studies, based on kidney biopsy materials, showed that blocking pro-inflammatory cells with the CCR5 chemokine receptor would inhibit inflammatory processes and prevent organ transplant rejection.³⁴

PD is the modality widely used in children with CKD. This research reveals that children on PD present lower expression of chemokine receptors, reflected by diminished MFI values, specially connected with CD4⁺, which might contribute to an increased hazard of inflammatory complications. Various inflammatory mediators have been extensively studied in patients on PD. It was confirmed that PD takes part in the inflammatory response by activating IL-6 with its soluble IL-6 receptor, which eventually results in the stimulation of T-cells proliferation and differentiation.³⁵ Musiał et al. claimed that the monocyte chemoattractant protein and macrophage colony-stimulating factor concentrations in children on PD are decreased in comparison with subjects on hemodialysis and healthy controls. This finding introduces PD as a weaker trigger of systemic cell migratory activity, whereas similar neopterin concentrations suggest comparable status of chronic inflammatory stimulation.³⁶

In conclusion, the presence of the phenomenon of earlier T-cells senescence in children with CKD on PD treatment could not be confirmed. Consequently, such research still requires further investigation. The higher percentage of T-cells with CCR7 surface receptor could be responsible for the increase of proliferation activity in this group of children.

Footnotes

Acknowledgements

None

Author contributions

MS was involved in protocol development, gaining ethical approval, patient recruitment and data analysis, drafted the article. ŁS and JB were involved in performing cytometric analysis and interpretation of data. BM was involved in literature search and protocol development. DZ and MT were involved in protocol development, patient recruitment, and data analysis. JK performed statistical analysis and was involved in data analysis. TS evaluated cytometric analysis and critically reviewed the content. All authors reviewed and edited the manuscript and approved the final version of the manuscript.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical approval

The approval of the SUM Bioethical Committee in Katowice NN-6501-131/06 from 20 September 2006 was obtained.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Polish Ministry of Science and Higher Education grant no. N407 008 32/0179.

Informed consent to participate

Parents and children over the age of 13 years have signed written informed consent for research.

Informed consent to publish

Written informed consent was obtained from the patient(s) for their anonymized information to be published in this article

Trial registration

Not applicable according to Polish guidelines.

ORCID iD

Maria Szczepańska

References

Han

. Constitutively active chemokine CXC receptors. Adv Pharmacol 2014; 70: 265–301.

Hall

Lefkowitz

. Regulation of G protein-coupled receptor signaling by scaffold proteins. Circ Res 2002; 91: 672–680.

Choi

. Biology and clinical relevance of chemokines and chemokine receptors CXCR4 and CCR5 in human diseases. Exp Biol Med 2011; 236: 637–647.

Panzer

Steinmetz

Stahl

RAK

, et al. Kidney diseases and chemokines. Curr Drug Target 2006; 7: 65–80.

Charo

Ransohoff

. Mechanisms of disease: the many roles of chemokines and chemokine receptors in inflammation. NEJM 2006; 354: 610–621.

Anders

Sayyed

Vielhauer

. Questions about chemokine and chemokine receptor antagonism in renal inflammation. Nephron Exp Nephrol 2010; 114: e33–e38.

Szczepańska

Sędek

Makulska

, et al. Expression of chemokine receptors on peripheral blood T cells in children with chronic kidney disease. Mediators Inflamm 2015; 2015: 536894.

Meijers

Betjes

Baan

, et al. T-cell ageing in end-stage renal disease patients: assessment and clinical relevance. World J Nephrol 2014; 3: 268–276.

Miao

Tang

Liu

, et al. CXCR3 blockade combined with cyclosporine A alleviates acute graft-versus-host disease by inhibiting alloreactive donor T cell responses in a murine model. Mol Immunol 2018; 94: 82–90.

10.

Paust

Riedel

Krebs

, et al. CXCR3+ regulatory T cells control TH1 responses in crescentic GN. J Am Soc Nephrol 2016; 27: 1933–1942.

11.

Gębura

Bogunia-Kubik

. Kliniczne znaczenie receptora chemokinowego CXCR4. Postępy Hig Med Dosw (online) 2012; 66: 252–266.

12.

Diomedi-Camassei

McDowell

De Ioris

, et al. Clinical significance of CXC chemokine receptor-4 and c-met in childhood rhabdomyosarcoma. Clin Cancer Res 2008; 14: 4119–4127.

13.

Song

Chang

, et al. Stem cell mobilizes targeting chemokine receptor CXCR4: renoprotective application in acute kidney injury. J Med Chem 2015; 58: 2315–2325.

14.

Maluf

Mas

Archer

, et al. Molecular pathways involved in loss of kidney graft function with tubular atrophy and interstitial fibrosis. Mol Med 2008; 14: 276–285.

15.

Zou

Cui

, et al. CXC Chemokine receptor type 4 antagonism ameliorated allograft fibrosis in rat kidney transplant model. Exp Clin Transplant 2017; 15: 448–452.

16.

Liao

Pirapakaran

Luo

. Chemokines and chemokine receptors in the development of lupus nephritis. Mediators Inflamm 2016; 2016: 6012715.

17.

Schwartz

Munoz

Schneider

, et al. New equations to estimate GFR in children with CKD. J Am Soc Nephrol 2009; 21: 1–9.

18.

Seder

Darrah

Roederer

. T-cell quality in memory and protection: implications for vaccine design. Nat Rev Immunol 2008; 8: 247–258.

19.

Freitas

GRR

da Luz Fernandes

Agena

, et al. Aging and end stage renal disease cause a decrease in absolute circulating lymphocyte counts with a shift to a memory profile and diverge in Treg population. Aging Dis 2019; 10: 49–61.

20.

Betjes

MGH

Langerak

van der Spek

, et al. Premature aging of circulating T cells in patients with end-stage renal disease. Kidney Int 2011; 80: 208–217.

21.

Chung

Kim

Sun

, et al. Increased interleukin-17 producing effector memory T cells in the end-stage renal disease patients. Immunol Lett 2012; 141: 181–189.

22.

Wurm

Steege

Rom-Jurek

, et al. CCR7 is important for mesangial cell physiology and repair. J Histochem Cytochem 2018; 66: 7–22.

23.

Kim

Doh

, et al. Clinical significance of CCR7⁺CD8⁺ T cells in kidney transplant recipients with allograft rejection. Sci Rep 2018; 8: 8827.

24.

Crépin

Legendre

Carron

, et al. Uraemia-induced immune senescence and clinical outcomes in chronic kidney disease patients. Nephrol Dial Transplant 2020; 35: 624–632.

25.

Effros

Walford

. T cell cultures and the Hayflick limit. Hum Immunol 1984; 9: 49–65.

26.

Larbi

Fulop

. From “truly naïve” to “exhausted senescent” T cells: when markers predict functionality. Cytometry A 2014; 85: 25–35.

27.

George

Mehta

Perez

, et al. Premature T cell senescence in pediatric CKD. J Am Soc Nephrol 2017; 28: 359–367.

28.

Szalai

Duba

Prohászka

, et al. Involvement of polymorphisms in the chemokine system in the susceptibility for coronary artery disease (CAD). Coincidence of elevated Lp(a) and MCP-1-2518 G/G genotype in CAD patients. Atherosclerosis 2001; 158: 233–239.

29.

Muntinghe

Gross

Bakker

, et al. CCR5 Delta32 genotype is associated with outcome in type 2 diabetes mellitus. Diabetes Res Clin Pract 2009; 86: 140–145.

30.

Harrison

Lukacs

. The chemokine receptors. Totowa, NY: Humana Press Inc., 2007.

31.

Segerer

MacK

Regele

, et al. Expression of the C-C chemokine receptor 5 in human kidney diseases. Kidney Int 1999; 56: 52–64.

32.

Moreno

Rubio-Navarro

, et al. Role of chemokines in proteinuric kidney disorders. Expert Rev Mol Med 2014; 16: e3.

33.

Yuan

Lee

Choi

, et al. Chemokine receptor Cxcr4 contributes to kidney fibrosis via multiple effectors. Am J Physiol Renal Physiol 2015; 308: F459–F472.

34.

Segerer

Nelson

Schlondorff

. Chemokines, chemokine receptors, and renal disease: from basic science to pathophysiologic and therapeutic studies. Am J Soc Nephrol 2000; 11: 152–176.

35.

Papanicolaou

Wilder

Manolagas

, et al. The pathophysiologic roles of interleukin-6 in human disease. Ann Intern Med 1998; 128: 127–137.

36.

Musiał

Zwolińska

. New markers of cell migration and inflammation in children with chronic kidney disease. Biomarkers 2019; 24: 295–302.