Dysregulated chemokine receptor expression and chemokine-mediated cell trafficking in pediatric patients with ESRD

Abstract

Background and objectives: Children and adolescents with ESRD on dialysis are susceptible to serious bacterial infections (SBI). Chemokines and chemokine receptors play a critical role in modulating macrophage and neutrophil function. This study examined the hypothesis that expression and/or function of these molecules is dysregulated in patients with ESRD, contributing to leukocyte dysfunction. Design setting, participants, & measurements: Pediatric patients, age 6 mo to 18 yr, with ESRD treated with either hemodialysis or peritoneal dialysis were enrolled in this prospective, nontherapeutic study. Blood was collected for plasma chemokine levels, chemokine receptor profiling by flow cytometry, and functional chemotaxis studies on neutrophils and mononuclear cells.

Results: ESRD in children was associated with reduced expression of the chemokine receptors CXCR1 and chemokine (C-C motif) receptor 2 (CCR2) on circulating neutrophils and monocytes, respectively. When ESRD patients were divided into two subgroups, those who were infection-free and those who had three or more SBI in the preceding year, the differences in chemokine receptor expression were statistically significant compared with control subjects only in those with recurrent infection. In addition to the effects of ESRD on baseline chemokine receptor expression, the hemodialysis procedure itself acutely lowered neutrophil CXCR1 and monocyte CCR2 expression. Furthermore, neutrophil and monocyte responsiveness to chemokine-mediated trafficking signals was impaired in all ESRD patients studied. This abnormality was independent of the level of chemokine receptor expression on the leukocytes.

Conclusions: The data presented in this study suggest that chemokine receptor dysregulation contributes to leukocyte dysfunction in patients with ESRD. This alteration is especially prominent in ESRD patients with recurrent infection.

Figure 1.

ESRD is associated with dysregulated expression of chemokine receptors on circulating neutrophils. Whole blood was collected and stained for CXCR1 or CXCR2 using phycoerythrin (PE)-conjugated antibodies specific for each receptor. All samples were co-stained with CD14-allophycocyanin (APC) to discriminate neutrophils (CD14−) from contaminating monocytes (CD14⁺). (A) Representative whole blood forward scatter/side scatter dot plots (to define R1/neutrophil gate) and two-color dot plots (R1/neutrophil gate only) for one healthy control, one patient with ESRD without infection over the preceding 12 mo (inf−), and one patient with ESRD having more than three infections over the preceding 12 mo (inf⁺). Numbers correspond to mean fluorescence intensity (MF) of chemokine receptor staining in neutrophil population (identified by dashed boxes). (B) Mean neutrophil CXCR1 expression in healthy controls versus patients with ESRD. (C) Mean neutrophil CXCR2 expression in healthy controls versus patients with ESRD. In panels B and C, the numbers in parentheses indicate the number of control or patient samples included in analysis. Where blood was obtained multiple times from a single donor all values were averaged and included as a single value for analysis. Data are plotted in box plot format where the box defines the 25th and 75th percentiles, the line inside the box marks the value of the 50th percentile, and the capped bars indicate the 10th and 90th percentiles. Data were obtained and analyzed as described in the Statistical Analyses section. The symbol * indicates P < 0.026 versus control.

Figure 2.

Cell surface expression of the macrophage chemokine receptor CCR2, but not CCR1 or CCR5, is significantly lower on blood monocytes from patients with ESRD as compared with blood monocytes from control individuals. Whole blood was collected and stained for CCR1, CCR2 or CCR5 using PE-conjugated antibodies specific for each receptor. All samples were co-stained with CD14-APC to identify monocytes (CD14⁺). (A) Representative whole blood two-color dot plots for one healthy control, one patient with ESRD (inf−), and one patient with ESRD (inf⁺). Numbers correspond to MF of chemokine receptor staining in CD14⁺ monocyte population (identified by dashed boxes). (B through D) Mean monocyte CCR1 (panel B), CCR2 (panel C), and CCR5 (panel D) expression in healthy controls versus patients with ESRD. Data were obtained and analyzed as described in the Statistical Analyses section. The symbol * indicates P < 0.033 versus control.

Figure 3.

Infection status regulates chemokine receptor expression. Whole blood was collected and screened for CXCR1 (panel A) or CCR2 (panel B) by staining with PE-conjugated antibodies specific for each receptor and then analyzing by flow cytometry gating on neutrophils (panel A) or CD14⁺ monocytes (panel B). Patients were divided into those without infection over the preceding 12 mo (inf−) and those having more than three infections over the preceding 12 mo (inf⁺). Subgroup analyses were performed using the Kruskal–Wallis nonparametric test followed by pairwise multiple comparisons using the Mann–Whitney test as described in the Statistical Analyses section. Significances are indicated as (*) P < 0.0042 versus control, (**) P < 0.0035 versus control.

Figure 4.

There is a time-dependent loss of chemokine receptor expression as a function of the dialysis process itself. The effect of the dialysis procedure itself on neutrophil CXCR1 expression (Figure 4A) and CCR2 on circulating monocytes (Figure 4B) was evaluated in a subset of hemodialysis patients by collecting EDTA anticoagulated blood at the start of hemodialysis and at specified time-points after initiation of treatment and analyzing CXCR1 expression by flow cytometry. Data from six independent patients are plotted here. For five patients for whom 60 to 90-min time point data were available, we compared those values to predialysis values using two-way ANOVA and found the percent decreases in CXCR1 and CCR2 expression to be significantly different as a function of time (CXCR1: P < 0.01; CCR2: P < 0.03), but not as a function of patient (P > 0.5 for both).

Figure 5.

Chemokine receptor function is impaired in ESRD. (A and B) Neutrophils were fluorescently labeled and assayed for responsiveness to IL-8 and N-formyl-methionyl-leucyl-phenylalanine (FMLP) in transwell chemotaxis assays. Data are reported as Chemotactic Index (fluorescence^EXP/fluorescence^CONTROL). (C and D) Mononuclear cells were assayed for responsiveness to monocyte chemotactic protein-1 (MCP-1)/CCL2 and FMLP in transwell chemotaxis assays. Data are reported as Chemotactic Index (# CD14⁺ cells^EXP/# CD14⁺ cells^CONTROL. In panels A and C, bars represent the means ± SD of all controls and patients analyzed and the numbers on the x-axis indicate the concentration of IL-8 or MCP-1/CCL2 in the bottom well of the chemotaxis chamber, respectively. In panels B and D, bars represent the means ± SD of three control and three patients analyzed, and chemoattractant concentrations were IL-8 (50 ng/ml) and FMLP (10⁻⁸M). All chemotaxis assays were performed in duplicate. Statistical analysis comparing migration in response to chemoattractants to migration in the absence of any chemoattractant was performed using one-way ANOVA with Bonferroni post test.