Erasmus University Medical Center, Internal Medicine, Division of Nephrology and Renal Transplantation, Rotterdam, The Netherlands
Correspondence and offprint requests to: D. A. Hesselink, Department of Internal Medicine, Division of Nephrology and Renal Transplantation, Room Ee 563 A, Erasmus MC, University Medical Center Rotterdam, Dr Molewaterplein 50, 3015 GE Rotterdam, The Netherlands. Email: d.a.hesselink{at}erasmusmc.nl
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Abstract |
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Methods. Using flowcytometry, we quantified mDCs and pDCs in peripheral blood of patients maintained on CIHD (n = 37), continuous ambulatory peritoneal dialysis (CAPD; n = 29), and patients with CKD not receiving RRT (n = 37). Twenty-nine healthy volunteers served as controls.
Results. Patients with CKD (n = 103) had lower pDC and mDC counts compared with volunteers: 4.2 vs 8.3 and 10.0 vs 13.8x106 cells/l, respectively (P 0.001). Within the CKD group, pDC counts did not differ between patients on CIHD, CAPD and those not receiving RRT (3.6 vs 5.0 vs 4.9x106 cells/l, respectively). In the latter group, pDC numbers correlated with the glomerular filtration rate (GFR; Spearman's r = 0.49; P<0.01). In contrast, mDC counts of patients on CIHD were lower compared with patients on CAPD (7.5 vs 10.1x106 cells/l; P = 0.039) and patients not receiving RRT (13.7x106 cells/l; P<0.001). Among non-dialyzing patients, no correlation existed between GFR and mDC numbers, which were comparable to those of volunteers, even when only non-dialyzing patients with a GFR below 15 ml/min were analysed.
Conclusions. Circulating DC counts are decreased in patients with CKD; for pDCs, this reduction is primarily related to the loss of GFR, whereas the dialysis treatment appears to affect mDC numbers.
Keywords: chronic haemodialysis; chronic kidney failure; chronic renal disease; continuous ambulatory peritoneal dialysis; immunodeficiency; renal replacement therapy
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Introduction |
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The exact mechanisms responsible for the impaired immunity of CKD are incompletely understood but several immune abnormalities have been described. First, patients with CKD have low numbers of circulating T-, B- and natural killer (NK) lymphocytes [5]. Second, there is evidence that T-lymphocyte function is aberrant in dialyzing patients. Several studies have demonstrated that phenotypically, T-cells are in an activated state and display altered proliferative responses after stimulation with mitogens or soluble antigens in vitro [5,6]. However, whether CKD causes an intrinsic T-cell defect is still a matter for debate as the observed alterations in T-cell response may have resulted from a defective antigen-presenting capacity of monocytes [7].
A cell type that has hitherto received little attention in the immunodeficiency of CKD is the dendritic cell (DC). DCs are antigen-presenting cells that are able to initiate and regulate T-, B- and NK lymphocyte immunity [8]. Physiologically, DCs play a key role in the protection against pathogens, tumour surveillance and the maintenance of tolerance to self [8]. DCs originate from bone marrow progenitors and travel via blood to nonlymphoid tissues, where they reside as immature DCs that are specialized in antigen uptake. Antigen uptake in combination with an inflammatory stimulus causes immature DCs to terminally differentiate, switching these cells into an immunostimulatory mode. This maturation process is characterized by the loss of the DC ability to capture antigens and by an increased expression of distinct chemokine receptors, MHC class I and II, and diverse adhesion and costimulatory molecules on the DC surface. This enables DCs to traffic to secondary lymphoid tissue and maximally stimulate effector cells from both the innate and adaptive immune system [8].
Recently, we demonstrated that patients on CIHD have reduced numbers of DCs in their peripheral blood as compared with healthy volunteers [9]. However, it was unclear whether this was a reflection of CKD or if this resulted from CIHD treatment. In this study, we therefore compared the numbers of the two main DC subsets present in human blood, the myeloid (m) and plasmacytoid (p) DC, between a group of patients on CIHD, a group of patients undergoing continuous ambulatory peritoneal dialysis (CAPD) and a group of patients with varying degrees of CKD not receiving renal replacement therapy (RRT). In addition, the maturation status and homing potential of these two DC subsets was studied by measuring the expression of CD83 and the chemokine receptor CCR7 on the DC surface. CD83 is a cell surface molecule that is upregulated upon antigen capture and maturation of DCs [8]. Like CD83, the expression of CCR7 increases with maturation and allows the DC to respond to the CCR7 ligands CCL19 and CCL21, enabling the cell to traffic to secondary lymphoid organs [10].
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Subjects and methods |
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Estimated GFR (ml/min/1.73 m2) = 186x(serum creatinine)1.154x(age)0.203x(0.742 if female)x(1.21 if African American).
Antibodies for staining
Fluorescence-activated cell sorter (FACS) analysis was performed using the following mouse anti-human monoclonal antibodies: fluorescein isothiocyanate (FITC)-conjugated CD34 (clone 8G12), FITC-conjugated lineage cocktail 1 [containing CD3 (SK7), CD14 (mP9), CD16 (3G8), CD19 (SJ25C1), CD20 (L27) and CD56 (NCAM16.2)], phycoerythrin (PE)-conjugated anti-IL-3 receptor
chain (CD123) (9F5), allophycocyanin-conjugated CD11c (S-HCL.3), peridinin chlorophyll protein-conjugated anti-HLA-DR (L243) (all purchased from Becton-Dickinson Biosciences, San Jose, CA, USA), PE-conjugated CD83 (HB15A17.11; DPC, Serotec, Oxford, UK), PE-conjugated anti-CCR7 (CDw197) (150503; R&D Systems Europe, Abingdon, UK), and PE-conjugated IgG2a (X39) and IgG2b (27-35) isotype control monoclonal antibodies (Becton Dickinson).
Immunofluorescence staining and flow cytometric analysis
Peripheral venous blood samples were collected in standard lithium-heparinized tubes and processed within 3 h of collection. Blood from patients undergoing CIHD was collected immediately before the start of a dialysis session. Whole blood samples were first incubated with the above-mentioned monoclonal antibodies for 30 min in the dark at room temperature. Next, erythrocytes were lysed by incubating the samples for 10 min with FACS lysing solution (Becton-Dickinson). Cells were then washed twice using FACS flow and subsequently 300.000 events were measured on a FACScalibur flow cytometer using CellQuest Pro software (Becton Dickinson). Cells that stained negative for the lineage cocktail (Lin) and positive for HLA-DR were gated and analysed for CD11c and CD123 expression. mDCs and pDCs were identified as Lin, HLA-DR+, CD11c+, CD123/low and Lin, HLA-DR+, CD11c, CD123high, respectively. Absolute counts of mDCs and pDCs were then obtained by multiplying the proportion of each DC subset within the total leukocyte population by the absolute number of white blood cells as determined on an automated microcell counter (Sysmex F-300; TOA Medical Electronics Co. Ltd, Kobe, Japan). For the expression of CD83 and CCR7 on mDCs and pDCs, CD123PE was replaced by CD83PE or CCR7PE. The Lin, HLA-DR+, CD11c+ cells were then considered as the mDC population and within this subset the percentage of CD83- or CCR7-positive cells was determined by comparison with their respective isotype control antibodies. The same was performed for the pDC population (Lin, HLA-DR+, CD11c cells). Determination of the percentage CD83- and CCR7-positive mDCs and pDCs was performed only if a minimum of 100 events was acquired for each DC subset.
Details on the reproducibility of the flow cytometric DC assay in our laboratory were published recently [13]. In addition, the performance of lymphocyte flowcytometry in our laboratory meets the proficiency standards of the Dutch Foundation for Quality Assessment in Medical Laboratories (SKML; http://www.skzl.nl).
Statistical analysis
All values are presented as median (range). To achieve a normal distribution, leucocyte and DC numbers, as well as the percentage CD83- and CCR7-positive mDCs and pDCs, were logarithmically transformed for statistical analysis. For comparisons between groups, the t-test, MannWhitney U-test, one-way ANOVA or KruskalWallis test were used, as appropriate. Post hoc analysis was performed using Bonferroni's test for multiple comparisons or MannWhitney U-test. P-values at <0.05 were considered statistically significant. For correlation analysis, Spearman's rank correlation coefficient (rs) was calculated.
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Results |
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Loss of GFR is related to decreased numbers of plasmacytoid but not myeloid DCs
Next, we investigated the relationship between the severity of CKD and DC numbers more closely. As depicted in Figure 1A, there existed an inverse relationship between serum creatinine and pDC counts (rs = 0.45, P<0.01). No such correlation was observed between mDC numbers and serum creatinine (rs = 0.06). A similar relationship between (the loss of) renal function and circulating pDC counts was observed when numbers of this cell type were correlated with the estimated GFR (Figure 1B; rs = 0.49; P<0.01). In contrast, there existed no significant correlation between mDC counts and GFR (rs = 0.15). When GFR was classified according to the National Kidney Foundation classification of CKD [12], a trend towards lower pDC counts with advancing stages of CKD was observed (overall P = 0.007; Figure 1C).
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CIHD but not CAPD, is related to decreased numbers of mDCs
To assess a potential (additional) effect of dialysis treatment, we compared DC counts of patients treated with CIHD or CAPD with non-dialyzing patients who only had a GFR below 15 ml/min (CKD class 5). Plasmacytoid DC counts were again not different between the CKD class 5 [3.2 (0.511.5)x106 cells/l] and the CAPD and CIHD groups (Table 2). In contrast, mDC numbers of non-dialyzing patients with CKD class 5 (10.9x106 cells/l) were comparable to healthy volunteers but significantly higher when compared with patients on CIHD (P = 0.04; Figure 2). Finally, no effect of residual diuresis on circulating DC numbers was observed within the CIHD and CAPD groups (data not shown).
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Discussion |
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In patients treated with CIHD or CAPD, both the plasmacytoid and the myeloid DC subset were affected, whereas in CKD patients not receiving RRT, only pDC numbers were significantly reduced compared with controls. The finding of a negative correlation between pDC counts and renal function among non-dialyzing patients indicates that the decreased number of circulating pDCs in patients with CKD principally results from the loss of GFR. However, the observation that non-dialyzing patients with diabetic nephropathy had lower pDC counts compared with patients suffering from other causes of renal insufficiency suggests that co-morbidity may also influence peripheral blood pDC counts. Importantly, the reduction of pDC counts among non-dialyzing patients was already apparent at a relatively mild degree of CKD. These observations are in line with the findings of DaRoza et al. [2] who observed a correlation between (the loss of) GFR and HBV vaccination response. In that study, patients with the worst kidney function were less likely to seroconvert after HBV vaccination compared with patients with moderate renal insufficiency [2]. Interestingly, mDC numbers were only significantly reduced in patients maintained on dialysis. Among non-dialyzing patients, no correlation was observed between mDC counts and renal function. In addition, residual diuresis of patients treated with either CIHD or CAPD did not affect mDC numbers. This suggests that, in contrast to the pDC subset, the reduction of mDC numbers in peripheral blood of patients with CKD results from dialysis treatment rather than the loss of GFR.
Our findings are in agreement with the results of several studies that investigated DCs in the skin of patients with CKD [1517]. Two types of cutaneous DCs can be distinguished: the Langerhans cell (LC) and the dermal DC, both of which are thought to differentiate from mDCs. The number of LCs in the skin of patients on CIHD and CAPD was found to be significantly lower compared with healthy volunteers, whereas LC counts were not different between non-dialyzing patients and control subjects [16]. In contrast, McKerrow et al. [15] did observe a reduction in epidermal LCs in patients with CKD not receiving RRT. With regard to dermal DCs, the results are less consistent: compared with healthy controls, dermal DC numbers were reduced in patients on CAPD, comparable in patients on CIHD and elevated in patients with CKD not on dialysis [17].
At present, the mechanism underlying the CKD-associated reduction in circulating DCs is unclear but it may involve an impaired generation of DCs from bone marrow progenitors. Indirect evidence for this possibility comes from the study by Ando et al. [18] who demonstrated that thrombocytopaenia is a common finding among patients on RRT. The principal cause for the decreased number of circulating platelets was a reduced megakaryocyte production in the bone marrow, possibly as a result of the presence of undialyzable marrow-suppressive toxins [18]. A second explanation for the low numbers of circulating DCs could be an increased peripheral DC turnover. Although no data on the lifespan of human DCs exist, several investigators have demonstrated that monocytes and lymphocytes from patients with CKD are more susceptible to undergo apoptosis in comparison with healthy volunteers [5,19]. Consistent with our observations on DCs, the rate of mononuclear cell apoptosis was higher in patients undergoing CIHD compared with non-dialyzing patients [19]. A third explanation could be a redistribution of DCs to peripheral tissue. Although the skin does not appear to be the site of such a redistribution [1517], increased DC migration to other peripheral tissues or secondary lymphoid organs remains a theoretical possibility.
The percentage of DCs expressing CD83 or CCR7 was not altered in patients with CKD, suggesting that under uraemic conditions, DCs do not undergo (increased) maturation in the circulation. However, under physiologic conditions, maturation of DCs only occurs after antigen uptake in combination with an appropriate inflammatory signal, and therefore this process is initiated in peripheral tissue and completed in the afferent lymphatics and secondary lymphoid organs [8]. A limitation of the present study is the lack of functional DC assays. Future work should address the effects of CKD and its treatment on DC function. However, as the number of circulating DCs in patients with CKD is low, isolation of adequate cell numbers to perform such assays will not be easy. Furthermore, the precise effect of dialysis treatment on circulating mDCs requires further investigation.
In conclusion, we show that in patients with CKD, DC counts are markedly reduced compared with healthy volunteers. The quantitative changes in circulating pDCs result mainly from the loss of GFR, whereas the reduction in mDC numbers appears to relate to the dialysis treatment itself. Our findings may provide an additional explanation for the immunodeficiency of CKD.
Conflict of interest statement. None declared.
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References |
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