Children with chronic renal failure have reduced numbers of memory B cells

Abstract

Reduced serum IgG and subclass levels have been demonstrated in children with chronic renal failure. To study possible causes of this reduction, we analysed B cell subset composition, T helper cell frequencies and immunoglobulin (Ig) production capacity in vitro in children with chronic renal failure, with or without dialysis treatment. B cell subsets were characterized by determining CD27, IgM, IgD and CD5 expression within the CD19⁺ population. Intracellular expression of interferon (IFN)-γ, interleukin (IL)-2 and IL-4 in PMA/ionomycin-stimulated peripheral blood mononuclear cells (PBMC) was used to evaluate T helper frequencies. The capacity of B cells to secrete Ig in vitro was determined by measuring IgG₁, IgG₂ and IgM in culture supernatants of anti-CD2/CD28 monoclonal antibody (MoAb)- or SAC/IL-2-stimulated PBMC. Memory B cell numbers (identified as percentage or absolute number of CD19⁺ IgM¯IgD¯ or CD19⁺CD27⁺ lymphocytes) were lower in children treated with haemodialysis (HD), peritoneal dialysis (PD) and children with chronic renal failure before starting dialysis treatment (CRF) compared to healthy controls (HC) (P < 0·05). Compared with HC, CD5⁺ (naive) B cells were reduced in HD-treated patients but not for PD or for children with chronic renal failure before starting dialysis treatment (CRF). No significant differences in CD4⁺ T helper cell subsets were found between the groups. However, CRF children had a higher percentage of IFN-γ producing CD8⁺ T lymphocytes compared to HC (P = 0·02). Finally, IgG₁, IgG₂ and IgM production in vitro was similar in the four groups. In conclusion, significantly lower numbers of memory type B cells were found in children with chronic renal failure compared to healthy controls. This reduction may contribute to the low Ig levels found in these children.

INTRODUCTION

Reduced serum IgG and subclass levels have been described in children treated with peritoneal dialysis [1–4]. Peritoneal loss, reduced synthesis or increased catabolism are possible explanations for this. In previous studies we have demonstrated that peritoneal loss is not the only explanation, as reduced serum IgG and subclasses levels were already present before the dialysis treatment had started [5]. Most IgG is produced by specialized B cells or plasma cells that have undergone class switching in the germinal centre in response to T cell-dependent antigens. During this response, B cells become activated by specific antigens, but depend on T helper cells-derived signals to differentiate into plasma cells and memory cells [6]. It is not clear whether the lower Ig levels in uraemic patients are caused by intrinsic B cell defects, disturbed helper T cell activity, or a combination of both. B cells express several surface molecules such as IgM, IgD, IgG, CD27 and CD5 that allow separation of naive (IgD⁺ or CD5⁺) and memory (IgD¯ or CD27⁺) type B cells [7–11]. However, no information is available on B cell differentiation in adult and paediatric patients with chronic renal failure, either treated or not with dialysis.

The T helper cells derived cytokines, interferon (IFN)-γ, interleukin (IL)-2 and IL-4 are involved in B cell maturation, Ig secretion and IgG subclass switching [12,13]. T helper type 1 cells (Th1) produce mainly IFN-γ and IL-2. IL-4 is particularly produced by T helper type 2 (Th2) cells [14]. The Th1/Th2 balance is crucial for an effective immune system and disturbances in the Th1/Th2 balance have been associated with diseases [14]. CD8⁺ (cytotoxic) T cells also produce IFN-γ, IL-2 and IL-4 [14]. Data on cytokine production in uraemia are controversial [15,16]. Most studies on cytokines in HD patients have focused on the effect of haemodialysis modalities and of the type of haemofilter on cytokine production and Th1/Th2 balance [17,18]. In PD patients, cytokine production has been studied mainly in peritoneal dialysis effluent in relation to peritonitis episodes, the toxicity of the dialysis solution and peritoneal permeability [19–21].

To explore further the possible causes of low IgG levels in uraemic children, we measured B lymphocyte immunoglobulin producing capacity in vitro, the maturational state of B cells and the T cell IFN-γ, IL-2 and IL-4 production in children with chronic renal failure.

PATIENTS AND METHODS

Eleven children treated with peritoneal dialysis (PD), 13 children on intermittent haemodialysis (HD) and 14 children with chronic renal failure, not yet dialysed (CRF) were analysed. The control group consisted of 13 healthy children (HC). The median (range) age, duration of PD or HD treatment and glomerular filtration rate (GFR) are listed in Table 1. The GFR in CRF patients was estimated using the Schwartz formula [22]. The primary renal disease of the patients is listed in Table 2. Four PD children were studied at four time-points: before starting PD, and after 1, 2 and 12 months of dialysis treatment. The longitudinal samples were analysed simultaneously. The study was approved by the Medical Ethical Review Committee of the hospital and written informed consent was obtained from the children and/or parents.

Table 1.

Patient characteristics

	PD	HD	CRF	HC
Number	11	13	14	13
Age(years)	10·3(3·5–15·3)	10·3(1·7–16·7)	10·0(0·5–18·4)	8·3(3·3–17·5)
Duration of dialysis(years)	4·0(0·1–7·2)	1·4(0·3–5·0)
GFR(ml/min/1·73 m2)			11(6–50)

GFR: glomerular filtration rate.

Table 2.

Primary renal disease

	PD	HD	CRF
Urological malformation	2	5	7
Glomerulopathy	4	0	5
Haemolytic uraemic syndrome	1	3	1
Metabolic disease	0	1	0
Congenital disease	2	2	0
Other diseases	1	1	1
Unknown	1	1	0

Cell isolation

Peripheral blood mononuclear cells (PBMC) were isolated from heparin blood by Ficoll-Isopaque density centrifugation. PBMC were frozen in 20% dimethylsulphoxide (DMSO) containing medium (75% wt/vol Earles's balanced salt solution, Tris buffered, 25% wt/vol fetal clone serum and penicillin/streptomycin) and stored in liquid nitrogen until use.

B cell phenotype assay

PBMC were washed with PBAP (phosphate-buffered saline solution supplemented with 0·5% wt/vol bovine serum albumin, 0·01% wt/vol sodium azide and 0·5 mmol/l potassium-EDTA) and centrifuged (500 g, 10 min). Subsequently, cells were incubated with saturating amounts of CD2-FITC (Beckton Dickinson (BD) Immunocytometry Systems, San Jose, CA, USA), CD19-Percp (BD), CD5-FITC (BD), CD27-FITC (Sanquin Blood Supply Foundation, CLB, Amsterdam, the Netherlands), anti-IgM-FITC (Pharmingen, BD) and anti-IgD-PE (Pharmingen, BD) labelled monoclonal antibodies (MoAb) for 30 min on ice in the dark. After incubation the cells were again washed with PBAP and centrifuged (500 g, 10 min). Flow cytometry analysis was performed directly thereafter with the FACSCalibur (BD). The lymphocyte population was gated on the basis of forward–sideward scatter. B lymphocytes were distinguished with CD19 MoAb. The percentage of subset-positive or -negative B lymphocytes were measured. A peripheral white blood cell count and differentiation was performed in all patients on a H3-Technicon counter (Technicon Instruments, Tarrytown, NY, USA). The absolute count was calculated as the percentage of subset-positive or -negative B cells obtained from the flow cytometry multiplied by the absolute B cell count. The absolute B cell count was calculated by the percentage of CD19⁺ B cells multiplied by the absolute lymphocyte number.

T cell function assay

This assay was performed with the BDIS FastImmune^™ Cytokine System (BD) and is based on the detection of intracellular cytokines in activated lymphocytes [23,24]. PBMC were stimulated with PMA (phorbol 12-myristate 13 acetate, final concentration 20 ng/ml) and ionomycin (final concentration 1 µg/ml) in the presence of BFA (Brefeldin-A, final concentration 10 µg/ml) (all from Sigma, St Louis, MO, USA). Cells were incubated for 4 h at 37°C, 7% CO₂. This was followed by cell-surface staining with CD3-FITC, CD4-APC, CD8-Percp, leucogate and isotype-matched negative control MoAb for 30 min on ice in the dark. Prior to the intracellular cytokine staining procedure cells were permeabilized with FACS Permeabilizing Solution (BD) for 10 min in the dark. Thereafter, saturating amounts of PE-conjugated anti-IFN-γ, anti-IL-2 and anti-IL-4 MoAb were added and incubated for 30 min at room temperature. Cells were fixed with Cellfix (BD). Flow cytometry analysis was performed on a FACS-Calibur (BD). The percentages of cytokine-producing lymphocytes and cytokine-producing CD4⁺ or CD8⁺ T cells were measured. T cell function was measured in eight PD, eight HD, nine CRF and nine HC children.

In vitro Ig production assay

Cells were resuspended in Iscove's modified Dulbecco's medium (IMDM) containing 10% fetal calf serum (FCS), 0·1%β2-mercapto-ethanol, human transferrin 20 µg/ml and penicillin/streptomycin. Thereafter, PBMC were cultured (20·000 PBMC/well), unstimulated or stimulated under two different conditions: a combination of CD2-triple (CD2 triple, ascites; CLB T11·1/1, final dilution 1 : 125, CLB T11·2/1, final dilution 1 : 125 and HIK27, final concentration 5 µg/ml) and CD28 (ascites, final dilution 1 : 1000) or a combination of SAC (Staphyloccal Aureus Cowan, final dilution 1 : 10000) and IL-2 (50 U/ml) for 8 days. Culture supernatant was collected and stored at −20°C until use. IgG₁, IgG₂ and IgM content in supernatant were measured by enzyme-linked immunosorbent assay (ELISA) [25]. The in vitro Ig production assay was performed in eight PD, eight HD, nine CRF and nine HC children.

Statistical analysis

The results are expressed as medians (range). Differences between the groups were tested with the Kruskall–Wallis one-way analysis of variance. In case of significance, the Mann–Whitney U-test was performed to analyse differences between two groups. Longitudinal data were analysed with a Friedman trend analysis and differences between two time-points with the paired Wilcoxon test. Two sided P-values < 0·05 were considered significant.

RESULTS

B cell subset distribution

The medians (ranges) of the lymphocyte numbers and subsets are listed in Table 3, and the relative B cell subset composition is shown in Fig. 1. The total lymphocyte count was lower in HD (P = 0·002) and CRF (P = 0·028) children compared with HC. The absolute CD19⁺ B lymphocyte number was reduced only in HD children (P = 0·018). The percentage of IgM¯IgD¯ B cells was lower in CRF children compared to HC (P = 0·03). No significant differences were found for the PD and HD group. The absolute number of IgM¯IgD¯ B cells was not significantly different among the groups. The percentage (P = 0·0005) and the absolute number of CD27⁺ B cells (P = 0·01) were lower in PD children compared with HC. This was also found in HD children (P = 0·023 and P = 0·004) compared with HC. The CRF group showed a trend towards lower percentages of CD27⁺ B cells, but this did not reach significance (P = 0·055). For CD5⁺ B cells, both percentage and absolute number were lower in HD (P = 0·008 and P = 0·008) children compared with HC (Fig. 1 and Table 3).

Table 3.

Number of total and B lymphocytes, and the phenotypic characteristics of the B lymphocytes

	PD	HD	CRF	HC
Lymphocytes	2·7(1·5–8·2)	2·06**(0·6–3·5)	2·1*(0·9–6·1)	2·8(2·1–6·5)
CD19+	0·64(0·13–1·1)	0·25*(0·06–1·33)	0·73(0·07–2·09)	0·56(0·35–0·96)
IgM–IgD–	0·08(0·04–0·17)	0·07(0·004–0·17)	0·07(0·004–0·25)	0·09(0·06–0·22)
CD27+	0·07*(0·03–0·16)	0·05**(0·01–0·15)	0·1(0·02–0·29)	0·13(0·06–0·39)
CD5+	0·07(0·02–0·52)	0·03***(0·01–0·16)	0·08(0·02–0·88)	0·12(0·04–0·68)

Results are given in median (range) absolute number ( ×10⁹/l). PD, peritoneal dialysis; HD, haemodialysis: CRF, chronic renal failure, not yet dialysed; HC, healthy controls.

^*P < 0·05

^**P < 0·01

^***P < 0·001.

Fig. 1.

The percentage of IgM¯/IgD¯, CD27⁺ and CD5⁺ lymphocytes of total B cells in patients and healthy controls (HC). PD, peritoneal dialysis; HD haemodialysis; CRF, chronic renal failure, not yet dialysed. *P < 0·05, **P < 0·01.

No significant changes of B cell subsets occurred in the longitudinal analysis during the first year of PD treatment (Fig. 2, data shown only for IgM¯IgD¯ B cells).

Fig. 2.

The longitudinal follow-up of the percentage of IgM¯/IgD¯ lymphocytes of total B cells during the first year of peritoneal dialysis (PD) treatment in four children.

T helper cell functions

No differences were found in the proportion IFN-γ, IL-2 and IL-4 producing total lymphocyte numbers (data not shown). However, in the CD4⁺ T cell population, HD children showed a significant lower percentage of IL-2-producing cells compared with CRF children (Fig. 3, P = 0·027). CRF children had more IFN-γ producing cells in the CD8⁺ T cell population compared with HC (Fig. 3, P = 0·015). Furthermore, HD children had a lower proportion IFN-γ-producing CD8⁺ T cells compared with CRF children (Fig. 3, P = 0·028). No differences were found in the T cell IL-4 production between the groups (Fig. 3).

Fig. 3.

The percentage of of T cell subsets (CD4⁺or CD8⁺) producing TH1-type cytokines (IFN-γ and IL-2) or TH2-type cytokines (IL-4). *P < 0·05.

In the longitudinal analysis, during the first year of PD treatment no significant changes of cytokine production were present.

In vitro Ig production

No significant differences were found in the in vitro IgG₁, IgG₂ or IgM production among all groups (data not shown). However, the range of Ig production was very wide. IgG₂ production could be detected only in the cell cultures stimulated with SAC and IL-2.

In the longitudinal analysis, during the first year of PD treatment no significant alterations were found in the Ig production.

DISCUSSION

The present study has shown that children with chronic renal failure had lower numbers of IgM¯/IgD¯ or CD27⁺ B lymphocytes, qualified as memory B cells. Differences in T helper cell subsets cannot explain the lower memory cell count because they were not significantly different between HD, PD and healthy children.

‘Immunological memory’ is important for a strong antibacterial and antiviral defence. B cells differentiate from lymphoid stem cells into mature B cells in the bone marrow, and through contact with antigen in secondary lymphoid tissues they form germinal centres and undergo differentiation into memory cells or plasma cells [26]. IgM is expressed on immature B cells. Both IgM and IgD expression occur on mature B cells. In the germinal centres, B cells undergo somatic hypermutation, isotype switching and affinity maturation. IgM and IgD expression is lost, whereas IgG, IgA or IgE appear on the surface of plasma cells and memory B cells [27]. T cells present in the germinal centres play an important role in this B cell differentiation process by cross-linking of the CD40 molecule on the B cell surface and CD40-ligand (CD40L) from T cells [28,29].

Both CD5 and CD27 were described originally as T cell surface markers. In humans, CD5⁺ B cells or naive B cells are the first B cells appearing in lymphoid tissues during early development and are present at very high frequency in the blood of newborns, diminishing throughout childhood and adulthood [30]. Furthermore, CD5 is expressed on the majority of peritoneal B lymphocytes [31]. CD5⁺ B cells secrete predominantly IgM [30]. Most CD5⁺ B cells are found among IgM⁺IgD⁺ B lymphocytes [32]. We did not find indications that any loss of CD5⁺ B cells by PD treatment resulted in a decreased number of those cells in the blood. Similar results were found by Donze et al. in adult CAPD patients [31]. Only HD children had lower percentage and absolute number of CD5⁺ B cells compared to HC.

CD27 is a marker for memory cells and is expressed on only a small proportion of B cells, in contrast to T cells [33,34]. It has been reported that the percentage of CD27⁺ B cells increases with age [8,9]. Low IgG production found in patients with X-linked hyper-IgM syndrome results from reduced numbers of IgD¯CD27⁺ memory B cells [35]. Data on memory B cells are not yet available in uraemic children and adults. The absolute number of IgM¯IgD¯ memory B cells was not significantly lower in PD, HD and CRF children compared to HC. However, the percentage of IgM¯IgD¯ B cells was lowest in CRF children. For CD27, both the percentage and absolute number of positive B cells were reduced, but in CRF children significance was not reached. The wide age range might influence the results, given that memory cells increase in number with age. The median age of the group of healthy children is lower; however, the difference is not significant. One might speculate that, if the median age of the healthy children had been similar to the patient groups, the results would have been more obvious as the PD, HD and CRF children (with a higher median age) already showed lower numbers of memory cells compared to the group of younger healthy children. In contrast to the literature, no correlation was found in our study between age and CD27⁺ or IgM¯IgD¯ B cells. Differences in the underlying disease, a previous kidney transplantation, infections and immunization status could influence the physiological changes of memory cells during normal life. Thus, dialysis patients have a lower number of memory B cells that might already be present before starting dialysis treatment. Possible explanations for this are a general suppression, suboptimal T helper activity or disturbances in B cell migration process caused by uraemia. Irrespective of the mechanism of memory B cell reduction, the consequence might be a lower capacity to mount a secondary immune response. Memory B cells produce IgG earlier after exposure to antigen compared to unprimed B cells. Bernasconi and co-authors reported that besides the antigen-driven short-term serological memory a long-term antigen-independent, polyclonal stimulation of memory B cells by environmental stimuli exists that maintains a constant level of plasma cells and serum antibodies [36]. The median serum IgG level in our patients (measured in eight PD, eight HD, nine CRF, nine HC) was lower (8·7 g/l) than the healthy controls (11·4 g/l). However, significance was not reached (P = 0·08). A significant correlation was found between the absolute CD27⁺ B cell number and serum IgG level (r = 0·4, P = 0·02). This implies a direct relationship between memory B cell number and serum IgG level irrespective of age. Further studies are needed to analyse the relation between B cell subset differences in uraemic patients and the lower IgG and subclasses levels as has been reported previously [5].

In contrast to other studies [37,38], we found that PBMC from the patients, unstimulated and after stimulation with anti-CD2/CD28 MoAb or SAC/IL2, produced equal amounts of IgG₁, IgG₂ and IgM in vitro compared to HC. The discrepancy between the studies can be explained by differences in methods such as stimulation conditions and patient number. Furthermore, the range of Ig levels was very wide and therefore significant differences can be found only when a large number of patients is considered. In addition, because CD27⁺ B cells are involved in Ig production in vitro, differences might have been found when Ig production by these cells would have been studied specifically in a larger number of patients. It should be noted, however, that in the in vitro B cell differentiation assay interacts with different cell types, cytokines and membrane receptors. Therefore, although dependent on the input of CD27⁺ B cells, Ig secretion may not necessarily correlate with the subset in a quantitative way.

IFN-γ is a cytokine released predominantly by CD4⁺ T cells of the Th1 type, CD8⁺ T cells and natural killer (NK) cells. This cytokine is involved in B cell differentiation, Ig and subclass production and IgG subclass switch [12,13,39]. Lower amounts of IFN-γ released by peritoneal lymphocytes from PD patients with high peritonitis incidence in comparison with patients with low peritonitis incidence and healthy people has been reported by Lamperi et al. [40]. The bacterial killing of peritoneal macrophages improved after treatment with IFN-γ. The toxicity of peritoneal dialysis solutions on immunological functions of peritoneal cells might reduce the production of cytokines by peritoneal lymphocytes during PD. We found no significant differences in IFN-γ production by PMA/ionomycin-stimulated peripheral blood T lymphocytes in CRF, PD and HD children, but we did not measure the cytokine production of peritoneal lymphocytes. We could not confirm the Th1/Th2 imbalance, reported previously in patients treated with dialysis [17]. Differences in methodology and age of the patients are possible explanations for this discrepancy.

In conclusion, children with chronic renal failure, especially those treated with dialysis, have lower numbers of memory B cells. This reduction may contribute to the low Ig levels found in these children.