29. Matsuyama K, Tomo T, Kadota J. Acetate-free blood purification can impact improved nutritional status in hemodialysis patients. J Artif Organs
2011; 14: 112–119
30. Carozzi S, Nasini MG, Caviglia PM et al. Acetate-free biofiltration. Effects
on peripheral blood monocyte activation and cytokine release. Asaio J 1992;
38: 52–54
31. Hmida J, Balma A, Lebben I et al. Clinical evaluation of acetate-free biofiltration at 84 ‰ in patients with chronic renal insufficiency. Tunis Med
2002; 80: 473–484
32. Aniort J, Petitclerc T, Créput C. Safe use of citric acid-based dialysate and
heparin removal in postdilution online hemodiafiltration. Blood Purif 2012;
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Received for publication: 1.2.2015; Accepted in revised form: 12.5.2015
Nephrol Dial Transplant (2015) 30: 1568–1576
doi: 10.1093/ndt/gfv092
Advance Access publication 1 May 2015
Encapsulating peritoneal sclerosis is associated
with T-cell activation
Michiel G.H. Betjes1, Meelad S. Habib1, Dick G. Struijk2, Deirisa Lopes Barreto2, Mario R. Korte3,
ORIGINAL ARTICLE
Alferso C. Abrahams4, Nicole M.A. Nagtzaam5, Marian C. Clahsen-van Groningen6, Willem A. Dik5 and
Nicolle H.R. Litjens1
1
Department of Nephrology and Transplantation, Erasmus Medical Center, Rotterdam, The Netherlands, 2Department of Internal Medicine,
Division of Nephrology, Academic Medical Center, Amsterdam, The Netherlands, 3Department of Internal Medicine, Division of Nephrology,
Albert Schweitzer Hospital, Dordrecht, The Netherlands, 4Department of Nephrology and Hypertension, University Medical Center Utrecht,
Utrecht, The Netherlands, 5Department of Immunology, Erasmus Medical Center, Rotterdam, The Netherlands and 6Department of Pathology,
Erasmus Medical Center, Rotterdam, The Netherlands
Correspondence and offprint requests to: Michiel G.H. Betjes; E-mail: [email protected]
production. In the years preceding the diagnosis of EPS, the
serum sCD25 concentrations increased while remaining at
stable levels in control PD patients. The peritoneum and ascites
of EPS patients showed a significant influx of T cells with relatively increased numbers of CD4+ T cells. These T cells were
fully differentiated and displayed a T helper 1 cell type with a
pro-inflammatory cytokine profile.
Conclusions. Increased serum sCD25 concentrations and peritoneal lymphocytosis in EPS patients indicate the involvement
of activated T cells in the pathophysiology of excessive fibrosis.
A B S T R AC T
Background. Encapsulating peritoneal sclerosis (EPS) is an excessive fibrotic response of the peritoneum that may occur after
long-term peritoneal dialysis (PD). The underlying pathophysiology is poorly understood, but involvement of peritoneal inflammatory T helper 1 cells may be pivotal.
Methods. Soluble interleukin-2 receptor alpha (sCD25) concentration was measured as a marker for T-cell activation in
serum and ascites from EPS patients and various control patient
groups. Peritoneal biopsies were stained for the presence of T
cells, and T cells isolated from ascites of EPS patients were characterized in detail for differentiation status and cytokine
expression.
Results. Serum sCD25 concentrations are significantly and specifically increased in EPS patients compared with haemodialysis, PD and predialysis patients. Peritoneal effluent of stable PD
patients contains very low levels of sCD25, while sCD25 levels
in ascites of EPS patients are high and indicative of local
© The Author 2015. Published by Oxford University Press
on behalf of ERA-EDTA. All rights reserved.
Keywords: biomarker, encapsulating peritoneal sclerosis, peritoneal dialysis, sCD25, T cells
INTRODUCTION
Encapsulating peritoneal sclerosis (EPS) is a rare but devastating complication of peritoneal dialysis (PD). The most important risk factor for EPS is duration of PD treatment, and
associations have been found with younger age, frequent
1568
M AT E R I A L S A N D M E T H O D S
Study population
Analysis of peritoneal biopsies, ascites and serum was performed on samples collected in the biobank of the EPS registry
at the Erasmus Medical Center [21]. All samples were collected
from the participating nephrology centres in the Netherlands.
Peritoneal biopsies in the non-EPS group were obtained from
stable PD patients (n = 8) prior to kidney transplantation at
the University Medical Centrum Utrecht. The non-EPS biopsies were matched for duration of PD with the EPS biopsies [respectively, 58.1 months (range 31.5–62.3) in EPS patients and
41.0 months (range 36.5–51.3) in non-EPS patients (P = 0.33
for difference in PD duration)].
The sera of the group of EPS patients (and the control PD
patients) with yearly measurements of sCD25, 4 years up to
the diagnosis of EPS, were kindly donated by the Academic
T cells and encapsulating peritoneal sclerosis
Medical Center in Amsterdam, Netherlands and have been described in detail before [22]. The diagnosis of EPS was made according to the criteria developed by the Ad Hoc Committee of
the International Society for Peritoneal Dialysis. EPS was defined as a clinical syndrome with symptoms of intestinal obstruction, with or without inflammation parameters and the
presence of compatible radiologic or macroscopic findings
such as peritoneal thickening, calcification and encapsulation
[23]. EPS patients in remission were defined as EPS patients
successfully treated with any combination of prednisone, tamoxifen or surgery, without symptoms of intestinal obstruction
and inflammation at time of inclusion in the study. Control
populations for serum sCD25 measurements consisted of a
random selection of stable haemodialysis (HD), PD and endstage renal disease patients (estimated glomerular filtration rate
< 15 mL/min), not taking immune suppressive medication
and/or with active autoimmune disease, from the Erasmus
Medical Center Nephrology Department. The clinical characteristics are shown in Table 1. In addition, serum sCD25 was
measured in patients with PD peritonitis or HD patients with
catheter sepsis as defined previously [24].
The study protocols were approved by the Medical Ethics
Committee (METC) of the Erasmus Medical Center and the
METC of the University Medical Center Utrecht in adherence
to the Declaration of Helsinki.
Immunohistochemistry and histological analysis
Briefly, 4-μm sections were cut from the formalin-fixed
paraffin-embedded tissue. Immunohistochemistry was performed on serial sections of the peritoneal membrane biopsies
on the Benchmark Ultra stainer (Ventana). Antibodies detecting CD3 (DAKO, Denmark) were used to detect all T lymphocytes, CD4 (Ventana) for T-helper cells, CD8 (DAKO) for
cytotoxic T-cell and CD25 (Cell Marque Sierra, Rocklin,
USA) for detection of the interleukin-2 receptor. After staining,
the overall histomorphological quality of the tissues was evaluated using a Zeiss microscope, by two observers including a
pathologist, who were unaware of the group to which the tissues
belonged. Immunohistochemical analysis was performed in a
blinded fashion using automatic image analysis of the digital
slides (KS-400 version 3, 1997, Carl Zeiss Vision GmbH) as previously described and validated [25]. The results were expressed
as a percentage of immunopositive staining in relation to the
whole surface area of the tissue.
Analysis of T-cell differentiation and cytokine production
A differential cell count of immune cells was performed on
the available cytospins of cells in the ascites from EPS patients.
Of three EPS patients, isolated cells from the ascites were present for more detailed analysis. Peritoneal lymphocytes were
immunophenotyped, and the functional capacity of T cells to
produce cytokines was assessed by multi-parameter flow cytometry [26].
To immunophenotype cells in the ascites, samples were divided into two parts and one part was stained with the 6-color
T/B/NK panel (BD, Erembodegem, Belgium) to determine percentages of monocytes, B, T (CD4+ and CD8+) and NK cells. In
addition, a more detailed analysis of T-cell subsets was
1569
ORIGINAL ARTICLE
peritonitis, kidney transplantation and probably cessation of
PD [1, 2]. The continuous presence of dialysis fluid in the peritoneal cavity acts as a chronic activating stimulus for cells of the
peritoneal membrane [3–5]. For reasons yet unknown, this response is aggravated and accelerated in EPS patients, leading to
a sclerotic cocoon that encapsulates the intestines completely or
more locally [6].
Several lines of evidence suggest that patients at risk for EPS
can be identified by a progressive loss of ultrafiltration [7, 8],
elevated intraperitoneal production of interleukin-6 and plasminogen activity inhibitor-1 (PAI-1) [9, 10] and increased
serum concentration of C-reactive protein (CRP) [11]. These
findings indicate the presence of a smouldering peritoneal inflammation with a systemic CRP response, which is present before acceleration to EPS occurs. Unfortunately, none of these
biomarkers and/or functional tests of the peritoneal membrane
are specific and sensitive enough for early identification of PD
patients who will develop EPS [10, 12].
The inflammatory peritoneal response is characterized by a
mononuclear cell infiltrate consisting of lymphocytes and
macrophages [13]. In particular, the presence of lymphocytes
is of interest as in recent years T cells have been identified as
key players in different animal models of peritoneal adhesion
formation and fibrosis [14–16]. However, the role of intraperitoneal T cells in the pathogenesis of EPS in PD patients has not
been studied.
The high-affinity, trimeric IL-2 receptor complex consists of
three major subunits; the interleukine-2 receptor α-chain (designated CD25), a β-chain (CD122) and the common cytokine
receptor γ-chain (CD132). Soluble (s)CD25 is produced by activated T cells and is released by a proteolytic cleavage of surface
CD25 by enzymes referred to collectively as ‘sheddases’, including neutrophil-derived elastase and metalloproteinase-9 [17].
Serum concentrations of sCD25 are increased in different
pathological conditions with significant T-cell activation such
as autoimmune diseases, sarcoidosis or lymphomas [18–20].
In this study, we investigated the presence and functionality
of T cells in the peritoneum and ascites of EPS patients and
tested serum sCD25 as a biomarker for EPS.
ORIGINAL ARTICLE
performed by staining the other part using AmCyan-labelled
anti-CD3 (BD), Pacific Blue-labelled anti-CD4 (BD) and
AlloPhycoCyanin-Cy7 (APC-Cy7)-labelled anti-CD8 (BD).
The T cells are defined as CD4+ or CD8+ and further dissected
into four different subsets as described before [27, 28] using
expression of CCR7 and CD45RO following staining using
PECy7-labelled anti-CCR7 (BD) and APC-labelled anti-CD45RO
(BD). In short, T cells are divided into naïve T cells (antigeninexperienced, CCR7+ and CD45RO− T cells) and memory T
cells (antigen-experienced). The memory T cells are progressively
more differentiated from central memory cells (CCR7+ and
CD45RO+), followed by effector memory T cells (CCR7− and
CD45RO+) and finally the terminally differentiated subset of
Temra cells (CCR7− and CD45RO−/CD45RA+). CD28 staining,
using Peridinin chlorophyll-Cy5.5 (PeRCPCy5.5)-labelled anti
CD28 (BD), was included as a marker of differentiation as this
marker is progressively lost with T-cell differentiation [29]. Moreover, CD25 staining was included as a marker for T-cell activation
[30] using PE-labelled anti-CD25 (BD).
The functional capacity of T cells to produce cytokines was
determined, performing an intracellular cytokine staining after
polyclonal stimulation, as described previously [31]. Cytokines
were stained intracellularly using APC-labelled anti-interleukin-2
(IL-2, BD) combined with fluorescein isothiocyanate (FITC)labelled anti-interferon-gamma (IFN-γ, BD) and phycoerythrin
(PE)-labelled anti-tumour necrosis factor-alpha (TNF-α; BD),
PE-labelled anti-IL-17 (EBioscience,Vienna, Austria), or PElabelled anti-IL-10 (BD). Percentages of cytokine+ CD4+ as well
as CD8+ T cells were determined by analysing the samples on
the fluorescence-activated cell sorter (FACS) Canto II (BD)
using FACSDiva software Version 6.1.2 (BD Biosciences).
Soluble CD25 measurement
Soluble CD25 was measured in serum samples using the Diaclone human sCD25 ELISA (Besançon, France) following the
manufacturer’s instruction. Intra- and inter-individual variabilities of this assay are 4.5 and 10.3%, respectively. Serum sCD25 is
very stable and not affected by at least five cycles of freezing and
thawing. A serum sCD25 concentration of 2500 pg/mL is considered the upper level of normal in healthy individuals.
Statistical analysis
The SPSS software version 20.0 was used for all statistical
analysis. Medians (25th–75th percentile) are presented of continuous variables and compared using the non-parametric
Mann–Whitney test. Categorical variables are presented as
numbers and/or percentage, and proportions were tested
using the χ² test. When appropriate, medians were given with
inter-quartile ranges (IQRs). The Spearman’s rank correlation
test was used to assess correlation coefficients. All probabilities
were two tailed; P-values of ≤0.05 were considered significant.
Table 1. Demographic and clinical characteristics of end-stage renal disease patient control populations
Age in years median (range)
Male/female
Dialysis vintage months median (range)
Underlying kidney disease
Diabetes
Hypertension
Polycystic kidney disease
Primary glomerulopathy
Other
a
Predialysis patientsa
Peritoneal dialysis patients
Haemodialysis patients
41 (32–47)
5/2
-
50 (21–72)
25/10
9 (5–78)
68 (27–85)
9/11
30 (2–144)
0
1
2
1
3
6
6
3
11
9
6
7
1
2
4
Patients with eGFR < 15 mL/min without dialysis treatment.
Table 2. Demographic and clinical characteristics of EPS cases
Age in years median (range)
Male/female
Duration of PD in months median (range)
Type of treatment at time of diagnosis
PD
Haemodialysis
Transplantation
Underlying kidney disease
Diabetes
Hypertension/nefrosclerosis
Polycystic kidney disease
Primary glomerulopathy
Other
Serum C-reactive protein concentration median (IQR)
Serum sCD25 concentration median (IQR)
Active EPSa
EPS in remission
59 (6–74)
7/4
62 (30–120)
58 (36–59)
4/0
55 (36–96)
0
8
3
0
3
1
3
4
2
4
2
89 mg/L (16–144)
17 100 pg/mL (13 800–25 400)
1
1
1
1
0
7 mg/L (2–11)
9168 pg/mL (6931–10 503)
IQR, inter-quartile range.
a
The diagnosis of EPS was made according to the criteria developed by the Ad Hoc Committee of the International Society for Peritoneal Dialysis [33]. EPS patients in remission were
defined as EPS patients successfully treated with any combination of prednisone, tamoxifen or surgery, without current symptoms of intestinal obstruction and inflammation at time of
inclusion in the study.
1570
M.G.H. Betjes et al.
R E S U LT S
Clinical characteristics of EPS patients
The patient characteristics of EPS patients included for analysis of sCD25 concentrations and peritoneal T-cell analysis are
shown in Table 2. Confirming previous reports, the EPS cases
were characterized by a relative long duration of PD treatment,
and the diagnosis of EPS was made when patients were
transferred to HD or after kidney transplantation ( posttransplantation EPS) [2, 32].
Serum sCD25 is increased at diagnosis in EPS patients
In 11 patients, the serum concentrations of sCD25 were
measured at the time of diagnosis of EPS. All patients were diagnosed with EPS after being transferred to HD (n = 8) or after
kidney transplantation (n = 3) and were in a pro-inflammatory
condition as reflected by a significantly elevated serum CRP
concentration (Table 2). As shown in Figure 1A, EPS patients
at time of diagnosis had a significantly (P < 0.001) elevated
serum sCD25 concentration (median 19 757 pg/mL, IQR
14209-27193) compared with patients on HD (median 7711
pg/mL, IQR 7284-9310; HD controls). EPS patients without
active disease had sCD25 serum concentrations (median 8402
pg/mL) that were not significantly different from HD patients.
Predialysis patients had similar serum sCD25 concentrations
(median 8200 pg/mL) as HD patients. As has been reported before [34, 35], patients with end-stage renal disease with or without
dialysis had a significantly higher serum sCD25 concentration
than healthy controls (upper limit of normal; 2500 pg/mL).
sCD25 concentrations in PD patients with
and without EPS
Remarkably, stable PD patients showed much more variation and on average a higher serum sCD25 concentration
compared with HD and predialysis patients (median 12 800,
IQR 9700-18150, Figure 1A, PD controls). Despite the increased serum sCD25 concentrations in stable PD patients,
EPS patients with active disease had on average significantly
higher sCD25 concentrations (P = 0.011).
Serum sCD25 concentrations were not associated with time
on PD (Figure 1B) or any other demographic or clinical parameter. Also, in the HD patient control group no relation was
found with dialysis vintage, age or gender (data not shown).
Confirming published data [36], serum sCD25 concentrations
were marginally increased in patients with PD peritonitis
ORIGINAL ARTICLE
F I G U R E 1 : (A) Serum sCD25 concentrations in patients with chronic kidney disease stage 5 ( predialysis, n = 7), stable haemodialysis patients
(HD controls, n = 14), patients with active (EPS active, n = 11) and encapsulating peritoneal sclerosis in remission (EPS remission, n = 4) and stable
peritoneal dialysis patients (PD controls, n = 35). (B) Serum sCD25 concentrations in stable PD patients in relation to duration of PD treatment
(n = 35). (C) Serum sCD25 concentrations in HD patients with catheter-related blood stream infection (HD + infection, n = 6) and PD patients
with peritonitis (PD + peritonitis, n = 5). (D) sCD25 concentrations in peritoneal effluent (n = 11; PD effluent) and in ascites of patients with active
EPS (n = 4).
T cells and encapsulating peritoneal sclerosis
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ORIGINAL ARTICLE
F I G U R E 2 : Serum sCD25 concentrations of patients on peritoneal dialysis (n = 7) in the years preceding the diagnosis of encapsulating peritoneal
dialysis [EPS patients, (A)] compared with a control group of peritoneal dialysis patients [PD patients, (B)]. The asterisk in (A) indicates that at
1 year before the diagnosis of EPS the serum sCD25 concentration is significantly higher (P < 0.05) in the EPS group.
(median 14 177 pg/mL) and in HD patients with catheterrelated blood stream infection (CRBSI, median 11 680 pg/mL,
P-value < 0.05 compared with stable HD patients, Figure 1C).
In contrast, serum CRP levels were substantially increased at
similar levels for EPS patients (median 89 mg/L), HD patients
with CRBSI (146 mg/L) and PD peritonitis (133 mg/L). The PD
effluent showed low levels of sCD25, which did not exceed
500 pg/mL in all cases tested (Figure 1D). In contrast, ascites
of four tested active EPS patients contained high concentrations
of sCD25 (median 23 450 pg/mL, IQR 22325-41225, Figure 1D)
with ascites sCD25:serum sCD25 ratios of 1.3, 1.7, 1.5 and 2.2,
respectively, indicative of intraperitoneal sCD25 production.
As shown in Figure 2, the average serum sCD25 concentration
increased in the years before EPS was diagnosed (Figure 2A) and
remained stable in the control PD patients (Figure 2B). One year
before the diagnosis of EPS, the difference was statistically significant between both groups (median 26 558 versus 17035 pg/mL,
P = 0.01).
Peritoneal lymphocytes and EPS
Lymphocytes were the predominant mononuclear cell type
in the ascites obtained from EPS patients as part of a diagnostic
procedure (Figure 3A and B). In three EPS cases, ascites was
available for isolation and subsequent analysis of peritoneal
lymphocytes by flow cytometry. The majority of lymphocytes
were T cells with CD4+ and CD8+ T cells present in equal numbers (Figure 3C). Most T cells were of the memory phenotype
(Figure 3D, typical example of CD4+ T cell subsets within ascites) with a clear dominance of highly differentiated effector
memory CD4+ and CD8+ T cells and terminally differentiated
CD8+ Temra cells (Figure 3E and F). Cell surface expression of
CD25 was low on both CD4+ and CD8+ T cells.
In Figure 4A and B, a typical flow cytometric example is
given of IL-2 and IFN-γ producing CD4+ (Figure 4A) and
CD8+ (Figure 4B) T cells. The cytokine profile of CD4+
(Figure 4C) and CD8+ (Figure 4D) T cells was pro-inflammatory
with high frequencies of TNF-α and IFN-γ but low frequencies of
IL-17 and IL-10 producing T cells. For CD4+ T cells, this was consistent with a pro-inflammatory T helper 1 cell signature.
Peritoneal biopsies of EPS patients (n = 8) showed a significant
increase in percentage of area stained for CD3+ T cells (Figure 5)
1572
compared with control (n = 8) PD patients (1.6 versus 0.3%, P <
0.001). In general, more CD4+ T cells were observed in the EPS
peritoneum, but the CD4/CD8 ratios were not significantly
increased in the EPS patient group (1.5 versus 1.0, P = 0.23). In
peritoneal biopsies of healthy controls, T cells are scarcely identified within the peritoneum (data not shown). Staining for CD25
showed almost absent expression of CD25-positive cells in peritoneal biopsies of EPS patients and control PD patients.
DISCUSSION
The present study shows that EPS patients have significantly increased serum sCD25 concentrations and intraperitoneal production of sCD25. Within the years preceding the diagnosis of
EPS, there is a significant rise in serum sCD25 concentrations.
These findings indicate that EPS is associated with activation of
lymphocytes shedding their CD25 into the circulation. This association is consistent with our observation that an influx of
lymphocytes is found in the peritoneum of EPS patients,
which is reflected in the relative lymphocytosis in the ascites.
The data from this study can only establish a strong association between T-cell activation and EPS but not a direct pathophysiological relationship. However, animal models have
shown that activated peritoneal lymphocytes are crucially involved in peritoneal adhesion formation and peritoneal sclerosis [14–16]. In particular, CD4 T cells have been identified
as pivotal cells for intraperitoneal adhesion formation [14,
15], and T cells producing IFN-γ have an essential role in the
development of peritoneal adhesions [15] and peritoneal sclerosis after multiple intraperitoneal challenges with staphylococcus epidermidis culture medium [16]. Our findings on sCD25
and Th1 cell infiltration in the peritoneum yield further support
to the experimental concept of peritoneal fibrosis driven by activated Th1 cells producing IFN-γ [15, 16]. In line with this concept, it has been shown that effector memory T cells capable of
mounting a Th1 response are the dominant T-cell type in the
peritoneal effluent of stable PD patients [33]. Of note, the
type of injury chosen to inflict peritoneal sclerosis in an animal
model is relevant as the absence of T cells actually increased
submesothelial thickness in a chlorhexidine gluconate model
of EPS [37].
M.G.H. Betjes et al.
ORIGINAL ARTICLE
F I G U R E 3 : Analysis of lymphocytes in the ascites from EPS patients. (A) It shows a typical example of lymphocytosis present in the ascites from
an EPS patient, and a differential cell count of immune cells in the ascites of EPS patients (n = 6) is given in (B). The different lymphocytes, i.e.
CD4+ and CD8+ T cells, (B) and NK cells, present within the ascites samples are further characterized using multi-parameter flow cytometry (C). A
detailed phenotypical analysis of T cells in ascites of EPS patients (n = 3) is shown in (D–F). In (D), an example of categorization of peritoneal
CD4+ T cell subsets, using the differential expression of CD45RO and CCR7, into naïve T cells (naïve), central memory T cells (CM), effector
memory T cells (EM) and terminally differentiated Temra cells (EMRA) (D) is given. In (E and F), the relative distribution of these T cell subsets
and percentages of CD28+ and CD25+ T cells in CD4+ (E) and CD8+ T cells (F) are given.
Although limited by the number of observations in our
study, it seems evident that the intraperitoneal lymphocytes
in EPS patients are highly differentiated memory T cells that
produce pro-inflammatory cytokines like IFN-γ and TNF-α
but little IL-17. The latter cytokine has been implicated in
peritoneal fibrosis [38], but our findings do not support a
role of T helper 17 cells in EPS. In addition, we noted an
influx of NK cells in the ascites of EPS patients which cannot
be readily explained. To date, conflicting reports are published
T cells and encapsulating peritoneal sclerosis
about the role of NK cells in inflammation and fibrosis depending on the type and location of the inflammatory process
[39].
The involvement of activated lymphocytes seems to be specific for EPS as PD peritonitis [36] and catheter sepsis do not
substantially increase serum sCD25 concentrations. Therefore,
the specific type of ( peritoneal) inflammation in EPS, which is
characterized by a mononuclear cell infiltrate, results in elevated
serum sCD25 concentrations. Peritoneal T cells within the
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ORIGINAL ARTICLE
F I G U R E 4 : Cytokine profiles of T cells present in the ascites of EPS patients (n = 3). Intracellular staining for cytokines was performed following a
6-h polyclonal stimulation using PMA and ionomycin. The upper panel shows typical dot plots of IFN-γ and IL-2 producing CD4+ (A) and CD8+
(B) T cells. The lower panel shows the percentage of CD4+ (C) and CD8+ (D) T cells expressing any of the cytokines, i.e. IL-2, IFN-γ, TNF-α, IL-10
and IL-17, as indicated on the X-axis.
inflamed peritoneum and harvested from EPS ascites hardly express CD25. These observations are consistent with the notion
that CD25 is released from the cell membrane of these activated
T cells and increases serum and ascites sCD25 concentration.
The lack of CD25 expression on peritoneal T cells is in line
with the observation that tissue obtained from other chronic inflammatory conditions, such as synovial tissue of patients with
rheumatoid arthritis, also contains few CD25-positive T cells
[40, 41]. To date, several enzyme candidates have been reported
to possess the capacity for cleaving CD25 including the endogenous enzymes elastase and matrix metalloproteinase-9
(MMP-9) [42, 43], which explains why inflammatory conditions involving activated T cells and macrophages (e.g. EPS, sarcoidosis and rheumatoid arthritis) are associated with increased
serum sCD25 concentrations [20]. Serum sCD25 may be a potential interesting biomarker to support the diagnosis of EPS,
particular in patients not on PD anymore. In addition, the significant increases in serum sCD25 in the years before the diagnosis of EPS may identify patients at risk for developing EPS
during PD treatment. Given the overlap in sCD25 values between control PD patients and EPS cases, the predictive value
will have limitations but may increase in combination with
1574
other potential biomarkers of EPS like effluent IL-6 and
PAI-1 concentrations [9].
Of interest is the observation that PD patients show large
variation in serum sCD25 concentrations, which are on average significantly increased compared with HD patients and
predialysis patients. This indicates that PD patients show
signs of lymphocyte-driven inflammation, which is most likely
derived from a peritoneum activated by dialysis fluid. The peritoneal surface is substantial and equals the surface of the skin
[44]. Therefore, even a mild peritoneal mononuclear cell infiltrate will comprise significant numbers of activated T cells.
Analysis of peritoneal biopsies of stable PD patients indeed
revealed the presence of T cells that are virtually absent in
the peritoneum of healthy individuals. PD treatment leads
thus to an influx of activated T cells into the peritoneum,
which is reflected by increased sCD25 serum levels. Combining
these observations, it seems evident that progressive peritoneal
inflammation involving T cells is preceding the development
of EPS. Peritonitis, kidney transplantation and/or cessation
of PD are triggers for accelerated and excessive peritoneal fibrosis [2, 32]. The involvement of T cells in the pathophysiology of EPS may explain the potential therapeutic effect of
M.G.H. Betjes et al.
immunosuppressive drugs like steroids in the treatment of
these patients [45].
There are obvious limitations to this study given the retrospective nature of the data collected and the limited number of
observations in some of the immunological assays. This may
have created a bias for which we cannot account. However, we
could not identify a systematic bias in the collection of biological
samples of EPS patients in the EPS Registry which was performed
on a regular basis, as described previously in detail [21].
In conclusion, the findings on sCD25 and activated peritoneal T cells strengthen the concept of a central role for T helper
cell type 1 in the pathogenesis of EPS.
C O N F L I C T O F I N T E R E S T S TAT E M E N T
None declared.
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F I G U R E 5 : A peritoneum biopsy of a patient with EPS showing the infiltration with T cells [brown dots in (B–D)]. (A) shows the HE-staining,
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Received for publication: 13.1.2015; Accepted in revised form: 16.3.2015
M.G.H. Betjes et al.