Departments of 1 Pediatrics and 5 Internal Medicine, Section of Nephrology, Taipei Veterans General Hospital, 3 Department of Pediatrics, Children Hospital, Changhua Christian Hospital, and 2 Institute of Clinical Medicine and 4 Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan, Republic of China
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Abstract |
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Methods. A cohort of 36 patients treated with CAPD and who had histories of peritonitis were divided into a group with rapid and a group with delayed response to antibiotics, and were followed for 3 years. CD4/CD8 T-cell ratios, T-cell cytokine mRNA expression patterns and transforming growth factor-ß1 (TGF-ß1) concentrations were examined in PDE during bouts of peritonitis. The change in 4 h D/P creatinine during the peritoneal equilibration test (PET) between year 0 and year 3 was expressed as D/P creatinine.
Results. The serial changes in T-cell subsets in PDE during peritonitis showed two patterns: (i) pattern 1, manifest as a progressive increase in the CD4/CD8 ratio, and associated with a rapid response to treatment; and (ii) pattern 2, manifest as a progressive decrease in the CD4/CD8 ratio, and associated with a delayed response to treatment. The major T-cell phenotypes in PDE during peritonitis were Th1-CD4+ and Tc2-CD8+, determined by cloning techniques, RTPCR and double immunofluorescence staining. TGF-ß1 in the effluent was undetectable in pattern 1 after 78 days, but remained detectable at 2 weeks in pattern 2. Pattern 2 patients had a significantly greater decrease (D/P creatinine: -0.198±0.086) in solute transport than pattern 1 patients (
D/P creatinine: -0.036±0.077, P<0.05).
Conclusions. These results suggest that a progressive decrease of the CD4/CD8 ratio in PDE correlates with a persistent expression of TGF-ß1, and plays a pathogenetic role in the evolution of peritonitis, PET deterioration and peritoneal fibrosis. Therefore, patterns of CD4/CD8 T-cell ratio in PDE may predict clinical outcomes of peritonitis in CAPD patients.
Keywords: CD4+ T cells; CD8+ T cells; peritoneal dialysis; peritonitis; TGF-ß1
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Introduction |
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Peritonitis is a local inflammatory disorder. A large amount of prior information suggests that the main cell type involved in it is the peritoneal macrophage [2]. However, more contemporary descriptions include all the resident cells of the peritoneal cavity in the coordinated response to an infection. Human peritoneal mesothelial cells, which line the surface of the peritoneal membrane, act as the target and primary responders to peritonitis. They produce inflammatory and chemotactic cytokines, prostaglandins, components of the fibrinolytic cascade, as well as lipids, proteoglycans and growth factors [35]. In addition, recruitment and infiltration of leukocytes are essential elements of the peritoneal immune response. Polymorphonuclear neutrophils (PMNs) are attracted to the peritoneal cavity by chemoattractants and engage in phagocytosis. While the activation of macrophages by T cell-derived interferon- (IFN-
) enhancing bactericidal activity is well recognized [6], information on the role of T lymphocytes in peritoneal immunity is limited.
T lymphocytes are classified into CD4+ and CD8+ cell subsets depending on their surface markers. CD4+ and CD8+ T cells differentiate into type 1 and type 2 T-cell subsets (Th1/Th2, Tc1/Tc2) with distinct cytokine patterns. Type 1 T cells produce interleukin (IL)-2, IFN-, IL-8 and tumour necrosis factor-ß (TNF-ß), and type 2 T cells produce IL-4, IL-5, IL-6, IL-10 and IL-13. The functions of T-cell subsets correlate with their distinctive cytokines. Type 1 cytokines activate cytotoxic and inflammatory functions, while type 2 cytokines are associated with strong antibody and allergic responses. The phenotype of peritoneal cavity lymphocytes (PCLs) has been analysed. A previous study demonstrated that the majority of peritoneal T cells were CD8+, and that a high percentage of CD8+ T cells secreted type 2 cytokines [7]. These Tc2-CD8+ T cells were modestly cytolytic and assumed a B-cell helper function for the synthesis of IgG and IgA. The outcomes of a wide range of disorders, including infections, allergies and autoimmune diseases, have been linked to the expression patterns of type 1- or type 2-like cytokines [8,9]. However, the relationship between the outcome of peritonitis and type 1- or type 2-like cytokine expression remains unclear.
Furthermore, a significant number of patients treated with CAPD gradually lose peritoneal membrane function, typically apparent as the loss of ultrafiltration and low solute clearance [10]. Partial or total disappearance of mesothelium, and fibrosis and thickening of the submesothelial tissue develop in a majority of these patients. Membrane fibrosis and failure are accelerated by repeated episodes of peritonitis, and certain organisms, such as fungi and Staphylococcus aureus, are particularly likely to cause rapid peritoneal failure [6]. Peritoneal fibrosis has been closely related to the proliferation of peritoneal fibroblasts and the deposition of extracellular matrix (ECM) in several studies. Transforming growth factor-ß1 (TGF-ß1) plays a major role in stimulating the deposition of ECM. The persistent expression of TGF-ß1 in dialysate may predict peritoneal fibrosis in CAPD patients with frequent bouts of peritonitis [11].
To clarify the role of peritoneal T lymphocytes in peritoneal immune defence mechanisms, this serial longitudinal study was designed to examine the changes in T-cell subpopulations and cytokine mRNA expression patterns during peritonitis in patients treated with CAPD. Our observations were correlated with responses to treatment and with outcomes. To determine the influence of peritonitis on peritoneal transport properties and peritoneal fibrosis, we also studied the peritoneal dialysing function and expression pattern of TGF-ß1 as a marker of prognosis.
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Subjects and methods |
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Patients recently entered in the dialysis programme
Peritoneal dialysate effluent (PDE) samples were collected from 10 new patients (six men, four women, mean age 41.25±13.57 years) admitted to our CAPD programme. Hourly, low-volume exchange of peritoneal dialysis fluid was performed in the first 48 h after the insertion of the peritoneal catheter. Thereafter, intermittent peritoneal dialysis was performed while the patient received CAPD training. PDE samples were collected on days 15 after the insertion of the peritoneal catheter, and at 1 and 3 months after the start of CAPD treatment.
Peritonitis and response to treatment
Peritonitis was diagnosed according to the criteria defined by the Ad Hoc Advisory Committee on Peritonitis Management [12]. Two of the three following criteria had to be fulfilled: (i) 100 or more white blood cells (WBCs)/mm3 of dialysate; (ii) clinical manifestations of CAPD peritonitis; and (iii) positive dialysate culture. Patients with evidence of tunnel-tract infection were excluded. Episodes of peritonitis were treated according to a standard protocol. The initial antibiotic regimen included vancomycin and a third-generation cephalosporin administered intraperitoneally, which was modified on the basis of cultures and drug sensitivities. The patients were divided into two groups according to their clinical response, whether rapid or delayed. A rapid clinical response was defined as the resolution of symptoms and signs of peritonitis, including disappearance of abdominal pain and clearing of the dialysate, within 72 h of onset of appropriate antibiotic therapy, followed by complete recovery within 710 days of treatment. A delayed clinical response was characterized by the persistence of symptoms and signs, positive dialysate culture and elevated WBC count in dialysate beyond 72 h of appropriate antibiotics treatment, and a fluctuating and protracted illness over 1014 days despite an appropriate antibiotic regimen.
Peritoneal equilibration test
The peritoneal equilibration test (PET) was performed as described by Twardowski et al. [13].
Briefly, a patient received an overnight dwell of 2.5% dialysis solution (Dianeal 2.5%, Baxter) for 812 h, which was then drained over 20 min with the patient in the upright position. With the patient in the supine position, 2 l of 2.5% dialysis solution was infused at a rate of 400 ml/2 min for 10 min. Children received a total volume of 40 ml/kg of dialysate. The patient was turned from side to side after the infusion of each 400 ml of solution, and every 2 min during the infusion to mix residual volume and the infused dialysis solution. Creatinine and glucose concentrations were measured in the dialysate at 0, 2 and 4 h, and plasma creatinine and glucose concentrations at 2 h.
Calculations
The correction factor in our laboratory is 0.000347.
All measurements were performed when the patients were euvolaemic, and >2 months after an episode of peritonitis. The PET schedule in our centre includes at least yearly follow-ups, which are used by the physicians to modify the treatment according to changes in dialysis efficiency. The follow-up duration was 3 years. A 4 h D/P creatinine at years 0 and 3 was calculated for each patient. The change in peritoneal solute transport (D/P creatinine) over time was calculated by subtracting the 4 h D/P creatinine at year 0 from that measured at year 3.
Recovery of cells and separation from drained dialysate
The dialysate was filtered through sterile gauze, cells were counted, and cytospin preparations were made for cell differential counts. The cells were separated by centrifugation at 400 g, 4°C for 10 min. The supernatant was stored at -70°C until analysis of TGF-ß1. The leukocyte-rich suspension was layered over a FicollTriosil density gradient and centrifuged at 600 g for 20 min [5]. The purity of the mononuclear cell (MNC) population was verified by WrightGiemsa staining, and cell viability by the trypan blue exclusion method, both being >95% [5]. The interface MNCs were incubated twice in a plastic Petri dish for 60 min at 37°C to remove the adherent peritoneal macrophages, monocytes and mesothelial cells. The non-adherent MNCs were then collected and filtered through a 70 µm nylon mesh cell strainer to remove cell debris and aggregates, and passed through a flow cytometer using CD3 monoclonal antibody (Ortho Pharmaceuticals, Raritan, NJ). Using the sorting technique, the CD4+ and CD8+ T cells were separated and collected.
Cell labelling procedure for flow cytometry
The cells were suspended in phosphate-buffered saline (PBS), containing 1% bovine serum albumin (BSA) and 0.1% NaN3, at 2x105 cells/100 µl, and directly conjugated monoclonal antibodies (mAbs) were added in volumes of 10 µl each. After 30 min of incubation at 4°C, peritoneal cavity lymphocyte (PCL) samples were prepared in a Q-Prep leukocyte preparation system (Beckman Coulter, Brea, CA) to lyse the erythrocytes and fix the stained cells. The various mAbs, labelled with fluorescein isothiocyanate (FITC) or phycoerythrin (PE), used for the staining of cell surface markers were obtained from Dianova-Immunotech (Hamburg, Germany): anti-CD2, -CD3, -CD4, -CD5, -CD7, -CD8, -CD11a, -CD11b, -CD16, -CD18, -CD19, -CD20, -HLA-DR, goat anti-human IgM (GaHIgM), goat anti-mouse Ig (GaMIg) and GaMIgM were from Becton Dickinson (San Jose, CA).
Flow cytometric analyses
The determination of the expression of CD3 and CD4/CD8 surface markers and sorting was carried out with a fluorescence-activated cell sorter (EPICS Altra HyPerSortTM system, Coulter).
CD4/CD8 ratio measurement
To measure the CD4/CD8 ratio, 1x106 cells were incubated on ice with a mixture of CD3FITC, CD4PC5 and CD8PE for 30 min. The cells were washed twice with PBS, then resuspended in 100 µl of PBS and analysed by flow cytometry. Dot plots were created. Percentages of CD8+ and CD4+ cells were measured with Coulter software, and CD4/CD8 ratios were calculated.
T-cell cloning
Using sorting techniques, CD8+ or CD4+ lymphocytes were isolated from PCLs by positive selection on either anti-CD8 or anti-CD4 mAbs. The purity of the CD8+ or CD4+ PCL preparations, determined by flow cytometric analysis, was >95%. Bulk cultures of either CD8+ or CD4+ PCLs were stimulated with oxidized allogeneic stimulator cells [antigen-presenting cells (APCs)]. APCs were prepared from buffy coats by Ficoll density gradient centrifugation, and were depleted of T lymphocytes by rosetting on sheep erythrocytes (E--APC). The E--APCs (4x107/ml) were irradiated (3500 rad) and then oxidized by incubation with neuraminidase (0.02 U/ml) and galactose oxidase (0.05 U/ml) in RPMI-1640 culture medium for 90 min at 37°C. Thereafter, the cells were washed three times in RPMI-1640 containing 0.02 M galactose.
Limiting dilution cloning of either peritoneal CD8+ or CD4+ T lymphocytes was carried out by stimulating CD8+ or CD4+ cells (600 cells/well to 0.3 cells/well) with irradiated and oxidized allogeneic E--APCs (5x104/well) in 96-well round-bottom tissue culture plates in the presence of 50 IU/ml of recombinant human IL-2 (rhIL-2). The medium was replaced every third day. Under these cultural conditions, the T-cell clones had probabilities between 85 and 95% of being derived from a single cell, calculated from the proportion of negative wells by Poisson statistics. The T-cell clones were maintained and expanded by restimulation with oxidized allogeneic cells every 1014 days.
Induction of type 1/type 2 cytokine mRNA expression from cloned T cells
Culture and induction experiments. To induce the expression of cytokine mRNA, either CD4+ or CD8+ T-cell clones were cultured in 5 cm diameter Petri dishes containing a total of 58x105 cells under RPMI-1640, supplemented with 10% human AB serum, and were then stimulated with recombinant human IL-2 (50 IU/ml) and lipopolysaccharide (LPS; 10 µg/ml). The cells, pelleted by centrifugation, were harvested 24 h after LPS stimulation.
Determination of cytokine mRNA expression. Cells were washed with PBS three times, and a proper volume of RNAzol B was added. They were mixed by repeated up and down pipetting, transferred into an Eppendorf tube containing a one-tenth volume of chloroform, vortexed vigorously for 2030 s and placed on ice for 15 min. After centrifugation at 14 000 g for 15 min, the upper aqueous phase was transferred to a new tube, and an equal volume of isopropanol was added and gently shaken. The sample was placed at -20°C for 1 h, then centrifuged at 12 000 g at 4°C for 15 min, and the RNA pellets were washed by 75% alcohol for 5 min, then centrifuged at 7500 g for 10 min. The pellets were dissolved in diethylpyrocarbonate (DEPC)H2O. After heating at 5060°C for 10 min and cooling on ice, the quantity and purity of the RNA preparations were determined by measuring their absorbances at 260 and 280 nm and confirming an A260/A280 ratio >1.9.
Reverse transcription. After the addition of 10 µg of RNA into DEPC-H2O (to a total volume of 39 µl), 1 µl of oligo(dT) (0.5 µg/ml) was added, then heated to 70°C for 10 min and immediately placed in an ice bath. TrisHCl, KCl, MgCl2, MnSO4, dithiothreitol (DTT), dNTP, RNase inhibitor and reverse transcriptase were added. The reaction was held at 37°C for 1 h, then at 65°C for 10 min, and placed in the ice bath to interrupt the reaction; the cDNA was stored at -70°C.
Polymerase chain reaction. The total amount of each sample was 50 µl, including 5 µl of reverse transcriptase product, 0.2 mM dNTP, 200 nM sense and antisense DNA probe (Table 1
), 15 mM MgCl2, 50 mM KCl, 10 mM TrisHCl (pH 9.0), 1% Triton X-100 and 2.5 U of Taq DNA polymerase. After being fully mixed, the samples were treated according to the following protocol: 94°C for 1 min, then 65°C gradually decreasing to 55°C, with 1 min pauses at each 0.5°C step, then 72°C for 1 min for a total of 21 cycles. This was followed by 94°C for 1 min, 55°C for 1 min, 72°C for 1 min, for a total of 19 cycles and, finally, 72°C for 5 min, then returned to 4°C and stored.
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Detection of cloned T-cell subsets and cytokines by immunofluorescence
Double immunofluorescence studies were performed to confirm the expression patterns of cloned T-cell subsets and the cytokines. CD4+ or CD8+ T-cell clones were cultured in 5 cm diameter Petri dishes with a total of 58x105 cells in RPMI-1640 supplemented with 10% human AB serum, and then stimulated with recombinant human IL-2 (50 IU/ml) and LPS (10 µg/ml). The cells, pelleted by centrifugation, were harvested 24 h after LPS stimulation and washed with PBS three times, and their density was adjusted to 5x105 cells/ml. Cytospins were prepared with 0.05 ml of the cell suspension in a Shandon cytocentrifuge (Shandon Southern Products Ltd, Runcorn, UK) and dried with cold air. Before staining, the slides were washed once with Hank's blanced salt solution (HBSS)0.1% saponin (Sigma). For staining, goat anti-human IFN- Ab (C-19) or goat anti-human IL-4 Ab (M-19) in a concentration of 2.5 µg/ml diluted in HBSSsaponin was added, and the slides were incubated for 30 min. After a single wash in HBSSsaponin, rhodamine-labelled donkey anti-goat IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) diluted at 1:100 in HBSSsaponin was added, and the slides were incubated for 30 min. After washing the cells three times in HBSS (without saponin), mouse anti-human CD4 Ab (18-46) or mouse anti-human CD8 Ab (32-M4) was added in a concentration of 5 µg/ml, and the slides were incubated for 30 min in HBSS. The slides were then washed three times in HBSS, and FITC-labelled donkey anti-mouse IgG (Santa Cruz Biotechnology, Inc.) at 1:200 was added. After 30 min, slides were washed again and mounted with buffered glycerol. All experiments included isotype-matched controls for both intracellular and extracellular proteins.
By using the double immunofluorescence method, the relative proportions of CD4+ (or CD8+) cells, fluorescing in green, IFN-+ (or IL-4+) T cells, fluorescing in red, and CD4+ IFN-
+ (or CD8+ IL-4+) T cells, fluorescing in both red and green, could be determined. These proportions were quantified by counting multiple high powered fields using an Olympus fluorescence microscope with epi-illumination and appropriate barrier filters for FITC and rhodamine. Approximately 200 cells were counted in each experiment, always by the same observer.
Measurement of TGF-ß1
Samples of the effluent for measurements of TGF-ß1 were treated immediately with 1 mM phenylmethylsulfonyl fluoride (Sigma) and centrifuged at 2000 r.p.m. for 5 min at 4°C. The supernants were then acidified with HCl to pH 2.5, and dialysed against three changes of PBS overnight. The samples were then divided into aliquots and stored at -20°C until assayed for TGF-ß1 activity. The concentrations of TGF-ß1 were measured by a commercial kit using an enzyme-linked immunoassay method (R&D System, Mckinley Place, MN). The lowest limit of sensitivity of that TGF-ß1 assay is 5 pg/ml.
Statistics
Data are presented as the mean±SD. Changes in the CD4/CD8 ratios in PDE over time were compared by analysing variance. The changes in TGF-ß1 and peritoneal solute transport, represented by D/P creatinine, were compared between the two groups by a two-sample t-test. A P-value <0.05 was considered significant. When the variance was not normally distributed, a non-parametric test was used to examine the difference.
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Results |
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Serial changes in the CD4/CD8 ratio in dialysate effluent during peritonitis
The serial measurements of the CD4/CD8 ratio made in the PDE during peritonitis followed two patterns (Figure 2): the first pattern (pattern 1, n=25) was characterized by a progressive increase in the CD4/CD8 ratio (Figure 2A
). The CD4/CD8 ratios on days 5, 6 and 7 were significantly different from those on day 1 (P<0.05). Overall, the patients who exhibited pattern 1 had favourable clinical courses. The WBC counts in their PDE decreased rapidly in response to antibiotics, and had totally cleared after 7 days, at which time the measurement of T-cell subsets was discontinued. The second pattern (pattern 2, n=11) was characterized by high initial CD4/CD8 ratios, which progressively decreased (Figure 2B
). The CD4/CD8 ratios on days 17 were significantly different from the ratio on day 14 (P<0.05). This second pattern was associated with a delayed clinical response to treatment in nine of the 11 patients. Symptoms and signs of peritonitis persisted beyond 72 h despite the administration of appropriate antibiotics, and high WBC counts in the dialysates were observed for >2 weeks.
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Clinical observations
The baseline characteristics of the pattern 1 and pattern 2 patient groups are shown in Table 2. The mean age of patients who exhibited pattern 1 was 40.2±14.1 years compared with 38.8±12.9 years for pattern 2 patients (NS). The ratios of male to female patients in the pattern 1 and pattern 2 groups were 13:12 and 6:5, respectively (NS), and they had been dialysed for a mean of 2.78±1.17 years (range 0.75.3 years) vs 2.92±1.08 years (range 1.14.5 years), respectively (NS). In the group exhibiting pattern 1, 59.9% of the microorganisms causing peritonitis were Gram-positive and 21.4% were Gram-negative; in 18.7% the cultures remained negative. In the group exhibiting pattern 2, the distribution of microorganisms was 61.2% of Gram-positive, 19.8% of Gram-negative, and 19.0% of subjects had negative cultures, not significantly different from the situation found with pattern 1.
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Cytokine expression patterns of peritoneal CD8+ cells
The cytokine expression patterns of peritoneal CD8+ cells, cloned from the PDEs of new patients and from the PDEs during peritonitis on day 2, were analysed. The expression of the mRNAs of IL-4, IL-5, IL-6 and IL-10 was strong in CD8+ cells, while the IL-2 and IFN- signals were weak. (Figure 3
, upper panel). The cytokine mRNA expression pattern of CD8+ cells cloned from patients with pattern 1 and pattern 2 was similar, indicating that the production of cytokines by these peritoneal CD8+ T cells was associated with a Tc2, rather than a Tc1 pattern.
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Cytokine expression patterns of peritoneal CD4+ cells during peritonitis
The cytokine expression patterns of peritoneal CD4+ cells cloned from PDEs during peritonitis were also analysed. After stimulation with LPS, the mRNA expression by peritoneal CD4+ cells of IL-2 and IFN- was prominent, whereas the expression of IL-4 and IL-10 was absent, and that of IL-5 and IL-6 was weak on day 2 after the onset of peritonitis (Figure 3
, lower panel). The peritoneal CD4+ cells cloned from pattern 1 and pattern 2 patients expressed similar cytokine mRNA patterns. This indicates that the majority of CD4+ cells during acute peritonitis were Th1 rather than Th2 cells.
Fluorescence microscopy on cytospins
To confirm the cytokine expression patterns of cloned T-cells from PDEs, cytospins were prepared and double immunofluorescence staining was performed. The purity of cloned CD4+ and CD8+ T cells by immunostaining with FITC-labelled Ab directed against CD4 (Figure 4B) or CD8 (Figure 4E
) was 97 and 96%, respectively. Cells were counterstained simultaneously with rhodamine-labelled Ab specific to type 1 (IFN-
) or type 2 (IL-4) cytokines. The CD4+ T cells showed a predominant IFN-
production, manifested as 85% positive immunostaining with anti-IFN-
Ab (Figure 4C
), indicating that the majority of peritoneal CD4+ T cells during acute peritonitis were Th1 cells. Double staining with anti-CD8FITC and anti-IL-4rhodamine was observed in 81% of CD8+ T cells (Figure 4F
), consistent with a phenotype of Tc2 for most CD8+ T cells during peritonitis. Isotype-matched controls were negative for both intracellular (IFN-
and IL-4) and extracellular proteins (CD4 and CD8).
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Serial changes in TGF-ß1 concentration in the dialysate effluent during peritonitis
The concentration of TGF-ß1 in the PDE between episodes of peritonitis was variable. In the pattern 1 group, TGF-ß1 concentration increased initially then rapidly declined. At 78 days after the onset of peritonitis, TGF-ß1 was no longer detectable in PDE (Figure 5). In the pattern 2 group, the TGF-ß1 concentration was high at the onset of peritonitis, then decreased gradually, but remained detectable for >2 weeks (Figure 5
). On days 7 and 8, significant differences were present between pattern 1 and pattern 2 in TGF-ß1 concentrations in dialysate effluents (P<0.05).
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Serial changes in peritoneal equilibration test in pattern 1 vs pattern 2 groups
Pattern 2 patients had a significantly greater decrease in solute transport than pattern 1 patients (D/P -0.198±0.086 vs -0.036±0.077, P<0.05, Figure 6
). This corresponded to a marked fall in 4 h D/P creatinine over the 3 years of follow-up in pattern 2 patients. Six of the pattern 2 patients developed peritoneal fibrosis, diagnosed by peritoneal biopsy, and were switched to haemodialysis. In contrast, in the pattern 1 patients, in whom TGF-ß1 in effluents became rapidly undetectable, the clinical course was consistently uneventful and uncomplicated after repeat episodes of peritonitis.
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Discussion |
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We observed that different patterns of T-cell subsets were associated with different therapeutic responses and outcomes of peritonitis. Pattern 1 exhibited a progressive increase in the peritoneal CD4/CD8 ratio and a generally favourable response to treatment. In pattern 2, the CD4/CD8 ratio at the onset of peritonitis was higher, then decreased progressively, and the response to treatment was usually delayed. The major T-cell phenotypes in PDE during peritonitis were Th1-CD4+ and Tc2-CD8+, as determined by cloning techniques, RTPCR and double immunofluorescence staining. Several investigators have described the impact of the various cytokines of T-cell subsets on the evolution of peritonitis in CAPD. Watson et al. reported significant and persistent IL-4 mRNA expression in recurrent peritonitis, but not in transient, resolving peritonitis [18]. Lu et al. found a persistent and prolonged increase in concentrations of IL-10 in persistently cloudy dialysate [19]. Shadrin et al. showed that enhancing Th1-cytokine in murine peritonitis, by the administration of recombinant IL-2, significantly prolonged the survival of the animals [20]. These observations suggest that the type 1 and type 2 patterns of T-cell cytokine expression play a role in the response to therapy of peritonitis complicating CAPD.
During peritonitis, denudation of the mesothelial surface and separation of the mesothelial cells have been noted. The normal repair process consists of re-mesothelialization. However, pathological conditions, such as delayed healing, prolonged or persistent inflammation, repeated tissue injury and defects in TGF-ß1 regulation, promote disorderly repair and contribute to peritoneal fibrosis and dialysis failure. TGF-ß1 has been implicated as a critical regulatory molecule in the development of fibrosis [9]. The roles that TGF-ß1 plays in the pathogenesis of peritoneal fibrosis may include induction of protease inhibitor synthesis and inhibition of ECM degradation protease synthesis. In a previous study, we demonstrated that TGF-ß1 was detectable during peritonitis in all dialysate samples, whether patients had high or low rates of recurrence of peritonitis [11]. That one of the cellular sources of the expression of the TGF-ß1 gene was peritoneal mesothelial cells was demonstrated by in situ hybridization using a 3H-labelled TGF-ß1 cDNA probe [11]. In the present study, we observed two patterns of TGF-ß1 expression during peritonitis. In the pattern 1 group, cell counts in the dialysate decreased rapidly during treatment, and the production of TGF-ß1 stopped after 78 days of illness. In contrast, in the pattern 2 group, cell counts in PDE remained high over 1014 days despite appropriate treatment. The persistent inflammatory response stimulated peritoneal mesothelial or other cells to produce TGF-ß1 and caused its prolonged expression into the peritoneal cavity for >2 weeks. These pattern 2 patients with persistent TGF-ß1 expression also had deteriorating PET during follow-up.
In the pattern 1 group, the gene expression and production of TGF-ß1 were transient and disappeared after a single episode of inflammation. Therefore, TGF-ß1 is beneficial in the process of post-inflammatory repair of the peritoneal membrane and prevents the development of peritoneal fibrosis. These results were consistent with the PET results. On the other hand, in the majority of pattern 2 patients, persistently elevated cell counts in the PDE and prolonged inflammation contributed to the persistent expression of TGF-ß1, which leads to peritoneal fibrosis and the deterioration of PET. These observations suggest that a persistent peritoneal inflammation may prolong the expression of TGF-ß1 genes, cause fibroblast proliferation and ECM accumulation, and perpetrate peritoneal fibrosis.
This study brings to light new and important information. First, the pattern of the CD4/CD8 ratio may be related to outcome in CAPD peritonitis. Secondly, a progressively increasing CD4/CD8 ratio during peritonitis may predict a favourable outcome, while a decreasing ratio may be a sign of unfavourable clinical evolution. The clinical outcome may be predicted early by measuring the peritoneal T-cell CD4/CD8 ratio. Thirdly, the prolonged expression of TGF-ß1 within the peritoneum may compromise the efficiency of dialysis and lead to peritoneal fibrosis in delayed response peritonitis. Longitudinal and cross-sectional studies have both suggested that a single or repetitive episodes of peritonitis may predispose to the loss of ultrafiltration [3] or the development of peritoneal fibrosis [20]. The link between initial inflammation and subsequent peritoneal fibrosis remains an important issue. Based on our observations, attempts to induce a progressive increase in CD4/CD8 ratio in PDE in the early stages of peritonitis may be warranted. This could be achieved by administering IL-12 or gene therapy to patients whose CD4/CD8 ratio and pattern of T-cell subsets might signal an unfavourable clinical evolution.
In conclusion, the results of this study suggest that a progressively decreasing CD4/CD8 ratio in PDE correlates with the persistent expression of TGF-ß1 in the dialysate, which may play a pathogenetic role in the outcome of peritonitis, PET deterioration and peritoneal fibrosis. Therefore, the pattern of the CD4/CD8 T-cell ratio in PDE may determine the outcome of peritonitis in CAPD patients. Future studies will concentrate on modulating the immune response during peritonitis in order to improve outcome and prevent the development of peritoneal fibrosis.
Conflict of interest statement. None declared.
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Notes |
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References |
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