Impairment of innate cellular response to in vitro stimuli in patients on continuous ambulatory peritoneal dialysis

Minoru Ando1, Asuka Shibuya1, Masako Yasuda2, Naoko Azuma2, Ken Tsuchiya2, Takashi Akiba2 and Kousaku Nitta2

1 Division of Nephrology, Tokyo Metropolitan Komagome Hospital and 2 Department of Medicine, Kidney Center, Tokyo Women's Medical University, Tokyo, Japan

Correspondence and offprint requests to: Minoru Ando, MD, Division of Nephrology, Tokyo Metropolitan Komagome Hospital, 3-18-22, Honkomagome, Bunkyo-Ku, Tokyo, 113-8677, Japan. Email: nephrol{at}cick.jp



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Most crucial in the initial stages of host defence against invading micro-organisms is innate immunity, in which peripheral mononuclear cells, in particular cytokines, are pivotal. Mortality from infections is high in dialysis patients, but it remains unclear if this arises from the ineffectiveness of innate immune mechanisms.

Methods. In 20 haemodialysis (HD) patients, 20 patients on continuous ambulatory peritoneal dialysis (CAPD), and 15 age-matched controls, we studied cytokine production by monocytes and helper T-cells in response to in vitro stimuli. The most important early-response cytokines for innate immunity, tumour necrosis factor (TNF)-{alpha} and interleukin (IL)-1ß, were tested in monocytes, and interferon-{gamma} and IL-4 were studied as indicators of polarization of helper T-cells into type 1 and type 2 cells. Peripheral blood cells stimulated with lipopolysaccharide or mitogen were labelled with anti-CD14+ and -CD4+ antibodies and then subjected to intracellular cytokine staining and flow cytometry.

Results. CAPD patients showed significantly reduced synthesis of TNF-{alpha} and IL-1ß and inhibited T helper phenotype development compared with HD patients and control subjects. In contrast, HD patients showed an unaltered monokine response and a marked polarization of helper T-cells towards the type 1 phenotype. We also found that a single HD treatment potentiated monocytes to synthesize TNF-{alpha}.

Conclusions. Circulating immune cells in CAPD patients may be hyporeactive against infections, indicating an unfavourable innate host defence.

Keywords: helper T-cell; IL-1ß; intracellular cytokine; monocyte; TNF-{alpha}



   Introduction
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Infections and their complications result in substantial morbidity and frequently in death among dialysis patients [1,2]. Accumulating data suggest that immune cellular dysfunction may actually be responsible for the high incidence of infections in these patients [3,4]. In particular, the innate immune system, the immediate activation of which is vital in inhibiting infections, may be defensively most crucial. Peripheral mononuclear cells play the most important role in innate immunity, since they initiate and orchestrate the innate immune response, primarily by producing cytokines. These cells are also responsible for activating the adaptive immune responses that gradually take over from the innate response [5,6]. However, cytokines and the innate immune responses have not been fully studied in dialysis patients.

It is becoming increasingly clear that the full expression of the innate host response requires the induction of early-response cytokines such as interleukin-1 (IL-1) and tumour necrosis factor-{alpha} (TNF-{alpha}). Once expressed, these cytokines activate other immune cells and amplify the immune response, thereby building up a force that attacks invading micro-organisms. The discovery of helper T-cell subsets—namely, type 1 (Th1) and type 2 (Th2)—has helped us to understand the mechanisms by which the innate immune response transits into an adaptive immune response. The development of an appropriate helper T-cell subset during infection is considered important. In particular, Th1 cytokines are believed to activate macrophages and neutrophils and to be involved in other cell-mediated immune responses. In contrast, Th2 cytokines tend to inhibit innate host defences, and instead promote humoral immunity [6,7].

We wondered if innate immunity works appropriately in dialysis patients when pathogens invade them. Most of the old studies that have tested serum cytokine levels have supported the hypothesis that monocytes are chronically activated in haemodialysis (HD) patients [8]; however, patients currently on HD, most of whom are not dialyzed with either complement-activating membranes or with LPS-contaminated dialysate, do not necessarily show evidence of overproduction of proinflammatory cytokines [9]. In addition, some authors have reported an impaired endotoxin-induced cytokine release from peripheral mononuclear cells of HD patients [10]. Consequently, we have attempted to assess cellular cytokine synthesis in response to in vitro stimulation, using the method that combines intracellular cytokine staining and flow cytometric analysis [11,12]. In this study, we focused on the synthesis of TNF-{alpha} and IL-1ß by peripheral monocytes and the polarization of helper T-cells into phenotypes in response to stimulation.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Subjects
The study included 20 patients on HD, 20 chronic ambulatory peritoneal dialysis (CAPD) patients and 15 normal controls, all matched for age. HD was performed three times a week for 4 h per treatment using conventional bicarbonate-buffered dialysate and regular heparin. The endotoxin concentration of the dialysates was measured by the limulus amebocyte lysate tests (Endospect® ES test; Seikagaku Corporation, Tokyo, Japan), and it was <1.0 EU/l. The HD patients were dialyzed using polysulfone (PS) and polymethylmethacrylate (PMMA) membranes. All CAPD patients were given daily exchanges of 8–10 l of peritoneal dialysate solution (Dianeal®; Baxter, Tokyo, Japan). The clinical profiles of the subjects are summarized in Table 1. Several drugs that can influence cytokine expression, such as steroids, statins and vitamins D, had been discontinued in the patients at least 1 month before the study. The chronic renal failure of the HD and CAPD patients was due to the following diseases: chronic glomerulonephritis (n = 20), nephrosclerosis (n = 12), polycystic kidney diseases (n = 3), unknown (n = 5). Any patient that had renal failure due to collagen diseases or diabetes mellitus and patients with hepatitis C virus RNA were excluded from the study, since the immune cells of such patients are considered to be functionally abnormal. The study was approved by the institutional review board of the hospital, and was conducted in accordance with the principles of the Declaration of Helsinki. Informed consent was obtained from all subjects.


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Table 1. Subjects

 
Reagents and antibodies
Brefeldin-A, lipopolysaccharide (LPS), phorbol 12-myristate 13 acetate (PMA) and ionomycin were purchased from Sigma Chemicals (Tokyo, Japan). The following reagents were purchased from Becton Dickinson (Tokyo, Japan): FACS lysing solution®, Permeabilizing solution®, FITC-conjugated anti-cytokine mAbs to IFN-{gamma} (anti-human IFN-{gamma} FITC) and TNF-{alpha} (anti-human TNF-{alpha} FITC), PE-conjugated anti-cytokine mAbs to IL-1ß (anti-Human IL1ß PE) and IL-4 (anti-human IL4 PE), isotype-matched Abs of irrelevant specificities (FITC- or PE-labelled mouse IgG), and 1% paraformaldehyde (Cell FixTM). PC5-conjugated monoclonal antibodies (mAbs) to CD4+ or CD14+ were purchased from Immunotech (Tokyo, Japan). RPMI 1640 medium, phosphate-buffered saline (PBS), and bovine serum albumin (BSA) were purchased from GIBCO (Tokyo, Japan).

Blood collection and mononuclear cell labelling
Blood samples were collected from HD patients before the first HD of the week and from CAPD patients when they visited the hospital for their regular medical examination. To measure the stimulation-induced cytokine production of these patients, we employed an intracellular cytokine staining technique, which uses the FASTIMMUNE Assay System® kit (Becton Dickinson) and is based on the method described by Picker et al. [11]. In brief, 1 ml of peripheral blood was diluted with 1 ml of RPMI 1640 medium and then incubated with 15 µg/ml of brefeldin-A, which disrupts intracellular Golgi-mediated transport and allows cytokines to accumulate, yielding an enhanced cytokine signal. Thereafter, 0.1 µg/ml of LPS was added to activate the monocytes or 40 ng/ml of PMA plus 4 µg/ml of ionomycin was added to activate the lymphocytes. The blood was then incubated for 4 h at 37°C in a CO2 incubator (5% CO2). Next, 500 µl of the activated blood was labelled with anti-CD4+ or CD14+ Abs over 15 min at room temperature.

Permeabilization and intracellular staining
FACS lysing solution® (4 ml) was added to the activated, cell-labelled blood and incubated for 10 min. The sample was then washed twice with PBS/0.1% BSA and the supernatant was removed. The Permeabilizing solution® (500 µl) was added next, and the specimen was left for 10 min in the dark. After they were washed twice, the cell pellets, suspended in 50 µl of RPMI medium, were incubated with the fluorescent anti-cytokine mAbs in tubes for 30 min in the dark. After separating the cells by centrifugation, the supernatants were removed and 500 µl PBS/1% paraformaldehyde was added. The samples were promptly analysed with a flow cytometer. The cytokines that had accumulated in the cells, namely, IL-1ß, TNF-{alpha}, IFN-{gamma} and IL-4, were identified by a specific mAb. Th1 cells were defined as IFN-{gamma}-positive and IL-4-negative CD4+ cells, while Th2 cells were defined as IFN-{gamma}-negative and IL-4-positive CD4+ cells. Isotype control mAbs of irrelevant specificities were added at matching concentrations in control samples to detect the non-specific binding of the mAbs to the cells.

Intracellular cytokine analysis by flow cytometry
The amounts of stimulants and the optimal stimulation time for cytokine synthesis had been determined by kinetic studies, using time-tracked cell cultures in the preliminary study. The samples were subjected to a flow cytometer (EPICS XL/MCL System II; Beckman Coulter, CA, USA) and analyzed with EXPO 32 (Beckman Coulter). The light scatter and fluorescence channels were set at logarithmic gains. First, only cells that were positive for CD14+ (monocytes) or CD4+ (helper T-cells) were gated (R1) on the SSC-FL3 plot. Each analysis involved 10 000 monocytes or helper T-cells within the gate. The cells labelled with specific anti-cytokine mAbs were profiled on the FL1-FL2 plot and compared with those in the samples that had been treated with non-specific mAbs (negative controls). Cutoff markers were set individually for each measurement on the basis of values in negative controls. The percentage of cells that were positive for each cytokine antibody was calculated on the quadrant diagram to assess the frequency of cells producing multiple cytokines, as previously described [11,12]. All experiments were performed in duplicate, and the mean values of data were used for analysis. Representative figures showing the analysis of the monocytes and helper T-cells from a healthy subject are shown in Figure 1.



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Fig. 1. Flow cytometric analysis of intracellular cytokine synthesis following in vitro stimulation of monocytes by LPS. Representative data in a healthy subject are shown. Cells positive for anti-CD14+ were gated as R1 on the diagram of SSC-FL3. Targeted for cytokine analysis were 10 000 monocytes within the R1 gate (left panel: A). The cells labelled with specific anti-TNF-{alpha} or anti-IL1ß mAbs were profiled on the diagram of FL1-FL2. Cutoff markers were set individually for each measurement using a negative control (right panel: B). The percentages of cells positive for each cytokine antibody (% positive cells) were calculated on the quadrant diagram to establish the frequency of different cytokine-producing cells. Light scatter properties were set on a logarithmic scale. The Th1 and Th2 polarization of helper T-cells after stimulation by PMA and ionomycin were analyzed by the slightly modified method.

 
Statistical analysis
The data are expressed as mean±SD unless otherwise stated. Statistical analysis was performed using the Kruskal–Wallis test to compare data between the three study groups and the Wilcoxon signed rank test to compare data before and after a single HD treatment. P<0.05 was considered to be statistically significant.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Laboratory data
The laboratory data of the subjects are shown in Table 2. The three groups did not differ from each other with respect to their total white blood cell, monocyte and lymphocyte counts. Also, no significant differences were observed between the dialysis groups with regard to serum parameters that are related to renal function (urea nitrogen and creatinine levels), nutrition (total protein, albumin, and lipids levels), and inflammation (erythrocyte sedimentation rate and C-reactive protein levels).


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Table 2. Laboratory data

 
Intracellular cytokine expression by monocytes in response to LPS stimulation
We measured the frequency of cells capable of producing different cytokines in response to LPS stimulation. It was difficult to determine the intracellular expression of cytokines without a stimulation step. Therefore, our method could not discriminate the ‘resting state’ of cytokine synthesis even between uraemic and control subjects, and it could not determine if uraemia activates cellular cytokine synthesis. The intracellular synthesis of IL-1ß and TNF-{alpha} by patient and control monocytes in response to LPS is shown in Figure 2. CAPD patients showed reduced synthesis of both IL-1ß and TNF-{alpha}, compared with the HD patients and the controls. In contrast, no significant differences were observed between the HD patients and controls with regard to either of the cytokines. Multiple linear regression analyses were performed to examine the influence of the laboratory parameters, including Kt/V, on intracellular cytokine responses in dialysis patients. Those laboratory parameters were not significantly correlated with in vitro cytokine responses (data not shown).



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Fig. 2. Intracellular cytokine synthesis following in vitro LPS stimulation of monocytes from HD (n = 20), CAPD (n = 20) and controls (n = 15). Data of TNF-{alpha} and IL-1ß are shown in (A) and (B), respectively. Intracellular cytokine synthesis was assessed by calculating the percentages of cells positive for each cytokine antibody (% positive cells). Data are shown as mean and standard deviation. P-values are for comparisons between the HD patients, the CAPD patients and the controls. *P<0.05, CAPD or HD vs controls; **P<0.01, CAPD or HD vs controls; and ##P<0.01, CAPD vs HD patients.

 
Intracellular synthesis of IFN-{gamma} and IL-4 by helper T-cells in response to mitogen
The data for the intracellular synthesis of IFN-{gamma} and IL-4 by helper T-cells are shown in Figure 3. Compared with CAPD patients and controls, HD patients showed a significantly stronger polarization of helper T-cells towards the Th1 phenotype, i.e., they had an elevated Th1:Th2 ratio. In contrast, the polarization of helper T-cells in response to mitogenic stimulation was blunted in the CAPD patients compared with the controls, resulting in a significant decrease in both Th1 cells and Th2 cells. Th1 cytokine levels in the CAPD patients were also lower than those in the HD patients. The Th1:Th2 ratio of the CAPD patients was not altered compared with the controls, but it was lower than that of the HD patients.



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Fig. 3. Polarization of helper T-cells on in vitro mitogen stimulation in HD (n = 20), CAPD (n = 20) and controls (n = 15). Data for Th1 and Th2 and the Th1:Th2 ratio are shown in (A), (B) and (C), respectively. Intracellular syntheses of IFN-{gamma} and IL-4 were assessed to differentiate helper T-cell subsets (Th1 and Th2); results are given as the percentage of cells positive for each cytokine antibody (% positive cells). Data are shown as mean and standard deviation. P-values are for comparisons between the HD patients, the CAPD patients and the controls. *P<0.05, CAPD or HD vs controls; **P<0.01, CAPD or HD vs controls; #P<0.05, CAPD vs HD patients; and ##P<0.01, CAPD vs HD patients.

 
Influence of a single HD treatment on the intracellular cytokine response
The data for the influence of a single HD treatment on the intracellular cytokine response are shown in Table 3. The experiment was performed before and after a single HD treatment only in the HD patients (n = 20), using the same biocompatible dialysis membranes (such as PS and PMMA) used for their regular HD. Blood was sampled before and soon after a 4 h HD. The intracellular monokine responses to LPS appeared to increase after a single HD treatment, but these differences were only statistically significant with regard to the synthesis of TNF-{alpha} (P<0.05). In contrast, the synthesis of IFN-{gamma} and IL-4 and the Th1:Th2 ratio were not changed after a single HD treatment.


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Table 3. Comparison of stimulated cytokine synthesis before and after HD treatment

 


   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
We found that, compared with HD patients and control subjects, monocytes derived from CAPD patients were hyporeactive to in vitro LPS stimulation. Moreover, the development of the helper T-cells into both subsets was significantly reduced in CAPD patients. These findings may suggest that the innate and adaptive immune responses of CAPD patients are blunted. In contrast, the HD patients had normal monokine responses, but they showed elevated Th1 responses and a significantly higher Th1:Th2 ratio compared with the CAPD patients and control subjects. In addition, in HD patients a single HD treatment provoked monocytes to synthesize TNF-{alpha} in response to stimuli, but it did not influence polarization of helper T-cells.

We found that the monocytes from the CAPD patients had an impaired cytokine response to LPS. It should be noticed that, in exerting their major physiologic and pathophysiologic effects, cytokines act as autocrine or paracrine entities. Therefore, the alteration of intracellular processing and local release of a cytokine may be more relevant to the pathogenesis of immune dysfunction in uraemia than the increased circulating levels of cytokines [13]. Intracellular cytokine staining is a better method than the conventional ones for observing the cellular potential for producing cytokines in response to stimulation. Our data may imply that the monocytes of CAPD patients are ‘exhausted’, possibly due to chronic uraemic activation or that they are refractory to pathologic stimulation. Insufficient or delayed cytokine response may blunt the immune reaction and increase the risk of infection in CAPD patients.

We confirmed that HD patients show a Th1 predominance, as the previous reports described [14,15] at the same time we showed that the T-cells of the CAPD patients were hyporeactive against stimulation. Among Th1 cytokines, IFN-{gamma} has a pivotal position in innate immune responses, since it may orchestrate a number of biological events that are related to the development of the innate and adaptive host responses and the transition from one to the other [6,7]. Thus, elevated Th1 levels and the consequent higher Th1:Th2 ratio in HD patients may protect these patients from infection, as long as the imbalance does not become too overwhelming. The decrease of Th1 cells in CAPD patients is not conducive to a strong innate immune response. Our data on the CAPD patients also may indicate that their humoral responses and antibody production are impaired, since they show a lower capacity to produce Th2 cells. Nevertheless, all previous studies and our present one have been unable to determine why dialysis patients have imbalanced helper T-cell functions. Notably, recent work has suggested that elevated levels of ß2-microglobulin may retard the differentiation of monocytes into dendritic cells, and that this compromises the Th1 cell response [16]. It will be of interest to determine whether elevated ß2-microglobulin levels are responsible for the impaired responsiveness of not only T-cells but also of monocytes in chronically uraemic patients.

To test the direct impact of dialysis modality on cellular cytokine response, we examined the change of the in vitro stimulation-induced cellular cytokine response before and after a single HD therapy. The polarization of the helper T-cells was not influenced by a single HD treatment at all, but monokine response appeared to be elevated, particularly in TNF-{alpha}. This suggests that HD in part potentiates the cellular cytokine synthesis response. It may be that even lesser degrees of cell activation, by contact with biocompatible membranes, may persist between haemodialysis sessions, and that it may elevate the cytokine response of HD patients to levels seen in the controls.

Our in vitro cytokine data suggest that CAPD patients may be more susceptible to infections than HD patients. Possibly supporting this are analyses of the old United States Renal Data System (USRDS), which have shown that mortality, in general, and deaths due to infections, in particular, are significantly higher in CAPD patients than in HD patients [17]. Nevertheless, the current USRDS has equivocal data. While they show that hospital days per patient-years at risk, adjusted all-cause mortality of long-surviving patients, and adjusted infection mortality are higher in CAPD patients than in HD patients, they also reveal that both admissions for pneumonia per patient-years at risk and adjusted hospitalization rates for septicaemia are lower in CAPD patients than in HD patients [1]. Specific host defences in CAPD—for instance, opsonisation via specific IgG antibodies—may be more important clinically than the reduced cellular cytokine response [18]. Other factors, such as racial and genetic differences, may account for the discrepancy between our in vitro results and the US data regarding a risk of infection. Additional clinical data, such as the correlation between infections and the in vitro cytokine response in a larger study population or longitudinal measurements will be needed to evaluate the in vivo significance of our in vitro data.

In conclusion, CAPD patients may have defects in their cytokine responses to invasive pathogens, leading to a disabled innate immune system. HD may serve as a partial aid to compensate for the hyporeactivity in cytokine synthesis. Our findings suggest that the dialysis modality may affect the cellular function associated with innate host defence.



   Acknowledgments
 
We sincerely thank Dr Yoko Ando in the Clinic of the Kantoh-Shinetsu National Tax Administration, Japan, for her help in collecting blood samples from healthy subjects. We also thank Drs Bengt Lindholm and Ernesto Rodriguez Ayala in the Karolinska University Hospital, Huddinge, Sweden for their valuable help.

Conflict of interest statement. None declared.



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 17.12.04
Accepted in revised form: 8. 6.05





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