Quantitative flow cytometry shows activation of the TNF-{alpha} system but not of the IL-2 system at the single cell level in renal replacement therapy

Iza C. van Riemsdijk-van Overbeeke, Carla C. Baan, Christiaan J. Knoop, Elisabeth H. M. Loonen, Robert Zietse and Willem Weimar

Department of Internal Medicine, University Hospital Rotterdam-Dijkzigt, The Netherlands



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Immunological dysfunction in patients on haemodialysis may be related to imbalanced cytokine systems, such as tumour necrosis factor (TNF)-{alpha} and interleukin (IL)-2. Despite activation of these systems, haemodialysis patients show high susceptibility for infections and malignancies, and have a poor immunological reaction to T-cell-dependent antigens, like hepatitis B vaccination. In this study we have determined the activation status of the two different cytokine systems, at the single cell level, using quantitative flow cytometry.

Methods. Using fluorescein isothiocyanate- or phycoerythrin-conjugated antibodies directed against TNF-R2 (CD120b), IL-2R{alpha} (CD25) and IL-2Rß (CD122), we measured the expression of these receptors at the single cell level in order to determine the level of activation of monocytes and T-lymphocytes.

Results. Significantly higher expression of the TNF-{alpha} receptor, TNF-R2, was present on both monocytes and T-lymphocytes in patients on renal replacement therapy (RRT) compared with pre-dialysis chronic renal failure (CRF) patients and controls, indicating activation of the TNF-{alpha} system. In contrast, IL-2R expression was comparable in all groups studied, which may reflect a non-activated state of the IL-2 system.

Conclusions. The present study illustrates an activated state of the TNF-{alpha} system in patients on RRT, at the single cell level, while the IL-2 system seems to be unaffected. These findings support the hypothesis that the interaction between the TNF-{alpha} and IL-2 cytokine systems is disturbed.

Keywords: activation; chronic renal failure; IL-2; quantitative flow cytometry; renal replacement therapy; TNF-{alpha}



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Patients with chronic renal failure (CRF) and on renal replacement therapy (RRT) suffer from a high susceptibility to infections and show a higher incidence of malignancies compared with healthy controls [1]. This immune incompetence may be due to imbalanced defence mechanisms in which cytokines derived from antigen-presenting cells (APC) and T-cells play a central role. Kimmel et al. reported that the immunological dysfunction in patients on haemodialysis (HD) is related to overall survival [2]. In patients with ESRF, on peritoneal dialysis (CAPD) or haemodialysis (HD), various cytokine systems are affected [35]. TNF-{alpha} is a pro-inflammatory cytokine which induces expression of MHC class I molecules, and activates the production of enzymes and adhesion molecules. It can induce programmed cell-death and is needed for T-cell proliferation. It is mainly produced by activated monocytes and macrophages and, to a lesser extent, by lymphocytes [6]; therefore it provides a pivotal role in the function of APC, whereas IL-2 is regarded as a central T-cell cytokine that promotes expansion of T-cells, augments the cytolytic activity of NK cells, and is involved in programmed cell death of activated T-cells and in the synthesis of immune globulins by B-cells [7]. Active TNF-{alpha} is unstable, and inadequate collection and processing of blood samples may result in undetectable circulating cytokines, while also the IL-2 protein measurements are influenced by mode of collection [8]. Measurements of free plasma TNF-{alpha} can be strongly influenced by the above-mentioned factors. In addition, variable results of TNF protein measurements using commercially available ELISA kits complicate comparisons between studies [9,10]. In contrast, measurements of the expression of membrane-bound activation markers by flow cytometry provide an elegant, reproducable and sensitive tool to determine the activated state of cytokine systems at the single cell level. In this study, we used flow cytometry to quantitate the expression of activation markers of both the TNF-{alpha} and IL-2 system: the TNF-R2 (CD120b) on lymphocytes and monocytes, and the IL-2R{alpha} (CD25) and IL-2Rß (CD122) chains on {alpha}/ß T-cell receptor (TCR)-positive T-cells.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Patients
In 11 patients (three males, eight females; mean age 45.6±19 years) with pre-dialysis CRF, eight (seven males, one female; mean age 44.5±14 years) on CAPD, and 12 (six males, six females; mean age 57.8±15 years) on HD, the activation markers of the TNF-{alpha} and IL-2 system were determined on peripheral T-lymphocytes and monocytes using quantitative flow cytometry. Mean serum creatinine was 552 µmol/l (CRF), 1031 µmol/l (CAPD) and 995 µmol/l (HD). The patients with pre-dialysis CRF had a mean creatinine clearance of 13±2.5 ml/min. In patients on RRT, the Kt/V was routinely determined using specific software (Baxter), in which the volume (V) is calculated according to the formula of Watson (in relation to length, gender, body weight and age). In HD patients and in those on CAPD, a Kt/V of 1.3±0.2 and 1.5±0.2 was obtained, respectively. Mean time on dialysis was 27.4±8.3 months for CAPD patients and 26.1±5.5 months for HD patients. The dialyser membranes used in the study were Polysulphone (F60 Fresenius, AG, Bad Homburg, Germany) and Hemophane (MA-12H, Kawasumi Laboratory Inc., Minamiohi, Tokyo, Japan). Both dialysers are known for their better bio-compatibility than cuprophane membranes [11]. Previously we have shown that patients dialysed with either of the two membranes showed no differences in activation parameters of the TNF-{alpha} system [12]. The HD patients were on bicarbonate dialysate. The dialysate is routinely cultured and no periods of contamination were found (<103 micro organisms per millilitre). The patients on CAPD used the Baxter twinbag system (2 l, four times daily). Four weeks before and during, and at least 4 weeks after blood collection the patients on CAPD showed no signs of peritonitis. Blood samples were collected during the infection-free period. In HD patients, blood samples were collected before the start of the haemodialysis procedure. At the time of blood sample collection no patients were known to have a malignancy. In CRF and CAPD patients, blood samples were collected during routine outpatient visits. Causes of renal failure were hypertension (n=10), membranous glomerulonephritis (n=8), Wegener's granulomatosis (n=2), focal segmental glomerulosclerosis (n=2), polycystic kidney disease (n=4), IgA nephropathy (n=1), amyloidosis (n=1) and unknown (n=3). Most patients used anti-hypertensive drugs, i.e. calcium-entry blockers, angiotensin-converting-enzyme (ACE)-inhibitors or ß-blockers. Subcutaneous recombinant erytropoietine treatment was started in all patients with haemoglobin levels of <6 mmol/l (9.7 g/dl). None of the patients used corticosteroids. Amongst the patients and controls there were no diabetics. All data were compared with those of nine healthy controls, who were not on medication (four males, five females; mean age 35.6 years; mean creatinine <100 µmol/l). Patient characteristics are summarized in Table 1Go.


View this table:
[in this window]
[in a new window]
 
Table 1. Patient characteristics

 

Sample preparation and flow cytometric analysis
Blood samples were collected in pyrogen-free tubes, containing EDTA in a final concentration of 1 mg/ml. Whole blood EDTA samples were monitored for the presence of the immune-competent cells: monocytes (CD14+) and lymphocytes ({alpha}/ß-TCR-positive). Surface activation markers were analysed by two-colour flow cytometry after staining with monoclonal antibodies directed against CD14 (Immunotech, Marseille, France) as a marker for the monocytes, and WT31 (Becton Dickinson, Mountain View, CA, USA) as a marker for the {alpha}/ß chain of the TCR. In these subsets, CD25 (IL-2R{alpha}, Becton Dickinson), CD122 (IL-2Rß, Becton Dickinson) and CD120b (TNF-R2, Immunotech) were monitored. The antibodies, except CD120b, were directly conjugated to fluorescein isothiocyanate or phycoerythrin (PE). For CD120b we used a two-step staining. After the first step with CD120b, cells were incubated with F(ab)2 goat-anti-Rat IgG PE. The staining procedure was performed by incubating 15 µl of 1/100 diluted CD120b antibody with 100 µl blood (30 min at 4°C). After washing in Hanks Balanced Salt Solution (HBSS, Gibco-BRL, Paisly, UK) with 0.1% bovine serum albumin (BSA, Sigma, St Louis, MO, USA) and 0.01% sodium azide (Merck, Darmstadt, Germany), the red blood cells were lysed by FACS Lysing Solution (Becton Dickinson). Samples were centrifuged and washed in Cell Pack (TOA, Hamburg, Germany). Flow cytometric analysis was performed on FACscan flow cytometer using Cell Quest software (Becton Dickinson). From each tube, 10 000 events ({alpha}/ß-TCR-positive, CD14+) in the gate were measured. In order to compare the measurements in time, the flow cytometer was calibrated using specific calibration beads (Calibration Beads Quantum 1000, Flow cytometry Standards Corp. San Jose, Puerto Rico, USA). Each bead contains a known amount of fluorochrome. The intensity of the fluorescence is converted to a standard curve using Quick Cal program for Quantum Beads (Becton Dickinson). The mean fluorescence is denoted as the molecular equivalent of fluorochrome, or MESF (Figure 1Go).



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 1. Standard curve fluorescence intensity of calibration beads. Upper panel: flow cytometric analysis of calibration beads, each of which contains a specific amount of fluorescine. Lower panel: standard curve of fluorescence intensity constructed after flow cytometric analysis of calibration beads, using Quick Cal program for quantum beads software. The intensity of the fluorescence is depicted as MESF.

 
The relationship between serum creatinine and membrane receptor expression was analysed for the whole group (patients and controls, n=40) using the Spearman rank correlation. Correlation between time on dialysis (CAPD and HD) and membrane receptor expression was also evaluated by the Spearman rank correlation test.

Statistics
Data are recorded as mean±SEM, or median and range. Absolute numbers of lymphocytes and monocytes in the patient groups were compared with the unpaired student's t test. Differences in receptor expression between patient groups were analysed using one-way analysis of variance (ANOVA), while differences between groups, separately, were analysed using the Mann–Whitney test. The Spearman r correlation coefficients were used to determine the relationship between serum creatinine, time on dialysis and membrane receptor expression (IL-2R{alpha} (CD25), IL-2Rß (CD122) and TNF-R2 (CD120b) on monocytes and lymphocytes) and age. P values <=0.05 were considered significant.



   Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
In patients with CRF (not on dialysis) and on RRT, with a mean time on dialysis of 26.1±5.5 months (HD) and 27.4±8.3 months (CAPD), we measured cytokine activation markers (CD25, CD122, CD120b) on immune-competent cells. Absolute numbers of {alpha}/ß-TCR-positive T-cells were significantly lower in HD patients than in healthy controls: (665±88 vs 979±99 cells/µl, P=0.02). In patients on CAPD (673±116 cells/µl) and CRF (738±172 cells/µl), the absolute number of {alpha}/ß-TCR-positive T-cells was comparable to the number of cells found in the control group (P=0.07 and 0.22, respectively). The absolute number of monocytes was comparable between all groups: 314±34 cells/µl (controls), 284±29 cells/µl (CRF), 374±59 cells/µl (CAPD) and 293±56 cells/µl (HD).

Expression of the activation markers of the IL-2 system (i.e. CD25, CD122) was in the same range for patients and controls. The mean expression of IL-2R{alpha} (CD25) varied from 779±132 MESF (controls) to 992±163 MESF (CAPD), 762±49 MESF (CRF) and 763±104 MESF (HD) (P=0.51 by ANOVA). The mean expression of IL-2Rß (CD122) ranged from 365±14 MESF (controls), to 409±19 MESF (CRF), 401±18 MESF (CAPD) and 431±38 MESF (HD) (P=0.40 by ANOVA) (Figure 2Go). In contrast to these comparable expression levels of the activation markers of the IL-2 system on T-cells, the expression levels of the activation marker of the TNF-{alpha} system TNF-R2 (CD120b) were significantly higher in patients on RRT, on both monocytes and on {alpha}/ß-TCR-positive lymphocytes (P=0.02 and 0.03, respectively, by ANOVA). The mean TNF-R2 (CD120b) expression on lymphocytes was 3153±508 MESF (controls) vs 3451±288 MESF (CRF) (P=0.65) CAPD: 5670± 997 MESF (P=0.06 vs controls), and in HD: 5466±893 MESF (P=0.01 vs controls). Mean TNF-R2 (CD120b) expression on monocytes was also higher in patients on CAPD and HD, but not in CRF compared with controls. MESF was 7285±1516 (CAPD) vs 2564±808 (controls) (P=0.005). MESF was 10233±3531 (HD) (P=0.02 by the Mann–Whitney test) (Figure 3Go).



View larger version (46K):
[in this window]
[in a new window]
 
Fig. 2. Expression of T-cell activation markers: CD25 (IL-2R{alpha} chain) and CD 122 (IL-2Rß chain) in patients with end-stage renal failure (ESRF), on peritoneal dialysis (CAPD) and haemodialysis (HD), measured with quantitative flow cytometry in peripheral blood. The quantity of fluorescence is depicted as MESF. Error bars indicate the SEM.

 


View larger version (37K):
[in this window]
[in a new window]
 
Fig. 3. Expression of the TNF-{alpha} activation marker: CD 120b (TNF-R2) on lymphocytes and monocytes in patients with end-stage renal failure (ESRF), on peritoneal dialysis (CAPD) and haemodialysis (HD) compared with healthy controls, measured by quantitative flow cytometry. The quantity of fluorescence is depicted as MESF. Error bars indicate the SEM.

 
Of the studied cytokine activation markers (CD25, CD122, CD120b on lymphocytes and monocytes), only CD120b on monocytes showed a correlation with serum creatinine. The r coefficient for CD25 was 0.08 (P=0.62), for CD122 it was r=0.16 (P=0.31), for CD120b on lymphocytes is was r=0.16 (P=0.31), and for CD120b on monocytes it was r=0.38 (P=0.03) according to the Spearman rank correlation. Except for CD120b expression on monocytes, we found no correlation between age and cytokine receptor expression: for CD25, r=-0.16 (P=0.32); for CD122: r=0.13 (P=0.42); for CD120 on lymphocytes, r=0.24 (P=0.16); and for CD120b on monocytes, r=0.43 (P=0.01), using the Spearman rank correlation. Also, time on dialysis was not correlated with the expression of the membrane receptors: r=0.12, P=0.68, using the Spearman rank correlation.



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
In the present study, we used quantitative flow cytometry to evaluate the expression of activation markers of the TNF-{alpha} and the IL-2 system in patients with CRF and on RRT. We determined the receptor expression for TNF-{alpha} and IL-2 on immune-competent cells, {alpha}/ß-TCR-positive lymphocytes and the CD14+ macrophages in order to differentiate which cells, T-cell or APC, are activated. To evaluate the activation of the IL-2 system we measured the expression of IL-2R{alpha} (CD25) and IL-2Rß (CD122). In this study, we were predominantly interested in the expression of the TNF-R2, because TNF-{alpha} action directed by TNF-R2 results in proliferation of T-cells. We found reduced absolute numbers of lymphocytes in the presence of comparable absolute numbers of monocytes in patients on HD vs all other groups. It is known that activated lymphocytes are sequestered in the capillaries of the lung after blood dialyser membrane contact [13]. Deenitchina et al. reported that this lymphopenia was the consequence of low numbers of CD4+ T-cells [14]. We found an increased expression of the TNF-R2 on both monocytes and lymphocytes from patients on RRT, which suggests activation of the TNF-{alpha} system [15]. This coincides with the high levels of soluble receptors and the significantly higher levels of free plasma TNF-{alpha} in these patients [16]. In contrast, in patients with CRF, TNF-R2 expression was comparable to the expression measured on immune-competent cells from healthy controls. Although the groups studied are small, it appears that the TNF-{alpha} system is not activated in CRF patients. As the creatinine clearance in the patients with CRF in this study was 13 ml/min, which is comparable to the creatinine clearance obtained in patients on RRT, i.e. CAPD or HD, renal insufficiency alone cannot be responsible for the differences in expression of the TNF-{alpha} activation marker, TNF-R2, found in these patients. Therefore, we think that the increased TNF-R2 expression is not caused by renal insufficiency itself, but by RRT, i.e. CAPD or HD. Because of the comparable expression of TNF-R2 on lymphocytes and monocytes in patients on CAPD and HD, it seems that other factors than the RRT itself result in TNF-R2 expression on lymphocytes or monocytes. After TNF-{alpha} binding to its membrane receptor, the extracellular domains are split off and can be identified as soluble TNF-R in peripheral blood. Other causes of shedding the membrane receptor are poorly understood. The sTNF-R is predominantly metabolized by renal clearance. Due to impaired renal clearance, levels of sTNF-R show a strong positive correlation with serum creatinine and renal function [12,17]. In the present study we found no correlation between the expression of CD25, CD122 and CD120b on lymphocytes and monocytes with serum creatinine per patient group. Nor did we find a correlation between time on dialysis (CAPD or HD) and the expression of the membrane receptors. To assess a possible correlation between the expression of the TNF-R2 (CD120b) on monocytes and progressive renal insufficiency, longitudinal data are needed. Age may be another confounding factor in this study. In the literature, conflicting data are reported concerning cytokine levels and production in aged subjects. Higher [1822] as well as lower [20, 23, 24] levels and production capacity of IL-2 and TNF-{alpha} in aged subjects have been described. In the present study, the mean age of the HD patients, in contrast to the other patient groups, was significantly higher compared with the controls (P=0.01 using ANOVA). There was no difference in the mean age between the patient groups. This supports the hypothesis that the increased expression of the TNF-R2 on cells of CRF, CAPD and HD patients is not the result of age, but rather the result of renal insufficiency. Moreover, no correlation was found between age and the various membrane receptor expressions studied: P=0.31 (CD25), P=0.42 (CD122) and P=0.16 (CD120b) using the Spearman rank correlation. Various drugs are known to influence TNF-{alpha} levels. Recombinant erytropoietine and ACE-inhibitors may induce TNF-{alpha} [25, 26]. In this study, the patients were on various medication (see Table 1Go). No differences in the use of ACE-inhibitors or erytropoietine between the various patient groups is present. Therefore the influence of the medication is not regarded as a dominant factor influencing the results of the TNF-R2 expression.

Despite the high expression of TNF-R2 on lymphocytes, the T-cells seem not to be activated as shown by normal expression of the IL-2 activation markers on the cell membrane. The interaction between the TNF-{alpha} system and the T-cells appears to be inadequate, which may result in the high incidence of infection and malignancy found in HD patients. Previously, we found that the phytohaemagglutinin (PHA)-stimulated TNF-{alpha} production by peripheral blood mononuclear cells (PBMC) was significantly reduced in patients on HD, while PHA-stimulated IL-2 production was comparable to the production by PBMC obtained from healthy controls [16]. Others reported differences between CAPD and HD patients in the production of specific T-cell cytokines. In CAPD, the mitogen phorbol-12-myrisate-13-acetate (PMA)-stimulated T-cell production of TNF-{alpha} and IL-2 was lower compared with the production by T-cells obtained from HD patients [27]. We suggest this impaired TNF-{alpha} production results from ‘exhaustion’ due to continuous activation of the TNF-{alpha} system. In HD patients, this is explained by chronic blood–membrane contact, and in CAPD patients by chronic, low-grade, subclinical intra-peritoneal inflammation [2830]. TNF-{alpha} activities, conducted by TNF-R1, are responsible for signalling apoptosis, while TNF-{alpha} activity conducted by TNF-R2 is responsible for signalling proliferation of thymocytes and cytotoxic T-cells [15]. A distinct difference in action is, however, not overt. Monoclonal antibodies directed against TNF-R2 can partially antagonize the same TNF responses that are induced by TNF-R1. Tartaglia et al. reported that specific activation of TNF-R2, in the presence of PHA, results in stimulation of human T-cells [30]. The increased expression of the TNF-R2 may thus result in T-cell activation and proliferation in the presence of TNF-{alpha} and mitogen.

In summary, we found an activated TNF-{alpha} system shown by high expression of the TNF-R2 on lymphocytes and monocytes. In previous studies, increased mRNA expression for TNF-{alpha} and elevated plasma TNF-{alpha} protein levels have already been found [12]. The IL-2 system seems not to be activated, as shown by the comparable expression of the T-cell activation markers, IL-2R{alpha} (CD25) and IL-2Rß (CD122), in patients and controls. The immunoregulatory activity of TNF-{alpha}, which results in induction of IL-2R expression and enhanced T-cell responses directed by IL-2, failed to induce T-cell activation in the patient groups we studied.

The present study shows that quantitative flow cytometry provides additional information about the activation status of cytokine systems at the single cell level, and clarifies the nature of the immune-competent cells involved in the immune responses in patients with CRF and on RRT. Our results show that interaction between APC (TNF-{alpha}) and T-cells (IL-2) does not result in activation or proliferation of the T-cells in spite of the increased expression of TNF-R2 on the membrane. This may contribute to the immune deficiency in patients on intermittent haemodialysis.



   Notes
 
Correspondence and offprint requests to: Dr I. C. van Riemsdijk-van Overbeeke, Department of Internal Medicine, University Hospital Rotterdam-Dijkzigt, PO Box 2040, 3000 CA Rotterdam, The Netherlands. Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

  1. Maisonneuve P, Agodoa L, Gellert R et al. Cancer in patients on dialysis for end-stage renal disease: an international collaborative study. Lancet1999; 354: 93–99[ISI][Medline]
  2. Kimmel PL, Phillips TM, Simmens SJ et al. Immunologic function and survival in hemodialysis patients. Kidney Int1998; 54: 236–244[ISI][Medline]
  3. Pereira BJ, Shapiro L, King AJ, Falagas ME, Strom JA, Dinarello CA. Plasma levels of IL-1 beta, TNF alpha and their specific inhibitors in undialyzed chronic renal failure, CAPD and hemodialysis patients. Kidney Int1994; 45: 890–896[ISI][Medline]
  4. Descamps-Latscha B, Herbelin A, Nguyen AT et al. Balance between IL-1 beta, TNF-alpha, and their specific inhibitors in chronic renal failure and maintenance dialysis. Relationships with activation markers of T cells, B cells, and monocytes. J Immunol1995; 154: 882–892[Abstract/Free Full Text]
  5. Girndt M, Kohler H, Schiedhelm-Weick E, Schlaak JF, Meyer ZBK, Fleischer B. Production of interleukin-6, tumor necrosis factor alpha and interleukin-10 in vitro correlates with the clinical immune defect in chronic hemodialysis patients. Kidney Int1995; 47: 559–565[ISI][Medline]
  6. Bazzoni F, Beutler B. The tumor necrosis factor ligand and receptor families. N Engl J Med1996; 334: 1717–1725[Free Full Text]
  7. Kelly CJ. T cell function in chronic renal failure and dialysis. Blood Purif1994; 12: 36–41[ISI][Medline]
  8. Loonen EH, Baan CC, Van Riemsdijk-Van Overbeeke IC. Measurements of IL-2 and sIL-2 receptors in plasma and serum. submitted
  9. Meager A, Bird C, Mire-Sluis A. Assays for measuring soluble cellular adhesion molecules and soluble cytokine receptors. J Immunol Methods1996; 191: 97–112[ISI][Medline]
  10. Kreuzer Ka, Rockstroh J, Sauerbruch T, Sprengler U. A comparative study of different enzyme immunosorbent assays for human tumor necrosis factor {alpha}. J Immunol Methods1996; 195: 49–54[ISI][Medline]
  11. Grooteman MP, Nube MJ, Van Limbeek J, Van Houte AJ, Daha MR, Van Geelen JA. Biocompatibility and performance of a modified cellulosic and a synthetic high flux dialyzer. A randomized crossover comparison between cellulose triacetate and polysulphon. ASAIO J1995; 41: 215–220[Medline]
  12. Van Riemsdijk-Van Overbeeke IC, Baan CC, Hesse CJ et al. TNF-alpha: mRNA, plasma protein levels and soluble receptors in patients on chronic hemodialysis, on CAPD and with end-stage renal failure. Clin Nephrol2000; 53: 115–123[ISI][Medline]
  13. Dinarello CA. Cytokines: agents provocateurs in hemodialysis. Kidney Int1992; 41: 683–694[ISI][Medline]
  14. Deenitchina SS, Ando T, Okuda S et al. Cellular immunity in hemodialysis patients: a quantitative analysis of immune cell subsets by flow cytometry. Am J Nephrol1995; 15: 57–65[ISI][Medline]
  15. Scheurich P, Thoma B, Ucer U, Pfizenmaier K. Immunoregulatory activity of recombinant human tumor necrosis factor (TNF)-alpha: induction of TNF receptors on human T cells and TNF-alpha-mediated enhancement of T cell responses. J Immunol1987; 138: 1786–1790[Abstract/Free Full Text]
  16. Van Riemsdijk-Van Overbeeke IC, Baan CC, Loonen EHM et al. Tachyphylaxis for TNF-{alpha} by T-cells resulting from activation of the TNF-{alpha} system during hemodialysis. Kidney Int2001; 59: 883–892[ISI][Medline]
  17. Van Riemsdijk-Van Overbeeke IC, Baan CC, Niesters HG et al. The TNF-alpha system in heart failure and after heart transplantation: plasma protein levels, mRNA expression, soluble receptors and plasma buffer capacity. Eur Heart J1999; 20: 833–840[Abstract/Free Full Text]
  18. Venjatraman JT, Fernandes G. Exercise, immunity and aging. Aging (Milano)1997; 9: 42–56[ISI]
  19. Sakata-Kaneko S, Wakatsuki Y, Matsunaga Y, Usui T, Kita T. Altered Th1/Th2 commitment in human CD4+ T cells with ageing. Clin Exp Immunol2000; 120: 267–273[ISI][Medline]
  20. Rink L, Cakman I, Kirchner H: Altered cytokine production in the elderly. Mech Ageing Dev1998; 102: 199–209[ISI][Medline]
  21. Molteni M, Della BS, Mascagni B et al. Secretion of cytokines upon allogeneic stimulation: effect of aging. J Biol Regul Homeost Agents1994; 8: 41–47[ISI][Medline]
  22. Pedersen BK, Bruunsgaard H, Ostrowski K et al. Cytokines in aging and exercise. Int J Sports Med2000; 21 [Suppl 1]: S4–S9[ISI][Medline]
  23. Bruunsgaard H, Pedersen AN, Schroll M, Skinhoj P, Pedersen BK. Impaired production of proinflammatory cytokines in response to lipopolysaccharide (LPS) stimulation in elderly humans. Clin Exp Immunol1999; 118: 235–241[ISI][Medline]
  24. Gon Y, Hashimoto S, Hayashi S, Koura T, Matsumoto K, Horie T. Lower serum concentrations of cytokines in elderly patients with pneumonia and the impaired production of cytokines by peripheral blood monocytes in the elderly. Clin Exp Immunol1996; 106: 120–126[ISI][Medline]
  25. Bryl E, Mysliwska J, Debska-Slizien A et al. The influence of recombinant human erythropoietin on tumor necrosis factor alpha and interleukin-10 production by whole blood cell cultures in hemodialysis patients. Artif Organs1998; 22: 177–181[ISI][Medline]
  26. Stenvinkel P, Andersson P, Wang T et al. Do ACE-inhibitors suppress tumour necrosis factor-alpha production in advanced chronic renal failure? J Intern Med1999; 246: 503–507[ISI][Medline]
  27. Zamauskaite A, Perez-Cruz I, Yaqoob MM, Madrigal JA, Cohen SB. Effect of renal dialysis therapy modality on T cell cytokine production. Nephrol Dial Transplant1999; 14: 49–55[Abstract]
  28. Zaoui P, Green W, Hakim RM. Hemodialysis with cuprophane membrane modulates interleukin-2 receptor expression. Kidney Int1991; 39: 1020–1026[ISI][Medline]
  29. Hory B, Racadot E, Saint-Hillier Y, Peters A, Perol C. Soluble interleukin-2 receptors in chronic renal failure. Am J Nephrol1991; 11: 276–280[ISI][Medline]
  30. Tartaglia LA, Goeddel DV, Reynolds C et al. Stimulation of human T-cell proliferation by specific activation of the 75-kDa tumor necrosis factor receptor. J Immunol1993; 151: 4637–4641[Abstract/Free Full Text]
Received for publication: 28. 4.00
Revision received 9. 2.01.



This Article
Abstract
FREE Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (1)
Disclaimer
Request Permissions
Google Scholar
Articles by van Riemsdijk-van Overbeeke, I. C.
Articles by Weimar, W.
PubMed
PubMed Citation
Articles by van Riemsdijk-van Overbeeke, I. C.
Articles by Weimar, W.