1 Department of Histology and Immunology and
2 Department of Nephrology, Transplantology and Internal Diseases, Medical University of Gdask, Gda
sk, Poland
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Abstract |
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Methods. Expression of T-cell surface molecules was measured in 14 HD patients ex vivo by flow cytometry of lymphocytes sampled from peripheral blood and in vitro using whole blood cell cultures stimulated either with phytohaemagglutinin (PHA) or with physiological as well as non-physiological doses of rHuEpo. The concentrations of the cytokines were measured in the supernatants from non- or PHA-stimulated cultures using bioassays (IL2, IL6, TNF) or ELISA tests (IL10, IL12).
Results. Compared with findings before the start of rHuEpo therapy the CD4+/CD8+ ratio increased after 1 year of follow-up, whereas the percentage of CD152+ peripheral blood lymphocytes decreased. The increase of the CD4+/CD8+ ratio was dependent on a decrease of the percentage of CD8+ cells. The decrease of CD152+ population affected mainly CD8+CD152+ cells. All these effects became apparent after 6 months of rHuEpo treatment. In vitro stimulation of whole blood cultures revealed that the addition of PHA up-regulated the percentage of CD152+ lymphocytes, while physiological concentrations of rHuEpo decreased the percentage of CD8+152+ T cells. None of the stimuli used affected the percentage of CD8+ T cells. The pattern of the cytokines shifted toward TH1 phenotype (increase of IL2 and 12 levels) with a decreased level of proinflammatory cytokines (decrease of IL6 and TNF levels).
Conclusions. The observed decrease of CD152+ lymphocytes together with the decrease of CD8+ cells may reflect the improved immune response observed in HD patients under rHuEpo treatment.
Keywords: CD4; CD8; CD152; flow cytometry; haemodialysis; recombinant human erythropoietin
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Introduction |
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The aim of our study was to assess an expression of the inhibitory CD152 molecule on CD4+ and CD8+ T lymphocytes from the HD patients during 1-year-long rHuEpo therapy. The CD152 ligand, known also as The cytotoxic T lymphocyte antigen 4 (CTLA-4) is the molecule that cross-links CD80/CD86 receptors on antigen presenting cells (APC). This molecule can bind the CD80 molecule with 20 times as high avidity as that of the CD28 [13], causing down-modulation of T-cell responses. This effect is realized by an arrest of the cell cycle of T lymphocytes [14], an inhibition of the IL2-receptor expression [15], a reduction of the synthesis of IL2 and interferon from TH1 clones, and that of IL3, IL4, IL5, and IL10 from TH2 clones [16]. Additionally, the CD152 molecule is likely to play a critical role in regulating of peripheral T-cell tolerance [15]. Lack of its expression in the knockout mice model has been responsible for splenomegaly, a massive lymphoproliferation, and multi-organ immune-dependent destruction [17]. An expression of the CD152 molecule on peripheral lymphocytes seems to be non-constitutive. Most of the molecule is localized in intracellular stores and its presence on the cell surface becomes augmented on activated T- and B-lymphocytes [18]. An experimental use of the fusion protein known as CTLA4-Ig caused an efficient inhibition of the immune system, due to its competition with CD28 and CD152 molecules for cross-linking CD80/86 receptors on APC [19]. This phenomenon has been applied for induction of allograft tolerance in many experimental and clinical models. While the administration of CTLA4-Ig was extremely effective in the survival of cardiac, renal, or pancreatic islet allografts in rodents [20], its application as the immunosuppressive monotherapy in primates failed [21]. This treatment was successful only in combination with another immunosuppressive agent, anti-CD154 antibody [21].
In our paper we postulate that a suppression of the immune response observed in HD patients may be associated with an increased representation of the CD152 molecule-bearing lymphocytes and rHuEpo may ameliorate this effect. Therefore, in HD patients we analysed lymphocytes with an expression of the CD152 molecule and followed up changes in their representation during 1-year-long rHuEpo therapy. Moreover, during an observation period the level of several cytokines produced by whole blood cell cultures of the patients were monitored in order to determine the status of immune system.
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Subjects and methods |
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Additionally, we examined similarly to the above-mentioned patients, a group of 31 healthy volunteers (45±23 years old). The criteria of health were based upon physical examination, carefully taken histories, and laboratory data. Chronic or acute inflammatory diseases, neoplasms, drugs known to affect immune system or laboratory data outside the norm from 6 months before the start until the end of the study excluded volunteers from the control group.
All the volunteers were informed about the purpose of the study, and gave their consent. Additionally, the study was approved by The Ethical Committee of the Medical University of Gdask.
Specimen collection and preparation
Fasting venous blood samples (5 ml) were collected aseptically into the heparinized tubes (Becton Dickinson Company, USA) for direct analysis by flow cytometry and for whole blood cell cultures before the HD session: before rHuEpo treatment, at 1 month, 6 months, and 1 year after start of the therapy.
Whole blood cell cultures
All cells were cultured on plastic 24-well plates (Corning, Science Products, USA) in triplicate. Five-hundred microlitres of the whole blood was diluted (1:1) with RPMI (Gibco, Life Technologies Inc., USA). The cultures were incubated for 48 h in a humidified atmosphere containing 5% CO2 at 37°C. As the stimuli were used: 1 µg/ml of phytohaemagglutinin (Pharmacia, Sweden) or physiological (0.05 IU/ml0.05 rHEpo) and non-physiological (1.0 IU/ml1.0 rHEpo) ranges of concentration of rHuEpo (Eprex, JanssenCilag, Switzerland). Control (non-stimulated) cultures were incubated for 48 h.
Staining of the samples
The samples of venous blood and cultures were vortexed and aliquoted into 12x75 mm plastic tubes (Falcon, Becton Dickinson Company, USA), 100 µl per tube. The cells were then stained with control, isotypic antibodies (IgG1 FITC and IgG1 PE), anti-CD4 (immunoglobulin G1 (IgG1
), R-phycoerythrin (PE)- or FITC-conjugated), anti-CD8 (IgG1
, FITC-conjugated), anti-CD152 (IgG1
, PE-conjugated) and with 2-colour combinations of the above mouse monoclonal antibodies (20 µg/test). All blood samples were stained with the single mAb and dual combinations of: CD4PE/CD8FITC (for the CD4+/CD8+ ratio), CD4FITC/CD152PE, CD8FITC/ CD152PE. For each set, the isotypic control was carried out using PE-conjugated IgG1
and FITC-conjugated IgG1
in a concentration equal with the examined probes. PharMingen, Becton Dickinson Company (San Diego, USA) supplied all the reagents for the staining of samples. After an incubation (30 min in the dark at a 4°C), probes were subsequently lysed and fixed using Immuno-prep reagents (Coulter, USA) with Q-prep Immunology Workstation (Coulter, USA).
Acquisition and analysis
Listmodes were acquired on Epics XL flow cytometer (Coulter, USA) and analysed using WinList, version 3.0 software (Verity, USA). Dead cells were excluded by forward and side-angle scattered light window. A lymphocyte gate was generated using CD14/CD45 leukogate. Dot plots of representative samples from the gate with cell surface CD4, CD8, and CD152 antigens were then performed. Typically, 10000 events were acquired in the gating region. The threshold level for positive cells was set for each sample at the interception between the histogram curves obtained from staining with the isotype control antibody and the specific monoclonal antibody.
Cytokine ELISA and bioassays
Supernatants from non-stimulated and PHA-stimulated whole blood cell cultures were analysed for IL2, IL6, and TNF using bioassays as described previously [9]. Levels of IL10 and IL12 were assessed by the ELISA (Medgenix Diagnostics, Belgium) technique according to the manufacturer's instructions.
Statistics
All results were analysed using computer program: Statistica version 5.5 (Statsoft, Poland). Findings were estimated by means of descriptive statistics, unpaired Student's t-test, ANOVA/MANOVA tests for repeated measures, and lower significant differences test (LSD) as a post hoc test.
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Results |
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In vitro data
Whole blood cell cultures were stimulated either with 1 µg/ml PHA or with two concentrations of rHuEpo: 0.05 IU/ml or 1.0 IU/ml. The control cultures were not stimulated. Looking at the CD4+/CD8+ ratio, we found that the results in vitro were closely related to those ex vivo. An increase of the CD4+ T cells percentage coincided with a decline of the CD8+ T cells percentage.
The percentage of CD8+ subpopulation decreased significantly in the stimulated cultures. This decrease was revealed in the all stimulated cultures during the study (MANOVA: PHA, F=4.17, P=0.05; 0.05 rHEpo, F=14.81, P=0.002; and 1.0 rHEpo, F=15.70; P=0.001). A comparison between the non-stimulated and stimulated cultures revealed that none of the used stimuli affected the percentage of CD8+ lymphocytes (MANOVA: PHA, F=1.03, P=0.34; 0.05 rHEpo, F=0.02, P=0.88; and 1.0 rHEpo, F=1.38, P=0.27). The decrease in the percentage of CD8+ T cells was significant in all stimulated cultures between points: before vs 1 year of rHuEpo treatment (LSD: PHA, P=0.01; 0.05 rHEpo, P=0.0004; 1.0 rHEpo, P=0.0003) (Figure 1A and Table 3
).
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Thus, both ex vivo and in vitro observations revealed that there was a similar tendency in the profile of the CD4+ and CD8+ cells. While the level of CD4+ lymphocytes increased during 1-year of rHuEpo therapy, that of CD8+ had decreased.
Percentage of CD152+ T cells during rHuEpo therapy
Ex vivo data
The percentage of lymphocytes expressing the surface marker CD152 declined during the follow up (ANOVA: F=5.81, P=0.02) (Figure 3A and Table 2
).
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In vitro data
The above phenomena concerning decreased percentages of CD152+ and CD8+CD152+ T cells ex vivo were also analysed in whole blood cell cultures. The results of this analysis showed a decrease of the percentage of CD152-bearing lymphocytes in the cultures in the course of rHuEpo therapy (Figure 3B and Table 3
). This effect was significant during the therapy, what was confirmed by MANOVA tests (PHA: F=5.62, P=0.03; 0.05 rHEpo, F=37.49, P=0.0001; 1.0 rHEpo, F=15.60, P=0.001). The post hoc analysis (LSD) revealed that the differences between points (before vs 1 year of rHuEpo treatment (P=0.0006), one month vs 1 year of rHuEpo treatment (P=0.01), and 6 months vs 1 year of rHuEpo treatment (P=0.0004)) were significant only for the cultures with high doses of rHuEpo.
The decrease of the CD8+CD152+ T cells percentage was responsible for a decline of the CD152+ lymphocytes in the stimulated cultures (MANOVA: PHA, F=4.98, P=0.05; 0.05 rHEpo, F=12.23, P=0.009; 1.0 rHEpo, F=7.30, P=0.01) (Figure 3C and Table 3
). Stimulation with PHA caused a significant increase, while that with 0.05 rHEpo caused a decrease of the CD8+CD152+ cells percentage as compared with the non-stimulated cultures. High doses of rHuEpo did not affect CD8+CD152+ subpopulation (MANOVA: PHA, F=14.85, P=0.01; 0.05 rHEpo, F=11.90, P=0.01; 1.0 rHEpo, F=0.37, P=0.56). The LSD tests revealed that the significant decreases in the percentage of CD8+CD152+ subpopulation were found between the points: before vs 1 year of rHuEpo treatment (LSD: PHA, P=0.01; 0.05 rHEpo, P=0.002; 1.0 rHEpo, P=0.005) and before vs 6 months of rHuEpo treatment (LSD: PHA, P=0.003; 0.05 rHEpo, P=0.008; 1.0 rHEpo, P=0.01). These results indicate a different effect of rHuEpo in relation to such non-specific stimuli as PHA.
The percentage of CD4+CD152+ subpopulation did not change in all stimulated cultures during the follow up (MANOVA: PHA, F=0.26, P=0.85; 0.05 rHEpo, F=0.33, P=0.88; 1.0 rHEpo, F=0.31, P=0.81) (Figure 3D and Table 3
). None of these stimuli affected the percentage of CD4+CD152+ cells (MANOVA: PHA, F=1.54, P=0.25; 0.05 rHEpo, F=0.48, P=0.50; 1.0 rHEpo, F=0.25, P=0.62).
Summing up, an analysis of the CD152+ population in the patients receiving rHuEpo therapy showed that treatment with rHuEpo was associated with a decrease in the CD8+CD152+ cells. This effect was visible both ex vivo and in vitro.
Percentage of CD152+T lymphocytes in the healthy volunteers and HD patients not receiving rHuEpo therapy
An analysis of the CD152+ subpopulations was additionally carried out in the group of healthy volunteers (n=31, 45±23 years old). A comparison between this population and HD patients receiving rHuEpo revealed that the level of CD152+ lymphocytes was statistically higher in the later group during the whole study. This difference was found ex vivo (Table 2) and in all in vitro cultures (Table 3
) (P<0.05, unpaired Student's t-tests).
A more detailed analysis of the CD152+ subpopulations disclosed that significant differences concerned only the CD8+CD152+ cells. A higher percentage of the CD8+CD152+ cells in HD patients receiving rHuEpo was noted at all points of ex vivo study (Table 2), while in the cultures stimulated with 0.05 or 1.0 IU/ml rHuEpo, the percentage of those cells decreased to the level observed in the healthy control group 6 months after beginning of the rHuEpo therapy (Table 3
) (P<0.05, unpaired Student's t-tests).
An analysis of whole CD8+ population in the healthy group and HD patients receiving rHuEpo revealed that in HD patients comparable levels of the CD8+ cells ex vivo as well as in vitro were attained 1 year after rHuEpo treatment. The earlier points of the study were characterized by a higher percentage of CD8+ lymphocytes in HD patients both, ex vivo and in vitro (Tables 2, 3
) (P<0.05, unpaired Student's t-tests).
The percentage of whole CD4+ lymphocytes did not differ between the healthy group and HD patients receiving rHuEpo at all the points analysed ex vivo (Table 2). The comparison of the cultured lymphocytes from the above-mentioned groups revealed that equal amounts of the CD4+ lymphocytes were noticeable at 1 year of rHuEpo treatment in the cultures stimulated with 0.05 rHEpo in vitro (P=0.04, unpaired Student's t-tests). All other cultures were characterized by a higher percentage of the CD4+ cells in healthy volunteers as compared to HD patients during the whole study (Table 3
) (P<0.05, unpaired Student's t-tests).
HD patients who did not receive rHuEpo were characterized throughout the study by a continuously high level of CD152+ cells, comparable with the values observed before the start of rHuEpo therapy in HD patients treated with this drug (Tables 2, 3
(only results of CD8+CD152+ cells are shown)). Also the amounts of CD4+ and CD8+ cells measured both ex vivo and in vitro were stable and similar to the results obtained in those treated with rHuEpo before beginning of the therapy (data not shown).
Levels of cytokines during the study
Levels of IL 2, IL12, (TH1 cytokines), IL6, TNF (proinflammatory cytokines) as well as IL10 (anti-inflammatory cytokine) were measured in supernatants from whole blood cell cultures stimulated with PHA. Results of this examinations revealed that levels of IL2 and IL12 increased significantly in supernatants of HD patients during the study (MANOVA: IL2, F=18.33, P=0.05; IL12, F=8.4, P=0.05). Statistically significant differences, assessed by LSD tests, concerned the points: before vs 6 months of rHuEpo therapy (P=7.5x10-5); before vs 1 year of rHuEpo treatment (P=2.0x10-5) for IL2; and before vs 1 year of rHuEpo treatment (P=0.04) for IL12 (Figure 4
).
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Levels of TNF and those of IL6 decreased significantly during the follow-up (MANOVA: TNF
, F=2.34, P=0.03; IL6, F=2.54, P=0.01). Significantly lower concentrations were noticed in the two last points of the study: before vs 6 months of rHuEpo treatment (LSD: TNF
, P=0.03; IL6, P=0.009) and before vs 1 year of rHuEpo treatment (LSD: TNF
, P=0.02; IL6, P=0.0002) (Figure 4
).
HD patients not receiving rHuEpo during the follow-up were characterized by stable levels of all cytokines with concentrations similar to those in the treated group before the start of rHuEpo therapy (Figure 4).
Summing up, long-term (612 months) rHuEpo therapy in HD patients restored the levels of TH1 cytokines IL2 and IL12 to the values seen in the healthy people. Simultaneously, rHuEpo therapy corrected a disturbed balance between the proinflammatory (TNF and IL6) and anti-inflammatory (IL10) cytokines.
Laboratory tests
Mean haemoglobin concentration before treatment was 8.9±0.9 g/dl and during the follow-up it was in the range 1012 g/dl. Mean levels of lymphocyte counts, CRP, C3, and C4 did not change during the therapy and did not correlate with the above changes in the cellular subpopulations. Iron status parameters, transferrin saturation and ferritin, were kept at appropriate levels during the observation period. Details of the data are presented in Table 1.
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Discussion |
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High percentages of the CD152+ lymphocytes in the peripheral blood could have additionally been up-regulated in our study by stimulation with PHA, suggesting that an elevated ex vivo percentage of the CD152+ lymphocytes in HD patients might have resulted from a constant activation of blood cells. HD patients were characterized by an extremely high CD152+ lymphoid populationtwo- or three-fold higher than that in healthy volunteers. On the other hand, the physiological concentration of rHuEpo in vitro decreased the percentage of CD8+CD152+ cells in whole blood cell cultures, suggesting that rHuEpo therapy was able to decrease a state of activation of blood lymphocytes. This line of thinking is in agreement with results obtained with cytokines. Before rHuEpo implementation, the level of TNF and IL6, the proinflammatory cytokines, was much above that produced by cultures of the healthy control subjects. One year after rHuEpo therapy, levels of these cytokines decreased, reaching values characteristic of the healthy. Simultaneously, a low level of anti-inflammatory IL10 increased at the same point of rHuEpo therapy. Thus, the immune conditions shifted from the stimulatory towards suppression. This was paralleled by a sign of a lower percentage of those cells with CD152 surface molecule.
Changes in lymphocyte subpopulations presented in our paper clearly indicate that the restoration of a haemoglobin level is not the only effect of rHuEpo treatment. An increase in the CD4+/CD8+ ratio, together with a decreased percentage of CD152+ cells, may improve an immune response against infections or vaccinations of HD patients, due to a higher amount of CD4+ T cells and a weaker CD152-mediated inhibitory signal. Treatment with rHuEpo reduced mainly the CD8+CD152+ subpopulation of HD patients, which was visible both in vitro and ex vivo. The fact that in mice, anti-CD152 monoclonal antibody was able to increase the production of IL2 by T lymphocytes [15] implies that a decreased representation of the CD8+CD152+ cells entailed a higher production of TH1 cytokines. Indeed, our study revealed an inverse relationship between the CD8+CD152+ and levels of IL2 and IL12 produced in PHA-stimulated whole blood cell cultures. Corrected levels of TH1 cytokines IL2 and IL12 may represent a change contributing to an improvement of a cellular immune response observed previously in rHuEpo-treated HD patients [8].
A better immune response following rHuEpo therapy has been reported by several authors [4,7]. A higher antibody titer after hepatitis B vaccination [22] and increased immune response to T-cell-dependent antigens [11] were found in HD patients treated with rHuEpo. The phenomenon of a better immunoglobulin profile connected with rHuEpo therapy [4] may be explained in the light of our results by an enrichment of helper CD4+ T cells and a decrease of suppressive CD152+ cells. A blockade of the reaction between the CD152 and the CD80/CD86 molecules by the CTLA4-Ig monoclonal antibody promotes synthesis of the cytokines known to be involved in the humoral response and switching the immunoglobulin classes [15]. The same kind of CD152-dependent down-regulation augments T-cell responses to antigens and intensity of superantigen-mediated toxicity [15]. Improved function of CD4+ cells in our study was confirmed by an increased level of TH1 cytokines in the cultures stimulated with PHA 1 year after the beginning of rHuEpo therapy. This effect may be referred to suppressive CD8+CD152+ T lymphocytes. Increased secretion of IL12 by APC was partially mediated by reduced cross-linking of CD80/CD86 receptors on APC with CD152 ligand on CD8+ cells. This lower inhibitory signal also permitted sufficient presentation of antigens by APC to CD4+ T helper lymphocytes, and finally caused an enhanced secretion of IL2-cytokine, which is produced in the human immune system exclusively by CD4+ T helper lymphocytes.
On the other hand, too high a concentration of the rHuEpo used in vitro in our study had depleted the cultures from CD4+ T cells, which may reverse a helper/cytotoxic equilibrium obtained by long-term rHuEpo therapy. This implies that excessive doses of rHuEpo may not be beneficial to HD patients. The differing effect of the two extremely different concentrations of rHuEpo indicates the necessity for careful selection of this drug dose in order to ensure its immunomodulating activity.
A wide range of immune functions is suppressed in HD patients [1,3]. Some defects may be better understood in the light of our results. For example, reduced mitogenic response of lymphocytes of HD patients to lectins [23] may have its source in an over-expression of the CD152 molecule and deficiency of IL2. Both defects were overcome in our patients in the late stage of rHuEpo therapy. In addition, an impaired function of APC cells has been reported in HD patients. This defect is predominantly connected with a decreased expression of CD80/CD86 receptors on monocytes [24]. A diminished cross-linking of CD80/CD86 with their ligand, CD28 on lymphocytes, additionally enhanced by a competitive inhibition caused by the other ligand, CD152, may finally hinder APC/lymphocyte co-operation and inhibit IL12 secretion. Until now, no data has been found in the literature indicating that rHuEpo may increase an expression of the CD80/CD86 molecules on APC cells. Our data suggest that an improved secretion of IL12 may be related to the decreased levels of the suppressive CD8+CD152+ lymphocytes. This change in the intensity of IL12 secretion was not of inflammatory origin, as was revealed in this study by decreased secretion of TNF and IL6 and constant levels of CRP, C3, and C4. In addition, iron deficiency was excluded.
An effect of rHuEpo on HD patients needs to be considered in relation to future allograft transplantation. There are reports indicating that during the first weeks after transplantation, functioning of the graft was worse in HD patients who were treated with rHuEpo as compared to untreated patients [4]. This effect may be a consequence of an improvement of immune functions. As our results suggest, CD8+ lymphocytes responsible for the cytotoxic functions represent subpopulation sensitive to rHuEpo treatment. On the other hand, renal biopsies done in cases of acute renal allograft rejection in humans revealed intense infiltration of this tissue by the CD8+CD152+ cells [25]. This fact, together with our results, supports the usefulness of rHuEpo therapy in eliminating the lymphocytes with such a phenotype prior to transplantation. Looking at the long-term allograft survival possibilities, it seems that a shift favouring CD4+ cells may bring an advantage.
Post hoc tests revealed that all the effects observed during rHuEpo therapy were obtained between 6 and 12 months after the beginning of rHuEpo therapy.
Treatment with the rHuEpo did not alter such late markers of the inflammation as plasma CRP, C3, and C4 components of the complement, as well as the total count of the lymphocytes. Therefore, these results indicate that the revealed changes are not effects of inflammation, but are essential consequences of the influence of rHuEpo on the lymphocyte subpopulations. It also means that appropriate laboratory monitoring of anaemia treatment using rHuEpo from the beginning of the HD therapy, or even earlier in the predialysis period, seems to be necessary due to imminent infections or planned transplantation.
Summing up, administration of rHuEpo to HD patients not only treats the anaemia but also results in a favourable immunomodulation. This therapy affects T lymphocytes and its full effect is visible after long-term treatment.
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Acknowledgments |
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Notes |
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* Current address: Mayo Foundation, Rochester, MN, USA.
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References |
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