chronic renal failure is accompanied by a deficiency state in both cell - mediated and humoral immunity which is deepened by hemodialysis ( hd ) . a direct contact of the patient s peripheral blood mononuclear cells ( pbmc ) with an artificial membrane increases the production of pro - inflammatory cytokines : tumor necrosis factor - alpha ( tnf- ) , interleukin ( il)-1 and il-6 [ 2 , 3 ] .
what s more , hd patients demonstrate a decreased production of il-2 and interferon gamma ( ifn- ) [ 4 , 5 ] . the deficient production of cytokines is probably related to changes in phenotype of cd4 lymphocytes from hd patients ,
which exhibit decreased expression of the major co - stimulatory cd28 antigen and main activation markers : cd25 and cd69 .
recombinant human erythropoietin ( rhepo ) administered to hd patients to correct the anemia state can influence both the phenotype of t lymphocytes and the production of cytokines .
our previous studies have shown that rhepo treatment normalizes the impaired expression of cd28 and cd69 antigens of cd4 lymphocytes .
additionally , several months of therapy restores the balance of cytokines by reducing the level of tnf- [ 8 , 9 ] and increasing the level of il-2 and il-10 [ 4 , 8 , 9 ] in whole blood cell cultures of hd patients during treatment .
the level of tnf- and il-10 changes immediately when the hemoglobin level exceeds the optimal value of 10  g / dl and is stable during treatment , while the level of il-2 increases continuously during treatment .
this observation suggests that the normalization of the levels of tnf- and il10 was reached shortly after the start of rhepo therapy .
increase of the level of il-2 has been achieved after several months of rhepo administration , so this change was secondary with respect to changes in the level of tnf- and il10 .
for example , the level of il-2 may depend on increased expression of cd28 on cd4 lymphocytes and/or decreased percentage of cd152 lymphocytes .
however , it is still not clear which cytokines are regulated by the presence of erythropoietin in the circulation . in order to better understand these mechanisms
, we decided to test how the presence of rhepo in cell culture influences cytokine production in stimulated cd4 lymphocytes from hd patients with a stable hemoglobin level and expression of cd28 , cd69 and cd25 antigens of stimulated cd4 lymphocytes similar to those observed in healthy individuals .
repetitive hd procedure has also been reported to induce apoptosis of t lymphocytes , which leads to t - cell lymphopenia sometimes observed in these patients .
therefore , we also tested if the presence of rhepo in cell culture can protect stimulated cd4 lymphocytes of hd patients from apoptosis .
for this study we have chosen 13 hd patients ( 11 men and 2 women , mean age 59    13.99  years ) with a hemoglobin level above 10  g / dl ( mean hemoglobin 12.53    1.57  g / dl ) and expression of cd28 , cd69 and cd25 antigens of stimulated cd4 lymphocytes similar to those observed in healthy controls .
7 hd patients received epoetin alpha or beta and the epoetin doses were adjusted to hemoglobin levels .
6 hd patients who did nt receive rhepo were also included in the study group .
these patients did not require rhepo administration , since the level of hemoglobin was maintained at the correct level .
they showed similar phenotype of stimulated cd4 lymphocytes and the level of th1 and th2 cytokines in culture supernatants as hd patients treated with rhepo and healthy controls ( data not shown ) .
none of the patients suffered from any infection , inflammation , malnutrition , malignancy or blood loss during the study .
the study has been approved by the ethical committee of the medical university of gdask .
thirty milliliters of venous peripheral blood from each hd patient was collected in tubes containing edta as the anti - coagulant agent before hd session . peripheral blood mononuclear cells ( pbmc ) were isolated from venous peripheral blood as previously described   and stimulated with an immobilized anti - cd3 antibody ( 125  ng / ml ) with or without the addition of rhepo ( epoetin alpha , 0.1 u / ml ) and incubated for 3  days at 37  c , 5  % co2 .
stimulated cells were then collected in order to estimate the percentage of apoptotic cd4 lymphocytes .
culture supernatants were collected and frozen at 80  c for the assessment of cytokine production .
collected cells were stained with the rpe - cy5-conjugated anti - cd4 monoclonal antibody ( dako , denmark ) and pe - conjugated annexin v ( bd pharmingen , usa ) and analyzed with flow cytometry on facscan ( becton dickinson , usa ) .
cytometric bead array ( cba , bd biosciences , usa ) was used to estimate the level of th1/th2 cytokines produced by stimulated pbmc .
data were analyzed with cyflogic , version 1.2.1 ( perttu terho and cyflow ltd ) .
statistical analysis was done using the statistica program , version 8 ( statsoft , poland ) .
the significance tests were chosen according to data distribution . the level of significance in all was p    0.05 .
the expression of erythropoietin receptor ( epo - r ) has been reported in many non - hematopoietic cells including lymphocytes .
we have recently shown that rhepo increases the phosphorylation of signal transducer and activator of transcription 5 ( stat5 ) in stimulated cd4 lymphocytes .
therefore , we examined whether the presence of rhepo in cell culture can protect stimulated cd4 lymphocytes of hd patients from apoptosis and we have shown that if present at a concentration similar to the physiological level rhepo promotes the survival of cd4 lymphocytes .
hd patients showed a significant decrease in the percentage of apoptotic cd4 cells ( cd4annexin v cells ) after stimulation with the anti - cd3 antibody combined with rhepo as compared to cells stimulated with the anti - cd3 antibody alone ( fig .  1 ) .
this fact has been observed in other cell types and our team is being the first to described it in t lymphocytes.fig .
1comparison of the percentage of cd4 cell apoptosis depending on the presence of rhepo in cell culture .
midpoints of figures present medians , boxes present the 25 and 75 percentile and whiskers outside visualize the minimum and maximum of all the data , p  <  0.05 , wilcoxon signed ranks test comparison of the percentage of cd4 cell apoptosis depending on the presence of rhepo in cell culture .
midpoints of figures present medians , boxes present the 25 and 75 percentile and whiskers outside visualize the minimum and maximum of all the data , p  <  0.05 , wilcoxon signed ranks test we also investigated the level of th1 cytokines ( il-2 , ifn- , tnf- ) and th2 cytokines ( il-4 , il-5 , il-10 ) in culture supernatants of lymphocytes stimulated with the anti - cd3 antibody with or without addition of rhepo .
we did nt observe any changes in the level of il-2 in culture supernatants of lymphocytes stimulated with the anti - cd3 antibody in the presence of rhepo .
since the phenotype of stimulated cd4 lymphocytes from these patients and the level of il-2 in culture supernatants is similar to those observed in healthy individuals , we suspect that the production of il-2 is dependent on the level of cd28 antigen on cd4 lymphocytes , as confirmed by a positive correlation between these parameters ( data not shown ) .
the presence of rhepo in cell culture increased the levels of ifn- and il-10 ( fig . 
2 ) , whose production can also be enhanced by il-2 [ 14 , 15 ] . since il-2 and epo act through receptors that belong to the same family and have common signaling pathways
the level of tnf- was decreased in the presence of rhepo in cell culture from hd patients ( fig .  2 ) .
it is not clear whether down - regulation of tnf- was caused by rhepo or rather il-10 , whose level was increased in the presence of rhepo .
not the first time we demonstrate the impact of rhepo on cytokine levels in hd patients .
our study shows that rhepo affects the production of cytokines which are found to be regulated through signaling pathways involving stat5 , for example .
the presence of rhepo appears to regulate levels of tnftnf- and il-10 , depending on the initial level of cytokines .
healthy controls had a lower percentage of apoptotic cd4 cells after stimulation with the anti - cd3 antibody compared to hd patients so the presence of rhepo in cell culture did nt influence it .
healthy controls also did nt show any changes in th1 or th2 cytokine levels when rhepo was added to cell culture but at the same time the level of tnf- was already decreased while the level of il-10 and ifn- was increased compared to hd patients ( data not shown).fig .
2comparison of the levels of th1 and th2 cytokines in culture supernatants of stimulated lymphocytes depending on the presence of rhepo .
graphs show the level of th1 ( il-2 , ifn- , tnf- ) and th2 ( il-4 , il-5 , il-10 ) cytokines in hd patients .
midpoints of figures present medians , boxes present the 25 and 75 percentile and whiskers outside visualize the minimum and maximum of all the data , p  <  0.05 , wilcoxon signed ranks test comparison of the levels of th1 and th2 cytokines in culture supernatants of stimulated lymphocytes depending on the presence of rhepo .
graphs show the level of th1 ( il-2 , ifn- , tnf- ) and th2 ( il-4 , il-5 , il-10 ) cytokines in hd patients .
midpoints of figures present medians , boxes present the 25 and 75 percentile and whiskers outside visualize the minimum and maximum of all the data , p  <  0.05 , wilcoxon signed ranks test
these results confirm one more time the role of rhepo signaling in t lymphocytes of hd patients . in our opinion
, rhepo protects cd4 lymphocytes from apoptosis and restores the balance of cytokines by reducing the level of tnf- and increasing the level of anti - inflammatory il-10 , though the mechanism of action of rhepo on t lymphocytes is still unclear . at the same time
these results suggest that improved production of il-2 is not directly dependent on rhepo presence but seems to be a consequence of a long - term rhepo treatment .
these observations confirm that rhepo administration not only has a beneficial effect on the red blood cells but also regulates the functioning of immune cells .
 recombinant human erythropoietin ( rhepo ) treatment of hemodialyzed ( hd ) patients normalizes the altered phenotype of cd4 + lymphocytes and restores the balance of th1/th2 cytokines . 
 we decided to test how the presence of rhepo in cell culture modulates cytokine production of cd4 + lymphocytes in hd patients with stable hemoglobin level and expression of activation antigens of stimulated cd4 + lymphocytes similar to those observed in healthy individuals . 
 we also tested whether the presence of rhepo in cell culture protects stimulated cd4 + lymphocytes of hd patients from apoptosis . peripheral blood mononuclear cells ( pbmc ) of hd patients 
 were stimulated with an immobilized anti - cd3 antibody with or without addition of rhepo . 
 the percentage of apoptotic cd4 + lymphocytes and the level of th1/th2 cytokines in culture supernatants were measured with flow cytometry . 
 hd patients showed a decrease in the percentage of apoptotic cd4 + cells after stimulation with the anti - cd3 antibody combined with rhepo . 
 the level of ifn- and il-10 was increased while the level of tnf- was decreased in the presence of rhepo in cell culture from hd patients . 
 these results confirm the role of rhepo signaling in t lymphocytes of hd patients . 
Introduction
Patients and Methods
Results and Discussion
Conclusions