Erythropoietin (EPO) is a physiological stimulator of erythropoiesis. One of the main eff ects of EPO is to prevent apoptosis of erythroid progenitor cells in the bone marrow. Th ese properties of EPO are widely used for treatment of various hematopoietic disorders including posttransplant conditions. Previously, it was found that activating EPO-eff ect on T-lymphocytes (TLC) accompanied by an increase in the number of fluorescent mitochondria (n m/c) and an increase in the total transmembrane potential on plasmatic (Δφp) and mitochondrial membranes (Δφm). However, it remains unclear which membrane potential is responsible for the EPO effect. Hence, we used specifi c inhibitors of oxidative phosphorylation in the respiratory chain. The aim of the present work was to assess the role of mitochondrial functions in EPO eff ects upon thymic lymphocytes.
We studied EPO (Eprex, Cilag) infl uence on fl uorescence of rat TLC aft er short-term incubation and treatment with some inhibitors: dinitrophenol (DNP-uncoupler of oxidative phosphorylation and inhibitor of respiratory chain), pentachlorphenol (PCP- uncoupler of oxidative phosphorylation), N,N -dicyclohexylcarbodiimide (DCCD- inhibitor of Ca2+- dependent mitochondria ATP-аse). Th e cells were then tested by electrical fi eld gradient sensitive probe DSM [4-(p-dimethylaminostyryl)- 1-methylpyridinium]. Rat TLC were isolated according to the standard method. Th e microfl uorimetric studies of DSM-stained TLC were performed by means of fl uorescent microscope "Lumam R-8", "LOMO", Russia) with thermostatic plate. Fift y to 70 single cells were measured per each specimen the mean fl uorescence intensity of TLC was calculated (F̃), as well as nm/c values. Statistical evaluation of the data was performed by the Spearmen range correlation.
EPO is able to partially recover the damage and polarization of the mitochondria membranes in TLC disturbed aft er exposure to specifi c ATP-ase inhibitor (DCCD). This in vitro approach may be used for screening other growth factors.
Erythropoietin, T-lymphocytes, energy activity, inhibitors, electrical field gradient sensitive probe DSM [4-(p-dimethylaminostyryl)-1-methylpyridinium].
Erythropoietin (EPO) was initially known as a physiological growth factor which is produced, mainly, by renal glomeruli, macrophages and some other cell types. EPO is shown to support survival and mitotic activity, as well as inhibit apoptosis of late erythroid precursor cells in bone marrow . Th erefore, recombinant erythropoietins-alpha (Eprex, Epobioicrine, Epostim etc.) are widely used to correct anemias in diff erent diseases and posttransplant conditions .
However, biological mechanisms of EPO action upon immune cells are still not clear. E.g., antioxidant in vivo eff ects of EPO (reduced lipid peroxidation in blood lymphocytes) are shown by Osikov et al. . Immunotropic eff ects of EPO are recently studied, due to its immunomodulatory eff ects under clinical and experimental conditions [4-6]. Response of lymphocytes and macrophages to EPO seems to be mediated by the EPO receptors on their surface . In vitro biological eff ects of EPO towards T-lymphoid cells were previously shown by Hisatomi et al.  who demonstrated suppression of IL-2 gene expression in TLC cells after their short-term (6 h) incubation with EPO. Hence, EPO is able to exert fast immediate action upon T-lymphoid cells over short incubation terms. Indeed, we have also revealed modulatory effect of EPO upon rat thymocytes using a specifi c potential-sensitive chemical probe . EPO was found to exert activating eff ect upon the TLC by changing total transmembrane potential of plasmatic (Δφp) and mitochondrial membranes (Δφm). This activation correlated with increased numbers of the probe-labeled fl uorescent mitochondria in exposed cells . To discern these mechanisms, we used specifi c inhibitors of oxydative phosphorylation, in order to assess the type of potential responsible for these EPO eff ects. Hence, the aim of this work was to evaluate the role of mitochondrial functions in EPO eff ects upon thymic lymphocytes in order to specify this response to EPO, either Δφm, or Δφp. To address this issue, we used specifi c inhbitors of phosphorylation in the respiratory chain which serve as important tools for studying energy supply in the living cells.
To address this issue, we used specifi c inhibitors of oxydative phosphorylation. In order to evaluate the role of mitochondrial functions for energy supply in thymic lymphocytes exposed to EPO. Hence, the aim of this study was to assess a restoring EPO eff ect upon the in vitro response of thymocytes aft er treatment with diff erent specifi c inhibitors of oxidative phosphorylation.
We have studied lymphoid cells isolated from thymuses of white Wistar rats (200-300 g), aft er gentle mincing of thymus glands . Th e cells were resuspended in standard Hank's solution at 2 to 3x107 cells/mL. Percentage of viable (dye-excluding) cells was determined by routine Trypan Blue staining.
To determine possible points for EPO actions, we used different inhibitors of the oxidative phosphorylation, i.e., dinitrophenol (DNP, Sigma, USA), an inhibitor of respiratory chain and uncoupler of oxidative phosphorylation; pentachlorophenol (PCP, Sigma USA), an uncoupler of oxidative phosphorylation; dicyclohexyl carbodiimide (DCCD, Sigma, USA), an inhibitor of mitochondrial membrane-bound ATP-ase domain. Erythropoietin (EPO) was purchased from Cilag (Eprex) was dissolved in Hank's solution. A potential- sensitive fl uorescent probe [4-(p-dimethylaminostyryl)- 1-methylpyridinium] (DSM) was used to test the energy potential of cells. DSM was produced and purchased from the Latvian Institute of Organic Synthesis . Th e thymocyte suspensions in Eppendorf-type tubes were pre-incubated with inhibitors for 10…20…40 min at 37°С, then EPO was added at the fi nal concentration of 2U/ml), followed by incubation for 30 min, addition of the DSM probe (1.5 μM), and post-incubated for 20 min. Final concentrations of inhibitors were as follows: DNP, 0.1 mM; PCP, 1.5 μM; DCCD, 0.1 mM, at a v/v ratio of <5% to the initial suspension volume. The control samples of cells were supplied with equal volumes of Hanks' solution, and, aft er 10…30 min. at 37°С, were incubated with DSM and EPO, as described above. Control and experimental TLC samples were then studied at the luminescent microscope LUMAM – R8 (LOMO, St. Petersburg, Russia) at a 900x magnifi cation, using a temperature-controlled stage for the count chamber. Th e fl uorescence was excited by a mercury lamp (λ= 405-436 nm). To measure light emission, a FMEL-1 photometric device was used, with an interference fi lter with a maximum transmission at 585 nm. Fluorescence intensity was manually measured for single cells localized in the vision fi eld, then transformed to a digital signal. Th e numbers of DSM-stained bright mitochondria, looking as intracellular yellow granules, were counted per each single cell (the nm/c values) . Fift y to seventy cells were studied per sample, and the mean fl uorescence intensity was calculated as conventional units (F̃, arbitrary units). The fluorescent signal did not quench over the measurement time. Photomicrographs of the DSM-stained cells were performed with a TSA 5.0 camera mounted in the LOMO R8 microscope, and analyzed by a Microanalysis View soft ware (from the same manufacturer). Statistical evaluation of the data was performed by the Spearmen range correlation criterion. A total of 8,000 cells have been studied in 160 samples. Each independent experiment included 3 to 5 measure points.
Initial amount of intact thymic lymphoid cells in cell suspensions was 92 to 96%. Several control experiments with have shown a decrease of nm/c and F̃ values for these cells aft er incubation with either inhibitor. Th e degree of such decrease depended on the type of inhibiting substance, and incubation terms (Table 1, Fig. 1 A, B, C). Th e most pronounced and faster eff ect was observed with DNP, i.e., fl uorescent mitochondria became virtually absent in DNP-treated cells as soon as aft er 10 min of exposure. F̃ values were also decreased by 20 min, with both nm/c and F̃ reduction. Fluorescent mitochondria disappeared by 40 min, with F̃ at the background levels. EPO addition did not restore F̃ and nm/c- parameters. Th e dynamics of thymocytes with absent mitochondrial fl uorescence aft er DNP exposure shown in Fig. 1A (NC-EM,%), like as absence of recovery aft er EPO addition. Th is eff ect may be caused by classical protonophore properties of DNP which irreversibly reduces both mentioned components of electrochemical gradient, thus causing the mitochondrial membrane depolarization.
Similar, but less pronounced decrease was observed 10 min aft er treatment with PCP, i.e., n m/c dropped by 37%, and F̃, by ~ 35%. Following 20-min incubation, we observed only ~ 26% nm/c and ~ 25% F̃ of control values. At 40 min., no fl uorescent mitochondria are seen.
Meanwhile, Fig. 1B shows increase of non-fl uorescent cell numbers (NC-EM, %) induced by PCP, followed by a recovery induced by EPO supplement. The inhibitory effect of PCP upon thymocytes proved to be partially reversible aft er addition of EPO (nm/c recovery by ~23%, and F̃ values by ~20%).
A 10-min. incubation of thymocytes with DCCD again retains only a part of fl uorescent cells (nm/c, 30%, and ~33% F̃). Only 18% nm/c and ~ 20% F̃ remain aft er 20 min. with DCCD, a mitochondrial membrane-bound ATP-ase. At 40 min. with DCDD, no fl uorescence was observed in the cells, with background F̃ values. However, 30-min. incubation with EPO has resulted into recovery of mitochondria-associated fl uorescence to 42% for nm/c and 38% for F̃ levels, as compared with control samples. Fig. 1C illustrates the dynamics (NC-EM, %) of de-energized thymocytes induced by DCCD followed by restoration of potential-coupled fl uorescence aft er EPO treatment.
Typical patterns of DSM-stained cells before and aft er treatment with mitochondrial ATP-ase inhibitor (DCCD) are shown in Fig. 2 A-C.
In our works, we have used inhibitor analysis, in order to assess the mechanisms which regulate anti-apoptotic eff ects of erythropoietin. Th e transmembrane potential of membranes in thymocytes was determined as fl uorescence intensity of DSM probe. Total DSM fl uorescence depends on a summary potentials of plasmatic and mitochondrial membranes. Earlier we have found that EPO acts upon thymic lymphoid cells by changing electric charge of cellular membranes. Th e EPO stimulatory eff ect is accompanied by increased nm/c, due to proton potential (Δφm), and/or external membrane potential (Δφp) .
Our data suggest that some metabolic eff ects of EPO are exerted via mitochondrial respiratory pathways. EPO was shown to reverse the de-energizing eff ects of DCCD, thus presuming functional changes of F0F1 membrane ATP which is specifi cally inhibited by the DCCD.
The mitochondrial F0F1-ATP-ase is a complex lipoprotein containing of hydrophilic catalytic center (F1), and a membrane domain (F0) . DCDD used in this work is a specific proton translocation inhibitor in the F0F1 ATP-ase , causing decrease in nm/c and general F̃ shown in our experiments, thus refl ecting a critical role of ATP-ase for sustaining the mitochondrial membrane potential. Th e reduced fl uorescence of DSM-induced mitochondria could be considered as mitochondrial de-energization, which proved to be reversible by EPO treatment. Th is fi nding may refl ect ability of EPO to restore functional integrity of mitochondrial membranes as a component of their eff ects upon immune system. The restored mitochondrial functions in the target cells for EPO allow to perform regulatory signaling in lymphoid cells, e.g., via phosphorylation of some transcription factors, e.g., STAT5 in lymphoid cells and tissues . Further studies in other lymphoid cell models could further elucidate the role of EPO as a regulator of mitochondrial function.
1. Thymocytes may represent a non-usual, but suitable model for evaluation of growth factor eff ects upon dividing, apoptosis- prone immune cells.
2. The in vitro testing of modifying EPO eff ects in thymocytes exposed to inhibitors of mitochondrial functions has revealed irreversible deletorious eff ect upon energetic potential of these cells. Erythropoietin (EPO) does not reverse the DNP-induced damage, but partially restores mitochondrial damage induced by DCDD, a specifi c inhibitor of the F0F1 mitochondrial membrane ATP-ase.
3. More extended studies are required, in order to screen positive eff ects of EPO in other non-erythroid cell types.
No potential confl icts of interests are reported.
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