ouabain was initially described as a compound extracted from plants , such as acocanthera ouabaio and strophanthus gratus , and has been used for the treatment of congestive heart failure due to its positive ionotropic effect on cardiac muscle . however , in 1991 , an endogenous analogue , biologically , structurally , and immunologically indistinguishable from the ouabain extracted from plants , was discovered in human plasma . nowadays , this endogenous ouabain is considered part of a new class of steroid hormones . under physiological conditions , levels of endogenous ouabain are relatively low and range between 10 and 10  m , whereas pharmacological levels are around 10  m. in stress situations , like physical exercise , these endogenous levels can also reach 10  m in humans [ 35 ] . additionally , increased levels of ouabain have also been associated with different pathological situation , such as hypertension , cardiac dysfunctions , chronic renal failure , and preeclampsia [ 1 , 4 , 610 ] . ouabain is secreted by hypothalamus and adrenal glands and is capable of binding to na , k - atpase , inhibiting na and k transport and triggering the activation of several cell signaling pathways [ 1113 ] . though the physiologic and pathophysiologic roles of ouabain are not well established , ouabain was shown to affect the immune system [ 14 , 15 ] and to have anti - inflammatory properties [ 16 , 17 ] . notwithstanding , ouabain was shown to modulate the release of proinflammatory cytokines by mononuclear cells . in fact , it was demonstrated that treatment of these cells with 10  m of ouabain promotes an increase of il-1 , tnf- secretion [ 1820 ] . furthermore , our group has demonstrated that after incubation with ouabain , a significant percentage of monocytes expressed lower levels of mcd14 and this appears to involve transactivation of egfr and activation of p38mapk . nevertheless , the effect of ouabain on other cell surface molecules or even the impact of ouabain on monocyte function has not been addressed yet . monocytes are crucial for immune responses , since they can perform effector functions such as antigen presentation , endocytosis , and the production of a diverse array of cytokines . thus , the aim of the present work was to verify whether ouabain was able to alter the activation state and function of monocytes , in an attempt to understand a possible role of this molecule on human monocytes . human peripheral blood samples were obtained from healthy volunteers ( < 40 years old ) and separated by ficoll - histopaque ( ge , sweden ) density gradient centrifugation ( 400  g , 30 minutes ) . after separation , mononuclear cells were collected , washed three times by centrifugation at 200  g for 10 minutes with phosphate buffered saline ( pbs ) , and resuspended in culture medium rpmi 1640 ( sigma , usa ) supplemented with 10% fetal calf serum ( fcs ) ( gibco , usa ) , inactivated at 56c for 1 hour . the study was approved by the ethics committee of the hospital universitrio clementino fraga filho - ufrj , and all volunteers gave written informed consent to this work . mononuclear cells were plated in 24-well plates ( tpp , switzerland ) at a concentration of 2  10  cells / well in a final volume of 1  ml and incubated for 2 hours in an atmosphere of 5% co2 at 37c . after this period , cells were washed twice with pbs for the removal of the excess of lymphocytes that , unlike monocytes , remain in suspension . finally , culture medium rpmi 1640 supplemented with 10% fcs was added to the culture . following the 2 hours of adhesion , cells were further incubated in the presence or absence of ouabain ( oua ) ( sigma , uk ) in an atmosphere of 5% co2 at 37c . to study surface molecules expression , cells were washed with pbs , removed with a cell scraper , and labeled with human fitc - conjugated anti - cd14 ( biolegend , usa ) or pe - conjugated anti - cd14 ( bd pharmingen , usa ) and pe - conjugated anti - hla - dr ( bd pharmingen , usa ) , pe - conjugated anti - cd86 ( bd pharmingen , usa ) or fitc - conjugated anti - cd80 ( bd pharmingen , usa ) , and fitc - conjugated anti - cd40 ( bd pharmingen , usa ) or pe - conjugated anti - cd69 ( bd pharmingen , usa ) for 30 minutes at 4c . cells were then washed twice with pbs + fcs 5% ( 200  g , 7 minutes ) and resuspended in the same solution in the cold . fluorescence measurements were performed using a facscan or a facscalibur flow cytometer ( beckton , dickinson and company , usa ) . fluorescence signals for no less than 10,000 cells were recorded in the monocyte gate , which was determined according to cell size and complexity parameters . using these parameters , after the 2-hour adhesion and removal of nonadherent cells , the remaining cells were incubated with 5  m of fluo-3am ( sigma , usa ) for approximately 10 minutes at 37c . this was followed by the addition of 10  m of ouabain to the culture and monocytes were further incubated for 5 , 15 , and 30 minutes . finally , cells were washed with ice - cold pbs and fluorescence intensity was analyzed by flow cytometry . after 24 hours of incubation in the absence or presence of 10  m of ouabain , monocytes were collected and cocultured with lymphocytes from a second donor for 5 days in 96-well round - bottom plates ( final volume of 200  l ) . the number of lymphocytes in this mixed culture was fixed at 10 cells per well in all conditions evaluated . for a 1  :  1 ( monocyte  :  lymphocytes ) ratio , 10 monocytes were added to the culture and , for a 1  :  10 ( monocyte  :  lymphocytes ) ratio , 10 monocytes were added to the culture . we also cultured monocytes ( 10 , as used in 1  :  1 ratio mixed cultures and 10 cells / well , as used in 1  :  10 ) and lymphocytes ( 10  cells / well , as used in both ratio mixed cultures ) alone as control proliferation . for a positive control , lymphocytes were cultured with 5  g / ml of pha ( data not shown ) . cell proliferation was assessed after these 5 days by [ h]thymidine incorporation . for that , an additional 18-hour pulse with 0.5  ci of [ h]thymidine per well was performed . after pulse , cells were collected on filter papers and nonincorporated [ h]thymidine was removed by washing the filter papers with water . after 24 hours of incubation in the absence or presence of 10  m of ouabain , culture supernatants were harvested and stored at 20c until cytokine assay . production of il-1 and tnf- was measured using human il-1 matched antibody pairs for elisa and human tnf- matched antibody pairs for elisa ( bender medsystems , austria ) . production of il-10 and tgf-1 was measured using human il-10 elisa and human tgf-1 elisa ( bioscience , usa ) . production of vegf was measured using human vegf elisa development kit ( peprotech , usa ) . after 24 hours of incubation in the absence or presence of 10  m of ouabain , cells were incubated with 1  mg / ml fitc - conjugated dextran ( sigma , usa ) for 1 hour at 37c or 4c ( for control endocytic activity ) . cells were then washed with ice - cold pbs to remove free dextran particles and fluorescence was analyzed by flow cytometry . the statistical analysis of the data was determined by two - tailed paired t - test or repeated measures anova with dunnett 's posttest , with the exception of cytokine production that was determined by wilcoxon matched pairs test . most experiments were performed at least 3 times with distinct subjects and , when this was not done , the number of experiments is indicated in the figure legend . to investigate the impact of ouabain on monocyte activation induced by adhesion , we analyzed the expression of cd69 , hla - dr , cd80 , cd86 , and cd40 . these molecules may be used as activation markers , since their expression may be induced or simply upregulated upon the activation of monocytes by different stimuli . our results show that hla - dr and cd86 were already expressed by the majority of peripheral blood monocytes , whereas cd80 and cd40 were present only in a very small percentage ( approximately 1% ) of these cells . in addition to that , we observed that monocytes cultured for 24 hours , in the absence of ouabain , expressed higher levels of hla - dr and cd86 per cell ( approximately a twofold increase ) compared to monocytes in the pbmc fraction . moreover , there was an increase in the percentage of monocytes expressing cd80 and cd40 ( sixfold for cd80 and sixteenfold for cd40 ) following the incubation of cells for 24 hours in the absence of ouabain . on the other hand , the percentage of monocytes expressing cd69 diminished ( approximately a threefold decrease ) ( data not shown ) . ouabain , at the concentration of 10  m , promoted an increase on the percentage of cd69 monocytes ( figure 1(a ) ) and a very small increment on the mean density of cd69 molecules per cell ( data not shown ) . an increase was also observed on the percentage of monocytes expressing cd80 after 24 hours of incubation with ouabain ( figure 1(b ) ) , whereas results with cd40 were inconclusive ( figure 1(c ) ) . hence , ouabain - treated monocytes expressed even higher levels of hla - dr and cd86 than control monocytes ( figures 1(d ) and 1(e ) ) . ouabain concentration of 10  m is the pharmacological dosage and may also be attained after exercise . lower concentrations , such as 10 and 10  m , have been described as capable of inducing physiological changes in other cell types and were also tested in the present work . as observed in figure 2 , the most marked differences on cell were obtained with 10  m , and this concentration was used throughout . modifications induced by ouabain on cell surface molecules expressed by monocytes were studied at different time - points . a significant increase our results suggest that 10  m of ouabain was able to modulate monocyte surface molecules expression . this specific concentration could induce an inhibition of na , k - atpase activity , which could , in turn , impact na - caexchanger and lead to an increase of calcium in the cytosol . a transitory increase in intracellular calcium levels was already apparent 5 minutes after 10  m ouabain exposure . hla - dr , cd86 , cd80 , and cd40 molecules are essential for antigen presentation . hla - dr and these costimulatory molecules interact with their respective receptors on lymphocytes and provide the necessary signals to induce proper t cell stimulation . since ouabain was able to increase the expression of some of these molecules , we sought to evaluate the effect of ouabain on the allostimulatory capacity of monocytes . our results show no significant difference between the proliferation of lymphocytes cocultured with control monocytes and ouabain - treated monocytes in the proportion of 1 monocyte to 1 lymphocyte ( figure 5 ) or 1 monocyte to 10 lymphocytes ( data not shown ) . previous studies have shown that ouabain modulates the production of proinflammatory cytokines , such as il-6 , il-1 , and tnf- [ 1820 ] , by mononuclear cells . however , the role of ouabain on the production of anti - inflammatory cytokines by these cells was not addressed so far . therefore , we next aimed to study ouabain effects on the secretion of different types of cytokines . in accordance with previous studies , ouabain promoted an increase in the secretion of il-1 and tnf- ( figures 6(a ) and 6(b ) ) . nevertheless , the levels of il-10 and vegf measured in the supernatants of cells cultured for 24 hours in the presence of ouabain were also higher than control levels ( figures 6(c ) and 6(e ) ) . regarding tgf1 , we observed variable results , as ouabain promoted an increase of tgf1 levels in 8 different supernatants and a decrease in 2 others ( figure 6(d ) ) . to further investigate the effect of ouabain on monocyte function , the endocytic capacity of monocytes was studied using dextran - fitc particles . our results show an increased dextran - fitc uptake by ouabain - treated monocytes , characterized by an increase in mean fluorescence intensity of these cells ( figure 7(a ) ) . on the other hand , no significant difference was observed on the percentage of dextran - fitc cells , suggesting that ouabain did not affect the number of monocytes engaged with the endocytosis of these particles ( figure 7(b ) ) . it has been previously described that the mannose receptor is related to dextran incorporation . however , in our experiments , no increase in cd206 ( also known as macrophage mannose receptor ) was observed per cell , there was not a significant difference in the number of cells expressing this receptor ( figures 7(c ) and 7(d ) ) . monocytes are mononuclear cells produced in the bone marrow and released in the bloodstream , where they represent approximately 10% of the leukocytes present in the peripheral blood [ 26 , 27 ] . circulating monocytes are a heterogeneous population and can differ in size , granulosity , and surface molecules expression and may also present distinct migratory and functional properties [ 2830 ] . monocytes can be further differentiatedinto macrophage and dendritic cells ; however , monocytes can be activated and can contribute to immunity without differentiation . a previous study from our group has shown that 10  m of ouabain decreases the expression of mcd14 on monocytes after 24 hours of culture . this effect could be a consequence of a reduced transcription of cd14 molecule or an increased cleavage of mcd14 into scd14 . if this last hypothesis is true , this could indicate an impact of ouabain on monocyte activation , since scd14 can be used as a biomarker of monocyte activation [ 32 , 33 ] . our present results supported the hypothesis that ouabain promoted a more activated state in monocytes by showing that , after 24 hours of culture , these cells expressed more cd69 , hla - dr , and costimulatory molecules . however , even though an increase of hla - dr and costimulatory molecules was observed , ouabain did not significantly affect the ability of these monocytes to activate and induce lymphocyte proliferation in allogeneic mixed lymphocyte reactions . our results demonstrated that ouabain increased the amount of dextran particles captured by monocytes , but was not able to affect the number of monocytes that were capable of this endocytosis . the mechanism involved in the increased endocytic ability appears to be independent of cd206 , also known as macrophage mannose receptor ( mmr ) , as ouabain did not increase the expression of this molecule . in accordance with this , kato and coworkers have shown a possible mmr - independent mechanism of dextran - fitc uptake in langerhans cells , as the uptake of dextran - fitc by these cells was not effectively inhibited by mannose . thus , it is plausible that ouabain could be increasing the expression of other molecules involved with particles uptake . regarding the production of cytokines , it has been previously shown that ouabain induced an increase in the production of il-1 and tnf- by mononuclear cells [ 1820 ] . in accordance with these studies in addition to that , there was also an enhancement in il-10 and vegf secretion ; however , we could not observe a clear pattern in tgf1 secretion . we hypothesize that this increase in the production of distinct types of cytokines promotes changes in their balance and may induce diverse responses by different cells . this could explain , at least in part , the complex anti - inflammatory and proinflammatory properties of ouabain . so far , it is not clear what the exact mechanisms that cause the observed effects are . similarly to what has already been described in other cell types , ouabain promoted an increase of intracellular calcium levels in monocytes . moreover , previous results from our group have shown a signaling cascade of ouabain in these cells that involves p38 activation and transactivation of egfr . ouabain regulated src - na , k - atpase interaction , activated src , induced transactivation of egfr , and also activated mapks . activated egfr is an important element in signal transduction networks , including networks of cytokines . therefore , it is possible that ouabain could also induce na , k - atpase inhibition as well as na , k - atpase - mediated signaling in monocytes . nevertheless , other cell signaling mechanisms induced by ouabain have been shown [ 3538 ] and may also be involved in monocyte ouabain - induced signaling cascade and could lead to the observed effects . taken together , these results indicate that ouabain induced a functional state of activation on monocytes , suggesting that it may have an immunomodulatory role on these cells .
ouabain is a steroid capable of binding to and inhibiting na+,-k+-atpase . 
 studies have demonstrated some actions of ouabain on immune cells , which indicated both pro- and anti - inflammatory properties of this molecule . nevertheless , its effects on human monocytes are still poorly understood . 
 thus , the present work investigated effects of ouabain in the activation and function of human adherent monocytes . 
 our results show that there is an increase in intracellular calcium levels already 5 minutes following monocyte treatment with 107  m of ouabain . 
 furthermore , monocytes expressed increased amounts of surface activation markers such as cd69 , hla - dr , cd86 , and cd80 and also presented an augmented endocytic activity of dextran - fitc particles after 24 hours of culture in the presence of ouabain . 
 however , monocytes treated with ouabain did not have an increased stimulatory capacity in allogeneic mixed leukocyte reaction . 
 ouabain - treated monocytes produced higher levels of il-1 and tnf- as reported before . 
 a novel observation was the fact that ouabain induced il-10 and vegf as well . 
 collectively , these results suggest that ouabain impacts monocyte activation and modulates monocyte functions , implying that this steroid could act as an immunomodulator of these cells .