as a therapy for hypercholesterolaemia , statins have been used clinically for over two decades . however , over the last decade , immunosuppressive effects have also been demonstrated which are independent of their cholesterol - lowering properties .
statins in vitro modulate cell adhesion through effects on endothelial cells and leukocytes , via blocking activation of lfa-1 and decreasing icam-1 and mcp-1 expression on activated leukocytes and endothelium [ 24 ] .
statins have been shown in vitro and in vivo to reduce leukocyte motility , migration , and infiltration . as compared to cyclosporine , statins were effective in reducing leukocyte infiltration in a rat model of allograft rejection .
they also inhibit the nf-b pathway , involved in transcriptional regulation of cytokines , chemokines , and adhesion molecules .
statins have been shown to upregulate suppressor of cytokine secretion ( socs ) 3 and socs 7 which , in turn , downregulate il-23 and il-6 production , thus decreasing il-17 production . clinical studies of statins in patients with immune - mediated diseases such as rheumatoid arthritis ( ra ) , multiple sclerosis , and organ rejection following transplantation have shown conflicting results due to different statins being used in those studies and often open or retrospective studies involving small patient numbers [ 9 , 10 ] .
immunomodulatory effects in experimental autoimmune encephalitis ( eae ) have been observed with atorvastatin attenuating murine eae , and it was found that atorvastatin upregulated il-4 , il-5 , and il-10 and downregulated il-2 , il-12 , ifn- , and tnf-. however , in another study investigating atorvastatin in murine experimental autoimmune uveitis ( eau ) , atorvastatin was found not to modulate the immune response despite histological grading suggesting mildly decreased inflammation , whereas in murine eau attenuated by lovastatin , there was a decrease in ifn- production but no effect on th2 cytokines . in a lewis rat model , eau
was decreased in severity by both atorvastatin and lovastatin , even when given after disease onset . with both of these statins
, there were decreases in clinical and histological disease scores , antigen responsiveness , and ifn- production .
simvastatin was reported to decrease cytokine production , including il-10 , in a murine model of collagen - induced arthritis   and in a murine lupus model , it decreased serum tnf- and ifn- levels but increased transcription of il-4 and il-10 .
overall , there appear to be a range of immune - related effects by statins depending on species , model , and cell type investigated .
anti - inflammatory therapy for intraocular inflammation often requires use of corticosteroids , yet these can have severe side effects in the eye including raising intraocular pressure , cataracts , and glaucoma . to reduce these effects ,
steroid - sparing agents are also used including cyclosporin a ( csa ) , mycophenolate , and rapamycin .
however , these drugs all have systemic side effects which limit their use in the long - term management of chronic disease . in chronic immune - mediated conditions such as ra and systemic lupus erythematosus ( sle )
, there is an associated increased premature atherogenesis and cardiovascular disease risk secondary to inflammatory processes [ 1719 ] .
in addition , patients with uveitis who are treated with steroids and immunosuppressive agents such as cyclosporine and mycophenolate have an increased risk of developing cardiac disease .
therefore , in these cases , the combined cholesterol - lowering and anti - inflammatory properties of statins may be clinically very useful .
the aim of this study was to determine whether individual statins exert immunosuppressive effects on t cells equivalent to those of conventional immunosuppressive agents in vitro .
the effects of atorvastatin , lovastatin , and simvastatin on normal human whole blood - derived t - cell viability , proliferation , and cytokine responses were studied and compared to dexamethasone , csa , mycophenolate , and rapamycin .
all drugs were dissolved in either dimethylsulphoxide ( dmso ) , rpmi 1640 ( dutch modification ) , and/or 100% ethanol .
, ny , usa ) was prepared as a 1  mm stock solution ; lovastatin ( calbiochem , nottingham , uk ) at 40  mm ; simvastatin ( msd , nj , usa ) at 10  m ; dexamethasone ( chauvin , kingstonupon - thames , uk ) at 400  g / l . ; mevalonate ( sigma - aldrich company ltd , dorset , uk ) at 0.5  m ; mycophenolate mofetil ( roche products limited , hertfordshire , uk ) at 10  mm ; rapamycin ( wyeth , pfizer , berkshire , uk ) at 0.1  mg / ml ; cyclosporin a ( sandoz , novartis pharmaceuticals , surrey , uk ) at 1  mg / ml . peripheral blood was obtained from 16 healthy donors ( all working at the institute / moorfields ) with informed consent ( mean age ( range ) , 34.3 ( 22.546.7 ) years ; six males ) .
exclusion criteria included a history of autoimmune disease , atopy , haematological disorder , or current usage of systemic medication .
the protocols used in this study were reviewed and approved by the local ethics committee .
all studies involving human subjects were conducted according to the tenets of the declaration of helsinki .
all assays were performed in rpmi 1640 ( dutch modification ) supplemented with 2  mm l - glutamine , 10  g / ml gentamycin , 20  m 2-me , nonessential amino acids , sodium pyruvate , and 10% human ab serum . peripheral blood mononuclear cells ( pbmc )
cells were incubated for 10  min in a 37c water bath with 7.5  m cfse ( molecular probes , uk ) in serum - free medium .
1  ml cold stop buffer ( 10% fcs in rpmi ) was then added before incubation at room temperature for 30  min .
cells were washed once with rpmi before being resuspended at 2  10/ml in t - cell medium for 5 days with or without atorvastatin , lovastatin , simvastatin , rapamycin , mycophenolate , csa , and dexamethasone .
t cells were stimulated with anti - cd3 ( 10  ng / ml ; clone hit3a ) and anti - cd28 ( 5  ng / ml ; clone cd28.2 ) antibodies ( pharmingen ) . all drugs or vehicle controls were added at start of culture , except csa , which was added 2  h prior to stimulation since no effect was seen with csa if added at time 0 .
two - color flow cytometry was performed ( facscan ; becton dickinson , oxford , uk ; bd ) .
gates were set on viable lymphocytes according to forward ( fsc ) and side scatter ( ssc ) .
listmode data was generated using cellquest acquisition software on 1525,000 events ( bd ) . for viability assays ,
all lymphocytes were gated to assess level of nonviable propidium iodide expressing ( pi ) cells . for proliferation , nonviable pi cells were excluded .
the total numbers of events were determined by analyzing the data using dot plots and rectangular regions to define the cell populations .
histograms were used to track the divisions of cfse - labeled cells enabling identification of the percentage of divided ( proliferated ) cells .
concentrations of statins required to achieve maximal inhibition of proliferation whilst maintaining viability were identified and used in a multiplex bead array to determine the effects of three statins on cytokine production .
100  l of heparinised whole blood was cultured with or without simvastatin , lovastatin , atorvastatin , dexamethasone , csa , mycophenolate , and rapamycin and stimulated with pma ( 50  ng / ml ) and ionomycin ( 1  g / ml )
supernatants were harvested at 18  h , centrifuged to remove cells , and stored at 70c .
multiplex bead cytokine arrays were conducted with a 10-plex kit ( bender medsystems , ebiosciences , hatfield , uk ) as per the manufacturer 's instructions .
supernatants were analyzed simultaneously for ifn- , il-1 , il-2 , il-4 , il-5 , il-6 , il-8 , il-10 , and tnf-. the lowest levels of detection were il-1 ( 6.2  pg / ml ) ; il-2 ( 6.3  pg / ml ) ; il-4 ( 5  pg / ml ) ; il-5 ( 5.8  pg / ml ) ; il-6 ( 14.8  pg / ml ) , il-8 ( 58.6  pg / ml ) ; il-10 ( 7.3  pg / ml ) ; ifn- ( 5.7 
il-17a was assayed by elisa ( r&d systems , abingdon , uk ) , with a minimum level of detection of 62.5  pg / ml .
whole blood cells were stimulated with pma / ionomycin , and 10  g / ml brefeldin a was added prior to culture for 18  h in the presence or absence of drugs .
cd3 t cells were stained intracellularly with mouse anti - human ifn--fitc ( clone 4s.b3 ) and rat anti - human il-10-pe ( clone jes3 - 9d7 ; both bd ) as previously described .
all intra - assay comparisons of means were analysed using student 's t - tests and for inter - assay data by mann - whitney u tests .
kruskal - wallis tests were used for multiplex cytokine detection analysis for 6 donors , with nonparametric post hoc comparisons . in experiments with csa , only 5 donors were included due to lack of available donors .
significance was reached when p < 0.05 , and this was achieved for several assays , despite relatively small sample sizes .
to determine whether the drugs had any effect on t - cell viability , cells were cultured in the presence of drugs , mevalonate , and vehicle controls for 72  h prior to staining for pi ( figure 1 ) .
a range of concentrations were investigated , and viability was generally high with the lowest viability observed at the highest concentrations of lovastatin and atorvastatin .
this was reversed with the addition of mevalonate , a downstream product of hmg - coa reductase ( p < 0.05 ) relative to lovastatin alone , suggesting that these higher drug concentrations reduced cell viability by inhibiting the mevalonate pathway and not via direct toxicity .
there was a dose - dependent inhibition of t - cell proliferation with all statins , with maximal inhibition at 50  m atorvastatin and lovastatin and 100  m simvastatin ( p < 0.01 ; figure 2(a ) ) .
the addition of mevalonate , fully restored proliferation ( figures 2(b)2(e ) insert ) confirming that these modulatory effects were statin - mediated .
a dose - dependent inhibition of anti - cd3/28-induced t - cell proliferation was also seen for the standard immunosuppressive agents with maximal inhibition at 100  m mycophenolate mofetil , 100  m rapamycin , ( figure 2(f ) ) , 100  g / ml dexamethasone , and 100  ng / ml csa ( data not shown ) .
six human donors ' pbmc were included in this study , and the responses were highly variable .
the mean background levels of cytokines secreted by unstimulated cells were subtracted from all wells .
the levels secreted by stimulated cells in the absence of exogenous drugs were as follows : tnf- ( 30.31  13.74  pg / ml ) , ifn- ( 0.66  0.47  pg / ml ) , il-10 ( 337.04  374.8 
pg / ml ) , il-4 ( 4.47  6.64  pg / ml ) , il-1 ( 447.46  236.07 
pg / ml ) , il-5 ( 14.53  13.66  pg / ml ) , il-17 ( 1186.50  599.27 ) , and il-2 ( 1.50  2.45  pg / ml ) .
following stimulation , there was variability in the cytokines produced among the donors , not unexpected in a mixed donor population .
nevertheless , for all six donors , simvastatin significantly increased il-1 ( figure 3 ; p < 0.05 ) , and all three statins inhibited il-17 production ( p < 0.01 ) . since not all donors ' cells exhibited the same cytokine response profile , trends were observed although these failed to reach significance .
atorvastatin reduced il-1 , il-4 , il-5 , and ifn- and increased il-10 , tnf- , and il-2 .
lovastatin increased il-1 and il-5 production and decreased il-10 , tnf- , and ifn- production .
mean levels of ifn- were relatively low , suggesting a low frequency of ifn- secreting cells in the normal donor population . due to the variable cytokine response profiles seen in individual donors
, we investigated whether there were any correlations between the cytokines that were common to the drugs in comparison with cells stimulated in the absence of drug ( pma / ionomycin alone ) .  
stimulation in the absence of statins induced a strong correlation between il-2 and ifn- and between il-6 and tnf-. in contrast , following atorvastatin treatment , il-6 showed a good correlation with il-2 , ifn- , and tnf- , and il-2 correlated strongly with ifn- and tnf- , as did ifn- with tnf-. in the simvastatin - treated group , il-6 correlated with il-2 , ifn- , and il-1 and il-2 with ifn-. finally , the lovastatin - treated group showed correlations of il-6 with ifn- , il-5 with tnf- , and ifn- with tnf-. atorvastatin was selected to investigate its effects on cytokine responses in combination with dexamethasone . in the presence of either drug alone or when added in combination , il-6 , il-8 , ifn- , and il-1 levels were all decreased although these did not reach significance , suggesting that the immunosuppressive effects of each of the drugs were maintained when added in combination ( figure 4 ) .
interestingly , the level of il-10 production was enhanced in the presence of dexamethasone , although this failed to reach significance .
for the production of il-2 , il-5 , and tnf- , the effects of atorvastatin were reversed upon addition of dexamethasone , or with dexamethasone alone , suggesting that this drug combination was antagonistic with respect to some cytokine responses . for the cytokine correlations ( table 1 )
, there was a baseline correlation between il-6 and tnf- following addition of atorvastatin but not in the presence of dexamethasone alone or combined with atorvastatin .
in contrast , the correlation of il-2 with ifn- was maintained in the presence of either atorvastatin or dexamethasone alone but was lost when the drugs were added in combination .
this suggests differential modulatory effects , with the combination of dexamethasone and atorvastatin exerting a distinct pattern of cytokine responses . for a more sensitive method of detecting ifn- ,
there was a significant decrease in intracellular expression of ifn- in cd3 t cells in the presence of the statins ( p < 0.05 for atorvastatin and lovastatin ; p < 0.01 for simvastatin ) .
dexamethasone , csa , mycophenolate , and rapamycin also demonstrated significant decreases in intracellular ifn- expression as compared to stimulated controls ( p < 0.01 ; figure 5 ) .
dexamethasone exerted the most significant reduction in expression of intracellular ifn- , whilst the combination of dexamethasone with atorvastatin also significantly reduced ifn- ( p < 0.05 ) .
statins have been demonstrated to exert anti - inflammatory effects in vitro : reduction in cd11b expression ; inhibition of ifn--induced mhc - ii expression on endothelial cells , macrophages , and therefore t - cell activation ; disruption of lipid rafts essential for t - cell activation . considering the use of statins as an adjunctive therapy for relapsing remitting multiple sclerosis
, the 2010 cochrane review on  statins for multiple sclerosis  concluded that data from double - blinded randomized trials would be needed before licensing for treatment of multiple sclerosis . in spite of the large amount of in vitro data indicating a potential anti - inflammatory role of statins
, there is little evidence for their direct role in modulating human t - cell cytokines .
the concentrations of statins required to inhibit t - cell responses to anti - cd3/28 stimulation used for this in vitro study were selected from titration experiments , and these concentrations have been used in other in vitro studies [ 2527 ] . due to the potency of the in vitro stimulants , higher , nonphysiological concentrations of statins were required , and therefore , we can not extrapolate the data to statin doses used clinically .
nevertheless , these studies can inform on the effects of statins on human t - cell responses . comparing the effects of drugs on t - cell proliferation ,
the statins inhibited proliferation to a comparable level to that achieved with conventional immunosuppressive drugs .
the viability of the cells in our study was reduced in the presence of these statin concentrations , but , following the addition of mevalonate , there was a recovery both in viability and in proliferation , confirming that the inhibitory effect was reversible , and a direct result of statin - mediated inhibition of the hmg - coa pathway .
of the conventional immunosuppressive drugs investigated , dexamethasone exerted the most potent immunomodulatory effect , and its ability to decrease production of il-1 , il-2 , il-6 , il-8 , ifn- , and tnf- is in agreement with previous studies [ 29 , 30 ] .
interestingly , dexamethasone treatment upregulated il-10 production , as reported by others . of course the study design does not permit us to determine the cellular source of the cytokines , and monocytes , which were likely to be present at low numbers , have previously been observed to respond to simvastatin by upregulating ifn- production which , in turn , led to a decrease in il-17 production .
the downregulatory effect of other immunosuppressive drugs ( csa , rapamycin , and mycophenolate ) on ifn- expression is in agreement with other studies [ 29 , 3234 ] .
these drugs also differentially inhibited other cytokines , with csa significantly decreasing levels of il-2 , il-4 , and il-5 ifn- but not il-10 production ( data not shown ) .
in contrast , a decrease in il-10 mrna expression has been reported in a study on csa - treated normal human whole blood following anti - cd3/cd28 t cell costimulation .
however , changes in il-10 mrna expression do not necessarily correlate with the level of protein production , which could explain the contradictory findings .
mycophenolate has recently been demonstrated to have a profound inhibitory effect on th17 cells , and we have not yet investigated this drug for its effects on th17 cells in our model ( in progress ) .
other discrepancies in cytokine responses observed across the literature are easily explained by the different genetic backgrounds of donors used in different studies .
all three statins in this study significantly reduced intracellular ifn- expression , as previously reported for mouse and human t cells [ 15 , 36 , 37 ] . for the other cytokines investigated in this study ,
intracellular expression levels of il-2 , il-4 , and il-10 were not significantly altered ( data not shown ) due to the variable responses between the individual donors and very low levels of il-4 and il-10 expression .
atorvastatin , when given to hypercholesterolemic patients , has been found to decrease pbmc production of tnf- il-1 , il-6 , il-2 , and ifn-  in vitro .
decreased serum il-8 and il-6 levels have been observed with atorvastatin postcoronary artery bypass grafts [ 40 , 41 ] .
more recently , atorvastatin has been reported to reduce the pathogenic production of il-6 and il-10 by activated t cells from sle patients thereby readjusting them toward a more tolerant phenotype . in our study , atorvastatin decreased production of il-17 and intracellular expression of ifn- , in agreement with data from a lewis rat model of experiment autoimmune neuritis   and in balb / c mice with experimental colitis , in which tnf- was also downregulated . in murine
eae treated with lovastatin , reduced levels of il-6 , tnf- , and ifn- with upregulation of il-4 , il-5 , and il-10 were found , suggesting a th2 polarization [ 44 , 45 ] .
we detected significant decreases in production of il-17 and in ifn- expression in response to lovastatin .
simvastatin decreased il-17 and ifn- expression and increased il-1. it has previously been found that simvastatin decreased tnf- in hypercholesterolemic patients   but had inconsistent effects on plasma il-6 levels .
simvastatin was not found to influence tnf- , il-6 , and il-1 receptor antagonist levels in an endotox in induced human in vivo model of low - grade inflammation   but decreased il-8 production by pbmcs of cad patients after 6 months of systemic treatment .
this contrasts with in vitro effects of simvastatin on anti - cd3/anti - cd28-stimulated pbmcs from ra patients with decreased ifn- and il-10 unaffected .
simvastatin also decreased levels of serum il-6 in mouse collagen - induced arthritis   and il-17 gene expression in human ms patients cd4 t cells . due to the clinical use of atorvastatin for controlling cholesterol levels ,
it was selected in this study for possible combinatory effects when added with dexamethasone . however , by comparing atorvastatin , dexamethasone , and dexamethasone / atorvastatin together , no significant differences were found in overall levels of cytokines produced .
however , the combination did affect cytokine correlations , suggesting that there might be an additive anti - inflammatory effect .
there is little in the literature to suggest contraindications in terms of side effects , and the combination has been used experimentally in a rat model to reverse dexamethasone - induced hypertension . in conclusion , different statins , with their distinct effects on cytokine responses
, could have clinical applications in specifically targeting those key cytokines relevant to each inflammatory disease process , perhaps as a corticosteroid - sparing therapy , while providing effective disease control .
 hmg - coa reductase inhibitors ( statins ) have been demonstrated to be immunomodulatory for human immune - mediated disease and in experimental models . 
 the aim of this study was to compare statin - mediated immunosuppressive effects on human t - cell responses in vitro with those of conventional immunosuppressives ( dexamethasone , cyclosporin a ( csa ) , mycophenolate , and rapamycin ) . 
 statins ( atorvastatin , lovastatin , and simvastatin ) were investigated for their modulatory effects on human pbmc viability , cytokine profiles , and t - cell proliferation . at concentrations that inhibited anti - cd3/28-stimulated t - cell proliferation ( p < 0.01 ) , simvastatin significantly decreased intracellular cd4 + t - cell expression of ifn- ( p < 0.01 ) to levels similar to those induced by conventional immunosuppressives . 
 atorvastatin and lovastatin also decreased ifn- expression , although to a lesser degree ( p < 0.05 ) . 
 all three statins reduced levels of il-17 production ( p < 0.01 ) . however , in response to anti - cd3/28 stimulation , simvastatin significantly upregulated il-1 production ( p < 0.05 ) . 
 the profile of cytokines produced in response to anti - cd3/28 stimulation was similar when both atorvastatin and dexamethasone were added as compared with dexamethasone alone , suggesting that atorvastatin can synergise with dexamethasone with respect to immunomodulation of cytokines . 
 this data supports the hypothesis of selective statin - mediated immunomodulatory effects on human immune cells . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion