atherosclerosis is a chronic inflammatory disease of the arterial wall in which immune responses play a crucial role . atherosclerotic plaques are characterized by the presence of an inflammatory cell infiltrate mainly composed of macrophages and t lymphocytes that modulate the atherosclerotic process by secreting inflammatory mediators . infiltrating t lymphocytes are activated t cells expressing cd25 on their surface   and predominantly expressing a th1 phenotype in advanced lesions [ 2 , 3 ] . in this context , identifying the antigens responsible for t lymphocyte activation in atherosclerosis may be relevant . accelerated atherosclerosis has been reported in patients with various autoimmune diseases [ 46 ] , suggesting an involvement of autoimmune mechanisms in atherogenesis . although infectious agents have been associated with the activation of immune mechanisms , several lines of evidence suggest that the main antigenic targets in atherosclerosis are modified endogenous structures . different self - antigens or modified self - molecules have been identified as target of humoral and cellular immune responses in patients with atherosclerotic disease thus behaving as dangerous signals able to activate proinflammatory responses . oxidative stress , increasingly reported in these patients , is the major event causing structural modification of proteins . oxidized low density lipoproteins ( ldl ) are the best characterized autoantigen . in particular , it has been demonstrated that about 10% of t lymphocytes infiltrating human atherosclerotic plaques are specific for oxidized ldl . in addition to ldl , other self - molecules modified by oxidative stress become target of autoimmune reaction in atherosclerosis [ 1214 ] . another two categories of autoantigens that have been implicated in atherosclerosis are the stress - induced heat shock proteins and antigens expressed by dying cells [ 15 , 16 ] . cell death in the atherosclerotic plaque may occur by apoptosis or by necrosis [ 17 , 18 ] . the uptake of apoptotic cells by macrophages and some subsets of dendritic cells may induce an anti - inflammatory response and play an important role in maintaining peripheral immune tolerance [ 19 , 20 ] . conversely , the uptake of necrotic cells or even a delayed uptake of apoptotic cells may result in immune activation and risk for the development of autoimmunity .   in a previous study , by the use of a molecular cloning strategy to identify endothelial autoantigens , we provided evidence of serum anti - actin antibodies in patients with carotid atherosclerosis and we suggested that actin is an autoantigenic molecule of potential clinical interest in carotid atherosclerosis . we designed this study to confirm and extend our previous results on the possible role of actin as target antigen of immune reactions in carotid atherosclerosis . for this purpose , we evaluated the proliferative response of circulating t lymphocytes obtained from patients and healthy subjects , stimulated in vitro with actin . we also investigated the ability of actin - specific circulating t lymphocytes to produce the pro - inflammatory cytokines ifn- and tnf- and the anti - inflammatory cytokines il-4 and il-10 . we enrolled 17 consecutive patients with asymptomatic severe or preocclusive carotid - artery stenosis 70% or with symptomatic stenosis undergoing endarterectomy ( cea ) at the sapienza university of rome . patients were grouped according to the histological type of their atherosclerotic plaques following the classification of stary et al . . thirteen patients had type v plaques and 4 patients had type vi plaques . in brief , type v plaques are defined as lesions in which prominent new fibrous connective tissue has formed . type vi plaques are defined as lesions in which disruption of the lesion surface , hematoma , or hemorrhage and thrombotic deposits have developed and may be referred to as complicated lesions . exclusion criteria for patients were recent infections ( < 1 month ) , autoimmune diseases , malignancies , and inflammatory diseases before enrollment . the inclusion criteria for healthy subjects were no history of myocardial infarction , coronary bypass , coronary angiography with angioplasty or stenting or both , cerebrovascular accident , or peripheral vascular disease all hematological variables including risk factors for atherosclerosis were in the range of  normal  values venous peripheral blood was drawn in heparin tubes from the 17 patients ( before surgery ) and from the 16 healthy subjects . peripheral blood mononuclear cells ( pbmc ) were separated from plasma by density gradient centrifugation ( lympholyte , cedarlane , on , canada ) and were used in the proliferation assay . triplicate cultures of pbmc ( 1  10  cells / ml ) were stimulated for 7 days with rabbit muscle actin ( sigma - aldrich , milan , italy , 20  g / ml ) , phytohemagglutinin ( pha , burroughs wellcome co. , beckenham , uk , 2  g / ml ) as a positive control of the assay , or human serum albumin ( hsa , sigma - aldrich , 10  g / ml ) as a negative control , or left unstimulated . endotoxin contamination in actin , as determined by the quantitative chromogenic limulus amebocyte lysate assay ( qcl-1000 , biowhittaker , walkersville , md ) , was < 0.03 endotoxin units/g of protein . to neutralize a possible endotoxin effect , all cells were cultured in the presence of polymyxin b ( 10  g / ml , sigma - aldrich ) . cell proliferation was assessed by h - methyl - thymidine incorporation assay as previously described . the proliferative response was expressed as stimulation indices ( si , ratio between the mean cpm in stimulated cultures and that in unstimulated cultures ) . the mean stimulation index in healthy subjects + 3 standard deviations was taken as the threshold level for positivity . ifn- , tnf- , il-4 , and il-10 concentrations in culture supernatants of circulating t lymphocytes were quantified with commercially available enzyme - linked immunosorbent assay ( elisa ) sets ( opteia set , bd biosciences , ca , usa ) as recommended by the manufacturer . the limits of detection were 1  pg / ml for ifn- , 2  pg / ml for tnf- , il-4 , and il-10 . mann - whitney u and wilcoxon nonparametric tests were used to investigate the significance of unpaired and paired data . all the covariates were examined in univariate analyses as predictors for actin - specific cellular response . fisher 's exact test and mann - whitney u test were used to evaluate the differences in discrete and continuous clinical characteristics between patients ' groups . in our selected healthy subject population , we determined a mean si value of 1.13 and a sd of 0.29 and we calculated the value of 2.0 as the cutoff level for positivity . actin induced a proliferative response in 8 of 17 ( 47% ) patients ' pbmc samples , with si ranging from 2.6 to 21.1 ( figure 1 ) . the difference between the si mean values in patients and healthy subjects was statistically significant ( 4.4 versus 1.1 , p = 0.02 by mann - whitney u test ) . univariate analysis showed that the presence of diabetes in patients was significantly associated with proliferative response to actin ( p = 0.04 , figure 2 ) . pbmc samples from patients produced higher concentrations of ifn- and tnf- than pbmc from healthy subjects ( figure 3 ) . in patients , ifn- and tnf- concentrations were higher in pbmc samples that proliferated in response to actin than in nonproliferating ones ( figure 3 ) . we found the presence of a positive correlation between ifn- concentrations and si ( p < 10 ; r = 0.71 ) ( figure 4 ) . in this study , we demonstrated for the first time a role of actin as a target autoantigen of cellular immune reactions in patients with carotid atherosclerosis . as observed for other candidate autoantigens , actin induced a proinflammatory th1 activation , characterized by high ifn- and tnf- expression . th1 response , not counteracted by an increase of anti - inflammatory il-4 and il-10 production , may contribute to tissue damage , endothelial dysfunction , and systemic inflammation . further characterization of these will establish whether they are a regulatory population able to counteract . our results support previous findings indicating that inflammatory autoimmune reactions are not exclusively localized within atherosclerotic lesions but can also contribute to systemic inflammation in patients with atherosclerosis [ 1214 ] . distinct cytokines promote pro- as well as antiatherogenic processes , thus modulating plaque development and clinical outcome [ 14 , 25 , 26 ] . ifn- and tnf- mediate proatherogenic processes by promoting monocyte activation and by influencing collagen synthesis and expression of adhesion molecules , tissue factor , and matrix metalloproteinases [ 27 , 28 ] . our finding on actin - specific t - cell activation is in line with a previous study where we identified actin as a candidate autoantigen of humoral immune response in patients with carotid atherosclerosis . it can polymerize in the presence of atp and its structure is remarkably conserved during evolution . anti - actin antibodies have been associated with various autoimmune diseases including systemic lupus erythematosus , a disease in which endothelial damage plays a key role . anti - filamentous actin antibodies characterize autoimmune hepatitis type 1 where the binding domain of -actinin on actin was shown to be a predominant actin epitope . anti - actin antibodies were also found in 5285% of patients with autoimmune hepatitis or chronic active hepatitis , in 22% of patients with primary biliary cirrhosis , and in patients with celiac disease and with autoimmune haemolytic anaemia [ 3134 ] . furthermore , nonmuscle -actinin 4 and cytoplasmic -actin were identified as immunodominant ovarian autoantigens involved in ovarian autoimmunity . an interesting and extremely important aspect in autoimmune diseases is to understand how abundant and highly conserved self - proteins can become the antigenic target of autoimmune reactions . it has been shown that apoptotic cancer cells may render actin immunogenic by exposing it on their surfaces [ 36 , 37 ] . in addition , many autoantigens , and in particular actin , represent a substrate for the proapoptotic cysteine proteases . the polypeptides produced in this way can be released into the extracellular space or can be presented as neoantigens , thus generating an autoimmune response [ 38 , 39 ] . interestingly , several studies have shown the presence of apoptotic cells , particularly macrophages and smooth muscle cells , in all stages of atherosclerosis development [ 40 , 41 ] .   in our study , we observed a positive association between the presence of diabetes and the response to actin . pancreatic beta - cell death by apoptosis , which can be induced by multiple stresses , contributes significantly to the pathogenesis of type 2 diabetes . the possibility that diabetes , characterized by increasing oxidative stress and apoptosis , may trigger the autoimmune response to actin is interesting and needs further investigation . a limitation of our study is that it does not provide a causal association between the t - cell response to actin and atherosclerosis in humans . in vivo experimental models our study takes research into the involvement of autoimmune responses in the pathogenesis of atherosclerosis , a small step ahead indicating actin as a candidate autoantigen target of cell - mediated immune responses in a proportion of patients with carotid atherosclerosis . our findings here call for further studies to identify epitopes on actin recognized by specific t lymphocytes .
atherosclerosis is a chronic inflammatory disease of the arterial wall associated with autoimmune reactions . in a previous study 
 , we observed the presence of actin - specific antibodies in sera from patients with carotid atherosclerosis . to extend our previous results we evaluated the possible role of actin as antigenic target of cell - mediated immune reactions in carotid atherosclerosis . 
 peripheral blood mononuclear cells ( pbmc ) from 17 patients and 16 healthy subjects were tested by cell proliferation assay and by elisa for cytokine production . 
 actin induced a proliferative response in 47% of patients ' pbmc samples , with si ranging from 2.6 to 21.1 , and in none of the healthy subjects ' samples ( patients versus healthy subjects , p = 0.02 ) . 
 the presence of diabetes in patients was significantly associated with proliferative response to actin ( p = 0.04 ) . ifn- and tnf- concentrations were higher in pbmc from patients than in those from healthy subjects and in pbmc proliferating to actin than in nonproliferating ones . 
 our data demonstrate for the first time a role of actin as a target autoantigen of cellular immune reactions in patients with carotid atherosclerosis . 
 the preferential proinflammatory th1 activation suggests that actin could contribute to endothelial dysfunction , tissue damage , and systemic inflammation in carotid atherosclerosis .